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Behavioral responses Dalbulus of leafhoppers to visual, chemical, and structural plant characteristics

Todd, Julie Lynne, Ph.D.

The Ohio State University, 1989

UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106

BEHAVIORAL RESPONSES OF DALBULUS LEAFHOPPERS

TO VISUAL, CHEMICAL, AND STRUCTURAL

PLANT CHARACTERISTICS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree of Doctor of Philosophy in the Graduate

School of the Ohio State University

Julie Lynne Todd, B.S., M.S.

*****

The Ohio State University

1989

D issertation Committee:

C.W. Hoy Approved by

L.V. Madden

L.R. Nault iA J f c & y Advisor P.L. Phelan Department of Entomology ACKNOWLEDGEMENTS

Free at last! But as the cage door opens, I see perhaps an even more disturbing existence than the life of a graduate student; one in which I have to be awake before 11:00 AM, and keep normal working hours. There are several people I should thank for giving me the opportunity to experience both worlds. If I had not been exposed to Dr. Ben Foote's enthusiasm for entomology, I would probably be writing novels about the slimy creatures that live under all our beds. My good fortune of working with people excited about their field was further extended to include Skip

Nault, who has helped me grow not only professionally, but also personally; I am now much more tolerant of long-winded people. I am also indebted to my co-advisor, Dave Denlinger, for his supportive comments, and the chance to occasionally be around flies.

To my committee members, Casey Hoy, Larry Madden and Larry Phelan,

I am grateful for comments concerning research projects and manuscripts, and for their discretion in waiting until I turned my back to shake their heads, and wonder how someone like me could get a Ph.D. I also appreciated having Dan Houston suffer through both my oral exams.

Many thanks to Rosa Maria Gimenez, Larry Phelan and Robin Taylor for making me laugh on rare occasions, and to John Flessel and Roger Williams for space in Thorne to conduct experiments. I appreciate all the technical assistance provided by Maxine Johnson, Mabel Kirchner, and Mary Beth Pavuk, and the help provided by Jackie Blackmer, Kirk Larsen and

Astri Wayadande on graphics that require two eyes. The unique friendships of Jackie Blackmer, Claudette Bibro, Andy Chappie, Carl Cruise, Marty

Hilovsky, Rosa Maria Gimenez, C larissa Maroon, Murdick McLeod, Erik Neff,

Larry Phelan, Bill Styer, Astri Wayadande, and Debbie Zombeck have been both challenging and supportive. I am very grateful Jackie Blackmer,

Astri Wayadande, and Ed Zaborski did not murder me before completing this dissertation for not always taking time to be the exceptional friends they've been to me. I enjoyed meeting Letty Nault and Art Bisges, and liv in g with Inger Hoelzle and Olcay Ture in the grad house. During my student life , food and other donations from my mother were appreciated.

I am very glad Dan Markowitz survived his brief transformation into a b ird to be around fo r this great event. His patience concerning my somewhat unrealistic need for personal space has no doubt added to the speedy completion of this research, and a challenging personal relationship.

Lastly, I thank Marion Harris at Kansas State for insightful conversations, and Tom Baker at UC Riverside for taking a giant leap of faith by hiring me as a postdoc in an area I know less about than Physics. VITA

January 31, 1961...... Born - Warren, Ohio

1983 ...... B.S., Biology Kent State University, Kent, Ohio

1983-1985 ...... Graduate Teaching A ssistant, Kent S tate U niversity, Kent, Ohio

1985 ...... M.S., Ecology, Kent State University, Kent, Ohio

1985-1986 ...... Research Technician, Department of Entomology, OARDC/OSU, Wooster, Ohio

1986-1987 ...... Graduate Teaching Associate Department of Entomology, The Ohio State University, Columbus, Ohio

1987-1989 ...... Graduate Research Associate, Department of Entomology, OARDC/OSU, Wooster, Ohio

PUBLICATIONS

Rogers, T.P., B.A. Foote, and J.L. Todd. Biology and immature stages of Chlorops certimus and Epichlorops exilis (Diptera: Chloropidae), stem borers of sedges, (submitted to Ann. Entomol. Soc. Am.)

iv Todd, J.L. and B.A. Foote. 1987. Spatial and temporal d istrib u tio n of shore flies in a freshwater marsh (Diptera: Ephydridae). Proc. Entomol. Soc. Wash. 89: 448-457.

Todd, J.L. and B.A. Foote. 1987. Resource partitioning in Chloropidae (Diptera) of a freshwater marsh. Proc. Entomol. Soc. Wash. 89: 803-810.

Todd, J.L., M.O. Harris, and L.R. Nault. Effects of color stimuli on host-finding by Dalbulus leafhoppers (Homoptera: C icadellidae). (submitted to Entomol. exp. appl.)

Todd, J.L ., L.V. Madden, and L.R. Nault. Spatial and temporal distribution and population dynamics of Dalbulus leafhoppers on maize (Homoptera: Cicadellidae): host associations and pest potential, (submitted to Ecol. Entomol.)

Todd, J.L., P.L. Phelan, and L.R. Nault. Interaction between visual and olfactory stimuli during host-finding by the leafhopper, Dalbulus maidis (Homoptera: Cicadellidae). (submitted to J. Chem. Ecology)

FIELDS OF STUDY

Major Field: Entomology

v TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... ii

VITA ...... iv

LIST OF TABLES...... vii

LIST OF FIGURES ...... ix

DISSERTATION ABSTRACT...... x

INTRODUCTION...... 1

CHAPTER PAGE

I. EFFECTS OF COLOR STIMULI ON HOST-FINDING BY DALBULUS LEAFHOPPERS ...... 4

Introduction ...... 4 M aterials and Methods ...... 6 R e s u lts ...... 14 D is c u s s io n...... 24

II. INTERACTION BETWEEN VISUAL AND OLFACTORYSTIMULI DURING HOST-FINDING BY THELEAFHOPPER, DALBULUSMAIDIS ...... 29

Introduction ...... 29 Materials and Methods ...... 31 R e s u l t s ...... 39 Discussion ...... 45

III. SPATIAL AND TEMPORAL DISTRIBUTION AND POPULATION DYNAMICS OF DALBULUS LEAFHOPPERS ON MAIZE: HOST ASSOCIATIONS AND PEST POTENTIAL ...... 51

Introduction...... 51 M aterials and Methods ...... 54 R e s u l t s ...... 60 Discussion ...... 73 EPILOGUE

LIST OF REFERENCES LIST OF TABLES

TABLE PAGE

1. Pre- and post-contact responses of three Dalbulus species to maize seedlings under laboratory conditions ...... 15

2. Effect of hue on contacts made by three Dalbulus species with vertical models presented in groups of four colors . . 19

3. Effect of value on contacts made by three Dalbulus species with vertical models ...... 22

4. Effect of hue on tenure of three Dalbulus species on vertical models ...... 23

5. Influence of plant extracts on contacts made by D. maidis with visual stim u li ...... 40

6. Influence of maize volatiles on contacts made by D. maidis with visual stim u li ...... 43

7. Post-contact responses of D. maidis to green light when exposed to maize volatiles ...... 44

viii LIST OF FIGURES

FIGURE PAGE

1. Spectral reflectance curves for paints compared to a seedling maize le a f ...... 9

2. Effect of hue on contacts made by three Dal bulus species with vertical m odels ...... 17

3. Observation chamber for testing the response of D. maidis to plant extracts ...... 33

4. Mean growth stages of maize in relation to the spatial and temporal distribution of three Dal bulus species ...... 57

5. Abundance of three Dalbul us species on maize over time . . . 61

6. Vertical distributions of three Dalbul us species on maize leaves over time ...... 65

7. Horizontal distributions of three Dal bulus species on maize structures over time ...... 68 DISSERTATION ABSTRACT

BEHAVIORAL RESPONSES OF DALBULUS LEAFHOPPERS

TO VISUAL, CHEMICAL, AND STRUCTURAL

PLANT CHARACTERISTICS

Julie Lynne Todd, Ph.D.

The Ohio S tate University, 1989

Professor Lowell R. Nault, Advisor

The effects of color stimuli on host-finding behaviors of three

Dal bul us species were determined by comparing responses to maize seedlings with those to vertical models varying in hue or value. Each species made more contacts with yellow than with other colors, and responses to a series of neutrals indicated contacts were elicited primarily because of hue, and not changes in value. Color also affected post-contact responses by significantly increasing the tenure of D. maidis with yellow. However, more leafhoppers made contacts with maize than with models, and individuals stayed longer on maize than on models.

The relationship between visual and chemical stimuli during host- finding by the maize-specializing leafhopper, D. maidis, was examined.

The number of contacts made by adult leafhoppers with green light increased in the presence of maize volatiles. More contacts were made with green light than with white light of similar intensity in the absence

x of olfactory stimuli; however, maize volatiles acted as a synergist by increasing the number of contacts with green light. Maize volatiles also significantly increased the stationary time of leafhoppers on green light, but not moving time, the distance traveled, or the speed while moving.

D. maidis exhibited a neutral response to volatiles from gamagrass, a marginal host, and a negative response to v o la tiles from a nonhost, sorghum.

The spatial and temporal distribution and population dynamics of three Dalbulus species were monitored on maize from the seedling stage through maturation and senescence in the greenhouse. Both D. maidis. a maize specialist, and D. gel bus, a gamagrass specialist occasionally found on maize in the field, completed two generations, but only D. maidis exhibited a population increase, and moved upward and into maize whorls in large numbers. D. ouinouenotatus. an exclusive gamagrass specialist in the field, produced one generation on maize, remained low on plants, and did not track the whorl. INTRODUCTION

Host-finding and accepting by a herbivorous insect involve a sequence of behavioral responses to both external and internal stimuli (Miller &

Strickler, 1984). Responsiveness to multiple plant stimuli (external) may aid the insect in rapidly and successfully discriminating host from nonhost plants. If the insect also possesses certain r-strategist characteristics, such as high fecundity, rapid development, and high mobility, its potential to become a pest may be enhanced relative to species lacking such characteristics. The Neotropical leafhopper genus

Dalbulus (Homoptera: Cicadellidae) is composed of 11 species which use maize, Zea mays L., or its wild relatives, the teosintes (Zea species) and the gamagrasses (Tripsacum species), as hosts (Nault and DeLong, 1980;

Nault, 1983; Nault, 1985). Only one species, D. maidis (DeLong &

Wolcott), is considered a serious pest in Latin America, primarily by serving as a vector of maize-stunting pathogens (Nault, 1980; Nault et al., 1981), but also by possessing the r-strategist characteristics mentioned above (Nault & Madden, 1985). Field and laboratory studies conducted on distribution, phylogeny, reproductive behavior, population dynamics, and vector ecology (Nault, 1985 and refs, therein) have provided considerable insight into how D. maidis differs from its nonpest congeners. However, nothing is known about host-finding or accepting by

Dalbulus species. Therefore, the three chapters in this dissertation

1 address the role of visual, chemical and structural plant characteristics

in host-finding and/or accepting by Dalbulus leafhoppers. In order to

provide more insight into differences between pest and nonpest Dalbulus. most studies were conducted using three species: D. maidis. a maize

specialist, D. gel bus DeLong, a gamagrass specialist that is occasionally

found on maize, and D. ouinauenotatus DeLong & Nault, an exclusive gamagrass specialist in the field (Triplehorn & Nault, 1985). All three

species can be reared and maintained in large numbers on seedling maize

in the laboratory (Nault & Madden, 1985).

My hypothesis was that responsiveness to multiple plant stimuli could

have a dramatic influence on host-finding and accepting by Dalbulus

leafhoppers; therefore, the goals of this research were: (1) to determine

the plant characteristics used by leafhoppers during host-finding and

accepting, and (2) to determine if behavioral responses of Dalbulus

leafhoppers to maturing maize could have an influence on pest potential.

Plants provide an array of visual, olfactory, gustatory, and mechanical stimuli that can be used by insects during host-finding and

accepting (Scriber & Slansky, 1981; Prokopy & Owens, 1983; V isser, 1986).

Within the Homoptera, the visual detection of plants has been extensively

examined for aphids, with many species attracted to yellow and green during orientation (Prokopy & Owens, 1983 and refs, th erein ). In

contrast, the behavioral responses of leafhoppers to color rarely have

been examined (Saxena & Saxena, 1974). Most of the studies on leafhopper

color responses have completely bypassed behavioral analyses, and focused

on the attractiveness of variously colored traps (Meyerdirk & Oldfield,

1985; A1verson et a l., 1977). Therefore, Chapter I examines the effects 3 of color stimuli on the pre- and post-contact responses of Dalbulus species to painted models differing in hue (dominant wavelengths) or value

(total amount of light reflected) by comparing the responses to those with maize seedlings.

Herbivorous insects also may find a host by responding to airborne chemicals (Visser, 1986 and refs, therein). As with visual stimuli, most of the research on host odor perception has been conducted with aphids

(Pettersson, 1970; Chapman et a l ., 1981; Visser & Taanman, 1987). Only two species of Empoasca leafhoppers have been demonstrated to respond to plant volatiles during orientation (Saxena & Saxena, 1974; Saxena et al.,

1974). Therefore, Chapter II examines the relationship between visual and olfactory stimuli during host-finding by D. maidis. and quantifies behavioral responses during both orientation and arrestment.

After finding a potential host, an insect receives contact stimuli that may aid it in determining the suitability of a plant for feeding or oviposition. The effect of intraplant variation (e .g ., in morphology and nutrition) on aphid population dynamics has been studied fo r several species (Summy & Gilstrap, 1982; Jepson, 1983; Webster et al., 1983). In contrast, studies on leafhopper within-plant distribution and population dynamics on a single plant in space and time rarely have been conducted

(S tilin g , 1980; Johnson et a l., 1988). Chapter III examines the spatial and temporal distrib u tio n and population dynamics of three Dalbulus species on maize from the seedling stage through senescence, and addresses how the behavioral responses of each species to maturing maize can explain field host associations and influence pest potential.

x ttX * CHAPTER I EFFECTS OF COLOR STIMULI ON HOST-FINDING BY DALBULUS LEAFHOPPERS Introduction

Phytophagous insects use a variety of cues during host-finding and accepting (Miller & Strickler, 1984), and although olfactory and gustatory cues have been considered of major importance in these processes (Seriher

& Slansky, 1981; Visser, 1986), the role of vision is becoming more widely recognized (Prokopy & Owens, 1983). Visual detection of plants by insects may involve the use of color stimuli. Color can be defined by three attributes referred to as hue, value, and saturation. Hue is determined by dominant wavelengths. Value (brightness, shade) indicates the total amount of light reflected over a range of wavelengths, and saturation

(chroma, tint) refers to the spectral purity of the reflected light.

The responses of homopterans to color have been extensively studied fo r aphids, with most species strongly attracted to green and yellow

(Kennedy e t a l ., 1961; Kring, 1967; Moericke, 1969; Hodgson & Elbakhiet,

1985). Studies on leafhopper color responses are rare, and the few studies th a t have been conducted have focused on evaluating the effectiveness of colored traps (Alverson et al., 1977; Meyerdirk &

Oldfield, 1985), or suggested that color may only influence pre-alighting behaviors (Saxena & Saxena, 1974).

4 The Neotropical leafhopper genus Dalbulus. composed of 11 species, is suggested to have evolved closely with maize (Zea mays L.) and its wild relatives, the teosintes (Zea species) and the gamagrasses (Tripsacum species) (Nault, 1985). Central and southern Mexico is the center of greatest diversity, and is believed to be the center of origin, for

Dalbulus and their hosts. Only one species, D. maidis (DeLong & Wolcott), is considered a serious maize pest throughout most of Latin America, primarily by serving as a vector of three maize-stunting pathogens, but also by differing from its nonpest congeners in several ecological traits

(Nault, 1985; Nault & Madden, 1985; Nault, 1990). Although more is known about Dalbulus than any other Neotropical leafhopper genus, behavioral differences in host-finding among species have not been studied, and may contribute to the pest status of D. maidis. Host-finding is defined according to Miller & Strickler (1984) as behavioral events leading from movement influenced by noncontact cues to sustained contact with a potential host.

In a series of laboratory experiments, I examined the effects of color stimuli on host-finding by D. maidis. a maize specialist, D. gel bus

DeLong, which uses both maize and Tripsacum. and D. ouinauenotatus DeLong

& Nault, a Tripsacum sp ecia list (Nault, 1985), by comparing responses to maize seedlings with those to painted models of various hues and values. 6

M aterials and Methods

Adult leafhoppers between 1 and 4 wk old were obtained from laboratory colonies established from individuals originally collected in

Mexico 2 to 5 yr prior to use in this study (Nault, 1985). Colonies were maintained in the laboratory for 20-60 generations on 4-6-leaf seedling maize (cv. Aristogold Bantam Evergreen), and kept in a rearing room held at 26 ± 2° C, 65-85% RH, and a 14L:10D cycle. Both D. maidis and D. gel bus are collected from maize in the fie ld , and although D. auinauenotatus has only been collected from Tripsacum species (Triplehorn & Nault, 1985), it can be reared and maintained on seedling maize in the laboratory (Nault

& Madden, 1985).

Unless specified, all experiments were conducted in a still-air

Plexiglas™ observation chamber (70 cm x 70 cm x 122 cm) located in a walk- in environmental chamber illuminated by 28 Cool White fluorescent bulbs

(1500-W). White sheets were placed over a metal framework inside of the walk-in chamber to provide a uniform background. The temperature inside the Plexiglas chamber was 26 ± 2° C and the RH was 50-65%. Experiments were replicated three times, using 50 unsexed individuals/replicate, unless otherwise specified. Replicates within each experiment were completed for one species before beginning experiments on another species.

Experiment 1: Pre- and post-contact responses to seedling maize.

Four 4-leaf maize seedlings (cv. Aristogold Bantam Evergreen) were spaced

24 cm from the chamber walls in a rectangular arrangement (21.5-cm x 23 cm). This arrangement was chosen to alleviate edge effects due to the constraints of the chamber. Each seedling was removed from its pot, and soil was washed from the roots. The root system was then placed through a 2.5-cm hole in the black Formica™ chamber floor into a beaker of water to prevent plants from wilting during the experiment. The holes were sealed with foam plugs. Leafhoppers were taken from colonies 1 h after lights on, and transferred into the chamber with an aspirator through a hole 80 cm above the chamber floor. Information was recorded every 0.5- h on the location of individuals in the chamber (e.g., upper, middle, lower th ird of the chamber, each 40 cm in height), and i f in the lower th ird , whether individuals were on or o ff seedlings. Data also were collected continuously after leafhoppers were released on the methods used to contact seedlings (e.g., jump/fly (unable to distinguish if the wings were opened) from the chamber walls and floor, or walk), u n til most individuals ( > 90%) were on seedlings.

To examine post-contact responses of leafhoppers to maize, one 4-leaf seedling was placed in the center of the chamber, and 20 male or female leafhoppers were released as described above. For the f i r s t 10 individuals contacting maize, data were collected on tenure fo r a 3 h period, with tenure defined as sustained contact with the seedling, regardless of on-plant movement. Individuals leaving the seedling before

3 h were removed from the chamber.

Experiment 2: Effect of hue on leafhopper contacts. Responses of

Dalbulus species to color stim uli were examined using painted models represented by 23 cm high x 1.9-cm diam wooden rods. Although all experiments were conducted under laboratory conditions, co lo r choices were similar to those observed under natural lighting conditions (J. L. Todd,

unpublished data). The effects of hue on leafhopper contacts were

determined by painting models with three coats of the following Testor™

paints (Rockford, IL.), or by mixing paints to obtain special

formulations: sea blue (B) (No. 1172), beret green (G) (No. 1171), light

green (LG) (special formulation), whorl green (WG) (special formulation), yellow (Y) (No. 1169), orange (0) (special formulation), and red (R) (No.

1150). The light green and whorl green hues contained increasing amounts

of yellow, respectively, with the latter closely resembling the color of

seedling maize foliage. The spectral reflectance of each paint and a

seedling maize leaf was measured (400-700 nm) as a percentage of the

reflectance from a white MgC03 standard in a Bausch and Lomb Spectronic 20

equipped with a reflectance attachment (Fig. 1).

The seven models were arranged randomly in the Plexiglas chamber in

a 16.5-cm diam c irc le , with 5 cm between models. The number of contacts with models was recorded for 3 h, with observations generally beginning

between 3 to 4 h a fte r lig h ts on. Models were placed in the chamber prior

to leafhopper release. In another set of experiments, models were

presented in groups^of four colors, and arranged similar to maize

seedlings (see experiment 1). The color sequence used for obtaining

groups was: B-G-LG-WG-Y-O-R. Starting with the first four colors, the

color group was changed by adding the next color to the rig h t in the above

sequence, and eliminating the color at the opposite end. The positions

of the models were randomized, and the number of contacts with models was

recorded for 3 h. Data also were collected on the methods (jump/fly, walk) individuals used to contact models for each group of four colors. Figure 1. Spectral reflectance curves for paints used in experiments 2

and 3 compared to a seedling maize le a f (B= blue, G= green,

LG= light green, WG= whorl green, Y= yellow, 0= orange, R=

red, ML= maize leaf).

9 % Reflectance 20 40 60 0 8 400 iue 1. Figure

450

500 aeegh (nm) Wavelength

550

600

ML 650

700 11

Based on the results of experiment 1, observations on D. maidis and D. gel bus began 3 to 3.5-h afte r release into the chamber, and observations on D. auinauenotatus began immediately. Data were collected in this manner to obtain information when leafhoppers were exhibiting host-finding behaviors (e.g., approaching and contacting models).

Experiment 3: Effect of value on leafhopper contacts. Black, white, yellow and a series of neutral gray models were used to assess the effects of value on leafhopper host-finding. Models were painted with three coats of the following Liquitex™ acrylic paints (Smith & Binney, Inc., Easton,

PA): mars black (BL1.5), neutral grays (NG3, 6, 7, and 8), yellow medium azo (Y8.2), and titanium white (W10). The numbers within parentheses represent value according to the Munsell system of color notation, and indicate that BL1.5 and W10 reflect the lowest and highest percentage of light, respectively, and that NG8 and Y8.2 are very similar in value.

Individuals were exposed to two grays, NG3 and NG6, along with BL1.5 and

W10, or to Y8.2 bracketed in value by NG7, NG8, and W10, and the number of contacts with models was recorded for 3 h. Observations on D. maidis and D. gel bus began 3.5-h after release into the chamber; D. ouinouenotatus was observed immediately. All observations were conducted between 3 to 6 h a fte r lig h ts on. Models were arranged in the chamber similar to maize seedlings (see experiment 1), and their positions were randomized for each replicate. The paints used in this experiment and experiment 2 did not appear toxic because the number of leafhoppers dying during the experiments (< 10%) was similar to that of leafhoppers released into the chamber when i t contained no models (J. L. Todd, unpublished d a ta ). Experiment 4: Effect of hue on post-contact responses. The four models that received the majority of contacts in experiment 2, light green, whorl green, yellow, and orange (see results, experiment 2), were used to assess the effect of hue on tenure of leafhoppers. The design of the experiment was a randomized complete block (n= 10), with a block represented by the four models placed singly in a 53 cm x 25 cm I.D.

Plexiglas cylinder in random order. One leafhopper was released into the cylinder through a hole located 20 cm from the cylinder floor using an aspirator. D. maidis and D. gel bus were released into the large Plexiglas chamber for 3.5 to 6.5-h prior to transfer to the cylinder for timing to predispose them to host-finding behaviors. D. ouinouenotatus were taken directly from colonies, placed in the cylinder, and observed immediately.

For each species, 10 individuals, five of each sex, were timed/color.

Tenure times were recorded using a stopwatch, and timing began when contact with a model was established, and ended when the individual jumped/flew from the model. A leafhopper was given 10 min to contact a model, and if contact was not made, the individual was removed and another individual was introduced.

Statistical Analyses. For experiment 1, significant differences regarding distribution within the Plexiglas chamber, the percentage of contacts made with maize seedlings by jumping/flying or by walking, and tenure time, were determined using Student's t-test (P < 0.05) for each species separately. For experiments 2-4, homogeneity of variances was determined using Bartlett's test (Sokal & Rohlf, 1981). The percentage data from experiments 2 and 3 were arcsin square-root transformed and subjected to Fisher's protected LSD if £ ratios were significant (Milliken 13

& Johnson, 1984). Duration data from experiment 4 were log transformed

before being subjected to Fisher's protected LSD. Significant differences

in tenure of males and females were determined using a two-way analysis

of variance (ANOVA) (P < 0.05). Statistical comparisons were not made

between species because our goal was to compare responses to maize

seedlings with those to painted models, and because leafhoppers had been

removed from natural conditions and maintained in the laboratory for many generations, perhaps diluting interspecific variation in color responses. Results

Experiment 1: Pre- and post-contact responses to seedling maize.

Within 0.5-h after release into the chamber, significantly more D. maidis and D. gel bus were located in the upper third of the chamber compared to the lower third near the four seedlings (Table 1). In contrast, significantly more D. auinauenotatus were located in the lower third of the chamber 0.5-h after release. The mean time until contacts were made with seedlings by D. maidis was 2.5-h after release, by D. gel bus. 1.5-h, and by D. guinauenotatus. 0.5-h. For all three species, more contacts were made with seedlings by jumping/flying than by walking (Table 1).

Most D. maidis and D. oelbus (> 90%) were located on seedlings within 8 h after release, and similar numbers of D. guinauenotatus were located on seedlings 1.5-h after release.

After contacting a seedling, males and females of each species exhibited no significant differences in tenure when observed continuously over a 3 h period (Table 1). Of the 10 individuals/sex observed, no D. maidis or D. gel bus left the seedling after contact. Similarly, there were no significant differences in tenure of male and female D. guinauenotatus. although one female and four males left the seedling before the end of the observations. 15

Table 1. Pre- and post-contact responses of three Dalbulus species to

maize seedlings under laboratory conditions.1

Location in chamber

0.5-h after release

(Mean 9i» in d s.l Mean % contacts Mean tenure (mini

SDecies UDDer lower .iump/flv walk male f emal e

D. maidis 79a 5b 77a 23b 180a2 180a

D. qelbus 69a 3b 87a 13b 180a 180a

D. auinauenotatus 10b 75a 85a 15b 131a 174a

1 Maize seedlings were 4-leaf stage. Experiments were conducted in a Plexiglas chamber divided into three sections (upper, middle, lower) (see text), although data for the middle section are not presented. Means within a species followed by the same letter are not significantly different at P i 0.05 (Student's t-test). Experiments on location in the chamber and leafhopper contacts were replicated three times using 50 individuals/replicate.

2 10 individuals/sex were timed for up to 3 h. 16

Experiment 2: Effect of hue on leafhopper contacts. When the seven models were presented simultaneously, the percentage of contacts with yellow was significantly higher than with other colors for each Dalbulus species (Figs. 2a-c). Whorl green, which was most similar to maize foliage in spectral reflectance (Fig. 1), elicited the second highest percentage of contacts, followed by orange and light green, respectively.

D. maidis contacted orange more than light green (Fig. 2a) while the other two species did not distinguish between these two colors (Figs. 2b, c).

Blue, green, and red received the least contacts, with no significant differences among these colors except for D. maidis. which made no contacts with red.

When models were presented in groups of four colors, a pattern of color choice emerged for each species, with the color reflecting maximally between 500-580 nm (Fig. 1) being contacted if yellow was not present

(Table 2). For example, consider the B-G-LG-WG group (Grl) for D. maidis. in which whorl green received significantly more contacts (69%) than the other colors. Similarly, with the 0-R-B-G (Gr6) and R-B-G-LG (Gr7) groups, orange (87%) and light green (93%) received significantly more contacts, respectively. When yellow was added to the group (Grs 2-5), it significantly dominated the other colors in eliciting contacts.

Because yellow in Gr5 received quantitatively more contacts compared to yellow in Grs 2-4 (Table 2), the percentages of contacts made with the yellow model in this group by jumping/flying or by walking were compared.

There were no significant differences in contacts made with the yellow model by jumping/flying or by walking for D. maidis (58% and 42%, respectively), and D. gel bus (56% and 44%, respectively), but D. Figure 2. Effect of hue on contacts made by a) D. maidis. b) D. gel bus.

and c) D. guinauenotatus with vertical models (B= blue, G=

green, LG= light green, WG= whorl green, Y= yellow, 0=

orange, R= red). Experiments were replicated three times

using 50 individuals/replicate. Means within a species

followed by the same letter are not significantly different

at P £ 0.05 by LSD.

17 Mean % Contacts Mean K Contacts

Figure 2. 19 Table 2. Effect of hue on contacts made by three Dalbulus species with vertical models presented in groups of four colors.1

Mean % contacts / group (Grl

Species Color Grl Gr2 Gr3 Gr4 Gr5 Gr6 Gr7

D. maidis B lc Od 4b lb

G 2c Id 5b 4b

LG 28b 3c 6c 93a

WG 69a 23b 19b 20b

Y 73a 68a 73a 91a

0 7c 6c 8b 87a

R Id lc 4b 2b

D. aelbus B lc 2c 3b lc

G 7c 2d lib 17b

LG 33b 15c lib 78a

UG 59a 22b 32a 27b

Y 61a 44a 58a 77a

0 13b 11c 18b 71a

R 4d 3c 15b 4bc 20

Table 2 (continued)

______Mean % contacts / group (Gr)

Species Color Grl Gr2 Gr3 Gr4 Gr5 Gr6 Gr7

D. ouinaue. B 0c lb 7b 2b

G lc 3b 17b 3b

LG 27b 15b 8b 92a

WG 72a lib 18b 17b

Y 71a 68a 61a 74a

O 6b 20b 23b 67a

R 2c lb 9b 3b

Models were 23 cm high x 1.9 cm diam. B= blue, G= green, LG= light green, WG= whorl green, Y= yellow, 0= orange, R= red. Experiments were replicated three times for each color group using 50 individuals/replicate. Means within a group for each species followed by the same letter are not significantly different at P < 0.05 by LSD. 21

Quinauenotatus made more contacts by jumping/flying than by walking (93% and 7%, respectively) (Student's t-test, P< 0.001).

Experiment 3. Effect of value on leafhopper contacts. Although yellow elicited the majority of contacts, the Dalbulus species tested may not have been stimulated by specific wavelengths, but by the amount of reflected light (Harris & Miller, 1983). However, when each species was exposed to black, white and gray models, the percentages of contacts on the models were not significantly different at P < 0.05 (AN0VA) (Table 3, neutrals). When yellow was presented with white and two gray models bracketing it in value (Table 3, neutrals and yellow), it received significantly more contacts by each species than any of the neutrals (P

< 0.001 for all three species).

Experiment 4: Effect of hue on post-contact responses. In addition to differentially eliciting contacts, hue also affects the duration of contacts by leafhoppers on models (Table 4). There were no significant differences between tenure of males and females within a species (D. maidis. F= 0.397, df= 1,32, £ > 0.25; D. aelbus. F= 0.901, df= 1,32, P >

0.25; D. Quinauenotatus. F= 2.20, df= 1,32, P > 0.10), so data were pooled. D. maidis spent significantly more time on yellow than on the other three colors. Both D. gel bus and D. guinauenotatus spent more time on yellow than on orange and light green, with whorl green intermediate. 22 Table 3. Effect of value on contacts made by three Dalbulus species with vertical models.1

Contacts on neutrals Contacts on neutrals

& yellow

SDecies BL1.5 NG3 NG6 W10 NG7 NG8 Y8.2 W10

D. maidis 17±172 40±30 42±24 1± 1 l±lb 2±lb 95±3a 2±lb

D. aelbus 37±11 25+ 7 31+16 7± 3 5+2b 6±4b 87±6a 2±lb

D. auinaue. 15+15 36±10 29+20 20+20 l±lb l±lb 97±la l±lb

Models were 23 cm high x 1.9-cm diam. BL= black, NG= neutral gray, Y= yellow, W= white. Experiments were replicated three times using 50 individuals/replicate. Numbers represent mean % ± SE. Means within a row followed by the same letter are not significantly different at £ 1 0.05 by LSD.

2 Data for neutrals are not significant at P i 0.05 (ANOVA). For D. maidis. F= 1.04, df= 3,8, P > 0.25); for D. gel bus. £= 2.24, df= 3,8, P > 0.10; for D. guinauenotatus. F= 0.560, df= 3,8, P > 0.50. 23 Table 4. Effect of hue on tenure of three Dalbulus species on vertical models.1

Mean tenure (mini on models

SDecies LG UG YO

D. maidis 2.8b 5.7b 10.4a 2.6b

D. aelbus 3.0b 4.9ab 9.2a 3.5b

D. Quinauenotatus 3.6b 6.4ab 10.1a 3.5b

Models were 23 cm high x 1.9-cm diam. LG= light green, WG= whorl green, Y= yellow, 0= orange. Ten individuals were timed/color. Means within a row followed by the same letter are not significantly different at P < 0.05 by LSD. 24

Discussion

Plant models can be very effective for examining the role of color stimuli in insect host-finding, and for separating relevant color attributes (Harris & Hiller, 1983; Prokopy et al., 1983). However, before responses to models can be clearly interpreted, their validity as plant mimics must be established. Data in the present study indicate that

Dalbulus species made contacts in similar times after release into the chamber, and in similar ways with both maize and models. Depending on the species, individuals began contacting maize within 0.5 to 2.5-h, and times to firs t contacts on models were observed to be similar. All species contacted maize more by jumping/flying than by walking. With models, D. auinouenotatus made significantly more contacts by jumping/flying than by walking, and although D. maidis and D. gel bus also tended to make more contacts by jumping/flying than by walking, the differences were not significant. Based on the similarities in responses of each species to maize and to models, the models served as effective substrates for examining responses of Dalbulus leafhoppers to color stimuli.

The strong responses of each Dalbulus species to yellow suggest they can discriminate foliage-like hues (500-580 nm) from nonfoliage-like hues

(< 500 nm and > 580 nm) (Prokopy & Owens, 1983). Whether or not this discrimination is adaptive for Dalbulus leafhoppers is not known.

Increased responsiveness to yellow aids some aphids in finding young, expanding leaves that are high in nitrogen, and often characterized by a 25 yellow color (Mooney & Gulmon, 1982). Because plants are primarily composed of carbohydrates, nitrogen is in limited supply for many phytophagous insects (McNeill & Southwood, 1978).

Yellow also represents a supernormal foliage-type stimulus to many phytophagous insects because it elicits a greater alighting response than colors more closely resembling preferred hosts (Prokopy & Owens, 1983).

Similarly, Dalbulus leafhoppers made more contacts with a yellow model than with a whorl green model (Figs. 2a-c), although the la tter color is similar to that of seedling maize foliage (Fig. 1). Yellow sticky cards have been used to monitor Dalbulus populations in maize in Mexico and

Costa Rica for several years (L. R. Nault, unpublished data). Although yellow was chosen due to its effectiveness in trapping other homopterans under field conditions (Roach & Agee, 1972; Meyerdirk & Moreno, 1984;

Summy et al., 1986), the present study confirms yellow would be the most effective trap color for monitoring Dalbulus populations. However, whorl green sticky cards may be more selective in that they would continue to trap Dalbulus species, but perhaps exclude other insect species strongly attracted to yellow. Irwin & Goodman (1981) conducted the only documented field study in which trap color closely matched that of a host plant. They used ermine green sticky traps to monitor aphid landing rates in soybean fields, and found the traps neither more nor less attractive to the aphid species under study than soybean foliage. A host plant- specific color response has only been shown for the mealy plum aphid,

Hvalopterous pruni (Geoffr.), which discriminates the foliage of its

Phraamites hosts from nonhost foliage on the basis of saturation

(Moericke, 1969). 26

The data in the present study also indicate that color continues to influence leafhopper behavior after contact with a model, as yellow elicited tenure by all three Dalbulus species as long or significantly longer than other colors (Table 4). However, color alone did not promote tenure similar to that on maize (Table 1), suggesting that volatile and/or contact chemicals may be necessary to maintain tenure, and stimulate acceptance behaviors such as feeding and oviposition (Backus, 1985).

Although all three Dalbulus species I examined were capable of discriminating between various hues and values under laboratory conditions, the degree to which each species uses color stimuli during host-finding in the field may be related to its flight behavior, and the habitat stability of its hosts. When compared to short-lived annuals such as maize, the perennial Tripsacum species provide a more stable habitat for leafhoppers not only between seasons, but also within a growing season. In the spring, growth of young Tripsacum shoots precedes germination of maize seed, and in the autumn, maize leaves senesce and die several weeks before those of Tripsacum (Nault, 1985). Although maize is grown year round with irrigation at low elevations in Mexico, much of the crop is seasonal. Heady & Nault (1985) suggested that these differences in host permanence may affect the flight behavior of Dalbulus species.

They showed that maize specialists were more likely to fly from their hosts when mechanically dislodged compared to Triosacum specialists, and that D. maidis from areas where maize is grown seasonally were more likely to fly than individuals from populations where maize is grown continuously. Because D. maidis and some populations of D. gel bus use maize (Nault, 1985; Triplehorn & Nault, 1985), they must periodically fly 27 to new host locations, and may use visual input (e.g., color stimuli) to aid in distinguishing foliage from nonfoliage. In contrast, Tripsacum specialists such as D. auinquenotatus are less mobile, and may leave their natal host plant only if competitive interactions develop and limit resource availability, or to escape from parasites and predators. As a consequence, visual cues may be used less often by Tripsacum specialists than by maize specialists, although the data show the la tte r also can discriminate between colors (Fig. 2c).

In the present study, I determined additional differences in flight behavior among maize- and Triosacum-specializina species that may be related to habitat stability, and indirectly affect the frequency with which Dalbulus species use color stimuli during host-finding. Not only

are maize specialists more likely to fly than Tripsacum specialists when dislodged from their hosts, they also are more likely to fly up than down

(Table 1). Although maize is generally concentrated in large areas

(cultivated fields), the same ground is not planted to maize from year to year. Flying up and above the plant canopy may increase the probability of D. maidis and D. gel bus dispersing with low level winds, and locating new hosts. This behavior also will expose them to color stimuli from a variety of plant habitats on a fairly regular basis.

In contrast, the Tripsacum hosts of D. guinauenotatus are more stable over time, although patchy in distribution. Individual plants or plant patches are often separated by great distances, and restricted to areas

along steep embankments or arroyos (Nault, 1985). This patchy and

infrequent distribution may make relocation of hosts after a flight less likely for Tripsacum specialists than for maize specialists, and explain 28 why significantly more D. guinauenotatus flew down rather than up when released into the chamber containing maize seedlings (Table 1). The propensity to fly down also may explain, in part, why D. guinauenotatus is not found on maize in the field, but can be reared and maintained on maize seedlings in the laboratory. Because Tripsacum specialists fly down, they will be exposed to color stimuli from other plant habitats less often than maize specialists, but may still use color stimuli during lateral movements within a Tripsacum plant or plant patch, perhaps to distinguish young foliage from that which is mature or senescing. CHAPTER II INTERACTION BETWEEN VISUAL AND OLFACTORY STIMULI DURING HOST-FINDING BY THE LEAFHOPPER, D. MAIDIS Introduction

Current research suggests differences exist between the two suborders of Homoptera, the Sternorrhyncha and the Auchenorrhyncha, in the importance of volatile chemicals during intraspecific communication and host-finding, with the Sternorrhyncha (aphids, whiteflies, mealybugs, psyllids) being much more responsive to odors than the Auchenorrhyncha

(leafhoppers, planthoppers, treehoppers, spittlebugs, cicadas). Olfactory perception of alarm pheromones (Nault & Phelan, 1984 and refs, therein) and sex pheromones (Carde & Baker, 1984 and refs, therein; Eisenbach &

Mittler, 1987) has been demonstrated for several sternorrhynchans.

Olfactory receptors responsive to plant volatiles have been identified from aphids (Bromley & Anderson, 1982; Yan & Visser, 1982), and several aphid species are known to alter their behavior when exposed to host and nonhost odors in the laboratory (Pettersson, 1970; Tamaki et al., 1970;

Visser & Taanman, 1987) and the field (Chapman et al., 1981). In contrast, intraspecific communication within the Auchenorrhyncha is primarily mediated by acoustic signals (Claridge, 1985; Heady et al.,

1986), although some treehoppers respond to alarm pheromones (Nault et al., 1974). Only a few auchenorrhynchans have been shown to possess host

29 30 odor receptors (Klein et a l., 1988), and to respond to plant volatiles during host-finding (Saxena & Saxena, 1974; Khan et al., 1988).

The Neotropical leafhopper genus Dalbulus is composed of 11 species that specialize on maize, Zea mavs L., and its wild relatives, the teosintes (Zea species) and the gamagrasses (Tripsacum species) (Nault,

1985). One species, D. maidis (DeLong & Wolcott), is considered a serious pest in Latin America, primarily by transmitting maize-stunting pathogens

(Nault, 1990). Virtually nothing is known about host-finding behaviors of Dalbulus leafhoppers. In Chapter I, the pre- and post-contact behaviors of three Dalbulus species to maize seedlings and to painted vertical models differing in hue (dominant wavelengths) or value (total amount of reflected light) were compared, and the data showed a strong orientation response of leafhoppers to yellow. Yellow influenced post contact behaviors by increasing the amount of time an individual maintained contact with a model. However, the models provided only color and perhaps structural stimuli, and did not e lic it contacts by as many leafhoppers as maize, and tenure after contact was significantly shorter on models than on maize. These data suggested that additional plant stimuli were needed by Dalbulus leafhoppers during host-finding and/or accepting. In the present study, I examined the response of D. maidis to volatiles from a host, maize, a wild maize relative and marginal host,

Tripsacum dactvloides (L.) L., and a nonhost, Sorghum bicolor (L.) Moench, and determined the relationship between visual and olfactory stimuli during host-finding. 31

Materials and Methods

Leafhoppers. Leafhoppers used in bioassays were between 1 and 4 wk post-adult eclosion, and obtained from colonies kept in a rearing room maintained at 26 ± 2° C and a 14L:10D cycle. Colonies were established from individuals originally collected in Mexico (Nault, 1985), and maintained on 6-leaf maize (cv. Aristogold Bantam Evergreen) for 20-60 generations in the laboratory. Leafhoppers were starved for 5 to 7 h prior to testing to predispose them to host-finding (Chapter I) by releasing them into a 87 cm x 88 cm x 68 cm plywood transfer hood painted black on all inner surfaces except the back wall, which was clear

Plexiglas™, and back-lighted by light from a 20-W, 61 cm Cool White fluorescent bulb reflecting off a white surface. The hood had one open side opposite the Plexiglas that was covered with a black curtain. For bioassays requiring sexed adults, leafhoppers were anesthetized with C02, and males and females were segregated into 30 cm x 7.2-cm diam butyrate tubes. The tubes were placed in the transfer hood until leafhoppers were conditioned for bioassays.

Source of volatiles. Extracts from the whorl leaves of three 4-leaf maize seedlings were used as a source of volatiles for most bioassays.

Whorl tissue was chosen because adults of D. maidis are found in high numbers in maize whorls in the field (Power, 1987) and in greenhouse studies (Chapter III). Seedlings were cut just below the whorl, and the leaves placed in a glass tissue grinder with 30 ml of HPLC grade hexane. 32

Extracts also were obtained from gamagrass, I. dactvloides. and seedling sorghum, S. bicolor. Gamagrass tissue of roughly similar age as maize was obtained by removing the youngest basal leaves that had not completely unfurled. Sorghum tissue was obtained in the same manner as maize tissue due to the morphological similarity between these plants. All extracts were stored at -10° C prior to use in bioassays.

Observation chamber. Bioassays were conducted in a 25.5-cm x 21 cm x 19 cm Plexiglas™ observation chamber (OC) spray-painted black (Dutch Boy

Flat Black 3727) on all outer surfaces except the top, which was le ft clear to facilitate observations (Fig. 3). Two 5-cm2 windows were cut in the front panel to hold narrow band or neutral density interference filters of the same dimensions that served as the source of visual stimuli. The windows were centered on the panel, and spaced 5 cm apart.

The chamber was located in a dark room unless otherwise specified. A

Kodak Ectagraphic III slide projector equipped with a 300-W projector bulb was placed 60 cm in front of the chamber, and the projector light was transmitted through the filter(s) so a reflected image appeared on the opposite chamber wall.

To determine the effects of volatiles on leafhopper host-finding behavior, the chamber was modified by replacing the side panels with nylon mesh screening, and by adding a mixing chamber (MC) to stabilize airflow and a holding chamber (HC) for the volatile source at the upwind end of the original chamber (Fig. 3). The holding chamber was constructed from

8 cm x 10 cm x 1 cm pieces of wood, and placed next to the screening. The top of the holding chamber was removable. The mixing chamber was the same Figure 3. Observation chamber for testing the response of D. maidis to plant extracts. 0C= observations chamber; MC= mixing chamber; HC= holding chamber; VT= vacuum tubing. The direction of airflow was from right (upwind) to left (downwind). Leahopper contacts with a reflected square of light were recorded by transmitting projector light through filter(s) placed in the downwind window.

33 _ 35 dimensions as the holding chamber, and wrapped tightly with three layers of fine cheesecloth. The two chambers were attached to each other, and to the original chamber using black duct tape. An exhaust fan was used to pull volatiles through the chamber at a rate of 2.5-cm/sec, and into

10 cm diam vacuum tubing (VT) attached to a separate box located at the downwind end of the chamber (Fig. 3). The movement of the odor plumes was estimated using titanium tetrachloride, and was not laminar but rather diffuse throughout the chamber.

General bioassay procedures. Adults were released 1 min prior to volatile presentation at the downwind end of the chamber using an

aspirator. A volatile source was provided by pipetting 1 ml of extract

(0.1 plant equivalents) onto 5.5-cm diam filter paper (excluding bioassay

1) that was attached with an alligator clip to a piece of wire in a rubber cork; the height at the middle of the filte r paper was 10 cm. The control consisted of filter paper treated with 1 ml of hexane. The volatile

source was placed in the holding chamber (Fig. 3), and data were collected on the number of contacts (excluding b'ioassay 6) with a reflected green square of light (excluding bioassay 3) produced by transmitting the projector light through a 540 nm narrow band interference filte r (Oriel

Corp., CT). Leafhopper response to reflected rather than transmitted

light was examined based on previous knowledge of Dalbulus host-finding behaviors (Chapter I). Preliminary observations indicated the window position of the filter did not elicit differential leafhopper contacts; therefore, all bioassays were conducted with the filte r placed in the downwind window. The upwind window was covered with a piece of black construction paper. All bioassays included a visual stimulus because 36 leafhoppers released into the chamber with only chemical stimuli (contact and/or volatile) did not move off the floor, and exhibited no upwind orientation. A contact was recorded if an individual walked or flew directly onto the green light, or turned back into the light after leaving momentarily. The number of contacts was recorded every minute for 15 min.

Bioassays were conducted between 6 to 12 h after lights on, and replicated

10 times unless otherwise specified, with a replicate consisting of a treatment and control run sequentially. A different group of 20 leafhoppers was used for each treatment and control, and after a bioassay, leafhoppers were returned to colonies.

Bioassay 1. Orientation of D. maidis to green light in the presence of contact and volatile maize chemicals was determined by placing a sheet of Trusite (nonglare glass) with the same inner dimensions as the chamber against the back wall, and by applying 1 ml of maize extract directly to the glass where the reflected green light appeared. After the chemical was applied, leafhoppers were released into the chamber, with the

Plexiglas side panels in place. The chamber and glass sheet were cleaned with acetone before running hexane controls. The bioassay was replicated

12 times.

Bioassay 2. Orientation of D. maidis to green light in the presence of volatiles from maize, gamagrass, and sorghum was quantified separately as described in the general bioassay procedures.

Bioassay 3. A 10 cm high x 1.9-cm diam wooden rod painted with three coats of titanium white (Liquitex™ acrylic paints, Binney & Smith, Inc.,

Easton, PA) was used to examine the response of D. maidis to a vertical form in the presence of maize volatiles. A model not exposed to volatiles 37 was used in the hexane control. The model was placed in the center of the chamber floor prior to leaf hopper release. The chamber was surrounded by white sheets to provide a uniform background, and illuminated by four 40-

W Cool White fluorescent bulbs hanging 90 cm above the chamber.

Bioassay 4. The relationship between visual and olfactory stimuli during the orientation phase of host-finding by D. maidis was determined by quantifying the number of contacts made with two visual stimuli, represented either bygreen light or by white light, in the presence of maize volatiles. The intensity of the white light was made similar to that of the green light by placing 0.1, 0.3, and 0.6 neutral density interference filters (Oriel Corp., CT) in the downwind window. A randomized complete block design (n= 10) was used with four treatments: green light + hexane; green light + extract; white light + hexane; and white light + extract. One block was conducted/day, with 20 individuals used for each treatment within a block.

Bioassay 5. Differences between the sexes in orientation to green light in the presence of maize volatiles were determined using a randomized complete block design (n= 10) with four treatments: male + hexane; male + extract; female + hexane; and female + extract.

Bioassay 6. The chemically modulated post-contact responses of D. maidis to green light in the presence of maize volatiles were quantified.

A piece of graph paper with 0.5-cm2 grids was photocopied onto a transparency, and the transparency was taped to the back wall of the chamber where the green light appeared. To aid in detecting leafhoppers walking towards the light, the grid extended 3 cm in each direction beyond the lighted area. The grid lines outside of the light were illuminated 38 by a 25-W incandescent red bulb positioned 10 cm above the chamber. The glass sheet (see bioassay 1) was placed in front of the grid to provide a smooth surface similar to that of the chamber walls. Leafhoppers were released individually into the chamber, and their behaviors were recorded using a RCA videocamera (Model # TC1005/U9) and a Panasonic videocassette recorder NV-8950. The camera was positioned so i t viewed the grid through the upwind window on the front panel of the chamber. Twenty leafhoppers were observed for both the treatment and the control. Data were collected on overall tenure within the green light, which was further subdivided into time spent stationary and time spent moving. The distance traveled was recorded by tracing the paths of individuals on an identical grid sheet photocopied onto a piece of paper, and by measuring their paths using an Inch Counter™. Two speeds were determined by dividing the distance traveled by either the time spent moving in the green light, or by dividing the distance traveled by the overall tenure in the green light, which included both stationary and moving time (overall speed).

Statistical Analyses. Homogeneity of variances was tested using the

^max tes* (Sokal & Rohlf, 1981). Data from bioassays 1-3 and 6 were log

(x + 1) transformed before being subjected to a t-te s t for paired comparisons (P < 0.05), or a two-way analysis of variance (ANOVA) (P <

0.05) (bioassays 4, 5). 39

Results

Bioassay 1. During a 15 min observation period, leafhoppers made

significantly more contacts with green light when maize extract was

applied to the glass sheet compared to hexane (t= 3.38, df= 11, P < 0.01)

(Table 5). Additionally, more than half of the first leafhopper contacts were made between 2 to 3 min after release when maize extract was present,

compared to 4 to 5 min when hexane was present. Although not measured

directly, observations indicated that the greater number of contacts with

the green light in the presence of maize extract (x= 68.7) compared to

hexane (x= 36.3) was due to more individuals making contacts rather than

to the same individuals repeatedly turning back into the light after

walking onto the black walls.

Bioassay 2. D. maidis made approximately two times as many contacts

with green light when exposed to maize volatiles as compared to hexane (t=

3.69, df= 9, P < 0.01) (Table 5). In contrast, the presence of gamagrass

volatiles did not influence the number of contacts leafhoppers made with

green light (t= 0.235, df= 9, P > 0.50). Fewer contacts were made with

the light when leafhoppers were exposed to sorghum volatiles compared to

hexane (t= 3.12, df= 9, £ < 0.02). When maize volatiles were present,

leafhoppers were observed moving towards the green light within 2 to 3 min

after release into the chamber compared to 7 to 8 min in controls. Most

leafhoppers oriented towards the light by walking across the chamber floor

until they were against the wall/floor juncture, and then proceeded to 40 Table 5. Influence of plant extracts on contacts made by D. maidis with

visual stimuli

Plant Visual x (± SE1 no. of contacts

Bioassav extract2 stimulus Extract Control

1 maize GL 68.7 ± 10.1 a3 36.3 ± 7.7 b

2 maize GL 33.1 ± 4.1 a 17.2 ± 3.3 b

TriDsacum GL 12.4 ± 1.1 a 12.9 + 1.9 a

sorghum GL 3.9 ± 0.7 a 8.8 ± 1.7 b

3 maize VWM 3.2 ± 0.8 a 3.6 + 0.4 a

Bioassays were conducted in a Plexiglas observation chamber with either a green light (GL) or a vertical white model (VWM) used as a visual stimulus (see text).

2 Plant extracts were obtained by crushing leaves of seedling maize, gamagrass, or sorghum, in hexane. Extracts were tested as contact/ volatile stimuli in bioassay 1, but only as volatiles in bioassays 2 and 3 (see te x t). Hexane served as the control.

3 Means within a row followed by the same letter are not significantly different at £ < 0.05 (t-test for paired comparisons). Bioassay 1 was replicated 12 times, and bioassays 2 and 3, 10times, using 20 individuals/replicate. Bioassays were conducted 15 min. 41 walk up the wall and onto the light. A few individuals flew directly onto the light.

Bioassays 3 and 4. Exposure to maize volatiles did not influence the response of D. maidis to a white vertical model (t= 0.789, df= 9, P

> 0.20) (Table 5), while the quality of reflected light (green vs. white) had a highly significant effect on leafhopper contacts (Table 6). In the absence of maize volatiles, leafhoppers made significantly more contacts with green light than with white light (F= 37.9, df= 1,36, P < 0.001).

An interaction also existed between visual and olfactory stimuli, with exposure to maize volatiles significantly increasing the number of contacts made with green light, but not white light of similar intensity

(F= 5.31, df= 1,36, P < 0.05).

Bioassay 5. There was no significant difference in the number of contacts with green lig h t made by males (x= 32.2) as compared to those made by females (x= 20.8) (F= 2.72, df= 1,36, P > 0.10). However, both sexes made more contacts with green light when maize volatiles were present compared to hexane (F= 6.68, df= 1,36, P < 0.025). The interaction between sex and maize volatiles was not significant (F= 0.513, df= 1,36, P > 0.25).

Bioassay 6. Regardless of whether stationary or moving, individuals spent more time inside the green light in the presence of maize volatiles

(x= 164.5 s) than in th e ir absence (x= 75.1 s) (t= 3.47, df= 19, P < 0.01)

(Table 7). When overall tenure was subdivided into stationary and moving time, significantly more time was spent stationary when individuals were exposed to maize volatiles (x= 155.9 s) compared to hexane (x= 66.7 s)

(t= 3.70, df= 19, P < 0.01). Maize volatiles did not have an effect on 42 the time spent in movement (i= 0.77, df= 19, P > 0.90), the distance traveled on the green light (t= 0.494, df= 19, P > 0.50), or the rate of locomotion when walking (t= 1.07, df= 19, P > 0.20). However, the overall rate of locomotion was significantly slower on the green light when maize volatiles were present compared to hexane (t= 3.09, df= 19, £ < 0.01)

(Table 7). 43 Table 6. Influence of maize volatiles on contacts made by D. maidis with visual stimuli.1

x (± SE) no. of x (± SE) no. of

contacts with green light contacts with white light

Maize Maize

Bioassav Sex extract2 Control extract Control

4 mixed 15.7 ± 2.0 a3 8.9 ± 1.0 b 3.2 ± 0.9 c 2.8 ± 0.8 c

5 male 32.2 ± 6.7 a 16.3 ± 3.4 b

female 20.8 ± 5.3 a 11.8 ± 2.5 b

mean 26.5 ± 4.4 a 14.1 ± 2.1 b

1 Bioassays were conducted in a Plexiglas observation chamber with volatiles pulled through the chamber at a rate of 2.5-cm/s. Visual stimuli were provided by shining projector light through filters (see te x t).

2 Extracts were obtained by crushing the whorl leaves of three 4-leaf seedlings in hexane. One ml of extract or hexane (control) was applied to filte r paper (see text).

3 Bioassays were replicated 10 times using 20 individuals/replicate. Data were subjected to a two-way AN0VA. Means within a row followed by the same letter are not significantly different at P < 0.05 by LSD. 44

Table 7. Post-contact responses of D. maidis to green light when

exposed to maize volatiles.

x (± SE1

Post-contact resDonse Maize extract Control

Overall tenure (s) 164.5 ± 25.9 a1 75.1 ± 12.1 b

Tenure stationary (s) 155.9 ± 24.8 a 66.7 ± 11.6 b

Tenure moving (s) 8.6 ± 1.8 a 8.4 ± 1.4 a

Distance traveled (cm) 10.2 ± 2.1 a 10.1 ± 1.8 a

Speed moving (cm/s) 1.1 ± 0.2 a 1.3 ± 0.2 a

Overall speed (cm/s) 0.07 ± 0.01 a 0.15 ± 0.02 b

1 Twenty leafhoppers were observed individually for the treatment (maize extract) and the control (hexane). Means within a row followed by the same le tter are not significantly different at P < 0.05 (t-test for paired comparisons). Discussion

Host-finding is generally considered a catenary process during which an insect can be influenced by both noncontact and contact plant stimuli

(Miller & Strickler, 1984). Orientation and settling can be mediated by both visual and/or olfactory stimuli (Prokopy & Owens, 1983; Visser,

1986). Sustained contact may require additional olfactory and/or gustatory stimuli which presumably provide the insect with specific information concerning suitability of the plant for feeding or oviposition

(Scriber & Slansky, 1981). The orientation phase of host-finding by D. maidis is mediated largely by visual stimuli. This conclusion is based on the observation that leafhoppers did not exhibit increased movement in the chamber or orient upwind when exposed solely to olfactory stimuli, and on the response to green light in the absence of olfactory stimuli (Table

6, bioassay 4). Similar results were obtained by Saxena & Saxena (1974) for the leafhopper, Empoasca devastans Distant, which oriented more strongly to green- than to white-illuminated surfaces. I suggest the low number of contacts by D. maidis with the vertical white model (Table 5, bioassay 3) does not provide substantial evidence that this leafhopper is unresponsive to shape stimuli during host-finding. In a laboratory choice te st (J. L. Todd, unpublished data), D. maidis made more contacts with a vertical model than a horizontal, circular, or square model of the same surface area if models were painted a green color closely matching that of seedling maize foliage (Chapter I). Host-finding behaviors mediated 46 primarily or solely by visual stimuli have been suggested for several aphid species after dispersal flights (Van Emden, 1972). Similarly, reorientation to a vertical green model by mechanically dislodged aphids occurs without olfactory stimuli (Phelan et al., 1976), and odors were demonstrated to play a minor role in orientation and settling by the greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Vaishampayan et al., 1975).

Although D. maidis may be responding primarily to visual stimuli during the early stages of host-finding (e.g., orientation and settling), the data indicate that olfactory stimuli also are important. In all bioassays, exposure to maize volatiles approximately doubled the number of contacts leafhoppers made with green light compared to hexane (Tables

5, 6). Volatiles from whorl tissue (Thompson et a l., 1974) and other maize structures (Buttery & Ling, 1984) have been identified, and consist mainly of alcohols, aldehydes, and a few terpenoid hydrocarbons. Similar volatiles were identified from the homogenized-1 eaf maize extract used in this study, and from intact leaves using GC-MS separation techniques (Todd

& Phelan, unpublished data). Exposure to contact and volatile maize chemicals simultaneously (Table 5, bioassay 1) may account for the higher number of contacts leafhoppers made with green light compared to other bioassays, in which only volatiles were present (Tables 1, 2, bioassays

2, 4, 5). Backus (1985) suggested that soon after arriving on a plant, an auchenorrhynchan will begin exploring the plant surface by labial dabbing, a behavior that could aid in determining a host from a nonhost.

Contact chemoreceptors have been identified on the labium of the brown planthopper, Nilaparvata luoens (Stal) (Foster et al., 1983). D. maidis 47 also possesses labial hairs (A. C. Wayadande, unpublished data) that may be responsive to maize chemicals. The higher number of contacts leafhoppers made with green light in the treatment, and the control, in bioassay 1 compared to the other bioassays also may be related to differences in orientation behaviors in still vs. moving air, respectively.

The low number of contacts made by leafhoppers with green light in bioassay 4 (Table 6) compared to the other bioassays (2, 5) in the presence of maize volatiles may be because the former bioassay was conducted too far out of synchrony with peak flight activity. By monitoring the number of D. maidis caught in suction traps over 24 h in a plant growth room, R. A. J. Taylor (unpublished data) has shown D. maidis exhibits two peaks in flight activity, one in the morning and one in the evening, when light intensity is low. Leafhoppers used in bioassay

4 were tested between 6 to 9 h after lights on, and leafhoppers used in other bioassays were tested between 9 to 12 h after lights on. However, despite discrepancies in the number of contacts leafhoppers made with green light in different bioassays, the doubling effect of maize chemicals was maintained in all bioassays.

The relative contributions of visual and olfactory stimuli in the orientation behavior of D. maidis were not equally weighted. The quality of the light (green vs. white) accounted for the majority of variability

in the data (Table 6), whereas variation due to volatile stimuli

irrespective of light quality was not significant (F= 3.95, df= 1,36, P

> 0.05). However, there was a significant interaction between maize volatiles and light quality, with maize volatiles acting as a synergist by increasing the number of contacts D. maidis made with green light, but not with quantitatively similar white light (Table 6). Chapman et al.

(1981) also provided evidence for an interaction between visual and olfactory stimuli during host-finding by the aphid, Cavariella aeaopodii

(Scopoli), which was trapped in higher numbers in yellow water traps baited with carvone compared to unbaited yellow traps or colorless traps.

These two studies provide the only documentation of an interaction between visual and olfactory stimuli during host-finding in the Homoptera. In the field, the responsiveness of D. maidis to green reflected light may aid

in distinguishing foliage from nonfoliage. However, because most plants are green, the use color stimuli alone during host-finding may result in leafhoppers making contacts with nonhosts. Therefore, the positive

interaction between host odor and color could be advantageous to D. maidis by aiding this leafhopper in distinguishing maize from nonhosts prior to contact.

In the present study, D. maidis exhibited a neutral response to gamagrass volatiles, and a negative response to sorghum volatiles (Table

5, bioassay 2). Although the constituents of gamagrass odor are not

known, the lack of a significant response of D. maidis to odors from this plant compared to maize suggests that gamagrass volatiles are either qualitatively or quantitatively different from those of maize. D. maidis has only been collected from gamagrass when maize in nearby fields has died back (Triplehorn & Nault, 1985). In the laboratory, D. maidis

reproduces on gamagrass, but the progeny are few in number, small in size, and take longer to develop than those on maize (Nault & Madden, 1985).

Therefore, a weak response to gamagrass odor in the field may be 49 advantageous because it will limit contacts, and thereby prevent some females from ovipositing in a plant that is not optimal for nymphal development and population growth. A chemical basis for resistance of sorghum to feeding by some homopterans may be due to HCN compounds and various phenolic acids (Dreyer et a l., 1981). Although these two plants look the same during the seedling stages, if sorghum volatiles act as repellents, they may provide a mechanism by which D. maidis avoids sorghum. The nonhost status of sorghum for D. maidis has been established in the laboratory (L. R. Nault, unpublished data).

The influence of host odors on the post-contact, nonfeeding behaviors of leafhoppers had not been documented prior to this study, which provides the first evidence that volatiles from a host can significantly increase leafhopper tenure on a green light (Table 7). When tenure was subdivided into stationary and moving time, individuals spent more stationary time on the green light in the presence of maize volatiles than in the presence of hexane (Table 7), perhaps because the volatiles stimulated leafhoppers to initiate probing. That probing is initiated soon after contact with maize seedlings is suggested by observations that D. maidis remains in its initial landing position on seedling maize for up to 3 h (Chapter I). By comparison, exposure to maize volatiles resulted in a mean tenure of 2.75- min for leafhoppers on green light, suggesting that gustatory and/or physical stimuli obtained during feeding are of primary importance in sustaining leafhopper contact with a host plant.

There were no differences between the sexes in the number of contacts made with green light (Table 6, bioassay 5), and although post-contact behaviors of the sexes were not observed separately, observations suggested that males spent more time moving within the green light than females in both the treatment and control. Greater male mobility was demonstrated for Dalbulus species by Heady & Nault (1985), who showed that males were more likely to fly when mechanically dislodged from maize seedlings than females. Hunt (1988) found that male Graminella niarifrons

(Forbes) leafhoppers engaged in more interplant flights than females when given access to oat seedlings (Avena sativa L.), a strategy for increasing the likelihood of locating a calling virgin female.

The present data, coupled with previous knowledge on the responses of D. maidis to visual stimuli (Chapter I), provide a more complete understanding of host-finding behaviors of this species. The strong orientation response of D. maidis to yellow and green may aid this leafhopper in distinguishing foliage from nonfoliage when flying above plant canopies, and when approaching a potential host, the differential responsiveness of D. maidis to plant odors may further aid it in orientation and settling. The specialization of D. maidis on maize may be enhanced by its positive response to maize volatiles, which not only effect orientation, but also post-contact behaviors. CHAPTER III SPATIAL AND TEMPORAL DISTRIBUTION AND POPULATION DYNAMICS OF DALBULUS LEAFHOPPERS O N MAIZE: HO ST ASSOCIATIONS AND PEST POTENTIAL Introduction

Few leafhopper species are serious pests due directly to feeding damage, but rather because they are vectors of plant pathogens (Conti,

1985). Dalbulus maidis (DeLong & Wolcott), the vector of corn stunt spiroplasma (CSS= Spiroplasma kunkeliih maize bushy stunt mycoplasma

(MBSM), and maize rayado fino virus (MRFV), is no exception (Nault, 1980,

1983; Nault et al., 1981). D. maidis is the most commonly found leafhopper species on maize (Zea mavs L.) in the Neotropics, and the pathogens it transmits are among the most serious constraints to maize production in Latin America (Nault et al., 1981; Gamez & Leon, 1985;

Power, 1987).

The genus Dalbulus is composed of 11 species which are thought to have evolved closely with maize and its wild teosinte (Zea) and gamagrass

(Tripsacum) relatives in Mesoamerica (Nault & DeLong, 1980; Nault, 1980,

1985). Four other Dalbulus species occur on maize in the field, but none has as wide a distribution (Triplehorn & Nault, 1985) or develops populations as large as D. maidis, the only member of the genus considered a serious pest (Nault, 1990).

51 52

Surprisingly, Dalbulus species that are found exclusively or principally on gamagrasses (Triplehorn & Nault, 1985) can be reared and maintained in large numbers on maize in the laboratory (Nault & Madden,

1985). Several of these species have been maintained continuously on maize without exposure to gamagrass for up to 9 yr. Among them are D. gel bus DeLong, a species that is considered a gamagrass specialist, but is occasionally found on maize in the field (Triplehorn & Nault, 1985), and D. auinauenotatus DeLong & Nault, an exclusive gamagrass specialist and myrmecophile in the field (Nault et al., 1983; Triplehorn & Nault,

1985). In the laboratory, D. maidis develops faster on maize than these two species, but in limited tests, it was found to be no more fecund

(Nault & Madden, 1985). Laboratory studies primarily have been conducted using seedling maize as a host (Nault, 1985 and refs, therein).

Similarly, for routine laboratory rearing, a continuous supply of maize seedlings is provided for feeding and oviposition. The apparent high suitability of maize as a host for D. gel bus and D. auinauenotatus in the laboratory is not reflected in field collections. All records of field hosts for Dalbulus species, however, were obtained in September and

October, after maize, teosintes, and gamagrasses had flowered (anthesis) and were beginning to senesce (Nault & DeLong, 1980; Nault et al., 1983;

Nault, 1985). Few D. gel bus and no D. auinauenotatus were collected from maize (Triplehorn & Nault, 1985), prompting us to ask the question: Is older or matured maize less suitable as a host for gamagrass specialists than seedling maize? The suitability of maize and gamagrass as field hosts for these species may be influenced by the duration of availability of green tissue for feeding and oviposition (Nault & Madden, 1985), or by differences in plant architecture, both of which could affect leafhopper within-plant distribution and population dynamics. Therefore, the present study was designed to compare the spatial and temporal distributions, and population dynamics of D. maidis. D. gel bus, and D. auinauenotatus on maize from the seedling stage through anthesis and senescence, and to determine if differences in behavioral responses of Dalbulus species to maturing maize could provide an understanding of field host associations, and serve as predictors of pest potential within the genus Dalbulus. 54

Materials and Methods

Leafhoppers were obtained from colonies kept in a rearing room at 26

+ 2° C and a 14L:10D cycle. Colonies were established from individuals originally collected in Mexico on maize or gamagrass hosts (Nault, 1985), and were maintained in the laboratory for 20-60 generations on 4-6-leaf seedling maize (cv. Aristogold Bantam Evergreen) prior to use in this study.

To initiate populations of the three Dalbulus species, 24 4-leaf maize seedlings (eight seedlings/species) were enclosed individually in

30.0 cm x 7.2-cm diam butyrate tubes. Ten females, 1 wk post-adult eclosion, were introduced into each tube with an aspirator, and given a

48 h oviposition access period. Five males also were introduced into each tube to insure that females were mated. The tubes were kept in a walk-in environmental chamber set at 26 ±'2° C and a 14L:10D cycle. After 48 h, all adults were removed, and each seedling was transplanted into a 23 cm x 25 cm diam plastic pot. The eight seedlings/species were divided into two groups of four seedlings, and each group was placed in a separate 2 m x 2 m x 2 m saran screen cage located in a greenhouse room with a daytime temperature of 35 ± 5° C and a nighttime temperature of 27 ± 2° C.

One seedling was placed in each corner of a cage so that leaves of adjacent plants would not overlap as plants grew. The cages were arranged in a randomized complete block design, with two rows of three cages, and one cage for each species randomly assigned a position in each row. Cages 55 within a row were 1.5-m apart, with 2.5-m between cages in adjacent rows.

The study was conducted from 1 July - 1 September, 1988, with the former date representing when females were released into tubes with seedlings for the oviposition access period (day 0), and the latter date representing complete senescence (collapse) of 50% of the plants, and the end of the study (day 62). Plants were inspected daily for emerging nymphs, and after first generation (FI) nymphs eclosed, data were collected on their abundance over time, developmental times to the adult stage, and spatial distribution on maize. Abundance and spatial distribution also were monitored for FI adults, and data collection was repeated for any species producing more than one generation before maize senescence. Unless otherwise specified, all references to temporal distribution of leafhoppers on maize refer to days post-oviposition access period.

The abundance of leafhoppers on maize was monitored daily between

0800 and 1200 h by counting all the individuals on the whole plant.

Nymphs generally were not disturbed when making observations; however, adults would occasionally fly from plants, especially second generation

(F2) adults, which were present on plants that were senescing. Nymphs were considered collectively, and not separated by instars. For species that produced more than one generation, remaining FI adults were removed from cages using an aspirator just prior (1 to 2 days) to F2 adult eclosion to avoid confusion between individuals of each generation.

The spatial distribution of nymphs and adults on maize over time is described both vertically and horizontally. Vertical distribution refers to the mean leaf position of leafhoppers on maize by counting individuals on each leaf blade (both surfaces), as well as the inner and outer sheaths of each leaf. Horizontal distribution refers to the abundance of leafhoppers on various maize structures: leaves (L); whorl (WH); outer sheath (OS); inner sheath (IS); reproductive structures (RS) (e.g., tassel and ears); and tille rs (T) (Fig. 4). Dalbulus population growth and development were monitored in accordance with stages of maize growth and development (Hanway, 1971). Leaves were numbered in ascending order from the bottom to the top of the plant by counting leaves emerged fully

(collar visible) from the whorl. Times for tassel formation and full emergence were noted for each plant, along with ear development.

Leafhopper abundance data were subjected to a multi-factorial analysis of variance (ANOVA) (P < 0.05) with the following factors: cages

(two/species), species (three), and time (days post-oviposition). Time was a repeated measure in the ANOVA. The analysis was done separately for each stage (nymph or adult) and generation (FI, F2). Vertical distributions (leaf position) of nymphs and adults on specific days were obtained separately by counting all individuals on each leaf (blade, outer and inner sheaths), multiplying that number by the leaf number (4, 5, etc.), summing the frequencies for all leaves, and dividing the resultant sum by the total number of individuals on all leaves for that day. Leaf positions are presented as mean leaf + SE. Percentage data for spatial distributions of nymphs and adults on maize structures were arcsin square- root transformed, and subjected to a multi-factorial ANOVA (P < 0.05), with the factors the same as for the abundance data. Horizontal distribution percentages of nymphs and adults within each generation on maize structures are combined, and presented as mean percent individuals Figure 4. Mean growth stages of maize in relation to the spatial and

temporal distribution of three Dalbulus species. Days 0-2

(not shown) represent the egg-laying period by 20

females/plant, with subsequent days referring to days post-

oviposition. L= leaves, IS= inner sheath, 0S= outer sheath,

RS= reproductive structures (tassel and ears), T= tiller, WH=

whorl.

57 58

Figure 4. ± SE. Although data were collected daily, abundances and spatial distributions of each species over time are presented at approximately 4- day intervals for clarity. Results

Dalbulus population growth and development on maize. D. maidis completed two generations before senescence and collapse of maize, with minimum developmental times from egg-to-adult of 22 days for the FI generation, and 23 days for the F2 generation (Fig. 5). The F2 developmental time was estimated by waiting 2 days after eclosion of the first adults to insure sexual maturation. FI nymphs began eclosing on day 8 (Fig. 4, maize 7-leaf stage), and their abundance peaked on day 12

(5T = 171 ± 17 nymphs) (maize 9-leaf stage). After day 12, nymphal population size decreased until day 22, when the firs t FI adults eclosed

(maize 13-leaf stage), and the last nymphs were counted on day 26 (maize

15-leaf stage) (Fig. 4). FI adults reached peak abundance on day 26 (x

= 84 ± 10 adults), and declined steadily in abundance until day 46 (maize in full tassel for about 11 days), at which time the remaining adults (x

= 6.5 ± 0.1 adults) were removed from cages. The firs t F2 nymphs eclosed after full tassel emergence on all but two plants, and their abundance increased dramatically to a peak on day 47 (5? = 955.5 ± 171.5 nymphs)

(maize in full tassel for about 12 days) (Fig. 5). Population size decreased sharply following peak abundance, until day 60, the last day nymphs were counted (3? = 42 ± 24 nymphs), because all plants with nymphs collapsed thereafter. The first F2 adults eclosed on day 47, and they peaked in abundance on day 60 (JT = 149 ± 42 adults), with only a slight decline in numbers by the last day of the study, day 62 (x = 119.5 ± 18.5 Figure 5. Abundance of three Dalbulus species on maize over time (note

changes in scale of y-axes). Numbers represent x ± SE. FIN

and F2N= firs t and second generation nymphs, respectively.

F1A and F2A= first and second generation adults, respectively.

Days 0-2 represent the egg-laying period on 4-leaf seedlings

by 20 females/plant (see text). The study ended 62 days post-

oviposition when all plants were tasseled and senescing.

61 a% ro

■ O ini 3331 » » ■ » • » ■ » » - « ■ ■ K> to m O ir* Mean Leafhoppers of No. Mean Leafhoppers of No. MeanLeafhoppers No. of O), * 5* 3 < o 6

Days post— • c -s a> CO * * “T | 63

adults).

D. gel bus also completed two generations before senescence and collapse of maize, although abundances for nymphs and adults of both generations were significantly lower compared to D. maidis (ANOVA, P <

0.05). Minimum developmental times for D. gel bus from egg-to-adult for

FI and F2 generations were 27 and 34 days, respectively (Fig. 5). FI nymphs began eclosing on day 10 (maize 8-leaf stage), and their populations peaked on day 14 (x = 98.5 ± 18.5 nymphs) (maize 10-leaf stage). Abundance decreased steadily until day 26, the first day of FI adult eclosion (Fig. 4, maize 15-leaf), and the last nymphs were counted on day 30. FI adults reached peak abundance on day 30 (x = 21 t 7 adults), when most tassels were fully emerged. Population size gradually decreased until day 53, the last day adults were counted (x = 5 ± 5 adults). F2 nymphs eclosed on day 44, about 10 days after full tassel emergence, and peaked in abundance on day 48 (x = 58 ± 19 adults). F2 adults eclosed 2 days prior to the end of the study on day 60, which also represented their peak abundance (x = 7.5 ± 5.5 adults).

In contrast to D. maidis and D. gel bus. D. ouinguenotatus produced only one generation on maturing maize, and based on ANOVA, abundances of nymphs and adults were significantly lower compared to the other two

Dalbulus species (£ < 0.05) (Fig. 5). FI nymphs began eclosing on day 12

(maize 9-leaf stage). Peak nymphal abundance was reached on day 16 (x =

23 ± 2.5 nymphs) (maize 11-leaf stage), and decreased to a mean low of 2.5

±1.5 nymphs on day 32 (tassels fully emerged on most plants and silking initiated) (Fig. 4). The first FI adults eclosed on day 32, and the last adults were counted on day 38 (x = 1.5 ±0.5 adults). Vertical distribution of Dalbulus leafhoppers on maize. D. maidis females did not oviposit in leaves below the third leaf of 4-leaf seedlings during the oviposition access period (Fig. 6). The mean leaf position of the firs t FI nymphs to eclose (day 8) was leaf 3 ± 0.0 on 7- leaf maize. Because oviposition was restricted to 2 days, all nymphs probably were eclosed by day 12, when peak abundance was reached (Fig. 5), and their mean leaf position was 3.6 ± 0 .1 . After day 12, nymphs began to move onto higher leaves, with a maximum mean leaf position of 4.9 ± 1.9 on day 26, when maize was at the 15-leaf stage (Fig. 4). The mean leaf position of the first FI adults to eclose was 4.3 ± 0.3 on day 22 when maize was at the 13-leaf stage, and at peak abundance (Fig. 5, day 26), most adults were on leaf position 4.8 ± 0.3. By day 30, all adults exhibited a mean leaf position > 12 on tasseled, 16-leaf maize (Fig. 6).

The mean leaf position of the first F2 nymphs to eclose was 13 ± 0.0 on day 34, although they eclosed on lower leaves or moved to lower leaves after day 34, with the lowest mean leaf position, 10.1 ± 0.7, on day 54.

Because of the extended oviposition period of FI adults (about 22 days), it was difficult to determine how many F2 nymphs were actually eclosing lower on the plant compared to those moving to lower leaves from upper leaves. F2 adults concentrated on the uppermost, youngest leaves (13-16), with a maximum mean leaf position of 13.0 ± 0.6 recorded on the last day of the study.

The mean leaf position of the firs t FI D. aelbus nymphs to eclose was 3.3 ± 0.1 of 8-leaf maize (Fig. 6). Assuming all nymphs were eclosed by day 14, there was an upward movement tendency (3.4 ± 0.8), and mean leaf position reached a maximum of 6.1 ± 1.1 on day 30, when maize was Figure 6. Vertical distribution of three Dalbulus species on maize

leaves over time, with time represented by days post egg-

laying period (see text). Mean leaf (+ SE) refers to the mean

leaf position of leafhoppers on a leaf blade and its

associated inner and outer sheaths (see Fig. 4) on plants with

a maximum of 16 leaves. Leaves numbered in ascending order

from bottom to top of plants. FIN and F2N= firs t and second

generation, respectively, and F1A and F2A= first and second

generation adults, respectively.

65 Mean leaf Mean leaf Mean leaf 16 2 1 16 2 1 Fgr 6.•Figure 0 ' 1 161— tassel-H 11 4 '

. quinquenotatus 0. 8 . maidis D. x as post-oviposition Days X X

gelbus 16 X e n ef stage leafMean X X X

32 X

40 X X X

56 X

64 - F2A i- F2N - F1A »- F1N F1N 66 67

tasseled with 16 leaves. As with D. maidis. the first FI D. gel bus adults

eclosed low (mean leaf position 4.0 ± 0.0) on 15-leaf maize, and after 4 days, concentrated on the upper leaves (> 13). The mean leaf position of

the firs t F2 nymphs to eclose was 12.2 ± 0.7, and at peak abundance (Fig.

5, day 48), they exhibited a mean leaf position of 10.0 ±0.1 on 16-leaf,

tasseled maize. Because the population size of F2 adults was low (Fig.

5), and the study ended 2 days after they began eclosing, we cannot draw

firm conclusions about their vertical distribution on maize; the few

adults present were located on the upper leaves (> 12) (Fig. 6).

The mean leaf position of the first FI D. auinouenotatus nymphs to

eclose was 2.8 ±0.1 on 9-leaf maize (Fig. 6), and at peak population size

on day 16 (Fig. 5), nymphs were about at the same position (5< = 2.3 ± 0.2

leaf position). They remained below leaf three for 8 days post-eclosion,

reaching a maximum mean leaf position of 4.8 ± 0.3 on day 32, when most maize was fully tasseled and silking (Fig. 4). As with D. gel bus F2

adults, the abundance of FI adults of D. ouinauenotatus did not allow for

firm conclusions concerning vertical distributions; the few adults present

stayed below leaf five (Fig. 6).

Horizontal distribution of Dalbulus leafhoppers on maize. During

peak abundance (Fig. 5, day 12), most D. maidis FI nymphs were on leaves

(Fig. 7), although some nymphs began moving off leaf blades, and into whorls (Fig. 4). After day 12, the number of nymphs on leaves steadily declined as more individuals moved into whorls, with 43.2 ± 3% of the

total nymphal population in whorls by day 22 (Fig. 7). Although FI adults

began eclosing on day 22, none was located in whorls. By day 26, all Figure 7. Horizontal distribution of three Dalbulus species on maize

structures over time, with time represented by days post-

oviposition by 20 females/plant (see text). Vertical lines

on top of bars represent + SE. Fl= firs t generation nymphs

and adults combined, F2= second generation nymphs and adults

combined. L= leaves, WH= whorl, 0S= outer sheath, IS= inner

sheath, RS= reproductive structures (tassel and ears), T=

tille rs (see Fig. 4). Obscured values for FI D. maidis on

leaves: day 26= 20.3 + 0.7%, day 30= 7.2 + 0.5%, and day 34=

19.3 + 14%.

68 j i 10 < Mean % FI D. maidis 12 16 20 22 26 30 34 38 42 46

g> a> o u m H o o o o o o Mean % F I D. gelbus V ■1 -4 1 (/> (/> F 1 D. quinquenotatus ■ P NT % o 8- 8 8 8 8 CD to 3 3 K) Q Q u> Mean Figure 7. Mean % F2 D. gelbus Mean % F2 O. maidis 100 0 2 40 60 80 iue . (continued) 7. Figure 8 2 7 4 0 62 60 54 47 42 38 4 3 4 8 3 0 62 60 53 48 44 as tovi ii n sitio o ip v st-o o p Days □ OS l 70 nymphs in whorls had eclosed as adults, and many of the adults on leaves had moved into whorls, thus FI adult abundance in whorls reached 79.7 ±

0.65% of the total adult population, and peaked 4 days later at 91.9 ±

1.5% (Fig. 7). By day 28, tassels were beginning to emerge on most plants. Ear development was firs t noted around day 32, and by day 34, the mean percentage of adults on the tassels or ears was 46.1 ± 4%. After whorls disappeared (> day 34), some adults were on outer leaf sheaths and reproductive structures, but most leafhoppers moved back onto leaves (Fig.

7). F2 nymphs and adults eclosed after full tassel emergence (about day

34), and were largely on leaves throughout the study (Fig. 7). During peak abundance (Fig. 5, day 47), most nymphs were on leaves (x= 83.9 ±

5.7%). Similarly, most adults (x= 53.2 ± 6.6%) were on leaves during their peak abundance (Fig. 5, day 54).

In contrast to D. maidis. fewer D. gel bus FI nymphs moved into whorls during their development. Nymphs were first observed in whorls 12 days after eclosion (day 22), whereas D. maidis nymphs moved into whorls 4 days after eclosion (day 12), and reached significantly higher abundances in whorls (P < 0.001 based on ANOVA). At peak abundance (Fig. 5, day 14),

D. gel bus nymphs were almost exclusively on leaves (x = 98.2 + 0.6%). At

FI adult peak abundance (Fig. 5, day 30), most individuals were on leaves

(?T = 64.3 ± 0.0%) or in whorls (1? = 32.5 ± 3.6%). The abundance of adults on reproductive structures, primarily the ears, peaked on day 38 (x = 12.5

± 12.5%). After day 42, all adults were on leaves (Fig. 7). At peak abundance, most F2 nymphs were on leaves (Fig. 5, day 48) (x = 63.8 ± 8%), followed by the reproductive structures (ears only) (X = 10.4 ± 7.8%)

(Fig. 7). During the 2 days that F2 adults were present, they were 72 primarily on the ears and leaves.

The horizontal distribution of D. auinauenotatus on maize was very different from the other two species (Fig. 7). The majority of FI nymphs eclosing on day 12 was on leaves (5T = 61.3 ± 7.5%), followed by the outer sheaths (5T = 34.9 ± 3.7%). By day 16, peak abundance was reached (Fig.

5), and no FI nymphs were on leaves; all individuals were concentrated in inner leaf sheaths (Figs. 4, 7). Tillers, usually two/plant, developed on both sides of the main stalks of all plants in D. auinauenotatus cages, and nymphs moved onto tillers at the 3-leaf stage, relocating inside leaf sheaths. Although FI adult abundance was low, the few individuals present exhibited a distribution similar to nymphs, staying hidden within leaf sheaths of lower leaves. Some adults also were found in the inner sheaths and whorls of tille rs, but not in main plant whorls. Discussion

This study has provided a rationale for the anomaly between laboratory and field associations of D. gel bus and D. auinauenotatus with maize, and further substantiated the successful utilization of maize by

D. maidis. Differences in duration of availability of green leaves between maize and gamagrass may have considerable impact on the population dynamics of Dalbulus species in the field. When compared to short-lived annuals such as maize, the perennial gamagrasses provide a more stable habitat for leafhoppers not only between seasons, but also within a growing season (Nault & Madden, 1985). In the spring, growth of young gamagrass shoots precedes germination of maize seed, and in the autumn, maize leaves begin senescing several weeks to months before those of gamagrass. D. gel bus and D. auinauenotatus have significantly longer developmental times than D. fflaidis on both seedling maize and gamagrass

in the laboratory (Nault & Madden, 1985). In the present study, D. gel bus and D. auinauenotatus also exhibited longer developmental times than D. maidis on maturing maize (Fig. 5). On gamagrasses, their preferred field hosts, longer developmental times would not be disadvantageous to survival because new tissue is available from tille rs at plant bases, and leafhoppers probably could complete two, and perhaps as many as three, generations before leaves die back in early winter. However, similar developmental times on maize will not provide enough time for the completion of multiple generations, or for populations to become large enough to perpetuate the species. These data showed that D. maidis was the only species that eclosed as FI adults several days before tassel formation; D. gel bus eclosed during tassel formation, and D. quinquenotatus eclosed after full tassel emergence (Figs. 4, 5). Because

F2 developmental times were even longer than those for the FI generation, only D. maidis eclosed as adults in large numbers prior to maize senescence (Fig. 5). These data suggest that the longer developmental times of the two gamagrass specialists compared to D. maidis severely limit their ability to establish and maintain large populations on maturing maize because their development is out of synchrony with maize phenology.

The duration of green tissue may not be the only plant attribute affecting Dalbulus population dynamics on maize and gamagrass.

Differences in the architecture of these two plants may have predisposed gamagrass specialists to adopt a different within-plant distribution on maize. Maize consists of a central stem with a single growing point, and meristematic tissue grows vertically until ears develop. In contrast, gamagrasses have many growing points, and meristematic tissue grows both vertically and laterally. Gamagrasses also continuously are producing tille rs at the plant base, thus establishing a dense lower canopy. The data indicate that FI nymphs and adults of D. maidis and D. gel bus moved upward as maize matured, but D. auinauenotatus nymphs and adults stayed low on plants (below leaf five) (Fig. 6). Plants in D. auinauenotatus cages were unique compared to those of the other two species in that the former were frequently occupied by ants, and produced tille rs. All eight plants in D. auinauenotatus cages tillered compared to none of the plants 75 in cages of the other species. In the laboratory, maize seedlings fed on by D. quinquenotatus develop red striping, which may be due to a toxin injected during feeding (L. R. Nault, unpublished data). Perhaps some component of D. quinquenotatus saliva also stimulates tillering in maize.

Most research on vertical distribution of Homoptera on their hosts has been conducted with aphids, with a general upward movement of individuals with plant age, regardless of whether the host is an annual or perennial

(Wyatt, 1965; Hodgson, 1978; Summy & Gilstrap, 1982; Hull & Grimm, 1983;

Jepson, 1983; Webster et al., 1983). Fewer studies have been conducted on the vertical distribution of leafhoppers on their maturing hosts, although upward movement to younger leaves increased for those species examined (Stiling, 1980; Johnson et al., 1988). The upward movement of

FI nymphs and adults of D. maidis and D. gel bus, and the significant downward movement of F2 nymphs of these species on maturing maize (ANOVA,

P < 0.05) (Fig. 6) suggests leaves vary in their suitability as feeding or oviposition sites over time, and that behavioral responses to changing leaf quality are important to leafhopper survival. The vertical movements of D. maidis and D. gel bus may be explained, in part, if these insects are tracking tissues high in nitrogen, a nutrient in limited supply to many phytophagous insects (McNeill & Southwood, 1978; Prestidge & McNeill,

1983). Soluble nitrogen, in the form of amino acids, may be in higher concentrations in the upper leaves, or leaves closest to maize ears at maturity (leaves 9-12) because materials are being mobilized and translocated to reproductive structures, and away from the pithy stem

(litis , 1987) and senescing tissues (Below et a l., 1981). Prestidge

(1982) showed that leafhopper species have different nitrogen 76 requirements, and suggested that greater mobility or other behavioral traits that increased the chances of locating optimum nutrient levels in graminaceous hosts would likely result in faster development, greater fecundity, and larger populations. If protein nitrogen is positively correlated with upper maize leaves, as was shown by Denno et a l . (1980) for the salt marsh grass, Spartina alterniflora Lois, these data suggest that the vertical displacement of D. maidis and D. aelbus on maturing maize enhances their survival compared to D. auinouenotatus. whose behavior of remaining on senescing tissue hastened its population extinction on post-anthesis maize (Fig. 6).

Although remaining low in the plant canopy is detrimental to the survival of D. Quinquenotatus on maturing maize, similar behavior on gamagrass has adaptive value. Because gamagrasses are continuously producing new tissue at plant bases, young tissue is easily accessible by horizontal movements over short distances. Movement of D. auinauenotatus horizontally rather than vertically was substantiated when nymphs and adults moved onto tille rs (Fig. 7). In comparison to gamagrass, the young, more nutritious tissues of maize are added vertically during growth, and leafhoppers must move up a considerable distance to stay in contact with these tissues. D. auinouenotatus may be adapted to move horizontally rather than vertically due to its long history of association with gamagrass (Nault, 1985; Nault, 1990), an association that has resulted in a synchronization between leafhopper behavior and plant growth pattern. D. auinauenotatus also is the only Dalbulus species that is tended by ants in the field and greenhouse (Nault et al., 1983; Triplehorn

& Nault, 1985; Nault, unpublished data). An association between ants and Homoptera has been widely recognized (Buckley, 1987 and refs, therein).

Ants often provide the homopterans with protection from predators and parasites, and remove honeydew that accumulates on the plant, while the ants receive a carbohydrate-rich food source from homopteran honeydew.

During this study, D. auinauenotatus was observed to produce more honeydew than the other two Dalbulus species. The excretion of large amounts of honeydew, coupled with the behavior of this species of remaining low on maize, may encourage ant attendance because the ants would not have to move far up on the plant to collect food, and risk displacement or exposure to predators. Myrmecophilous Homoptera also tend to aggregate in feeding clusters (Buckley, 1987). Compared to D. maidis and D. gel bus.

D. auinauenotatus is gregarious, with individuals clustering together in the lower canopy of gamagrass (Nault et al., 1983; Triplehorn & Nault,

1985). During the present study, D. auinauenotatus also clustered at the bases of maize, in contrast to D. maidis and D. gel bus. The clustering behavior of D. auinauenotatus mav facilitate ant attendance. However, the benefits of ant attendance to D. auinauenotatus may be masked on maturing maize because leafhoppers remain low on maize where leaves are senescing or dead. The lack of suitable food sources may not be compensated for by increased colony hygiene. In the present study, ants probably were only removing honeydew because cages were kept free of potential predators.

Although the data indicated vertical displacement of two Dalbulus species on maturing maize, horizontal distribution among a variety of above-ground structures may be more important in determining leafhopper survival and population growth (Figs. 5, 7). One reason why D. maidis is so successful in utilizing maize as it grows may be because it tracks the maize whorl (Fig. 7). High numbers of D. maidis are consistently found in maize whorls in the field (Power, 1987). The importance of the maize whorl in the population dynamics of Homoptera has been studied for the corn planthopper, Perearinus maidis (Ashmead), which shows a preference for ovipositing in whorl leaves prior to tasseling (J. Buth and L. R.

Nault, unpublished data), and the corn leaf aphid, Rhopalosiphum maidis

(Fitch), which develops much more rapidly when enclosed within whorl leaves surrounding the tassel than on exposed leaf blades (Foott, 1977).

The whorl may be an important resource for Dalbulus species for several reasons. Compared to males, females of D. maidis. and to a lesser extent,

D. elimatus (Ball), another maize specialist, move into whorls more quickly, and in higher numbers, suggesting the whorl may serve as a prime oviposition site for these species (J. L. Todd, unpublished data).

Expanding whorl leaves may be easier for the ovipositor to penetrate

(Heady et al., 1985) than the tougher, more lignified tissues of older leaves. Changes in leaf quality with age have been shown to influence egg distribution of the threehorned alfalfa treehopper, Spissistilis festilus

(Say), on soybean, with preferred oviposition sites shifting upward with plant maturity (Daigle et al., 1988). Whorl tissue also may have a higher

N:C ratio than maturing leaves prior to tasseling because the leaves have not yet completely unfurled and reached their full photosynthetic potential (Larcher, 1980). Denno et a l. (1980) demonstrated that the planthopper, Prokelisia maroinata (Van Duzee), prefers to colonize and feed on the youngest, terminal leaves of its Spartina hosts prior to seed head formation because these leaves have a higher protein content than the basal, older leaves. Moving into the whorl also may serve as a way to escape from enemies. Although little is known about the predators and parasites of Dalbulus species, some enemies may be avoided by moving into the whorl rather than by remaining on exposed leaf blades, even if the primary reason for movement is differential nutritional quality between maize structures. All Dalbulus species in whorls face upward, and although this behavior is probably a response to light, it may position leafhoppers so that they can rapidly leave the whorl if a potential enemy approaches.

Nault (1990) suggested that in addition to D. maidis. the most serious maize pest in Latin America, there are seven other Dalbulus species that could achieve pest status. By monitoring the spatial and temporal distribution and population dynamics of two of these species, D. gel bus and D. auinouenotatus. on maize from the seedling stage through senescence, I suggest their pest potential is limited. Although these species can successfully utilize seedling maize in the laboratory, these data suggest that maturing maize is not suitable for their survival, and that their populations will not reach sizes that could lead to the significant spread of maize-stunting pathogens. EPILOGUE

The three chapters in this dissertation provide some of the first detailed information on host-finding and accepting by leafhoppers, and on the role of various plant characteristics in these processes. Chapter I indicated that color influences both pre- and post-contact behaviors of

Dalbulus species. As with many phytophagous insects, these leafhoppers were attracted to yellow and green; however, the data are unique because they provide the first evidence that color influences post-contact behaviors (tenure) of leafhoppers. Chapter II provided the first evidence for an interaction between visual and olfactory stimuli during host-finding by a leafhopper, and also for an effect of host volatiles on post-contact, nonfeeding behaviors. In Chapter III, a rationale was provided for the anomaly between laboratory and field associations of gamagrass-specializing Dalbulus with maize by demonstrating these species exhibit different within-plant distributions and developmental times than maize-specializing Dalbulus on maturing maize. The data suggest that behavioral responses of gamagrass specialists to maturing maize may limit their ability to produce large populations, and thereby have a significant

impact on the spread of maize-stunting pathogens.

Host-finding and accepting by Dalbulus leafhoppers in the field has not been studied; however, the data in this dissertation provide insight

into how the behavioral responses of three Dalbulus species to plant

80 characteristics might influence host-finding and accepting under natural conditions. A leafhopper flying above plant canopies may use color stimuli to distinguish foliage from nonfoliage. Host plants reflect light maximally between 500-580 nm (green to yellow/green) (Prokopy & Owens,

1983); therefore, the strong response of these leafhoppers to green and yellow would orient them to plants (Chapter I). Although the data in

Chapter I indicated that value (intensity) alone was not important during host-finding, value may be important in combination with hue (interaction effect). For example, Dalbulus species may be more attracted to the brighter yellow areas of maize (e.g., whorl tissue, and young or senescent leaves) than to mature leaves that reflect a lower percentage of light in the 500-580 nm range (Mooney & Gulmon, 1982). The principle vector of

CSS, D. maidis. also may be attracted to infected maize whose leaves have a characteristic chlorotic striping (Nault, 1980), or to the whorl leaves of maize infected with MBSM, which are much more yellow in appearance than the older, surrounding red leaves (Nault, 1980). Although not tested directly, the green (G) model used in Chapter I was similar in color to that of some bean foliage (Phaseolus species), and leafhoppers were not attracted to this model compared to the other green models, light green

(LG) and whorl green (WG), which contained increasing amounts of yellow, respectively (Fig. 1). Intercropping of maize and bean in Mexico and

Costa Rica (Power, 1987) is common. Perhaps the number of Dalbulus leafhoppers landing on maize in fields intercropped with beans would be reduced compared to fields of pure maize because the darker green foliage of beans would disrupt orientation behaviors. 82

Although color may draw leafhoppers closer to foliage, color stimuli alone probably are not a very discriminating characteristic during host- finding. Host plants appear green; therefore, responsiveness to other plant stimuli would be advantageous in fine-tuning host selection. In

Chapter II, I showed that D. maidis was differentially responsive to plant volatiles. Leafhoppers showed a positive response to maize volatiles, a neutral response to gamagrass volatiles, and a negative response to sorghum volatiles, as measured by the number of contacts made with green light. These data suggest that in the field, D. maidis flying or walking close to a plant may be able to distinguish stimulatory from deterrent airborne chemicals, and adjust its behavior accordingly. For example, a positive response to maize volatiles may induce landing and arrestment on maize, a plant which is suitable for feeding and the development of large populations (if the plant is found during the seedling stage) (Chapter

III). The neutral response to gamagrass volatiles may be advantageous because D. maidis will be less likely to land on gamagrass, a marginal host (Nault & Madden, 1985), than on maize if both plants are available.

Sorghum, although morphologically very similar to maize during the seedling stages, may repel D. maidis by emitting volatiles that deter orientation behaviors. My data suggest that close range chemo-orientation is a mechanism by which D. maidis can distinguish host from nonhost plants prior to contact. The distance over which airborne chemicals are perceived is not known. However, even if responsiveness is only operating at close-range, D. maidis may benefit by wasting less time and energy landing on plants not suitable for feeding and/or oviposition. The positive response of D. maidis to maize volatiles provides further insight 83 into the pest status of this species. Preliminary data indicated the

Tripsacum specialist, D. charlesi Triplehorn & Nault, which can be reared on maize in the laboratory, was not attracted to a foliage cue (green light) when exposed to maize volatiles (J. L. Todd, unpublished data).

Once Dalbulus leafhoppers have found a potential host, the behavioral responses they exhibit to intraplant variation (e.g., in nutrition and morphology) with plant maturity can have a dramatic influence on reproductive success and population growth. Data from Chapter III indicated that although D. maidis. D. gel bus, and D. auinouenotatus can be reared and maintained on seedling maize in the laboratory, the latter two species will not be successful on maize as it grows, and probably will emigrate from maturing maize in the field. The data indicate that if D. maidis locates a maize field when the plants are seedlings (4-leaf), it can utilize plants until senescence, and attain large population sizes

(Fig. 5). D. auinauenotatus does poorly on maturing maize, and if given no alternative food source, populations of this species probably will go extinct (Fig. 5). D. gel bus is intermediate between D. maidis and D. auinauenotatus with regard to utilization of maturing maize for feeding and population growth (Fig. 5). Although I did not examine the mechanisms underlying the differential success of these three Dalbulus species on maize, the spatial distributions, both vertical and horizontal, suggest maturing maize undergoes changes in morphology and/or nutrition that can only be dealt with successfully by D. maidis. The pest potential of D. gel bus and D. auinauenotatus (Nault, 1990) therefore is limited because they will not reach population sizes large enough to damage maize by direct feeding, or more importantly, by spreading maize-stunting pathogens over a wide area. Although one D. maidis leafhopper can transmit maize-stunting pathogens (e.g., CSS) to many plants within a field, the ability of this species to produce large populations on maize compared to the other two species may promote disease spread to other fields if leafhoppers disperse to new host locations when local maize fields senesce. Because D. gel bus and D. Quinquenotatus are less efficient vectors than D. maidis (Nault, 1985 and refs, therein), more leafhoppers would be needed to spread the pathogens, both within and between maize fields. LIST OF REFERENCES

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INFORMATION to USERS the Most Advanced Technology Has Been Used to Photo­ Graph and Reproduce This Manuscript from the Microfilm Master

INFORMATION to USERS the Most Advanced Technology Has Been Used to Photo Graph and Reproduce This Manuscript from the Microfilm Master