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Identifying Host-Strain Behavioral Differences of Fall Armyworm in Florida (Lepidoptera: Noctuidae)

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IDENTIFYING HOST-STRAIN BEHAVIORAL DIFFERENCES OF FALL ARMYWORM IN FLORIDA (LEPIDOPTERA: NOCTUIDAE) By CHARLES J. STUHL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Charles J. Stuhl

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To my family: Without them, I would not be the man I am today

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ACKNOWLEDGMENTS 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 iv

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as well. I acknowledge the U.S. Department of Agriculture, Agricultural Research Service for employment, and the opportunity to achieve my goals. v

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TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .........................................................................................................viii ABSTRACT .......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 2 OVIPOSITIONAL PREFERENCE OF HOST-STRAINS TO CORN AND STARGRASS...............................................................................................................6 Introduction...................................................................................................................6 Materials and Methods.................................................................................................8 Strain Isolation and Plant Growth.........................................................................8 Oviposition Bioassay.............................................................................................9 Statistical Analysis..............................................................................................10 Results.........................................................................................................................10 Discussion...................................................................................................................10 3 LARVAL PREFERENCE OF HOST-STRAINS TO CORN AND STARGRASS..18 Introduction.................................................................................................................18 Materials and 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 SUMMARY LIST OF REFERENCES...................................................................................................47 BIOGRAPHICAL SKETCH.............................................................................................53 vi

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LIST OF TABLES Table Page 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 olfactometer 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 vii

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LIST OF FIGURES Figure Page 2-1. Fall armyworm 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. Small Petri dish bioassay............................................................................................37 3-2. Choice cage bioassay..................................................................................................38 3-3. Y-tube olfactometer used in volatile study.................................................................39 3-4. Petri dish passing-over study......................................................................................40 3-5. Wind tunnel layout.....................................................................................................41 3-6. Wind tunnel bioassay..................................................................................................42 viii

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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) By Charles J. Stuhl December 2004 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 (COI) 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. nlemfuensis 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 ix

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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 experiment. 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 Y-tube. 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. x

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CHAPTER 1 INTRODUCTION Fall armyworm (FAW), Spodoptera frugiperda (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 of FAW 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 (Chittenden 1901). 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. 1

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2 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 similarity. 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.

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3 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, Vickery 1929). 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 modern 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).

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4 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 (Berdegu 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

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5 species may not be equipped with the inherent response to host-plants (Carlsson et al. 1999). 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.

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CHAPTER 2 OVIPOSITIONAL PREFERENCE OF HOST-STRAINS TO CORN AND STARGRASS Introduction 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. 1983). 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 6

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7 (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 parents 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

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8 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., #Mk16) lined with a 7 cm x 7.6 cm coffee filter (Bunn, BCF). Holes of ~5.0 mm were placed in the top position 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 souffl 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

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9 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-F10) 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). Oviposition Bioassay 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 chambers environment was set at 23.9 2C, ~ 80% RH with a 14/10 day/night cycle. Two plastic souffl 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

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10 surface of the enclosure was also inspected as a possible surface for oviposition. Six replicates were performed for each strain. Statistical Analysis Analysis of variance (PROC MIXED, Contrasts, Littell et al. 1996) was used to examine variation among oviposition substrates. Results 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). Discussion 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

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11 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 (Hbner) 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 females 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 corn-strain females. 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

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12 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 females ovipositional preference (Pitre et al. 1983).

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13 Table 2-1. Number of corn and rice-strain egg masses recovered in the oviposition bioassay Mean number of egg masses Substrate Corn-strain Rice-strain 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 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.

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14 Figure 2-1. Fall armyworm collection sites

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15 Figure 2-2. Strain isolation bioassay container

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16 Rice-strain Corn Strain 569 bp 497 bp 72 bp 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

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17 Figure 2-4. Dead FAW corn-strain male being used as an oviposition site

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CHAPTER 3 LARVAL PREFERENCE OF HOST-STRAINS TO CORN AND STARGRASS Introduction 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) [Spodoptera frugiperda (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). 18

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19 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

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20 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 Revco 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

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21 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 l-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 #4740C10) 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.

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22 Passing-Over Tests 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 Revco 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

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23 larvae passing through corn to stargrass and through stargrass to corn. The number of larvae on each plant was counted 24 h after introduction. Statistical Analysis 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 Choice Tests 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).

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24 Passing-Over Tests 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 3-7). 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. Discussion 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

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25 (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 dont show an orientation response to specific host-plants (Saxena and

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26 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 Hanson 1984). 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

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27 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 females preference for grass in the ovipositional study and larval preference in the wind tunnel indicates strain orientation toward bermudagrass.

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28 Table 3-1. Mean ( SEM) number of corn or rice-strain larvae collected from corn or stargrass sections in the Petri dish choice bioassay Strain Host-plant Corn Rice Corn 14.4 1.21 a 16.5 0.83 a Stargrass 3.3 1.17 b 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.

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29 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 Empty 5.1 5.1 0.0431 Stargrass 95.9 2.2 Cornstrain Empty 4.1 2.2 0.0495 Corn 99.6 0.3 Empty 0.4 0.3 0.0431 Stargrass 85.7 7.2 Ricestrain Empty 14.2 7.2 0.0495 For each comparison, n = 3, df = 1.

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30 Table 3-3. Percent larvae ( SEM) of both strains selecting either corn or stargrass in the choice cage bioassay Host-plant % Larvae P value Corn 38.2 7.5 Corn-strain Stargrass 61.8 7.5 0.1172 Corn 53.2 8.8 Ricestrain Stargrass 46.8 8.8 0.6015 For each comparison, n = 5, df = 1.

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31 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 Soil 31.6 5.1 0.0209 Stargrass 64.2 7.1 Cornstrain Soil 34.9 7.4 0.0433 Corn 81.1 6.6 Soil 18.9 6.6 0.0209 Stargrass 67.8 5.7 Ricestrain Soil 32.1 5.7 0.0209 For each comparison, n = 3, df = 1.

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32 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 Stargrass 31.8 15.0 0.1102 Corn 53.8 9.0 Rice-strain Stargrass 46.2 9.0 0.5653 For each comparison, n = 7, df = 1.

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33 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 dish bioassay First exposure Plant section selected Larvae P value Corn 18.5 0.47 Corn to Stargrass Stargrass 0.7 0.21 0.0001 Stargrass 16.6 0.79 Stargrass to Corn Corn 2.0 0.72 0.0001 For each comparison, n = 10, df = 1.

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34 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 dish bioassay First exposure Plant section selected Larvae P value Corn 9.0 1.56 Corn to Stargrass Stargrass 8.5 1.61 0.7035 Stargrass 14.2 0.94 Stargrass to Corn Corn 1.8 0.49 0.0001 For each comparison, n = 10, df = 1.

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35 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 First exposure Plant selected % Larvae P value Corn 58.4 8.37 Corn to Stargrass Stargrass 41.6 8.37 0.3472 Stargrass 46.7 8.13 Stargrass to Corn Corn 56.9 8.08 0.4647 For each comparison, n = 5, df = 1.

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36 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 Stargrass 30.4 6.97 0.0163 Stargrass 67.4 15.5 Stargrass to Corn Corn 32.6 15.5 0.4647 For each comparison, n = 5, df = 1.

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37 Figure 3-1. Small Petri dish bioassay

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38 Figure 3-2. Choice cage bioassay

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39 Figure 3-3. Y-tube olfactometer used in volatile study

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40 Figure 3-4. Petri dish passing-over study

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41 Figure 3-5. Wind tunnel layout

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42 Figure 3-6. Wind tunnel bioassay

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CHAPTER 4 SUMMARY 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 identified. 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-strains 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, 43

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44 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 adults 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

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45 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

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46 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 thuringiensis 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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LIST OF REFERENCES Adamczyk, J. J., J. W. Holloway, B. R. Leonard, and J. B. Graves. 1997. Susceptibility of fall armyworm collected from different plant hosts to selected insecticides and transgenic Bt cotton. J. Cotton Sci. 1: 21-28. Becerra, J. X., and D. L. Venable. 1999. Macroevolution of insect-plant associations: The relevance of host biogeography to host affiliation. Proc. Natl. Acad. Sci. 96: 12626-12631. Berdegu, M., S. R. Reitz, and J. T. Trumble. 1998. Host-plant selection and development in Spodoptera exigua: do mother and offspring know best? Entomol. Exp. Appl. 89: 57-64. Berger, R. S. 1963. Laboratory techniques for rearing Heliothis species on artificial medium. Iowa State Col. J. Sci. 37: 163-172. Berlocher, S. H., and J. L. Feder. 2002. Sympatric speciation in phytophagous insects: moving beyond controversy? Annu. Rev. Entomol. 47: 773-815. Carlsson, M. A., P. Anderson, E. Hartlieb, and B. S. Hansson. 1999. Experience-dependent modification of orientational response to olfactory cues in larvae of Spodoptera littoralis. J. Chem. Ecol. 25: 2445-2454. Chang, N. T. 1985. Fall armyworm (Lepidoptera: Noctuidae) orientation and preference for selected grasses. Florida Entomol. 68: 296-303. Chittenden, F. H. 1901. Fall armyworm and variegated cutworm. USDA Tech. Bull. 29, 64 p. Claycomb, G. B. 1954. Notes on the habit of a moth, Laphygma frugiperda (Smith and Abbot). Proc. Louisiana Acad. Sci. 17: 50-51. de Boer, G., and F. E. Hanson. 1984. Foodplant selection and induction of feeding preferences among host and non-host-plants in larvae of the tobacco hornworm Manduca sexta. Entomol. Exp. Appl. 35: 177-193. de Boer, G., and F. E. Hanson. 1987. Differentiation of roles of chemosensory organs in food discrimination among host and non-host-plants by larvae of the tobacco hornworm, Manduca sexta. Physiol. Entomol. 12: 387-398. 47

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48 Diehl, S. R., and G. L. Bush. 1984. An evolutionary and applied perspective of insect biotypes. Annu. Rev. Entomol. 29: 471-504. Futuyma, D. J., and T. E. Philippi. 1987. Genetic variation and covariation in responses to host-plants by Alsophila pometaria (Lepidoptera: Geometridae). Evolution 41: 269-279. Fuxa, J. R. 1989. Seasonal occurrence in Spodoptera frugiperda larvae on certain host-plants in Louisiana. J. Entomol. Sci. 24: 273-285. Glover, T. 1856. Insects frequenting the cotton-plant. U.S. Comm. Patents Rpt. 1855 (Agr.): 64-115, illus. Hanson, F. E., and V. G. Dethier. 1973. Role of gustation and olfaction in food-plant discrimination in the tobacco hornworm, Manduca sexta. J. Insect Physiol. 19: 1019-1034. Hsiao, T. H. 1974. Chemical influence on feeding behavior of Leptinotarsa beetles. pp. 237-248. In L.B. Browne [ed.], Experimental analysis of insect behavior. Springer-Verlag, New York. Ishikawa, S., T. Hirao, and N. Arai. 1969. Chemosensory basis of host-plant selection in the silkworm. Entomol. Exp. Appl. 12: 544-554. Jallow, M. F., M. P. Zalucki, and G. P. Fitt. 1999. Role of chemical cues from cotton in mediating host selection and oviposition behaviour in Helicoverpa armigera (Hbner) (Lepidoptera: Noctuidae). Australian J. Entomol. 38: 359-366. Knipling, E. F. 1980. Regional management of the fall armyworm a realistic approach? Florida Entomol. 63: 468-480. Leonard, M. C. 2003. The Floridians. http://www.floridahistory.org/floridians/textpg.htm Accessed Oct. 2004. Levy, H. C., A. Garcia-Maruniak, and J. E. Maruniak. 2002. Strain identification of Spodoptera frugiperda (Lepidoptera: Noctuidae) insects and cell line: PCR-RFLP of cytochrome oxidase C subunit I gene. Florida Entomol. 85: 186-190. Littell, R. C., G. A. Milliken, W. W. Stroup, and R. D. Wolfinger. 1996. SAS system for mixed models. SAS Institute Inc., Cary, NC. Luginbill, P. 1928. The fall armyworm. USDA Tech. Bull. 34, 91 pp. McMillian, W. O., C. D. Jiggins, and J. Mallet. 1997. What initiates speciation in passion-vine butterflies? Proc. Natl. Acad. Sci. USA. 94: 8628-8633.

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49 Meagher, R. L., and M. Gallo-Meagher. 2003. Identifying host-strains of fall armyworm (Lepidoptera: Noctuidae) in Florida using mitochondrial markers. Florida Entomol. 86: 450-455. Meagher, R. L., and R. N. Nagoshi. 2004. Population dynamics and occurrence of Spodoptera frugiperda host-strains in southern Florida. Ecol. Entomol. 29: 614-620. Mislevy, P., W. F. Brown, L. S. Dunavin, D. W. Hall, R. S. Kalmbacher, A. J. Overman, O. C. Ruelke, R. M. Sonoda, R. L. Stanley, Jr., and M. J. Williams. Florona stargrass. Univ. of Florida, IFAS, Circ. S-362. 13 p. Mitchell, E. R. 1979. Migration by Spodoptera exigua and Spodoptera frugiperda, North American style, pp.386-393. In R. L. Rabb and G. G. Kennedy [eds.], Movement of highly mobile insects, University Graphics, N.C. State University, Raleigh. Nagoshi, R. N., and R. L. Meagher. 2003a. Fall armyworm FR sequences map to sex chromosomes and their distribution in the wild indicate limitations in interstrain mating. Insect Mol. Biol. 12: 453-458. Nagoshi, R. N., and R. L. Meagher. 2003b. FR tandem-repeat sequence in fall armyworm (Lepidoptera: Noctuidae) host-strains. Ann. Entomol. Soc. Am. 96: 329-335. Nagoshi, R. N., and R. L. Meagher. 2004. Seasonal distribution of fall armyworm (Lepidoptera: Noctuidae) host-strains in agricultural and turf grass habitats. Environ. Entomol. 33: 881-889. Nuessly, G., K. Pernezny, P. Stansly, R. Sprenkel, and R. Lentini. 1999. Florida corn insect identification guide. http://fciig.ifas.ufl.edu/index.htm Accessed Oct. 2004. Pashley, D. P. 1986. Host-associated genetic differentiation in fall armyworm (Lepidoptera: Noctuidae): a sibling species complex? Ann. Entomol. Soc. Am. 79: 898-904. Pashley, D. P. 1988. Quantative genetics, development, and physiological adaptation in host-strains of fall armyworm. Evolution 42: 93-102. Pashley, D. P. 1993. Causes of host-associated variations in insect herbivores: an example from fall armyworm. pp. 351-359. In K. C. Kim and B. A. McPheron (eds.), Evolution of insect pests/patterns of variation. John Wiley & Sons, New York, 496 p.

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50 Pashley, D. P., T. N. Hardy, and A. M. Hammond. 1995. Host effects on developmental and reproductive traits in fall armyworm strains (Lepidoptera: Noctuidae). Ann. Entomol. Soc. Am. 88: 748-755. Pashley, D. P., S. J. Johnson, and A. N. Sparks. 1985. Genetic population structure of migratory moths: the fall armyworm (Lepidoptera: Noctuidae). Ann. Entomol. Soc. Am. 78: 756-762. Pashley, D. P., T. C. Sparks, S. S. Quisenberry, T. Jamjanya, and P. F. Dowd. 1987. Two fall armyworm strains feed on corn, rice and bermudagrass. Louisiana Ag. 30: 8-9. Pencoe, N. L., and P. B. Martin. 1981. Development and reproduction of fall armyworm on several wild grasses. Environ. Entomol. 10: 999-1002. Pencoe, N. L., and P. B. Martin. 1982. Grass hosts of fall armyworm larvae: preference and methods of determination. J. Georgia Entomol. Soc. 17: 118-126. Pitman, W. D., S. S. Croughan, and M. J. Stout. 2002. Field performance of Bermudagrass germplasm expressing somaclonal variation selected for divergent responses to fall armyworm. Euphytica 125: 103-111. Pitre, H. N., J. E. Mulrooney, and D. B. Hogg. 1983. Fall armyworm (Lepidoptera: Noctuidae) oviposition: crop preferences and egg distribution on plants. J. Econ. Entomol. 76: 463-466. Price, P. W., C. E. Bouton, P. Gross, B. A. McPherson, J. N. Thompson, and A. E. Weis. 1980. Interactions among three trophic levels: influence on plants on interactions between insect herbivores and natural enemies. Annu. Rev. Ecol. Syst. 11: 41-65. Quaintance, A. L. 1897. The fall armyworm: southern grass worm. Florida Agr. Expt. Sta. Bull. 40: 507-512. Quisenberry, S. S., and F. Whitford. 1988. Evaluation of bermudagrass resistance to fall armyworm (Lepidoptera: Noctuidae): influence of host-strain and dietary conditioning. J. Econ. Entomol. 81: 1463-1468. Ramaswamy, S. B. 1988. Host finding by moths: sensory modalities and behaviors. J. Insect Physiol. 34: 235-249. Rojas, J. C., A. Virgen, and L. Cruz-Lpez. 2003. Chemical and tactile cues influencing oviposition of a generalist moth, Spodoptera frugiperda (Lepidoptera: Noctuidae). Environ. Entomol. 32: 1386-1392.

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51 Saxena, K. N., and L. M. Schoonhoven. 1978. Induction of orientational and feeding preferences in Manduca sexta larvae for an artificial diet containing citral. Entomol. Exp. Appl. 23: 72-78. Saxena, K. N., and L. M. Schoonhoven. 1982. Induction of orientational and feeding preferences in Manduca sexta larvae for different food sources. Entomol. Exp. Appl. 32: 173-180. Schoonhoven, L. M. 1969. Gustation and foodplant selection in some lepidopterous larvae. Entomol. Exp. Appl. 12: 555-564. Showler, A. T. 2001. Spodoptera exigua oviposition and larval feeding preferences for pigweed, Amaranthus hybridus, over squaring cotton, Gossypium hirsutum, and a comparison of free amino acids in each host-plant. J. Chem. Ecol. 27: 2013-2028. Singer, M. 1984. Butterfly-host-plant relationships: host quality, adult choice and larval success. In: pp. 81-88. R. Vane-Wright and P. R. Ackery (eds.), The biology of butterflies. Academic Press, New York. Smits, P. H., M. C. Van Velden, M. Van Devrie, and J. M. Vlak. 1987. Feeding and dispersion of Spodoptera exigua larvae and its relevance for control with a nuclear polyhedrosis virus. Entomol. Exp. Appl. 43: 67-72. Sparks, A. N. 1979. A review of the biology of the fall armyworm. Florida Entomol. 62: 82-87. Thompson, J. N. 1988. Evolutionary ecology of the relationship between oviposition preference and performance of offspring in phytophagous insects. Entomol. Exp. Appl. 47: 3-14. Thomson, M. S., and J. N. All. 1984. The use of oviposition on artificial substrates as a survey tool for the fall armyworm. Florida Entomol. 67: 349-357. Thorsteinson, A. J. 1960. Host selection in phytophagous insects. Annu. Rev. Entomol. 5: 193-218. Tingle, F. C., and E. R. Mitchell. 1977. Seasonal populations of armyworms and loopers at Hastings Florida. Florida Entomol. 60: 115-122. Veenstra, K. H., D. P. Pashley, and J. A. Ottea. 1995. Host-plant adaptation in fall armyworm host-strains: comparison of food consumption, utilization, and detoxication enzyme activities. Ann. Entomol. Soc. Am. 88: 80-91. Vickery, R. A. 1929. Studies on the fall armyworm in the Gulf coast district of Texas. USDA Tech. Bull. 138, 64 pp.

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52 Walton, W. R., and P. Luginbill. 1916. The fall army worm, or grass worm and its control. USDA Farmers Bulletin 752. White, R. W., and P. Busey. 1987. History of turfgrass production in Florida. Proc. Florida State Hort. Soc. 100:167-174. Whitford, F., S. S. Quisenberry, and D. J. Moellenbeck. 1992. Nutritional response by rice and corn fall armyworm (Lepidoptera: Noctuidae) strains dietary component substitution in artificial diets. J. Econ. Entomol. 85: 1491-1496. Whitford, F., S. S. Quisenberry, T. J. Riley, and J. W. Lee. 1988. Oviposition preference, mating compatibility, and development of two fall armyworm strains. Florida Entomol. 71: 234-243. Zacharuk, R. Y., and V. D. Shields. 1991. Sensilla of immature insects. Annu. Rev. Entomol. 36: 331-354.

PAGE 63

BIOGRAPHICAL SKETCH 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. 53


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Title: Identifying Host-Strain Behavioral Differences of Fall Armyworm in Florida (Lepidoptera: Noctuidae)
Physical Description: Mixed Material
Copyright Date: 2008

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Full Text











IDENTIFYING HOST-STRAIN BEHAVIORAL DIFFERENCES OF FALL
ARMYWORM IN FLORIDA (LEPIDOPTERA: NOCTUIDAE)















By

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


2004

































Copyright 2004

by

Charles J. Stuhl

































To my family: Without them, I would not be the man I am today















ACKNOWLEDGMENTS

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

Page

L IST O F TA B LE S ................ ....... ... .. .... .. ............. ........ .. ..vii

LIST OF FIGURES ..... .......... ......... ........ .... .... .............. viii

ABSTRACT.................................. .............. ix

CHAPTER

1 INTRODUCTION ................... ............................ .......... .. .......... 1

2 OVIPOSITIONAL PREFERENCE OF HOST-STRAINS TO CORN AND
STARGRASS .......................................................6

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


Table Page

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

Figure Page

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
























viii















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)

By

Charles J. Stuhl

December 2004

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

experiment.

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

Y-tube.

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.














CHAPTER 1
INTRODUCTION

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

(Chittenden 1901).

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

similarity.

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,

Vickery 1929).

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






5


species may not be equipped with the inherent response to host-plants (Carlsson et al.

1999).

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.















CHAPTER 2
OVIPOSITIONAL PREFERENCE OF HOST-STRAINS TO CORN AND
STARGRASS

Introduction

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.

1983).

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).

Oviposition Bioassay

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.

Statistical Analysis

Analysis of variance (PROC MIXED, Contrasts, Littell et al. 1996) was used to

examine variation among oviposition substrates.

Results

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).

Discussion

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

corn-strain females.

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).






13


Table 2-1. Number of corn and rice-strain egg masses recovered in the oviposition
bioassay

Substrate Mean number of egg masses'
Corn-strain Rice-strain
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


Miamli-Dade County


a -


Figure 2-1. Fall armyworm collection sites




























Figure 2-2. Strain isolation bioassay container

























V I


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















CHAPTER 3
LARVAL PREFERENCE OF HOST-STRAINS TO CORN AND STARGRASS

Introduction

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.









Passing-Over Tests

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.

Statistical Analysis

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

Choice Tests

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).









Passing-Over Tests

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

3-7).

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.

Discussion

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

Hanson 1984).

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.






28


Table 3-1. Mean ( SEM) number of corn or rice-strain larvae collected from corn or
stargrass sections in the Petri dish choice bioassay

Host-plant Strain
Corn Rice

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.






29


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.


0.0431
Rice- Empty 0.4 0.3
strain Stargrass 85.7 + 7.2
0.0495
Empty 14.2 + 7.2
For each comparison, n = 3, df = 1.






30


Table 3-3. Percent larvae ( SEM) of both strains selecting either corn or stargrass in the
choice cage bioassay






31


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
0.0209
Corn- Soil 31.6 5.1
strain Stargrass 64.2 + 7.1
0.0433
Soil 34.9 + 7.4
Corn 81.1 6.6
0.0209
Rice- Soil 18.9 6.6
strain Stargrass 67.8 5.7
0.0209
Soil 32.1 + 5.7
For each comparison, n = 3, df = 1.






32


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
dish bioassay
First exposure Plant section selected Larvae P value'
Corn to Corn 18.5 0.47
StargrassStargrass0.70.210.0001
Stargrass Stargrass 0.7 + 0.21


Stargrass to Stargrass
Corn Corn
SFor each comparison, n = 10, df = 1.


16.6 + 0.79
2.0 + 0.72


0.0001






34


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
dish bioassay
First exposure Plant section selected Larvae P value'
Corn 9.0 + 1.56
Corn to Stargrass 0.7035
Stargrass 8.5 1.61


Stargrass
Stargrass to Corn Srr
Corn
For each comparison, n = 10, df = 1.


14.2 + 0.94
1.8 + 0.49


0.0001






35


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


First exposure

Corn to Stargrass


Plant selected
Corn
Stargrass


Stargrass
Stargrass to Corn Srr
Corn
For each comparison, n = 5, df = 1.


% Larvae
58.4 + 8.37
41.6 + 8.37
46.7 + 8.13
56.9 + 8.08


P value'

0.3472

0.4647






36



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


Stargrass
Corn


1 For each comparison, n = 5, df = 1.


67.4 15.5
32.6 15.5


0.4647




































Figure 3-1. Small Petri dish bioassay








Ui,
V"' '
r:


Figure 3-2. Choice cage bioassay


mmm












































Figure 3-3. Y-tube olfactometer used in volatile study


F































-.-',. ?-~-


Figure 3-4. Petri dish passing-over study










52 cm





Corn Grass Corn Grass


Height = 15 cm


Figure 3-5. Wind tunnel layout


Airflow

0.250 m/s





























Figure 3-6. Wind tunnel bioassay















CHAPTER 4
SUMMARY

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

identified.

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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BIOGRAPHICAL SKETCH

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.