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Studies of leafhopper probing behavior and its role in maize chlorotic dwarf virus transmission
Wayadande, Astri Cassandra, Ph.D. The Ohio State University, 1991
UMI 300 N. ZeebRA Ann Aibor, MI 48106
STUDIES OF LEAFHOPPER PROBING BEHAVIOR AND ITS ROLE IN
MAIZE CHLOROTIC DWARF VIRUS TRANSMISSION
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Astri Cassandra Wayadande
The Ohio State University
1991
Dissertation Committee: Approved by
L. R. Nault
P. L. Phelan
R. E. Gingery Adviser / D. J. Horn Department of Entomology ACKNOWLEDGMENTS
This dissertation could not have been completed were it not for the help and support that I received from many people. I am indebted to my advisor and mentor, Dr. Lowell "Skip" Nault for his guidance and lively discussions which changed the direction of my research interests. I am also grateful to my committee members Drs. David Horn, Roy Gingery and co-advisor Larry Phelan for advice given throughout the research. I also thank Drs. El-Desouky
Ammar, Randy Hunt and Elaine Backus for their valuable discussions about virus transmission and Larry Madden for statistical advice.
I come away from Ohio State with not only a degree, but also with many close friends who made my graduate experience a positive one. I thank Jackie
Blackmer, Julie Todd, Andrew Chappie, Ed Zaborski, Clarissa Maroon, Sharon
Wainshilbaum and the other graduate students for their friendship and support. I am especially grateful to Susan Heady, Robin Taylor and Bill Styer for their friendship and for encouraging me when I needed it the most. Finally,
I am thankful for the support of my family, especially my mother, Frances, and husband, Art Bisges, who walked the dogs when I could not. VITA
December 1,1960 ...... Born - Porterville, California
1983...... B.S. University of California, Davis, California
1987...... M.S. University of Missouri, Columbia, Missouri
1987-1991...... Graduate Research Associate Ohio State University - OARDC, Wooster, Ohio
1991-present...... Research Associate, Oklahoma State University Stillwater, Oklahoma
PUBLICATIONS
Wayadande, A C. & E. A. Backus. 1989. Feeding behavior of the potato leafhopper, Empoasca fabae (Harris) (Homoptera: Cicadellidae) on phostmet- and chlordimeform-treated alfalfa. J. Econ. Entomol. 82: 766-772.
FIELD OF STUDY
Major Field: Entomology TABLE OF CONTENTS
ACKNOWLEDGMENTS ...... ii
VITA...... iii
LIST OF TABLES...... vi
LIST OF FIGURES...... viii
CHAPTER
PROLOGUE...... ;...... 1
I. LEAFHOPPER PROBING BEHAVIOR ON MAIZE AND JOHNSONGRASS...... 4
Introduction...... 4 Materials and Methods ...... 6 Results...... 10 Discussion...... 29 Chapter I References...... 36
II. LEAFHOPPER PROBING BEHAVIOR ASSOCIATED WITH MAIZE CHLOROTIC DWARF MACHLOVIRUS TRANSMISSION TO MAIZE...... 41
Introduction...... 41 Materials and Methods ...... 44 Results...... 50 Discussion...... 59 Chapter II References...... 65 III. LEAFHOPPER TRANSMISSION OF MAIZE CHLOROTIC DWARF MACHLOVIRUS ISOLATES FROM MAIZE AND JOHNSONGRASS AND FROM MAIZE DOUBLY-INFECTED WITH TWO MCDV STRAINS...... 68
Introduction...... 68 Materials and Methods ...... 71 Results...... 74 Discussion...... 79 Chapter III References...... 84
EPILOGUE...... 87
APPENDICES
A. Salivary Inactivation Experiments...... 92
LIST OF REFERENCES...... 97
v LIST OF TABLES
TABLE PAGE
1. Association of electronically monitored waveform patterns with the distal end of the salivary sheaths of Graminella nigrifrons and Dalbulus maidis in maize tissue...... 15
2. Excretion rate and pH of honeydew droplets produced during salivation, x-waveforms, phloem ingestion, non-vascular probing, and non-sieve element ingestion by electronically monitored Graminella nigrifrons and Dalbulus maidis on maize...... 16
3. Number of Graminella nierrifrons. Amblvsellus errex. Graminella oquaka. and Dalbulus maidis leafhoppers which produced salivation, x-waveform, phloem ingestion, non-vascular probing, and non-sieve element ingestion patterns when electronically monitored for 180 minuts on maize or johnsongrass...... 20
4. Average time probing, salivating, producing x-waveforms, ingesting from phloem, probing non-vascular tissues, and ingesting from non-sieve elements for Graminella nigrifrons Amblvsellus errex. Graminella oquaka. and Dalbulus maidis when electronically monitored for 180 minutes on maize or johnsongrass...... 21
5. Number of probes, duration of salivation, x-waveform, phloem ingestion and total probing of viruliferous Graminella nigrifrons that did or did not transmit MCDV during electronically monitored inoculation test feeds on maize of up to 45 min...... 54
6. X-waveform variable means used to differentiate MCDV vectors and non-vector leafhoppers by cluster analysis...... 60
vi 7. Percent of maize test plants inoculated with MCDV Ml, M8, or both by single Graminella nigrifrons females which had been exposed previously to healthy, singly- or doubly-infected maize plants...... 78
vii LIST OF FIGURES
FIGURE PAGE
1. Electronically recorded patterns of Graminella nigrifrons probing maize...... 11
2. Comparison of representative x-waveforms and phloem ingestion of A) Graminella nigrifrons. B) Amblvsellus grex. C) Graminella oquaka and D) Dalbulus maidis electronically recorded on maize...... 13
3. Center sections of x-waveform sequences of A. Graminella nigrifrons and B. Dalbulus maidis electronically monitored on maize...... i...... 14
4. Average time of x-waveform sequences of Graminella nigrifrons. Amblvsellus grex. Graminella oquaka. and Dalbulus maidis electronically monitored for 180 min on maize and johnsongrass...... 22
5. Kinematic diagram of probing behavior transitions for Graminella nigrifrons electronically monitored for 180 min on maize and johnsongrass...... 24
6. Kinematic diagram of probing behavior transitions for Amlvsellus grex electronically monitored for 180 min on maize and johnsongrass...... 25
7. Kinematic diagram of probing behavior transitions for Graminella oquaka electronically monitored for 180 min on maize and johnsongrass...... 26
8. Kinematic diagram of probing behavior transitions for Dalbulus maidis electronically monitored for 180 min on maize and johnsongrass...... 27
viii FIGUREPAGE
9. Waveforms from Graminella nigrifrons leafhoppers recorded while feeding on maize ...... 49
10. Representative middle sections of x-waveform sequences of A. Graminella nigrifrons and B. Dalbulus maidis electronically monitored on maize...... 51
11. Mean transmission rate of single Graminella nigrifrons females exposed to MCDV infected maize for 24 hr followed by a 0.25, 0.50, 1, 2, 4, 8, 16, and 24 hr inoculation access period on maize test plants...... 52
12. Relationship between Graminella nigrifrons x-waveform duration and percent transmission to maize...... 56
13. Representative three minute excerpts of center sections from x-waveforms sequences of ten electronically monitored leafhoppers...... 58
14. Dendrogram of MCDV vectors Graminella nigrifrons. Graminella oquaka. Graminella sonora. Amblvsellus grex. Stirellus bicolor and non-vectors Dalbulus maidis. Dalbulus quinquenotatus. Ollarianus strictus. Eucelidius variegatus. and Macrosteles quadrilineatus based upon cluster analysis of x-waveform characteristics...... 61
15. Percent of maize test plants inoculated with maize chlorotic dwarf virus mild and white stripe isolates of the type strain and the Ml strain by single Graminella nigrifrons. Amblvsellus grex. and Dalbulus maidis leafhoppers acquiring from maize or johnsongrass sources...... 76 PROLOGUE
Leafhoppers are important vectors of plant pathogenic viruses and
bacteria. During feeding, they may acquire or transmit plant pathogens when
their straw-like mouthparts pierce plant cells. Unlike aphid-borne viruses, which include those transmitted in a non-persistent, semipersistent or persistent manner, most leafhopper-borne viruses are persistently transmitted
(Nault & Ammar 1989). The exceptions are three semipersistently transmitted viruses of agronomic grasses, rice tungro machlovirus (RTMV), rice tungro badnavirus (RTBV) and maize chlorotic dwarf machlovirus
(MCDV).
MCDV has a 30 nm isometric particle and a ssRNA (Gingery 1988). It infects several grasses including maize and its overwintering host johnsongrass (Gordon et al. 1977). The distribution of MCDV is limited to the area of overlap between johnsongrass and its primary field vector, Graminella nigrifrons (Forbes). MCDV symptoms in maize include slight to moderate stunting caused by shortening of the internodes, intermittent clearing of the secondary and tertiary veins, overall chlorosis, and sometimes reddening, yellowing, or tearing of the leaves. Within the infected plant, MCDV is
1 limited to the phloem and phloem parenchyma, where it replicates and sometimes forms inclusion bodies (Ammar et al. 1987). MCDV does not replicate in the leafhopper vector.
MCDV is a foregut-borne virus. MCD virus-like particles (VLP’s) were found embedded in a densely staining matrix attached to the cuticular lining of the precibarium, cibarium, and pharynx of G. nigrifrons which had fed upon
MCDV infected corn 0 or 4 hours earlier, but not 4 days earlier. No VLP’s were observed in the foreguts of leafhoppers fed on healthy corn (Ammar&
Nault 1991). The densely staining material is thought to be the helper component, thought to be a viral-gene produect which aids in virus attachment to the leafhopper cuticle (Ammar & Nault 1991). MCDV can be transmitted immediately after acquisition. MCDV is retained for up to 72 hr, although longer retention occurs at lower temperatures (Nault, unpublished).
If acquired by nymphs, MCDV transmissibility is lost after molting (Nault et al. 1973). Although MCDV is found in the phloem, it is not transmitted by
Dalbulus maidis Delong & Wolcott, a vector of other phloem-limited pathogens. When other leafhopper species were tested as MCDV vectors,
Nault & Madden (1988) showed that leafhoppers closely related to G. nigrifrons within the tribe Deltocephalini and recent Eucelini were the best vectors. It was reported that MCDV virions attached to vector species but not non-vectors (Childress & Harris 1989). This was recently refuted by evidence that MCDV attached to D. maidis (Ammar & Nault 1991). This finding gave support to an alternative hypothesis, that leafhopper feeding
behavior was determining vector specificity.
This dissertation represents research primarily done to explore the
feeding behavior of leafhoppers and its role in the transmission of MCDV. In
Chapter I, I characterized the probing activities of four leafhopper species
using an electronic monitoring device. Specifically, I looked for quantitative
and qualitative differences in probing behavior between four leafhopper
species feeding on maize or johnsongrass. In Chapter II, I focused upon the phloem-associated probing behavior of G. nigrifrons to determine precisely when MCDV inoculation occurs during the feeding process. I also analyzed the waveform pattern associated with MCDV inoculation and identified similarities and differences among patterns of vector and non-vector species.
Finally in Chapter III, MCDV hosts, isolate and vectors were compared to determine their effect on transmission efficiency. CHAPTER I
Leafhopper Probing Behavior on Maize and Johnsongrass
INTRODUCTION
Leafhoppers and other homopterans feed by inserting stylets into plant tissues where they feed on plant sap. Because feeding occurs beneath the plant epidermal layers, stylet tips and associated feeding behavior cannot be observed. The electronic feeding monitor (McLean 1965) has enabled researchers to learn many details of in situ homopteran feeding. Compared to aphids (McLean & Kinsey 1967, Campbell et al. 1982, Dorschner et al.
1990), only a few leafhopper species have been studied using the alternating current electronic monitoring system (AC-EMS). These include, a xylem feeder (Crane 1970), a mesophyll feeder (Hunter & Backus 1989, Wayadande
& Backus 1989) and four species that feed primarily from the phloem (Kawabe
& McLean 1978, Kawabe & McLean 1980, Triplehorn et al. 1984, Rapusas &
Heinrichs 1990). Several AC-EMS waveforms have been associated with phloem feeders. Salivation, probing of non-vascular tissue and ingestion from
4 phloem, xylem, and non-vascular tissues are the probing behaviors associated with four waveforms. The behavior(s) associated with the x-waveform are uncertain, however, phloem ingestion is always preceded by this waveform.
Because so few leafhopper species have been monitored, it is difficult to make generalizations about their feeding behavior. Previous EMS studies with leafhoppers have involved feeding behaviors associated with host plant selection and resistance. Before now, no one has used EMS to study virus transmission by these insects.
Maize chlorotic dwarf machlovirus (MCDV) is a semipersistently transmitted (Nault et al. 1973) phloem-restricted virus (Ammar et al. 1987) transmitted in the field by Graminella nigrifrons (Forbes) (Gordon & Nault
1977). The virus has been experimentally transmitted by eight additional deltocephaline leafhopper species (Nault & Madden 1988). Sixteen other leafhopper species tested failed to transmit MCDV in laboratory tests. Vector leafhopper species are more closely related to one another than non-vectors.
It has been suggested that vector specificity for MCDV may be due, in part, to differences in feeding behavior (Nault & Madden 1988, Nault & Ammar
1989, Ammar & Nault 1991). Thus in this paper, I examine in detail, using the newly modified AC-EMS, the feeding behavior of four leafhopper species.
Based on these studies, I report that the feeding behaviors of three vector species G. nigrifrons. Amblvsellus grex (Oman), and Graminella oquaka
DeLong are similar to one another and differ from Dalbulus maidis (DeLong 6 & Wolcott), a species that does not transmit MCDV.
MATERIALS AND METHODS
Leafhoppers and Plants
Leafhoppers were reared in organdy covered cages (D’arcy & Nault 1982) in a room held at 27 ± 2 C, under 16:8 L:D photoperiod. G. nigrifrons and A. grex. were reared on oats, Avena sativa (variety unknown). G. nigrifrons was collected from grasses near Wooster and A. grex from grasses in Utah County,
Utah. G. oquaka and its host plant, Panicum virgatum L., were collected in
1987 near Brewster, OH. Laboratory colonies of G. oauaka were supplemented yearly from collections made at the same site. G. oauaka was reared on mature P. virgatum grown from rhizomes. The D. maidis colony was started from adults collected on maize near Tepexpan, Mexico and was reared on sweet corn (variety ’Aristogold Evergreen Bantam’). Adult females
1-3 wk post-eclosion were used in all tests. Leafhoppers were caged on the recording host for a 24 hr acclimation period prior to electronic monitoring.
Maize and seedling johnsongrass were grown in a greenhouse and used in experiments after reaching the 5-6 leaf stage. Experimental Procedures
G. nigrifrons. G. oquaka. and A. grex were electronically monitored on
maize and johnsongrass. D. maidis was monitored on maize only. A 2.5 cm
segment of 12 um diam gold wire tether (Sigmond Cohn Inc., Mt. Vernon, NY)
was attached with silver conductive paint (Ladd Industries, Burlington Vt.) to
the pronotum of adults immobilized on a stage by a gentle vacuum. Tethered
leafhoppers then were placed onto the abaxial surface of a severed leaf of the
recording host for a 1 hr acclimatation period.
Five to six-leaf maize or johnsongrass plants were severed at their bases
and placed in a glass vial containing the voltage input electrode. Leaves were laid flat onto a Plexiglas^ holder so that leafhoppers could feed on the abaxial surface. An alligator clip on the holder held leafhoppers attached to a 2.5 cm copper stub glued to the gold wire tether. Leafhoppers were monitored electronically with the Insect Feeding Monitor (IFM) (Electronic Instruments
Laboratory, University of Missouri, Columbia, MO) for three hr. The IFM is a differential amplifier with two input electrodes; noise from the reference electrode is automatically subtracted from the signal of the insect electrode.
A 70 mv current with a carrier frequency of 125 Hz was applied to the plant by the input electrode. Because of the low voltage, it was not necessary to modify the signal by logarithmic scaling. After amplification, the signal was sent to a strip chart recorder (Servagor 430, ABB Metrawatt, Bloomington,
CO) operated at 100 mV sensitivity and chart speed of 3 cm/min. Eighteen or 19 leafhoppers were monitored for each species/host combination using a
completely randomized design.
Waveform patterns on strip charts were identified and measured using a
standard metric ruler. Differences in the number of leafhoppers producing
specific patterns were determined using Chi-square analysis. Differences in
probe number and duration of salivation, x-waveform behavior, phloem
ingestion, non-vascular probing, non-sieve element ingestion and total probing
were tested using analysis of variance (Minitab, Inc.). Means were compared
with the Least Significant Difference mean separation test if there was a
significant F-value. Only leafhoppers which produced patterns were included
in the analyses. When necessary, data were subjected to square root or log
transformation to stabilize heterogeneity of variance.
To describe changes in behavior during probing, transitional matrices were constucted in which each cell in the matrix (Nij) was the number of times behavior i was preceded by behavior]. First order transitions were tested for randomness by the G test statistic (Sokal & Rohlf 1969) applied to a 2 x 2 collapsed table around each cell (Hancock et al. 1989). Transition probabilities
> 0.02 were used to construct kinematic diagrams for each species-host combination. Specific transitions were compared among species-host combinations by chi-square analysis of 2 x 2 collapsed tables around cells containing the transition being analyzed (Paynter et al. 1990). To associate waveform patterns with probing behaviors in specific plant tissues, G. nigrifrons and D. maidis leafhoppers producing specific waveforms were interrupted and plant tissues examined for sheath saliva. Plant tissues
(2x2 mm) were excised and fixed in 0.1 M phosphate buffer containing 3% gluteraldehyde, 2% paraformaldehyde and 1.5% acrolein for a minimum of 3 d then dehydrated in increasing percentages of ethanol and tertiary-butyl alcohol (30%-100%). Tissues were then infiltrated with ParaplastR, embedded, sectioned at 12 um, and stained with safranin and fast green for examination under the light microscope at 100 to 400 X. Salivary sheaths and xylem vessels stain red and other tissues stain green.
To further relate waveforms to probing behavior, honeydew pH and rate of droplet production was studied. Honeydew pH was determined by collecting droplets with a glass microsyringe pulled by a Micropipette Puller
(Model Ml) (Industrial Science Associates, Ridgewood, NY) and spotting them onto pH indicator paper (Micro-Essential Laboratories, Brooklyn, NY). In some cases, leafhoppers were allowed to excrete directly onto the indicator paper. Buffers of known pH were used as standards. Honeydew droplet excretion rates were calculated by counting droplets and dividing by the number of minutes in the observation period. 10 RESULTS
Waveform Descriptions and Associated Probing Activities
G. nigrifrons. A. grex. G. oquaka. and D. maidis produced five waveform
patterns while feeding on maize and johnsongrass. Similar waveforms have
been reported for other leafhopper species (Kawabe & McLean 1978, 1980,
Rapusas & Heinrichs 1990) and G. nigrifrons (Triplehorn et al. 1984). In this
paper I use the same terms to describe the salivation, phloem ingestion and
non-sieve element sap ingestion waveforms. I also use the term x-waveform
to describe the stereotypic pattern of unknown activity which preceeds
phloem ingestion (McLean & Kinsey 1967, Triplehorn et al. 1984) and refer to that activity as x-waveform behavior. I use the term non-vascular probing for the pattern previously known as the ’R’ waveform (Sogawa 1972, Rapusas
& Heinrichs 1990). The rationale for this is explained later in the discussion.
All leafhopper species produced identical salivation, non-vascular probing and non-sieve element ingestion waveforms. Representative patterns associated with these behaviors are shown in in Fig. 1. X-waveforms and phloem ingestion patterns were similar among G. nigrifrons. A. grex. and G. oquaka but were different qualitatively from those produced by D. maidis (Figs. 2,3).
Interpretation of behaviors associated with waveforms is supported by salivary sheath termination points (Table 1), honeydew excretion rates and droplet pH (Table 2). When leafhoppers produced salivation waveforms i—NSI —■ ------X — S —------NVP------NSI 1 - S ---
Figure 1. Electronically recorded patterns of Graminella nigrifrons probing maize. S = salivation, NSI = non-sieve element ingestion, NVP = non-vascular probing, X = x-waveform. Waveforms are read right to left, small arrow denotes probe initiation, bar insert = 60 sec. Note drop-off in amplitude of the NSI (large arrow) relative to the x-waveform. Compare this to the mid-amplitude phloem ingestion waveform following x-waveform in Fig. 2 Figure 2. Comparison of representative waveforms and phloem ingestion of
A. Graminella nigrifrons. B. Amblvsellus grex. C. Graminella oquaka. and D.
Dalbulus maidis electronically monitored on maize. S = salivation, X = x- waveforms, PI = phloem ingestion. Waveforms read right to left, bar insert
= 60 sec.
12 .------PI X s
p i
UU j UU
-PI i— ------section * A phr I phr II.
section
i------1
Figure 3. Center sections of x-waveform sequences of A. Graminella nigrifrons and B. Dalbulus maidis electronically monitored on maize. Phr I = phrase I
(smooth phrase) and Phr II = phrase II (spiking phrase), bar insert = 60 sec. Table 1. Association of electronically monitored waveform patterns3 with the distal end of the salivary sheaths of firaminoHa niprifrnns and Dalbulus maidis in maize tissue.
tali vary sheath termination points In aaiie tissues
wavefora aesophyll or mclesr or pattern______N______xylea phi oca bundle sheath______col tench vaa not fotsid
fi* niarifrona salivation 10 0 0 7 0 3
x*wavefora IS 0 7 3 1 4
phioca 12 0 8 0 0 4 Inecstion
non-vsscular 29 0 1 14 0 14 probing non'sieve eleaent 17 Q 1 10 0 6 Ingestion
J>. aaidis salivation 10 0 0 6 0 4
x-uavefora 4 0 2 0 O ' 2
phioca 3 0 3 0 0 0 ingestion
non-vascular 20 0 1 10 0 9 probing
non* si eve eleaent 25 0 0 14 0 11 ingestion
a Leafhopper feeding was internpted by reaoving leaf hoppers during indicated behavior, then leaf tissue on
which the leafhopper was feeding was excised and processed for observation by thick section light atcroscopy. Table 2. Excretion rate and pH of honeydew droplets produced during salivation, x-waveforms, phloem ingestion, non-vascular probing, and non-sieve element ingestion by electronically monitored flraminella nierrifrons and
Dalbulus maidis on maize.
no. of tiaes no. of leafhoppers
wavefora behavior was which secreted n n no. oean pH
pattern recorded honeydew drops/.in ♦- SO ♦- so
0. nlarifron* salivation 32 5 • -- -
x-wevefora 28 0 -- --
phloea ing. 19 19 0.70 ♦- 0.31 7.02 ♦- 0.15
non-vatc. prob. 10 5 * ----
non-si eve eleaent 21 K 0.53 ♦- 0.37 6.70 ♦- 0.2* ingestion
B- m id lt salivation 15 1 b ---
x-wavefora 10 0 -•--
phloea ing. 9 9 0.62 ♦- 0.38 6.90 ♦- 0.23
non-vasc. prob. H 1 b ----
non-sieve eleaent 22 9 0.38 ♦- 0.25 6.50 ♦- 0.71 ingestion
■ five tiaes • tingle droplet was excreted but only when following periods of ingestion froa phloea or non- sieve eleaent tissue
b one tia e • single droplet was excreted but th is was followinga period of ingestion froa non-vascular tissue 17 (Fig. 1), sheaths terminated in non-vascular tissues (mesophyll parenchyma and bundle sheath cells). D. maidis and G. nigrifrons infrequently produced honeydew droplets when the salivation waveform was recorded (Table 2).
When non-vascular probing was recorded, salivary sheaths for both leafhopper species usually terminated in the mesophyll or bundle sheath (Table 1) and the behavior infrequently was associated with the excretion of honeydew
(Table 2). Phloem and non-sieve element sap ingestion for G. nigrifrons and
D. maidis were associated with honeydew droplets of neutral to basic pH
(Table 2). Honeydew droplets excreted by leafhoppers and planthoppers when they feed from phloem have a neutral to basic pH, whereas when they feed from xylem the honeydew is acidic (Auclair et al. 1982, Kimmins 1989).
Although droplet rate for G. nigrifrons was stable at 0.7/min after 1 hr sustained phloem ingestion, non-sieve element ingestion rates were more difficult to measure because a series of droplets rarely were produced during brief (avg. 6.1 min) non-sieve element ingestion bouts.
Although there was no unique waveform pattern associate^ with xylem ingestion, G. oquaka occasionally produced 6-10 (pH 4-5) droplets per min during the non-sieve element ingestion pattern. One leaf tissue containing the salivary sheath of a leafhopper producing rapid, low pH droplets was examined and the sheath was found terminated in the xylem. G. nigrifrons.
A. grex. and D. maidis never produced rapid, low pH droplets during ingestion waveforms in this study. However, in another study G. nigrifrons produced 18 rapid, acidic droplets on young maize seedlings during the non-sieve element
ingestion pattern (3-4 leaf stage)(Wayadande, unpublished) and was thought
to be xylem ingestion.
X-waveforms were produced in sequences. I adopt the terminology used
by Heady & Denno (1991) to describe acoustic signals of planthoppers for
describing the x-waveform sequences of these insects. G. nigrfrons (Fig. 2A),
A. grex (Fig. 2B), and G. oquaka (Fig. 2C) sequences consisted of 5-25 repeated
sections. Each section (1 section = 1 waveform) was comprised of two phrases, a smooth phrase and a spiking phrase (Fig. 3A). Section duration averaged 60 sec and increased with each successive section culminating in either phloem ingestion or transition to another pattern. D. maidis x- waveform sequences (Fig. 2D) consisted of 80-120 repeated single phrase sections, each approximately 5-10 sec, with 1-3 intermittent spikes througout the sequence.
Two ingestion patterns followed x-waveforms. The phloem ingestion pattern of G. nigrifrons. A. grex. and G. oquaka consisted of a mid-amplitude, level waveform with regularly occurring spikes, one every 20-30 sec (Fig 2A-
C). Between these spikes were 4-8 smaller spikes. D. maidis phloem ingestion pattern was also mid-amplitude, but flat without spikes or other characteristics (Fig. 2D). Unlike phloem ingestion which was always preceded by x-waveforms, non-sieve element ingestion was preceded by salivation, x- waveforms, or non-vascular probing. Non-sieve element ingestion was 19 distinguished from the phloem ingestion pattern by its low amplitude and
’noisy’ irregular appearance (Fig. 1 see arrow).
Comparison of Probing Behavior on Maize and Johnsonerrass
G. nigrifrons. A. grex. G. oquaka and D. maidis displayed little difference
in total probing duration during the 180 min access period (F = 2.89, df =
6,121 NS) regardless of host. D. maidis probed more frequently than other
species on maize. Because of the high rate of probing, average duration of D.
maidis probes was correspondingly shorter than for other species (F = 3.60, p < 0.005). All leafhoppers salivated during probing (Table 3) however, salivation duration was different among species. A. grex salivated significantly more when feeding on maize than on johnsongrass and salivated more than any other species (Table 4).
Significantly fewer D. maidis produced x-waveforms than G. nigrifrons
(Table 3). Host plant did not affect total duration of x-waveform behavior, however, there were differences between the four species probing maize. G. oquaka x-waveform behavior was shorter than for G. nigrifrons while average
D. maidis behavior was longer (Table 4). Because leafhoppers often initiated more than one x-waveform sequence in the 180 min access period, individual x-waveform sequences were analyzed separately. When examined individually,
D. maidis x-waveform sequences were longer than those of G. nigrifrons. A. grex. and G. oquaka on maize (Fig. 4). Table 3. Number of Graminella nigrifrons. Amblvsellus grex. Graminella nnnaka. and Dalbulus maidis leafhoppers which produced salivation, x-waveform, phloem ingestion, non-vascular probing and non-sieve element ingestion patterns when electronically monitored for 180 min on maize or johnsongrass.
Hunber of leafhoppers producing pattern
phloem non-vascular non-sieve elerne
Species Host N salivation x-waveform ingestion probing ingestion
G. nigrifrons maize 19 19 a 19 a 18 a 12 a 17 a
G. nigrifrons johnsongrass 18 18 a 15 ab 15 a 14 a 16 a
A. grex maize 18 18 a 12 ab 11 ab 16 a 18 a
A. grex johnsongrass 18 18 a 16 ab 12 ab 16 a , 18 a
G. oquaka. maize 18 18 a 14 ab 10 ab 14 a 17 a
G. oquaka johnsongrass 18 18 a 14 ab 12 ab 15 a 14 a
D. maidis maize 19 19 a 6 b 5 b 18 a 17 a
frequencies in columns followed by different letters are significantly different, confidence interval test, = 12.590, df = 6, p < 0.05)
Oto Table 4. Average time probing, salivating, producing x-waveforms, ingesting from phloem, probing non-vascular tissues, and ingesting from non-sieve element tissues for Graminella nigrifrons. Amblvsellus grex. Oflminplla oquaka. and Dalbulus maidis when electronically monitored for 180 min on maize (M) or johnsongrass (J).
aaan a SE (ain)
(n )b
to ta l phloea non* vascular non-sievs eleaent
specias boat b- probing salivation a-uevefora in g estio n probing ingestion
S- nianfcsm N 19 171.1 a 2.5 ac 14.3 a 2.9 a 15.3 a 1.7 c 96.9 a 11.5 ate 30.0 ♦ 9.3 a 33.3 * 0.4 b (19) (19) (19) (10) (12) (17)
C. n io rifro n . J 10 160.0 a 3.4 a 12.2 a 4.1 a 13.7 a 1.6 c 127.5 a 0.3 c 39.7 ♦ 13.3 a 10.3 * 2.7 a
(10) (10) (15) (15) (M> (16)
&• U U H 10 155.0 « 5 .9 a 42.5 « 4.3 c 14.5 a 1.6 be 64.7 a 14.0 a 47.4 « 10.6 a 19.4 e 2.9 * (10) (10) (12) (11) (14) (10)
4* »r»« 4 10 146.2 a 0 .6 a 31.5 a 5.4 b 14.4 a 2.0 c 09.1 a 14.0 wb 35.4 ♦ 9 .2 a 11.4 * 2 .7 e
(10) (10) (15) (12) (16) (10)
fi. cart. N 10 165.2 a 4.0 a 30.3 a 6.3 b 9.2 a 2.1 ab 09.9 a 19.3 ate 74.6 ♦ 15.0 a 20.7 ♦ 2 .7 b
(10) (10) (13) (10) (U > (17)
fi. M a t i J 10 151.1 a 7.0 a 10.23 a 4.6 a 0.0 a 1.0 a 93.4 a 15.2 abc 55.0 ♦ 12.0 a 14.4 « 3.7 ab
(10) (10) (13) (12) (15) (14)
£ . n a id is N 19 163.5 a 2 .0 a 10.54 a 2 .0 a 27.7 a 5.3 d 54.5 a 13.5 a 50.7 ♦ 0 .0 a 66.2 ♦ 7 .9 c
(19) (19) (6) (5 ) (10) (19)
* ruber ot insects recorded
b (n) ■ lu tx r of Insects fren uhich behaviorm u recorded
c aaan* in coltane followed by different iattara era significantly different, least aignificant diffaranca asan separation last: total probing f ■ 2.96,
df • 6,121, p « 0.05; salivation P • 4.06, df • 6, 121, p < 0.000; A-uevstora P • 4.91, df • 6, 06, p « 0.000; phloea (ngetilon I • 2.55, dt • 6, 76, p to * 0.027; non-vascular probing f • 1.75, df ■ 6,90, K; nnvvsscuiar ingestion P ■ 14.00, df ■ 6,112, P * 0.000. t-a 22
•ma 20 B a 15 a ► »® » X *
o O. aifrifroma
Figure 4. Average time of x-waveform sequences (summed x-wave durations/number of sequences performed) of Graminella nigrifrons.
Amhlvsellus grex. Graminplla oquaka. and Dalbulus maidis electronically monitored for 180 min on maize and johnsongrass. D. maidis was monitored on maize only. Vertical lines indicate standard error of the mean, numbers indicate sample size, and means with different letters are significantly different (F = 13.42, df = 6, 217, p < 0.05), least significant differences mean separation test. 23 Phloem was the predominant ingestion site for G. nigrifrons. A. grex. and
G. oquaka. On maize, phloem ingestion comprised 66% or more of all
ingestion and more than 75% of ingestion on johnsongrass for these species.
Not all leafhoppers ingested from phloem; but of those that did, there were
significantly fewer D. maidis compared to G. nigrifrons that did (Table 3).
Fewer A. grex and G. oquaka ingested from phloem than G. nigrifrons.
however, these differences were not significant. Duration of phloem ingestion
did not differ between species monitored on maize compared to johnsongrass,
nor were there differences between the two hosts for each species (Table 4).
Frequency and duration of non-vascular probing did not differ between
leafhopper species or between hosts. Of the leafhoppers monitored on maize,
D. maidis ingested significantly longer from non-sieve element tissues than did
other species. Only G. nigrifrons showed a host response by ingesting from non-sieve element tissues longer on maize than on johnsongrass (Table 4).
Feeding Transitions During Probing
Kinematic diagrams illustrating conditional probabilities of behavioral transitions were used to follow the sequence and of behaviors during the 180 min access period. (Figs. 5-8). Leafhoppers behaved in a similar manner and so a general description of the transitions is applicable to all four species. For many leafhoppers, sustained phloem ingestion lasted several hours. If more than 30 min of sustained phloem ingestion was recorded, it was considered a 24
Qraaiaella nigriftoai maize Qrtaiaella aigrlfront johmonirau
146 79
1.0 .06 1.0 .13 .09 .41 .57
.07 .21
.19 .55
.35 X-WAVE NCN-VASC. INGESTION 35 .04 .51 .06 .06
Figure 5. Kinematic diagram of probing behavior transitions for Graminella nigrifrons electronically monitored for 180 min on maize (N = 19 leafhoppers) and johnsongrass (N = 18 leafhoppers). Values enclosed in circles and boxes are the number of times a behavior was recorded. Numbers by arrows are the proportion of insects changing from one behavioral state to another indicated by the arrows. Transition from non-probing state (double-lined box) to the probing state always begins with salivation. 25
Aablytclltu grex maiz* Aabljrtellat grex jobmonirui
219 158
.09.99 1.0 .16 .64 .72 NCW-VASC SALIVATION . 361 ^ 14 80 16 58 .25 .26 .66 .69
X-WAVE .39 .22 38
.28 .47 .48
.07 .07
Figure 6. Kinematic diagram of probing behavior transitions for 18
Amblvsellus grex electronically monitored for 180 min on maize and johnsongrass. See Fig. 5 caption for more information. 26
Qramiaella oquaka maize QramiaeJla oquaka johnaonfiais
208 153
1.0 .09 14 .08 .25.99 .49 .55 NON-VASC. SALIVATION . 319 > 15 .22 .14 .58 .31
.04 .40 .22
.12 .32 .56
.20
Figure 7. Kinematic diagram of probing behavior transitions for 18 flraminella oquaka electronically monitored for180 min on maize and johnsongrass. See Fig. 5 caption for more information. 27
Datbulua maidia maize
371
1.0 .68 .27 .23
.07 .23 .02 .29
NOW-VASC. NGESTIOI 157 .70
Figure 8. Kinematic diagram of probing behavior transitions for 19 Dalbulus maidis electronically monitored for 180 min on maize. See Fig. 5 caption for more information. 28 terminal behavior. The type and order of behavior preceding x-waveform
behavior was variable. Probes (stylet insertion into plant tissue) always began
with salivation followed by stylet withdrawal, non-vascular probing, non-sieve
element ingestion, or x-waveform behavior. Transitions between non-
vascular probing and non-sieve element ingestion were common. Although
probes may begin early on with a high degree of behavioral switching, the
final sequence of behaviors culminating in sustained phloem ingestion was
stereotypic: salivation always preceded x-waveforms which always preceded
phloem ingestion. However, phloem ingestion did not always follow x-
waveform sequences. A significant proportion of transitions from x-waveforms were to non-sieve element ingestion or salivation for all species. The probability of changing from one behavior to another was approximately the same for most of the possible transitions of all species-host combinations (Figs.
5-8). However, there were differences between species on the same host.
There were differences between hosts, especially for phloem-associated probing. Once an insect salivated, the probability of changing to x-waveform behavior was the same on maize and johnsongrass for G. nigrifrons. G. oquaka. and A. grex (Figs. 5-7) However, D. maidis was much less likely to produce x-waveforms (Fig. 8) than G. nigrifrons on maize (X2 = 55.34, df =
1, p < 0.001). G. nigrifrons feeding on johnsongrass was more likely to follow x-waveform behavior with phloem ingestion than when feeding on maize (X2
= 4.23, df = 1, p < 0.05) (Fig. 5). There were no differences in x-waveform 29 to phloem ingestion probabilities between maize and johnsongrass for A. grex and G. oquaka. There were also no differences between maize and johnsongrass in the probability of phloem ingestion continuing for more than
30 min for each species (e.g. G. nigrifrons-maize compared to G. nigrifrons-
n johnsongrass) or between G. nigrifrons and D. maidis on maize (X = 0.126, df = 1, p = 0.05).
DISCUSSION
Previous electronic monitoring studies have shown that most sheath- feeding leafhoppers produce identical or nearly identical waveform patterns associated with salivation, phloem ingestion, and non-sieve element ingestion
(Crane 1970, Kawabe & McLean 1978,1980, Triplehorn, et al. 1984, Rapusas
& Heinrichs 1990). The leafhoppers in this study also produced similar patterns associated with these behaviors. In the earliest studies with leafhoppers Hordnia circellata (Baker) (=Graphocephala atropunctata) (Crane
1970) and Macrosteles fascifrons Stal (Kawabe & McLean 1978) workers did not report x-waveforms. Thus, at first it was believed that unlike aphids, leafhoppers did not produce x-waveforms prior to phloem ingestion until this behavior (Xip) was described for Nephotettix cinciteps (Kawabe & McLean
1980). G. nigrifrons x-waveforms were previously reported by Triplehorn et al. (1984). In this study, I show that G. oquaka. erex.A- and D. maidis. also 30 produce x-waveforms.
The x-waveform was first described by McLean & Kinsey (1965) as a repeating pattern with unknown associated probing behavior(s). Salivary sheaths of aphids interrupted during x-waves always terminated in phloem.
McLean & Kinsey (1967) and McLean (1977) postulated that aphids secrete enzyme bearing watery saliva to prevent callose formation and/or to taste small quantities of phloem sap during x-waveforms. In this study, light microscopy showed that salivary sheaths of three D. maidis and seven of eleven G. nigrifrons terminated in the phloem during x-waveforms.
Honeydew droplets rarely were observed when leafhoppers produced x- waveforms, suggesting that little, if any, fluid intake occured.
The termination of aphid stylets or salivary sheaths in the phloem suggest that x-waveform behavior is produced in response to phloem contact
(Scheller & Shukle 1986, McLean & Kinsey 1967, Nault & Styer 1972). In this study, some of the salivary sheaths excised during G. nigrifrons x-waveforms did not terminate in the phloem, but rather in nearby cells. Also, not all of the x-waveforms were followed by phloem ingestion (Figs. 5-8). Thirty-five percent G. nigrifrons x-waveforms were followed by non-sieve element ingestion, suggesting that the x-waveform pattern is not exclusively associated with phloem probing. I propose that leafhopper x-waveforms represent behavior(s) performed by the insect in response to stimulus received when stylets puncture phloem sieve elements. However, leafhoppers occasionally 31 produce x-waveforms while probing other cell types.
The non-vascular probing waveform was previously described as a pattern of unknown behavioral activity (Kawabe & McLean 1978,1980, Sogawa 1973).
Rapusas & Heinrichs (1990) interpreted this pattern (’R’) as probing without ingestion because N. virescens did not excrete honeydew when the pattern was recorded. I found that G. nigrifrons and D. maidis rarely produced honeydew when the pattern was recorded. I prefer to call the pattern non- vascular probing because it reflects stylet position during probing without inferring specific ingestion activity. This same pattern was indistinguishible among the four species and also in ten other sheath-feeding leafhoppers and planthoppers (Kawabe & McLean 1978, Kimmins 1989, Wayadande, unpublished) and strongly resembled the lb pattern produced by the mesophyll-feeding leafhopper, Empoasca fabae Harris. The lb pattern was correlated with puncturing and draining of individual parenchyma cells by E. fabae feeding on faba bean leaves (Hunter & Backus, 1989). In the present study, examination of cells in plant tissues penetrated by stylets during non- vascular probing were not significantly damaged, suggesting that puncturing and ingesting of cell contents was not occurring.
Plasticity in Leafhopper Probing Behavior
Prior to electronic monitoring studies, little was known about leafhopper probing activity, other than what could be inferred from transmission of tissue-specific plant pathogens (Purcell 1979, Tonkyn & Whitcomb 1987 and references therein) and from light microscopic examination of plant tissues containing salivary sheaths (Smith & Poos 1931, Day et al. 1952, Alivizatos
1982). The prevailing view that homopterans were specific with respect to tissue selection served as the basis for erecting three feeding guilds among the
Homoptera; phloem feeders, xylem feeders, and mesophyll feeders (Tonkyn
& Whitcomb 1987). Although this classification may be useful for ecologically separating the Homoptera, it infers that homopterans may be inflexible in their choice of probing sites. Ullman & McLean (1988) proposed that this generalized approach to homopteran feeding strategies should be reevaluated in light of results that show no preference from among several tissues by the pear psylla (Ullman & McLean 1988). The present study also shows that leafhoppers spend a significant proportion of time ingesting from more than one tissue. Ingestion duration (Table 4) for G. nigrifrons. G. oauaka. and A. grex. showed preference (ranging from slight to strong) for phloem over non sieve element tissue sap, whereas D. maidis did not. However, although the number of D. maidis (6 of 19) and proportion of probes locating phloem was low (< 1%), the likelihood of sustained ingestion in phloem was high (p =
0.70). It is possible that D. maidis requires a longer settling time than the other species before phloem-probing behavior is initiated and that the 180 min access period was too short to show this. 33 Analysis of behavioral transitions has been used to describe insect courtship behavior (Phelan & Baker 1990, Birch et al. 1989, Hancock et al.
1989) and host acceptance (Drost & Carde 1989, Paynter et al. 1990). Ullman and McLean were the first to apply conditional probabilities to homopteran feeding with pear psylla. They showed that although overall ingestion duration was the same from phloem, xylem, or non-vascular tissues, the psyllid showed a higher probability of sustained ingestion when probing from vascular tissues (Ullman & McLean 1988). My analysis confirms speculation that homopteran tissue site selection is a non-random process (Ullman &
McLean 1988), and not the hit-or-miss strategy proposed by Day & McKinnon
(1951). As a way to describe leafhopper feeding, conditional probabilities showed that there a ’pattern’ of successive behaviors leading to phloem ingestion. No such pattern was apparent with non-sieve element ingestion.
Furthermore, analysis of transitions showed that leafhoppers can probe and ingest from more than one tissue during the same probe, indicating that leafhoppers have flexibility in their choice of tissues during a probe. Tissue choice flexibility enables leafhoppers to exploit alternate tissues and is especially important when the insect is feeding on a suboptimal host (Khan
& Saxena 1985, Kimmins 1989, Rapusas & Heinrichs 1990). 34 Probing behavior and its Relationship to Virus Transmission.
Maize and johnsongrass were suitable feeding hosts for the three MCDV vectors, including the Panicum virgatum specialist, G. oquaka. This result was surprising because G. oquaka transmitted MCDV from johnsongrass to johnsongrass but not maize to maize (Nault & Madden 1988). The explanation for failure of G. oquaka to transmit MCDV from maize was poor survivorship of leafhoppers on maize. Probing frequency, total probing duration and ingestion, specifically phloem ingestion, are often used as measures of host suitability for homopterans (Backus 1985). This study showed that maize and johnsongrass were acceptable experimental, short term feeding hosts for G. oquaka. and that phloem contact does not preclude it from transmitting MVDV to maize. On johnsongrass, G. oquaka made fewer x-waveforms, but a higher proportion of these were followed by phloem ingestion, though not significantly (Fig. 7). Because these leafhoppers were monitored for only 180 min, extrapolation of these results to the 24 hr AAP or 24 hr LAP used by Nault & Madden (1988) might not be warrented. It is possible that forced long-term feeding on maize, such as what was imposed by the 24 hr AAP and 24 hr IAP might have impaired the phloem-finding or feeding capacity of this species.
MCDV is a phloem-limited virus (Ammar et al. 1987) and it is likely that phloem-associated probing by leafhopper vectors is necessary for transmission.
Many leafhoppers probe phloem (Tonkyn & Whitcomb 1987), yet MCDV 35 vectors are clustered primarily with the leafhopper tribe Deltocephalini and
recent Eucelini (Nault & Madden 1988). Among the experimental vectors,
including A. grex and G. oquaka. transmission efficiency varied and none were
as efficient as G. nigrifrons (Nault & Madden 1988). In a separate study,
individual A. grex were 20% less likely to transmit MCDV from both maize
and johnsongrass than G. nigrifrons (see Chapter 3). In this study, I showed
that 40% fewer A. grex. and 50% fewer G. oquaka than G. nigrifrons located
and ingested from phloem sieve elements (Table 3). Although these differences were not statistically significant, they may be biologically significant and may suggest that fewer phloem contacts might result in less
MCDV acquisition or inoculation compared to G. nigrifrons.
In this study, D. maidis contacted and ingested from phloem less often than G. nigrifrons. Lower phloem probing frequency or ingestion duration is unlikely to explain vector specificity, however, because this species transmits phloem-limited pathogens, including maize rayado fino marafivirus (Gamez
1988) and two com stunting mollicutes (Nault 1980). D. maidis acquires and retains MCDV on the same attachment sites in the foregut as in vector species (Ammar & Nault 1991). Thus, some factor(s) other than the ability to probe phloem is determining vector specificity. Perhaps the differences I recorded in phloem probing behavior between the three vector species and the non-vector, especially in the behaviorally complex x-waveform, can explain transmission success. 36
CHAPTER I REFERENCES
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Ammar, E. D., & D. T. Gordon. & L. R. Nault. 1987. Ultrastructure of maize chlorotic dwarf virus infected maize and viruliferous leafhopper vectors. Phytopathology 77: 1743 (abstract).
Ammar, E. D. & L. R. Nault. 1991. Maize chlorotic dwarf virus-like particles associated with the foregut in vector and non-vector leafhopper species. Phytopathology 81: 444-448.
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Birch, M. C. D. Lucas, & P. R. White. 1989. The courtship behavior of the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae), and the role of male hair-pencils. J. Insect Behavior 2: 227-239.
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Campbell, B. C., D. L. McLean, M. G. Kinsey, K C. Jones, & D. L. Dreyer. 1982. Probing behavior of the greenbug (Schizaphis graminum. Bio type C) on resistant and susceptible varieties of sorghum. Entomol. Exp. Appl. 31: 140-146. 37 Childress, S.A.&K.F. Harris. 1989. Localization of virus-like particles in the foreguts of viruliferous Graminella nigrifrons leafhoppers carrying the semi-persistent maize chlorotic dwarf virus. J. Gen. Virol. 70:247-251.
Crane, P. S. 1970. The feeding behavior of the blue-green sharpshooter, Hordnia circellata (Baker) (Homoptera: Cicadellidae). PhD Dissertation, University of California, Davis.
Choudhury, M. M. & E. Rosenkranz. 1983. Vector relationship of Graminella nigrifrons to maize chlorotic dwarf virus. Phytopathology. 73: 685-690.
Cochran, W. G. 1954. Some methods for strengthening the common X tests. Biometrics. 10: 417-441.
D’Arcy, C. J. & L. R. Nault. 1982. Insect transmission of plant viruses and mycoplasmalike and rickettsialike organisms. Plant Dis. 66: 99-104.
Day, M. F. & A. McKinnon. 1951. A study of some aspects of the feeding of the jassid Orosius. Aust. J. Sci. Res. 4: 125-135.
Day, M. F., H. Irzykiewicz, & A. McKinnon. 1952. Observation on the feeding of the virus vector Orosius argentatus (Evans), and comparisons with certain other jassids. Aust. J. Sci. Res. 5: 128-142.
Dorschner, K.W.&C.R Baird. 1989. Electronically monitored feeding behavior of Phorodon humuli (Homoptera: Aphididae) on resistant and susceptible hop genotypes. J. Insect Behavior 2: 437-447.
Drost, Y. C. & R. T. Carde. 1990. Influence of experience on the sequential and temporal organization of host-acceptance behavior in Brachvmeria intermedia (Chalcidae), and endoparasite of gypsy moth. J. Insect Behavior 3: 647-661.
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Gamez, R. & P. Leon. 1988. Maize rayado fino and related viruses. 1988. In The Plant Viruses, R. Koenig, ed. Plenum Pub. 38 Gordon, D. T. & L. R. Nault. 1977. Involvement of maize chlorotic dwarf virus and other agents in stunting diseases of Zea mays in the United States. Phytopathology 67: 27-36.
Hancock, R. G., W. A. Fosterm & W. L. Yee. 1989. Courtship behavior of the mosquito, Sabethes cvaneus (Diptera: Culicidae). J. Insect Behavior 3: 401-416.
Harris, K. F. B. Treur, J. Tsai, & R. Toler. 1981. Observations of leafhopper ingestion-egestion behavior: Its likely role in the transmssion of noncirculative viruses and other plant pathogens. J. Econ. Entomol. 74: 446-453.
Heady, S. E. & R. F. Denno. Reproductive isolation in Prokelisia planthoppers (Homoptera: Delphacidae): Acoustic differentiation and hybridization failure. J. Insect Behavior.
Hunter, W. B. & E. A. Backus. 1989. Mesophyll-feeding by the potato leafhopper, Empoasca fabae (Homoptera: Cicadellidae): Results from electronic monitoring and thin-layer chromatography. Environ. Entomol. 18: 465-472.
Kawabe, S. & D. L. McLean. 1978. Electronically recorded waveforms associated with salivation and ingestion behavior of the aster leafhopper, Macrosteles fascifrons Stal (Homoptera: Cicadellidae). Appl. Entomol. Zool. 13: 143-148.
Kawabe, S. & D. L. McLean. 1980. Electronic measurement of the probing activities of the green leafhopper of rice. Entomol. Exp. Appl. 27: 77-82.
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McLean, D. L. 1977. An electrical measurement system for studying aphid probing behavior. In Aphids as Virus Vectors, K F. Harris & K Maramorosch, eds. Academic Press, New York, NY. 39 McLean, D. L. & M. G. Kinsey. 1965. Identification of electronically recorded curve patterns associated with aphid salivation and ingestion. Nature 205: 1130-1131.
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Nault, L. R. & W. E. Styer. 1972. Effects of sinigrin on host selection by aphids. Entomol. Exp. Appl. 15: 423-437.
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Rapusas, H. R. & E. A. Heinrichs. 1990. Feeding behavior of Nephotettix virescens (Distant) on rice varieties with different levels of resistance. Environ. Entomol. 19: 594-602.
Scheller, H. V. & R. H. Shukle. 1986. Feeding behavior and transmission of barley yellow dwarf virus by Sitobion avenae on oats. Entomol. Exp. Appl. 40: 189-195. 40 Smith, F. F. & F. W. Poos. 1931. The feeding habits of some leafhoppers in the genus Empoasca. J. Agr. Res. 43: 267-285.
Sokal, R. F. & F. J. Rohlf. 1969. Biometry. W. H. Freeman & Co., San Francisco.
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Triplehorn, B. W., L. R. Nault, & D. J. Horn. 1984. Feeding behavior of Graminella nierrifrons (Forbes). Ann. Entomol. Soc. Am. 77: 102-107.
Ullman, D. E. & D. L. McLean. 1988. The probing behavior of the summer- form pear psylla. Entomol. Exp. Appl. 47: 115-125.
Wayadande, A. W. & E. A. Backus. 1989. Feeding behavior of the potato leafhopper (Homoptera: Cicadellidae) on chlordimeform- and phosmet- treated alfalfa. J. Econ. Entomol. 82: 766-772. CHAPTER H
Leafhopper Probing Behavior Associated with Maize Chlorotic Dwarf Machlovirus Transmission to Maize
INTRODUCTION
Most leafhopper-borne plant viruses are persistently-transmitted and have a circulative or propogative relationship with their vectors (Nault & Ammar
1989). The maize chlorotic dwarf machlovirus (MCDV) is one of only three semipersistently-transmitted, foregut-borne viruses transmitted by leafhoppers (Nault et al. 1973, Nault & Ammar 1989, Brunt et al. 1990.
Recently, much has been learned about the mechanisms involved in leafhopper transmssion of MCDV (Hunt et al. 1988, Nault & Madden 1988,
Nault & Ammar 1989, Childress & Harris 1989, Ammar & Nault 1991). In this chapter, I report on how feeding behavior affects transmission and can explain vector specificity.
The principle field vector of MCDV is Graminella nigrifrons (Forbes)
(Nault et al. 1973, Gordon & Nault 1977), but several other leafhopper species are experimental vectors. Nault & Madden (1988) tested 25 leafhopper
41 42 species from the subfamily Deltocephalini and found that nine transmitted the virus. Moreovever, they discovered that vector species were related phylogenetically to one another. Most leafhoppers from the tribe
Deltocephalini and the morphologically advanced Eucelini were efficient vectors, provided that the virus test plant, maize, was a developmental host for the leafhopper species. Leafhoppers from the more distantly-related, primitive Eucelini and Macrostelini were inefficient vectors or did not transmit MCDV. Nault & Madden (1988) proposed that the reason why some
Deltocephalini species do not transmit MCDV or transmit virus inefficiently, is that these insects do not feed from the phloem where virus inclusions are found (Ammar et al. 1987). Feeding from the phloem cannot be the reason why some of the Macrostelini species do not transmit MCDV. Dalbulus maidis
(DeLong & Wolcott) and Dalbulus elimatus (Ball) transmit the phloem- limited, propogative maize rayado fino marafivirus (MRFV) (Nault et al. 1980), but these leafhoppers and seven other Dalbulus species are not MCDV vectors. Nault & Madden (1988) suggested that that perhaps D. maidis and other Macrostelini fail to transmit MCDV because virus does not bind to attachment sites in the foreguts of non-vectors as it does in vector species.
Ammar & Nault (1991) later found that binding is not the reason why D. maidis does not transmit MCDV. They examined the foreguts of MCDV- exposed leafhoppers from three vector species and D. maidis. In all four species they found virus-like particles (VLP) embedded in a densely staining 43 matrix attacted to the lining of the precibarium, cibarium, and pharynx. The
VLP were similar in size and shape to MCD virions. VLP were seen in leafhoppers exposed 6 hr earlier to MCDV-infected plants but not in leafhoppers three days earlier or in leafhoppers exposed to MRFV-infected or healthy plants. The densely staining matrix was thought to be the putative helper component needed for leafhopper transmission of MCDV (Hunt et al.
1988). These results suggest that that virus acquisition and binding occur in
D. maidis as it does in vector species and that other factors, perhaps those associated with inoculation feeding can explain failure of this species to transmit MCDV.
The electronic monitor has proven to be an invaluable tool for studying feeding behavior in leafhoppers and other homopterans. Wayadande (Chapter
1) monitored the feeding behavior of D. maidis and three MCDV vector species, G. nigrifrons. Graminella oquaka DeLong, and Amblvsellus grex
Oman. I found that all four species fed from non-vascular tissues as well as the phloem. Penetration of the phloem by the stylets was associated with the recording of x-waveforms, a rhythmic, repeating pattern that always preceeds phloem ingestion by aphids (McLean & Kinsey 1967, Nault & Styer 1972,
Campbell et al. 1981), leafhoppers (Triplehorn et al. 1984, Rapusas &
Heinrichs 1990), and planthoppers (Velusamy & Heinrichs 1986). I also reported that the x-waveform of D. maidis was distinct from that of the three vector species and that perhaps behavior(s) associated with the x-waveform 44 could explain vector specificity.
In this study, I present evidence that MCDV is transmitted to maize by
electronically-monitored, viruliferous G. nigrifrons only when x-waveforms are
recorded. I show that longer periods of phloem-associated, x-waveform
probing result in higher transmission rates. I report that x-waveforms of five
MCDV leafhopper vector species are qualitatively similar to one another, but
significantly distinct from D. maidis and four other leafhopper species that do not transmit MCDV. Finally, I conclude that the x-waveform behavior associated with MCDV inoculation is ’extravasation’ (McLean & Kinsey 1984) and that this behavior may be rare or absent in D. maidis and other
Macrostelini.
MATERIALS AND METHODS
Leafhopper and Virus Maintenance
Leafhoppers were reared in organdy covered cages (D’arcy & Nault (1982) in a room held at 27 C ± with a 16:8 L:D photoperiod. G. nigrifrons.
Graminella sonora Ball, A. grex. and Macrosteles quadrilineatus Forbes, were reared on Avena sativa L. (oat variety unknown), G. oquaka on Panicum virgatum L., and Eucelidius variegatus (Kirschbaum) and Stirellus bicolor Van
Duzee on Lolium multiflorum Lam., (variety unknown). D. maidis. Dalbulus quinauenotatus DeLong and Nault, and Ollarianus strictus Ball were reared 45 on Zea mavs L. (sweetcom ’Aristogold Evergreen Bantam’). Origin of leafhopper colonies was discussed previously (Nault & Madden 1988). Voucher specimens of species used in this study are deposited at the Ohio State
Collection of Insects and Spiders.
The type isolate of the MCDV-type strain was orignally obtained from johnsongrass rhizomes collected in 1972 (Nault et al. 1973). Virus was maintained in sweetcom by inoculating 3-4 leaf seedlings every two weeks with MCDV-exposed G. nigrifrons. Infected plants were used as a virus source
12-16 d after inoculation.
Inoculation Access and MCDV Transmission.
To test for the relation of inoculation access time of G. nigrifrons to transmission rate of MCDV, G. nigrifrons females were placed on MCDV- infected source plants for a 48 hr acquisition access period (AAP).
Leafhoppers then were placed individually into 5 cm x 15 cm tube cages fitted over 3-4 leaf seedling corn, inbred OH28, for each of 1, 2, 4, 8, 16, and 24 hr inoculation access periods GAP). For 15 min and 30 min IAPs, leafhoppers were placed directly on plants and individually observed and timed before removal. Plants were placed in the greenhouse and rated for symptoms after
12-14 d. Twenty leafhoppers were tested for each time interval except for the first replication of 15 and 30 min where only ten were tested. The experiment was repeated three times. Percent transmission was calculated 46 and correlated with IAP duration using Pearson’s Product Moment (Minitab,
Inc.).
Electronic Monitoring of Leafhopper Probing.
Adult female G. nigrifrons were caged on MCDV-infected maize plants for a 48 hr AAP, then prepared for electronic monitoring. Leafhoppers were tethered with 12 um diam, 2.5-3.0 cm long gold wire and then placed on 3-4 leaf maize seedlings and monitored using an Insect Feeding Monitor (IFM)
(Scientific Instruments Laboratory, University of Missouri, Columbia, MO).
The IFM generates an alternating current (AC) signal which passes through the insect when the stylets penetrate plant tissue. The IFM uses a differential amplifier by which the signal from the reference electrode
(background noise) is subtracted from the insect electrode before amplification. Leafhoppers were recorded approximately 45 min or until the waveform(s) of interest were performed. After the recorded IAP, tethers were removed and leafhoppers were immediately placed on a second 3-4 leaf maize plant for a 48 hr IAP to determine vector status. Recorded and second IAP test plants were placed in a greenhouse and symptoms read for analysis.
Behaviors associated with waveforms were previously determined by correlations with salivary sheath termination points and honeydew analysis
(Chapter 1). 47 For analysis, leafhopper probing behaviors were placed into three groups:
1) probing that included penetration to ingestion from non-vascular or xylem tissue (Fig 9A) 2) probing that included phloem penetration (x-waveforms) and phloem ingestion (Fig. 9B), and 3) probing that included phloem penetration, but excluded phloem ingestion (Fig. 9C). Probe number and time of salivation, x-waveform behavior, phloem ingestion, and total probing duration were compared between IAP transmitters and non-transmitters using t-tests.
Frequency of behavior associated with transmission was analyzed with chi- square contingency tests. Leafhoppers which transmitted MCDV during the recorded IAP or second IAP were identified as viruliferous and included in the analyses.
X-waveform patterns were recorded from ten Deltocephaline species. D. maidis. 0. strictus. and D. auinquenotatus were recorded on maize. G. sonora and M. quadrilineatus were recorded on maize or oats. E. variegatus and S. bicolor were recorded on ryegrass, whereas G. nigrifrons. G. oquaka. A. grex, were recorded on maize or Sorghum halapense L. The x-waveform pattern for leafhopper species does not differ from one host to another (Wayadande,
Chapter I, unpublished) therefore patterns produced on different hosts can be compared. The terms I use to describe x-waveform patterns are taken from those used by workers interpreting oscilligraphic waveforms produced by insect acoustic signals (Heady & Denno 1991). The x-waveform sequence (Fig.
9B,C) is comprised of repeated sections. Section characteristics were Figure 9. Waveforms from Graminella nigrifrons leafhoppers recorded while
feeding on maize. Waveforms read right to left, bar insert = 60 sec. A.
Leafhopper probe begins with salivation (S), then non-vascular probing (NVP),
then more salivation and finally non-sieve element ingestion (NSI). B.
Leafhopper probe begins with salivation, then x-waveforms (X) and finally
phloem ingestion. C. Leafhopper probe begins with salivation, then x-
waveforms. Probe was interrupted (arrow) before phloem ingestion could
commence.
\ 48
50 identified for each species. They included 1) sections with one (Fig 10B) or
two (Fig. 10A) phrases 2) whether section duration increases in time later in
the x-waveform sequence 3) average duration of last complete section 4)
section delineated by major and minor spikes (Fig. 10A) 5) number of spikes
per section and 6) amplitude of the waveform around the midline < or > 10
mv (Fig 10A,B). Although x-waves infrequently were produced by several of
these species, at least six sequences from a minimum of three insects per species were included in the analysis. Euclidean distances from standardized means were calculated and used to form a complete-linked hierarchal dendrogram using the Cluster option in Systat, Inc. (Wilkinson 1989).
RESULTS
Inoculation Access and MCDV Transmission
G. nigrifrons transmitted MCDV at all time intervals with transmission rate increasing with longer IAPs (Fig. 11). Based upon these results, a 30 to
60 min IAP was selected for subsequent studies for electronically monitored
IAP’s. Longer periods were not considered because of the large number of insects which had to be recorded to obtain an adequate sample of viruliferous leafhoppers. Also longer feeding periods result in more switching from one feeding behavior to another making it difficult to know which behavior was associated with MCDV inoculation. 51
section A phr I .Phr II. ..maj
> E-midline o
section
Figure 10. Middle sections of x-waveform sequences of A. Graminella nigrifrons and B. Dalbulus auinauenotatus electronically monitored on maize.
Patterns read right to left, bar insert = 60 sec. Phr I = smooth phrase, Phr
II = spiking phrase, min = minor spikes, maj = major spike. Note amplitude around section midline and number of phrases and spikes per section. 52
50 I 09 1 00 „ c 30-- « l. I- > 2 0 -- oO 2 10 -- #
0.1 1 10 100 Log IAP Duration (hr)
Figure 11. Mean transmission rate of single Graminella nigrifrons females exposed to MCDV infected maize for 24 hr followed by a 0.25. 0.50,1, 2, 4, 8,
16, and 24 hr inoculation access period on maize test plants. N = 50 for 0.25 and 0.50 hr, and N = 60 for all others. Vertical lines indicate standard error of the mean. Pearson’s product moment, R = 0.91 p < 0.05. 53 Probing Behavior Associated with MCDV Transmission
Fifty-eight of 148 G. nigrifrons monitored in this study transmitted
MCDV either during the recorded IAP or during the second 48 hr IAP. Only data from these 58 viruliferous insects were considered in the analyses.
Sixteen of 58 G. nigrifrons transmitted MCDV during the recorded IAP, of which all probed phloem, indicating that MCDV inoculation was dependent on phloem contact (X2 = 12.53, df = 1, p = 0.001). Leafhoppers which salivated, probed non-vascular tissues or xylem and not phloem failed to transmit. X- waveform duration of IAP transmitters was significantly longer than that of non-transmitters, but there were no differences in mean probe number, time of salivation or phloem ingestion between the two groups (Table 5).
Half of the 16 recorded transmitters were interrupted before the x- waveform sequence was completed and before phloem ingestion could begin.
Leafhoppers interrupted before x-waveform patterns were completed were compared to leafhoppers allowed to continue and ingest from phloem.
Transmission rate by interrupted leafhoppers did not differ from those allowed to ingest from the phloem (X = 0.36, df = 1, NS). There were no differences in x-waveform duration between interrupted leafhoppers (n = 8, x = 14.7 min) and those allowed to ingest from phloem (n = 8, x = 13.4 min)(F = 0.15, p = 0.70), thus the x-waveform durations of both types of IAP transmitters could be pooled. The 36 insects (Table 5) that produced x- waveforms were grouped by duration of x-waveforms (0-5 min, 5-10 min, 10-15 Table 5. Number of probes, salivation, x-waveform behavior, phloem ingestion, and total probing of viruliferous
Graminella nigrifrons that did or did not transmit MCDV during electronically monitored inoculation test feeds on maize seedlings of up to 45 min.
mean (min) ± SE
number of mean no. phloem total probing
leafhopperB probes salivation x-waveform ingeBtion duration
Transmitters 16 6.5 ± 1.0 ab 9.0 1 1.3 a 14.3 l 1.6 a 19.5 ± 4.3 a 42.9 1 2.4 a
(16) (16) (16) (8) (16)
Non-transmitters 42 4.6 ± 0.6 a 8.3 ± 0.9 a 7.8 ± 1.1 b 15.2 t 3.0 a 3^.8 t 1.8 b
(42) (42) (20) (8) (42) T value, P 1.78, 0.08 0.38, 0.70 3.23, 0.003 0.72, 0.48 2.49, 0.016
* number of leafhoppers performing behavior
b meana In columns followed by different letters are significantly different, t-test
Ox4*- 55 min, and >15 min) and correlated with transmission rate. Linear trend analysis (Cochran 1954) showed that longer x-waveform patterns were associated with higher transmission rates (X = 6.83, df = 1, p < 0.01) (Fig.
12).
Analysis of MCDV Vector and Non-vector X-waveforms
Representative 3 min middle segments of x-waveform sequences from five leafhoppers species that transmit MCDV (G. nigrifrons. G. oauaka. G. sonora.
A. grex. and S. bicolor) and five species that do not (D. maidis. D. quinquenotatus. M. quadrilineatus. E. variegatus. and O. strictus) are illustrated in Fig. 13. MCDV vectors all produced multiple high amplitude, biphrasic sections with two or more spikes per section. X-waveform sequences varied between 5-15 min and the number of sections per sequence ranged from 4-20. In contrast, from species that do not transmit MCDV, x-waveforms typically were low amplitude, repeated monophrasic sections without spikes.
The two Dalbulus species produced similar x-waveform patterns, however, D. quinquenotatus produced longer sequences comprised of 3-4 times the number of monophrasic sections than D. maidis. Some D. quinquenotatus sequences lasted over one hr and contained several hundred sections. Generally, O. strictus andM- quadrilineatus x-waveform sequences were comprised of short, repeated humps or peaks which lasted several seconds to several minutes.
Eucelidius variegatus’ x-waveforms shared few characteristics with those 56
80
§ 70-- • *4 09 .2 60-- 0 g 50-- u H 40--
Q 3 0 ”
S 2 0 "
# 1 0 --
5-10 10-15 > 150-5 X-waveform Duration (min)
Figure 12. Relationship between Graminella nigrifrons x-waveform duration and percent transmission to maize. Points calculated from number of transmitters / number of insects which produced x-waveforms of indicated duration. Pearson’s product moment, R = 0.93, p < 0.05. Figure 13. Representative three min excerpts of center sections from x- waveform sequences of ten electronically monitored leafhoppers: A.
Graminella nigrifrons. B. Amblvsellus grex. C. Graminella oquaka. D.
Graminella sonora. E. Stirellus bicolor. F. Eucelidius variegatus G. Dalbulus maidis. H. Dalbulus quinquenotatus. I. Macrosteles quadrilineatus. and J.
Ollarianus strictus. Waveforms read right to left, bar insert = 60 sec.
'57 G. nigrifrons E varlegatus
A g r e x .
VVtYVVV***VyV V ^^
G o q u ak a D quinquenotatus
G sonora M quadrilaatus w m 0 w m 0
S bicolor O strictus
rrt Figure 13 Cn 00 59 produced by other species (Fig. 13). Cluster analysis of the six x-waveform characteristics showed significant differences between MCDV vectors G. nigrifrons. G. sonora. A. grex. G. oquaka. S. bicolor, and non-vectors D. maidis. D. quinquenotatus. M- quadrilineatus. O. strictus. and E. variegatus (Table 6). Vectors were clustered into one branch clearly separated from non-vector species (Fig. 14). DISCUSSION Results from the timed inoculation feeding periods are similar to an earlier report by Choudhury & Rosenkranz (1983) that long inoculation access by vectors result in higher transmission rates. However, I report higher transmission rates for 15 and 30 min inoculation access than do Choudhury & Rosenkranz (1983). The probable reason for this is that I used single G. nigrifrons and recorded inoculation access only when leafhoppers were in contact with plants. Choudhury & Rosenkranz (1983) used groups of leafhoppers introduced on caged plant. They did not observe whether insects settled immediately or instead settled elsewhere in the cage. This may explain Choudhury & Rosenkranz’ (1983) low transmission rates for IAP’s under 30 minutes. The increase in transmission rate with increase in inoculation access feeding is typical for what has been reported for semipersistently transmitted closteroviruses, badnaviruses, caulimoviruses Table 6. Mean of x-waveform variables used to differentiate MCDV vector and non-vector leafhoppers by cluster analysis. mean variable MCDV vectorB MCDV non-vectors N df F-ratio P phrase3 2.00 1.00 5 1 286.88 0.000 section 2.00 1.20 5 1 31.98 0.000 lengthen section time 1.21 0.31 5 1 24.61 0.001 major/minor 1.88 1.00 5 1 111.75 0.000 spikes spike no. 6.28 0.00 5 1 36.11 0.000 amplitude 2.00 1.25 5 1 12.66 0.007 phrase » sections comprised of one (1.0) or two (2.0) phrases section lengthen • sections increase over time no (1.0) yes (2.0) section time ■ duration of last complete section major/minor spikes - sections begin and end with major and minor spikes no (1.0) yes (2.0) spike no. *■ number of spikes per section amplitude « signal amplitude around the midline < 10 mV analyzed using the KHeans procedure in the cluster option of the Systat Statistical Program, Systat Inc. O0 5 61 G nigrifrons <9.9) A g rex (8.8)---- G oc/uaka (9.9) — G sonora (6.4) S bicolor (1 0 3) £ variegatus (6.4) M. quactiiineatus (7.6) O. strictus (9.7) D m aidis (9.6)-i D. quinquenotatus (7,4)J Euclidean Distance I------1------1------1------1------1 0 1 2 3 4 5 Figure 14. Dendrogram of MCDV vectors Graminella nigrifrons. Amblvsellus grex. Graminella oauaka.Graminella sonora. Stirellus bicolor, and non-vectors Dalbulus maidis. Dalbulus quinquenotatus. Eucelidius variegatus. Ollarianus strictus and Macrosteles quadrilineatus based upon cluster analysis of x- waveform characteristics. 62 (Raccah et al. 1976, Ling & Tiongco 1979, Matthews 1991). It is generally believed that longer inoculation feeding periods increase the probability of phloem contact hence transmission rate (Matthews 1991). I have shown that G. nigrifrons must probe phloem to transmit MCDV. Phloem associated probing consists of two behaviors, x-waveform behavior and phloem ingestion (Chapter 1). MCDV inoculation occurs during x-waveforms, but not phloem ingestion. In previous electronic monitoring studies of vectors of phloem-limited viruses, separation of the two behaviors was not considered. The aphid Sitobion avenae F. transmitted barley yellow dwarf luteovirus (BYDV) more often when it penetrate 2-3 sieve elements compared to a single phloem contact (Scheller & Shukle 1986). BYDV is a circulative virus and is transmitted when aphids salivate the in phloem. Scheller and Shukle’s (1986) data suggest that inoculation occured during the x-waves, but they did not specify whether phloem ingestion was excluded in the analysis of the data. Feeding studies have been done with the vector of rice tungro machlovirus (RTMV), another semipersistently transmitted virus (Khan & Saxena 1985, 1988, Heinrichs & Rapusas 1984). Since rice tungro is transmitted in the same manner as MCDV, inoculation probably also occurs during x-waveforms and not during phloem ingestion. Behaviors associated with the homopteran x-waveform are largely unknown. McLean & Kinsey (1967) speculated that a combination of salivation and fluid uptake were occuring during aphid x-waveforms. Aphids 63 secrete salivary enzymes, some of which may deactivate P proteins responsible for callose formation in the sieve plate. X-waveform peaks are thought to be associated with salivation whereas the plateau (smooth) region may be uptake of plant sap through the food canal (McLean & Kinsey 1967, McLean 1977). Imbibed plant sap is ’tasted’ when it passes over the precibarial chemosensilla (Wensler & Filshie 1969, Backus 1985), and in leafhoppers, x-waveform behavior may, in part, inform the leafhopper if stylet tips have penetrated phloem (Chapter 1). At present, there is no direct evidence that x-waveform behavior consists of salivation and sampling of phloem sap. The inoculation of MCDV during the x-waveform is strong evidence that this behavior includes extravasation. Extravasation is the expulsion of foregut contents from the homopteran food canal (McLean & Kinsey 1984). The mechanics of extravasation are not understood, but back pressure created by the closing of the coelomic valve or precibarial valve may force fluid within the food and buccal canal to be expelled through the stylets. For MCDV transmission to occur, virions attached to the foregut cuticle must detach and be expelled with plant sap from the opening of the food canal during extravasation (Garrett 1973, Harris et al. 1981, McLean & Kinsey 1984, Ammar & Nault 1991). Results from analysis of 10 leafhopper species clearly show that the x- waveforms of the MCDV vectors have chracteristics that differ significantly from leafhoppers that fail to transmit MCDV. The most notable of these 64 characteristics are the x-waveform sequences that the consist of repeated sections with two phrases and a 10-20 mV deflection around the section midline (Fig. 10). The RTMV vectors, Nephotettix virescens (Rapusas & Heinrichs 1990) and Nephotettix cincticeps (Kawabe & McLean 1980), also produce biphrasic x-waveform sections which are similar to those produced by MCDV vectors. Absence of high amplitude spikes during non-vector x- waveforms suggests that these leafhopper species do not extravasate, and thus, are unable to transmit MCDV. 65 CHAPTER II REFERENCES Ammar, E. D., D. T. Gordon, & L. R. Nault. 1987. Ultrastructure of maize chlorotic dwarf virus infected maize and viruliferous leafhopper vectors. (Abstr.) Phytopathology 77: 1743. Ammar, E. D. & L. R. Nault. 1991. Maize chlorotic dwarf virus-like particles associated with the foregut in vector and nonvector leafhopper species. Phytopathology 81: 444-448. Brunt, A., K Crabtree, & A. Gibbs. 1990. Viruses of Tropical Plants. CAB International, Redwood Press, Whiltshire, UK Campbell, B. C., D. L. McLean, M. G. Kinsey, K C. Jones & D. L. Dreyer. 1982. Probing behavior of the greenbug (Schizaphis graminum. biotype C) on resistant and susceptible varieties of sorghum. Entomol. Exp. Appl. 31: 140-146. Childress, S. A. & K F. Harris. 1989. Localization of virus-like particles in the foreguts of viruliferous Graminella nigrifrons leafhoppers carrying the semi-persistent maize chlorotic dwarf virus. J. Gen. Virol. 70: 247-251. Choudhury, M. M. & E. Rosenkranz. 1983. Vector relationship of Graminella nigrifrons to maize chlorotic dwarf virus. Phytopathology 73: 685-690. Cochran, W. G. 1954. Some methods for strengthening the common X tests. Biometrics. 10: 417-441. Elnager, S. & A. F. Murant. 1976. Relations of the semipersistent viruses, parsnip yellow fleck and anthriscus yellows, by their vector, Cavariella aegopodii. Aim. Appl. Biol. 84: 153-167. Harris, K E., B. Treur, J. Tsai, & R. Toler. 1981. Observations of leafhopper ingestion-egestion behavior: Its likely role in the transmission of noncirculative viruses and other plant pathogens. J. Econ. Entomol. 74: 446-453. 66 Heady, S. E. & R. F. Denno. Reproductive isolation in Prokelisia planthoppers (Homoptera: Delphacidae): Acoustic differentiation and hybridization failure. J. Insect Behavior. Hunt, R. E., L. R. Nault,&R.E. Gingery. 1988. Evidence for infectivity of maize chlorotic dwarf virus and a helper component in its leafhopper transmission. Phytopathology 78: 499-504. Garrett, R. G. 1972. Non-persistent aphid-borne viruses. In Viruses and Invertebrates, A. G. Gibbs, ed. North-Holland Publ. Co., Amsterdam. Gordon, D. T. & L. R. Nault. 1977. Involvement of maize chlorotic dwarf virus and other agents in stunting diseases of Zea mavs in the United States. Phytopathology 67: 27-36. Ling. K C. & E. R. Tiongco. 1979. Transmission of rice tungro virus at various temperatures: A transitory virus-vector interaction, m: Leafhopper Vectors and Plant Disease Agents, K. Maramorosch&KF. Harris, eds., Academic Press, New York, NY. Matthews, R. E. F. 1981. Plant Virology. Academic Press, New York, NY. McLean, D. L. 1977. An electrical measurement system for studying aphid probing behavior. In Aphids as Virus Vectors, K F. Harris & K Maramorosch, eds. Academic Press, New York, NY. McLean, D. L. and M. G. Kinsey. 1967. Probing behavior of the pea aphid, Acvrthosiphon pisum. I. Definitive correlation of electronically recorded waveforms with probing activities. Ann. Entomol. Soc. Am. 60: 400-406. McLean, D. L. & M. G. Kinsey 1984. The precibarial valve and its role in the feeding behavior of the pea aphid, Acvrthosiphon pisum. Bull. Entomol. Soc. Am. 30: 26-31. Nault, L. R. 1980. Maize bushy stunt and corn stunt: A comparison of disease symptoms, pathogen host ranges, and vectors. Phytopathology 70:659- 662. Nault, L. R., W. E. Styer, J. K Knoke,& H. N. Pitre. 1973. Semipersistent transmission of leafhopper-borne maize chlorotic dwarf virus. J. Econ. Entomol. 66: 1271-1273. 67 Nault, L. R & W. E. Styer. 1972. Effects of sinigrin on host selection by aphids. Entomol. Exp. Appl. 15: 423-437. Nault, L. R & L. V. Madden. 1988. Phylogenetic relatedness of maize chlorotic virus leafhopper vectors. Phytopathology 78: 1683-1687. Nault, L. R & E. D. Ammar. 1989. Leafhopper and planthopper transmission of plant viruses. Ann. Rev. Entomol. 34: 503-530. Raccah, B., G. Loebenstien, & M. Bar-Joseph. 1976. Transmission of citrus tristeza by the melon aphid. Phytopathology 66: 1102-1104. Rapusas, H. R & E. A. Heinrichs. 1990. Feeding behavior of Nephotettix virescens (Homoptera: Cicadellidae) on rice varieties with different levels of resistance. Environ. Entomol. 19: 594-602. Triplehorn, B. W., L. R Nault, and D. J. Horn. 1984. Feeding behavior of Graminella nigrifrons (Forbes). Ann. Entomol. Soc. Am. 77: 102-107. Velusamy, R. & E. A. Heinrichs. 1986. Electronic monitoring of feeding behavior of Nilaparvata lugens (Homoptera: Delphacidae) on resistant and susceptible rice cultivars. Environ. Entomol. 15: 678-682. Wensler, R J. & B.K Filshie. 1969. Gustatory sense organs in the food canal of aphids. J. Morph. 129: 473:-492. Wilkinson, L. 1989. Systat. Evanston,111., Systat, Inc. CHAPTER HI Leafhopper Transmission of Maize Chlorotic Dwarf Machlovirus Isolates from Maize and Johnsongrass and from Maize Doubly- Infected with Two MCDV Strains INTRODUCTION Maize chlorotic dwarf machlovirus (MCDV) is a leafhopper-borne virus transmitted in a semipersistent maimer. Like other foregut-borne viruses (Pirone 1977, Hibino 1983, Murant et al. 1976), MCDV transmission is dependent upon a helper factor. Hunt et al. (1988) showed that the principle field vector, Graminella nigrifrons (Forbes) was unable to transmit the purified white stripe isolate of MCDV (MCDV-WS), but could do so if previously fed on corn infected with the mild isolate. A densely staining material adhering to the foregut of G. nigrifrons embedded with MCD virus like particles was postulated to be the MCDV helper component (Ammar & Nault 1990). Hunt et al. (1988) also tested if maize dwarf mosaic potyvirus A (MDMV-A) helper component assisted in MCDV transmission. G. nigrifrons 68 69 which first fed on MDMV-A infected corn, then on purified MCDV, failed to transmit MCDV, suggesting that helper component is specific for viral groups. There are two known strains of MCDV. The type strain possesses three capsid proteins which produce a characteristic banding patterns by polyacrylimide gel electrophoresis (Maroon 1989). Recently, Gingery and Nault (1990) isolated a second strain, MCDV-M1. The two strains are differentiated by the migration rates of the second and third coat proteins (CP2 and CP3). CP2 of the Ml strain is 2,000-2,200 Da larger than CP2 of the type strain and CP3 was 500-900 Da larger than type strain CP3 (Gingery & Nault 1990). Difference in CP2 and CP3 molecular weight may indicate a difference between the two strains in structure or function of the these capsid proteins, possibly in transmissibility by leafhoppers. Transmission characteristics of MCDV were previously determined using the mild isolate of the type strain (MCDV-mild) (Nault et al. 1973, Nault & Madden 1988, Ammar & Nault 1991). This isolate was experimentally transmitted by several leafhopper species closely related to G. nigrifrons (Nault & Madden 1988). MCDV mild was not transmitted by distantly related Dalbulus maidis DeLong & Wolcott or Macrosteles quadrilineatus. which are vectors of other plant viruses. It is not known if vector specificity extends to other MCDV isolates and strains. This paper reports transmission rates of the mild and white stripe isolates of the type strain and the Ml strain of MCDV by G. nigrifrons. an experimental vector Amblvsellus grex Ball, and D. 70 maidis. Differential acquisition from maize compared to johnsongrass was also tested using the same isolates and vectors. Corn infected with the Ml strain shows faint veinal chlorosis and very mild stunting. Doubly-infected plants with both the Ml strain and and other type strain isolates are severely stunted with moderate chlorosis, leaf twisting, and leaf tearing, indicating a synergistic relationship between both strains (Gingery & Nault 1990). This phenomenon also occurs between the two unrelated causal agents of rice tungro, rice tungro machlovirus (RTMV) and rice tungro badnavirus (RTBV) (Hibino et al. 1979, Hibino 1983). RTSV produces helper factor necessary for transmission of RTBV, which cannot be transmitted from singly-infected plants (Hibino & Cabauatan 1987). Although the MCDV Ml and type strains are not dependent upon each other for transmission, the interaction between these two strains, if any, during transmission has not been fully characterized. To this end, transmission of MCDV Ml and the M8 isolate of the type strain from doubly-infected maize is also described. 71 MATERIALS AND METHODS Leafhopper Maintenance and Virus Source. Leafhoppers G. nigrifrons and A. grex were reared on Avena sativa (variety unknown), and D. maidis was reared on sweetcorn, Zea mavs L. (variety ’Aristogold Evergreen Bantam’) in 35 x 12 x 30 cm mesh covered cages in a rearing room held at 28 C under a 16:8 L:D photoperiod. Collection sites for leafhoppers was described previously in Chapter 1. Voucher specimens were deposited in the Ohio State University Collection of Insects and Spiders. Adult females, 1-3 wk post- eclosion were used in all tests. MCDV-type and white stripe isolates were collected from johnsongrass and maintained in maize (Aristogold Evergreen Bantam sweet corn) for several years. Ml and M8 were isolated from infected johnsongrass rhizomes collected in 1989 near Portsmouth, Ohio and maintained in maize (Gingery & Nault 1990). All isolates were maintained by biweekly transfers from infected to healthy maize by G. nigrifrons. For all experiments, the maize inbred, OH28, was used for source and test plants because symptoms appear 1-2 d earlier and vein clearing is more easily seen, especially for the very mild Ml strain. Three to four leaf stage maize or johnsongrass seedlings were inoculated and used as source plants after 12-14 d. Doubly-infected source plants were obtained by caging 10 Ml and 10 M8 exposed G. nigrifrons together on single 4-5 leaf OH28 seedlings for a 24 hr inoculation access 72 period (IAP). Leafhoppers were removed and plants were placed in the greenhouse until symptoms appeared. Doubly-infected plants could not be differentiated from M8 singly-infected plants until 3-4 weeks when severe stunting and leaf tearing became evident. Doubly- or singly- (Ml or M8) infected plants were used as virus sources in the second experiment. Type. Ml. and White Stripe Transmission Test. Effects of host, isolate, and leafhopper species on MCDV transmission efficiency were tested. G. nigrifrons. A. grex. and D. maidis were placed in a 3 x 4.5 x 1.5 cm plastic box cage clipped onto an infected OH28 maize or johnsongrass plant for a 24 hr acquisition access period (AAP). Leafhoppers were then placed individually into 3 x 15 cm cylindrical tube cages placed over 3-4 leaf 0H28 maize seedlings for a 24 hr IAP. Leafhoppers were removed, plants were sprayed with a pyrethroid insecticide, and then placed in a greenhouse and assessed for symptoms after 10-12 d. Each combination of host, vector and isolate was tested for a total of 18 treatments. Twenty leafhoppers were tested for each treatment with five replications per treatment (total 100 leafhoppers/treatment) using a completely randomized design. Transmission data were converted to percentages, subjected to an arcsin p transformation (p = R/N) and subjected to analysis of variance (Minitab, Inc.). Means were compared using Least Significant Differences when a significant F-value occurred. 73 Transmission from Ml and M8 Doubly Infected Plants. Ml and M8 assortment during transmission was tested by allowing G. nigrifrons to acquire virus from Ml and M8 singly- or doubly-infected maize. Four types of source plants were used for acquisition: doubly infected Ml + M8, Ml alone, M8 alone, and healthy maize. Thirty-five to forty G. nigrifrons were placed into box cages clipped onto source plants for a 24 hr AAP. Leafhoppers were then individually caged on OH28 seedlings for a 24 hr IAP. Insects were then removed and plants were placed in the greenhouse. Symptoms were assessed weekly starting 10 d after inoculation and the number of plants infected with Ml, M8, or both was recorded. Thirty leafhoppers were tested per treatment and the experiment was replicated four times for a total of 120 leafhoppers tested per treatment. Expected Ml and M8 transmission rates from doubly-infected plants were estimated from Ml and M8 transmission rates from singly-infected plants. Overall expected and observed transmission rates and expected and observed rates from doubly-infected plants were compared using Chi-square contingency tests. 74 RESULTS Type. Ml. and White Stripe Transmission Test. Analysis of the data revealed that there were significant differences in transmission efficiency of the three leafhoppers. G. nigrifrons transmitted all isolates more efficiently than A. grex (F = 38.87, df = 1, p < 0.0001) (Fig. 15A, B, C). D. maidis failed to transmit the three isolates from maize or johnsongrass (Fig. 15A, B, C). There was no difference between maize and johnsongrass as a source of virus for leafhopper transmission (F = 1.76, df = 1, p = 0.191), nor was there a difference in transmission between the isolates (F = 1.53, df = 2, p = 0.228). There was, however, a significant plant host x isolate interaction (F = 7.40, p < 0.002, df = 2). The mild isolate was transmitted at higher rates when johnsongrass was the virus source (Fig. 15A) (LSD = .146, p < 0.05) by both G. nigrifrons and A. grex. White stripe, however, was more efficiently transmitted from maize than johnsongrass (LSD = .146, p < 0.05) by G. nigrifrons (Fig 15C). Transmission was the same when A. grex acquired white stripe, regardless of the virus source. Although Ml transmission was higher from johnsongrass than from maize, the difference was not significant. Unlike the mild and white stripe isolates, Ml was transmitted from both maize and johnsongrass with equal efficiency when G. nigrifrons was the vector. There were no significant host x vector, vector x isolate, or host x vector x isolate interactions. Figure 15. Percent of maize test plants inoculated with maize chlorotic dwarf virus mild and white stripe isolates and Ml strain by single Graminella nigrifrons. Amblvsellus grex. and Dalbulus maidis leafhoppers acquiring from maize or johnsongrass sources. A. Mild isolate of the type strain, B. Ml strain, C. White stripe isolate of the type strain. Vertical lines indicate standard deviation of the mean. 75 V 6 0 76 7 0 6 0 5 0 AO 3 0 20 10 O G. roorifror* a . crox D. maidis G. nffrfrora A. 7 « x D. maidis 80 70 od 60 3 so • 40 5 30 # 20 10 G. rigrifrara A. grax D. maidis malz* )o(r»ofgM> Figure 15 77 Transmission from Ml and M8 Doublv-Infected Plants. From singly-infected plants, 26.6% of G. nigrifrons transmitted Ml, a rate almost identical to that obtained in the previous experiment (Table 7). M8 was transmitted by 39.9% of the leafhoppers and those exposed to healthy plants failed to transmit. Insects acquiring virus from doubly-infected plants transmitted one or both isolates to 48.3 % of the test plants. This was not different than the expected rate based upon Ml and M8 transmission rates from singly-infected plants (26.6 + 39.8 - 10.2) (Chi-square = 1.32, df = 1, NS). However, less than half of the leafhoppers exposed to doubly-infected plants transmitted both isolates to the same test plant (Table 7). Ml and M8 isolate transmission rates from doubly-infected plants (24.1%, Ml; 42.6%, M8) did not differ from rates obtained from singly-infected plants. However, leafhoppers acquiring virus from doubly-infected plants transmitted both isolates more often to the same plant than would be expected if Ml transmission was independent of M8 transmission (Chi-square = 9.15, df = 3, p < 0.05). Most of the Ml transmissions occured in conjunction with M8, whereas slightly more than half of the M8 transmissions were to singly- infected plants (Table 7). Table 7. Percent of maize test plants inoculated with MCDV Ml, M8, or both by single(rraminella nigrifrons females which had been previously exposed to healthy, singly- or doubly-infected maize plants. percent transmission Inoculum Source Ml M8 Ml + M8 Ml 26.3 0 0 M8 0 39.8 0 Ml + M8 5.8 24.1 18.3 Healthy 0 0 0 N = 118 plants tested for Ml and M8 sources N = 120 for Ml + M8 and healthy sources 79 DISCUSSION The field vector of MCDV is G. nigrifrons and in this study it was shown to be a more efficient vector than A. grex of all isolates from both virus source host species. D. maidis failed to transmit the Ml and white stripe isolates. This finding suggests no difference in Ml, mild and white stripe particle characteristics which may determine vector specificity. Plant host, however, is important to transmission efficiency of specific isolates. Johnsongrass, which is the overwintering host of MCDV in the field, served as a better acquisition host for the type isolate. In the field, initial infection results from leafhoppers moving from infected johnsongrass to corn seedlings. Although primary infection rates from leafhoppers acquiring virus from johnsongrass sources are unknown, secondary spread of MCDV from maize to maize is low (Gingery 1988, Madden et al. 1990). The data from this study suggest that MCDV-mild transmission from johnsongrass to maize occurs more readily than from maize to maize transmission and may partially explain low rates secondary spread in the field. There are several possible explanations why MCDV-mild transmission is higher when acquired from johnsongrass than from maize. Mild-infected johnsongrass is symptomless in the field, but under laboratory conditions vein clearing, but not stunting, is pronounced. To check the possibility that the virus titer in infected johnsongrass was higher than in infected maize, a 80 quantitative ELISA (enzyme-linked immunosorbent assay) revealed no difference in UV absorbance values from both infected hosts (Wayadande, unpublished). Unless a large proportion of the detected coat protein consisted of empty capsules, the ELISA results indicate that virus titers in the two infected hosts are approximately equal and thus, do not explain differential transmission rates. Alternatively, it is possible that some plant-associated factor facilitated acquisition, such as greater virion availability in the phloem or induced factors which influenced helper component activity. It is also probable that tissue site selection, and thus MCDV acquisition, by the feeding leafhopper may be influenced by the host. MCDV is phloem-limited and vectors must ingest from sieve elements to acquire virus. When electronically monitored, phloem ingestion was longer (although not significantly) when feeding on johnsongrass than on maize for G. nigrifrons (Triplehorn et al. 1984, Chapter 1) and A. grex (Chapter 1). Longer phloem ingestion probably leads to greater virion acquisition which may explain higher MCDV mild acquisition from johnsongrass. However, longer phloem feeding from johnsongrass does not explain greater white stripe transmission from maize observed in this study. Although not examined in this study, it is possible that MCDV white stripe titer is higher in infected maize than johnsongrass. Laboratory grown maize and johnsongrass infected with the type isolate show the same degree of symptom severity. Maize infected with the white stripe isolate shows much 81 more severe vein clearing and overall symptom expression than white stripe infected johnsongrass. This observation suggests that white stripe may be more virulent in maize and that more virions are available for acquisition than in infected johnsongrass. Transmission from Ml and M8 Doublv-Infected Corn. Insect transmitted viruses use vector mobility for transport to new and uninfected hosts. Some poorly transmitted viruses may have associated with viruses (or other pathogens) which have evolved mechanisms for efficient vector transmission (Hu et al. 1988, Hibino 1983, Hobbs & McLaughlin 1990, Pirone 1977 and references therein). Rice tungro causal agents RTMV and RTBV are morphologically distinct and serologically unrelated (Omura et al. 1983, Hibino 1987). Leafhopper vectors of the rice tungro complex, including Nephotettix virescens Distant and Nephotettix nieropictus Stal, transmit both RTMV and RTBV from doubly-infected plants, but cannot transmit from RTBV singly-infected plants (Hibino 1979, Hibino 1983) suggesting that RTBV was not originally transmitted by RTMV vectors. How RTBV was originally transmitted is not known, but other badnaviruses are transmitted by more sedentary mealybug vectors (Brunt et al. 1990). If RTBV was also originally transmitted by mealybugs, it has probably gained greater dispersal by its association with RTMV and its leafhopper vectors. 82 Maize chlorotic dwarf strains may benefit from the same kind of association in doubly-infected plants. Unlike the rice tungro complex, Ml and M8 are related serologically (Gingery & Nault 1990) and there is no evidence that one strain is dependent upon the other for leafhopper transmission. From doubly-infected plants the transmission frequencies for Ml and M8 strains were equal to those obtained from singly-infected plants. This suggests that Ml and M8 do not interfere with each other or compete for replication sites or resources when inoculated at the same time. However, Ml and M8 did not sort independently; a higher proportion of the leafhoppers which acquired from doubly-infected plants inoculated the test plants with both Ml and M8 than would be expected if there was no association between strains. These data suggest that there was some type of association between Ml and M8 strains, a phenomenon observed with other interacting viruses (Langenberg 1989, Langenberg & Purcifull 1990, Langenburg et al. 1990), which facilitated simultaneous inoculation of both strains. This type of association might be advantagous to both genomes because of the phenotypic expression of the doubly infected plant. Ml + M8 doubly- infected maize is more stunted and yellowed than maize infected with Ml or M8 alone. Leafhoppers and other homopterans are attracted to yellow (Prokopy 1983 and references therein, Todd 1990) and plants yellowed by virus infection (Baker 1960, Macias & Mink 1969). Yellowed or diseased plants are associated with higher nitrogen content (Prokopy 1983) and are 83 often better developmental hosts to homopterans, including G. nigrifrons. than healthy plants (Baker 1960, Hunt & Nault 1990). Ml and M8 doubly-infected plants may be more attractive to potential vectors, thus increasing the likelihood of acquisition of both viruses. This is plausible only for acquisition from maize and not from johnsongrass where MCDV singly- and doubly- infected plants are asymptomatic in the field. 84 CHAPTER III REFERENCES Ammar, E. D. & L. R Nault. 1991. Maize chlorotic dwarf virus-like particles associated with the foregut in vector and non-vector leafhopper species. Phytopathology Baker, P. F. 1960. Aphid behavior on healthy and on yellows-virus-infected sugar beet. Ann. Appl. Biol. 48:384-391. Brunt, A., K. Crabtree, & A. Gibbs. Viruses of Tropical Plants. CAB International, Redwood Press, Whiltshire, UK Choudhury, M. M. & E. Rosenkranz. 1983. Vector relationship of Graminella nigrifrons to maize chlorotic dwarf virus. Phytopathology 73:685-690. Gingery, R. E. 1988. Maize chlorotic dwarf and related viruses In The Plant Viruses, Vol. 3, R Koenig, ed., Plenum Publishing. Corp. Gingery, R. E. & L. R Nault. 1990. Severe maize chlorotic dwarf disease caused by double infection with mild virus strains. Phytopathology 80:687-689. Hibino, H. & P. Q. Cabauatan. 1987. Infectivity neutralization of rice tungro- associated viruses acquired by vector leafhoppers. Phytopathology 77:473-476. Hibino, H. 1983. Transmission of two rice tungro-associated viruses and rice waika virus from doubly or singly infected source plants by leafhopper vectors. Plant Dis. 67:774-777. Hibino, H., N. Saleh, & M. Roechan. 1979. Transmission of two kinds of rice tungro-associated viruses by leafhopper vectors. Phytopathology 69:1266- 1268. Hobbs, H. A. & M. R. McLaughlin. 1990. A non-aphid transmissible isolate of bean yellow mosaic virus that is transmissible from mixed infections with pea mosaic virus-204-1. Phytopathology 80:268-272. 85 Hu, J. S., W. F. Rochow, P. Palukaitis, & R R Dietert. 1988. Phenotypic mixing: mechanism of dependent transmission for two related isolates of barley yellow dwarf virus. Phytopathology. 78:1326-1330. Hunt, RE.&L.R Nault. 1990. Influence of life history of grasses and maize chlorotic dwarf virus on the biotic potential of the leafhopper Graminella nigrifrons (Homoptera: Cicadellidae). Environ. Entomol. 19:76-84. Hunt, R. E., L. R. Nault&RE. Gingery. 1988. Evidence for infectivity of maize chlorotic dwarf virus and a helper component in its leafhopper transmission. Phytopathology. 78: 499-504. Langenberg, W. G. 1989. Soilborne wheat mosaic virus antigen binds to cylindrical inclusions of potyviruses in doubly infected cells of wheat. Phytopathology 79:1265-1271. Langenberg, W. G., S. A. Lommel & D. E. Purcifull. 1989. Sorghum chlorotic spot virus binds to potyvirus cylindrical inclusions in tobacco leaf cells. J. Ultrastruct. Mol. Struct. Res. 102:47-52. Langenberg, W. G. & D. E. Purcifull. 1989. Interactions between pepper ringspot virus and cylindrical inclusions of two potyviruses. J. Ultrastr. Mol. Struct. Res. 102:53-58. Macias, W. & G. I. Mink. 1969. Preference of green peach aphids for virus- infected sugarbeet leaves. J. Econ. Entomol. 62:28-29. Madden, L. V., J. K Knoke & R. Louie. 1990. Spread of maize chlorotic dwarf virus in maize fields by its leafhopper vector, G. nigrifrons. Phytopathology 80:291-298. Maroon, C. M. 1989. Serological relationships between capsid proteins of maize chlorotic dwarf .virus - mild. Master’s thesis, Ohio State University, Columbus. Murant, A. F., I. M. Roberts, & S. Elnagar. 1976. Association of virus-like particles with the foregut of the aphid Cavariella aegopodii transmitting the semi-persistent anthriscus yellows and parsnip yellow fleck. J. Gen. Virol. 31:47-57. Nault, L. R., W. E. Styer, J. K. Knoke & H. N. Pitre. 1973. Semipersistent transmission of leafhopper-borne maize chlorotic dwarf virus. J. Econ. Entomol. 66:1271-1273. 86 Nault, L. R. & L. V. Madden. 1988. Phylogenetic relatedness of maize chlorotic dwarf virus leafhopper vectors. Phytopathology 78:1683-1687. Omura, T., Y. Saito, T. Usugi & H. Hibino. 1983. Purification and serology of rice tungro spherical and rice tungro baciliform viruses. Ann. Phytopathol. Soc. Jpn. 49:73-75. Pirone, T. P. 1977. Accessory factors in nonpersistent virus transmission. In Aphids as Virus Vectors, K F. Harris & K Maramorosch, eds. Academic Press, New York, NY. Prokopy, R. J. 1983. Visual detection of plants by hervivorous insects. Ann. Rev. Entomol. 28:337-364. Rosenkranz, E. 1969. A new leafhopper-transmissible corn stunt disease agent in Ohio. Phytopathology. 59:1344-1347. Todd, J. L. 1990. Importance of color stimuli in host-finding by Dalbulus leafhoppers. Entomol. Exp. Appl. 54:245-255. Triplehorn, B. W., L. R. Nault & D. J. Horn. 1984. Feeding behavior of Graminella nigrifrons (Forbes). Ann. Entomol. Soc. Am. 77:102-107. EPILOGUE Among the phytophagous Heteroptera, no group is as diverse in their feeding habits as the family Cicadellidae. They use two different feeding strategies, the more advanced lacerate and flush feeding and salivary sheath feeding (Backus 1988). Electronic monitoring has shown that the sheath feeding strategy can be further subdivided into xylem specialization (Crane 1970) and feeding from other tissues, primarily the phloem. The difference between the latter two is that xylem feeders are morphologically and physiologically adapted to feed from this tissue and infrequently probe and ingest from other tissues (Crane 1970) whereas "phloem" ingesting sheath feeders are morphologically less specialized and appear to be more flexible in their choice of host tissues (Khan & Saxena 1988, Rapusas & Heinrichs 1990, Chapter I). Unfortunately, these generalizations are based upon a relatively small sample of electronically monitored leafhoppers. This work represents a major step toward understanding the feeding process of this diverse group of economically important insects. By describing and quantifying probing behavior, we better understand both the behavioral ecology of these insects and the mechanisms of plant pathogen transmission. 87 88 From other published accounts of electronically monitored, sheath feeding leafhoppers (Crane 1970, Triplehorn et al. 1984, Khan & Saxena 1985, Rapusas & Heinrichs 1990) and the leafhoppers monitored in this work, it appears that several behaviors (deduced from the appearance of the waveforms) are highly conserved. Salivation, non-sieve element ingestion and non-vascular probing patterns are indistinguishible when compared among leafhoppers in the subfamily Deltocephalinae. Salivation and non-sieve element ingestion patterns are also identical to aphid patterns. It is interesting, however, that no aphids have been reported to produce the non-vascular probing pattern. Unlike leafhoppers which probe intracellularly, aphid stylet follow an intercellular pathway (Pollard 1977). The non-vascular probing pattern may be associated with a probing style that penetrates cells, or alternatively, may be an artifact of the large diameter of the penetrating leafhopper stylet bundle compared to the smaller aphid bundles. In aphids, the appearance of individual x-waveforms is similar among species from three subfamilies and several tribes, suggesting that the behavior which is the basis for x-waveform configuration may be the same for all aphids. This is not so for the Cicadellidae. Leafhoppers in two subfamilies, the Typhlocybinae and the Cicadellinae do not produce x-waveforms although they occasionally probe phloem (Kabrick & Backus 1990, Crane 1970). Within the subfamily Deltocephalinae, there is a wide variety of x-waveform configurations (Chapter II). These patterns were always produced prior to phloem ingestion. 89 However, because the appearance of these patterns differ between the Deltocephalini (Graminella spp.. A. grex), Eucelini (S. bicolor. N. virescens. E. variegatus), and Macrostelini (Dalbulus spp.. M. quadrilineatus. O. strictus). the underlying repetoire of behaviors is likely to be different between these Deltocephaline tribes. Cluster analysis of the x-waveforms separated the Deltocephalini species from species in the other two tribes. Behavioral analysis of acoustic signals has been used to identify and separate species within a single genus. (Heady & Nault 1988). This type of analysis of probing behavior may also be useful in determining broad phylogenetic relationships at the tribal level. Two Eucelini leafhoppers, S. bicolor, and N. virescens (Rapusas & Heinrichs 1990) which produced biphrasic x-waveforms with spikes, would likely cluster with the Deltocephalini, suggesting that perhaps these two species are more closely related to the Deltocephalini than other Euceline leafhoppers. Speculation about the origin of the signal associated with the homopteran x-waveform and phloem ingestion has almost always included penetration of the sieve element. Unlike other patterns, aphid x-waveforms were never produced in the absence of phloem (i.e. on artificial diet), and thus it has been assumed that phloem sap or the penetration of the sieve element plasma membrane must account for part of the unique pattern (Backus, personal communication). Leafhoppers, however, produce x-waveforms in tissues other than phloem (Chapter II). If aphid and leafhopper x-waveform behaviors are 90 the same, this implies that aphids are better able to locate sieve elements than leafhoppers before x-waveform initiation. Since the first report of x-waveforms (McLean & Kinsey 1965) the activities associated with the pattern were unknown. McLean & Kinsey hypothesized that x-waveforms represented a combination of salivation and ingestion. I have demonstrated that G. nigrifrons extravasates during x-waveforms, transmitting MCDV from attachment sites in the foregut. Failure of D. maidis to transmit virus from these sites strongly suggests that this species does not extravasate. Other x-waveform activities may be elucidated using phloem-limited circulative viruses, i.e. geminiviruses and luteoviruses. When it was first determined that MCDV was transmitted by some leafhoppers and not others and that transmission efficiency varied among vector species, it was first thought that specific attachment of virions to vectors, aided by a helper component, determined vector specificity (Nault & Madden 1988). This and other hypotheses including specific detachment (Ammar & Nault 1991) and salivary inactivation (Appendix A) were refuted (Ammar & Nault 1991) or unsupported (Appendix A). This research provides the best evidence that feeding behavior is, in part, determining MCDV vector specificity. Other factors which must be considered are ability of a potential vector species to use MCDV host plant(s) and specificity of the helper component (Nault & Madden 1988). Many experimental vectors, such as S. bicolor, may never transmit MCDV in the field because they are not normally 91 associated with johnsongrass or maize. Similarly, Pereerrinus maidis. a delphacid planthopper which feeds on maize, is unable to transmit MCDV because the helper component does not attach to the foregut (Ammar & Nault 1991). This multifaceted approach to understanding MCDV transmission will be useful in identifying potential vectors of this disease. Identification of inoculation behavior may also be useful in identifying resistance factors for both MCDV and other semipersistently transmitted viruses. APPENDIX A Salivary Inactivation of Maize Chlorotic Dwarf Machlovirus Homopteran saliva contains enzymes used to digest away cell wall and/or to start digestion of plant sap just before and during fluid uptake into the food canal. Although these enzymes have not been fully characterized, some species’ saliva contains pectinases, carbohydrases and proteinases (Miles 1972). It has been suggested that saliva of homopteran non-vectors may contain some factor which inactivates virus particles (Miles 1972). In the course of looking for possible mechanisms of MCDV vector specificity, I tested for inactivation of virus by the non-vector D. maidis. I also attempted to test for activation of virus by G. nigrifrons. Attempts to block transmission by G. nigrifrons or to force D. maidis to transmit were done both in vivo and in vitro. In vivo assays were done by crowding non-viruliferous leafhoppers onto a small area of a plant for a pre feeding period. The area was flooded with saliva so that when the next leafhopper was placed into the area, the virus particles would come into contact with saliva during fluid uptake or after extravasation into the plant. 92 93 Each experiment was done with different objectives, and so each is described separately. Expt. 1. Blocking white stripe transmission by G. nigrifrons. Small clip cages were placed on healthy test plants and either 20 D. maidis or 20 G. nigrifrons were placed inside for a 6 hr pre-feed. Control plants had no pre-feed. After removal, two MVDV-WS exposed G. nigrifrons were placed in the same cage for a 18 hr inoculation access period (LAP). Results: control 3/13 plants were infected G. nigrifrons pre-feed 0/13 " D. maidis pre-feed 2/13 " Expt. 2 Same experiment with only 10 pre-feed leafhoppers and 1 MCDV-WS exposed G. nigrifrons. Results: control 4/11 plants were infected G. nigrifrons pre-feed 4/15 " D. maidis pre-feed 3/15 " tube cage control 4/11 " Expt. 3. Same experiment but with increased numbers of pre-feeding leafhoppers. 200 G. nigrifrons or D. maidis nymphs were places on a 2 cm area of MCDV-WS infected corn for a 6 hr pre-feed. G. nigrifrons adults were 94 then placed into the cage for a 18 hr AAP. G. nigrifrons were then placed on test plants (2/plant). The experiment was run twice. Results: G. nigrifrons pre-feed 0/14, 5/12 plants infected D. maidis pre-feed 2/17, 4/15 " Expt. 4 Salivary activation - Forcing D. maidis to transmit MCDV-WS. 150 G. nigrifrons nymphs were caged onto a 2 cm section of corn infected with MCDV-WS for a 6 hr pre-feed. Thirty adult D. maidis were added to the cage for an 18 hr simultaneous acquisistion access period (AAP). D. maidis were placed on healthy test plants (5 / plant) for a 48 hr IAP. G. nigrifrons which had eclosed to adults were also placed 5/plant on test plants. This was repeated three times with the same results. Result: D. maidis acquisition 0/10, 0/6, 0/6 plants were infected G. nigrifrons acquisition 0/14, 1/6, 1/6 " After several of the in vivo tests were completed, it became apparent that the treatments were having no effect on transmission by either G. nigrifrons or D. maidis. There was no indication that saliva was coming in contact with the virus using the plant as the medium. To better control the experimental conditions, I changed to an m vitro system, using Parafilm membranes and purified virus in artificial feeding solutions. MCDV-WS was 95 purified using the protocol of Hunt et al. (1987) and resuspended in 0.05M Tris buffer + 5% sucrose. Virus was exposed to leafhopper saliva by pre feeding on the membranes for 4 hr. Test leafhoppers were given a 24 hr AAP on MCDV-mild to acquire helper component, and then placed onto the membranes for a 2 hr AAP and then placed onto test plants (5/plant). This experiment was repeated twice. Results: D. maidis pre-feed, then G. nigrifrons AAP 4/10, 0/20 WS infected plants G. nigrifrons pre-feed, then D. maidis AAP 0/10, 0/20 " G. nigrifrons pre-feed, then G. nigrifrons AAP - , 2/20 " buffer control 0/10, 0/20 " Expt. 6 Activation or inactivation of virus in vivo. Membranes with 0.05M tris buffer + 5 % sucrose were exposed to 15-20 G. nigrifrons or D. maidis adults for 6 hr pre-feeds. MCDV-exposed G. nigrifrons or D. maidis were then placed onto the membranes for 2 hr, and then placed 5/plant onto corn seedlings. This was repeated twice. Results: D. maidis pre-feed, then G. nigrifrons 8/10, 6/15 plants infected G. nigrifrons pre-feed, then G. nigrifrons - , 5/16 " G. nigrifrons pre-feed, then D. maidis 0/10, 0/22 " no pre-feed, then G. nigrifrons 9/10, 3/9 " 96 From these preliminary experiments, I was unable to show that the pre feeding treatments had any effect on later transmission by either G. nigrifrons or D. maidis. There are several explanations why no effect was observed. It may be that leafhoppers ejected only very small quantities of saliva into the plant or membrane sachets, thus, virus probably did not come into contact with purported salivary enzymes. It is also possible that insects may have ingested recently ejected saliva during the pre-feeding exposures, leaving little saliva for the following leafhoppers to encounter. It is also possible that saliva plays little or no role in either inactivating or activating MCDV. Because of the inconclusive results, the salivary inactivation hypothesis remains unsupported. However, I believe that the experimental design and techniques used in this study were inadequate to test the hypothesis. One must first isolate the saliva of these two insects and identify salivary components to show the true effect of saliva on MCDV transmission. LIST OF REFERENCES Alivizatos, A. S. 1982. 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