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Biology of maize chlorotic dwarf virus and mobility of its leafhopper vector, Graminella nigrifrons (Homoptera: Cicadellidae)
Lopes, Joao Roberto Spotti, Ph.D.
The Ohio State University, 1993
UMI 300 N. ZeebRd. Ann Arbor, MI 48106
BIOLOGY OF MAIZE CHLOROTIC DWARF VIRUS AND MOBILITY
OF ITS LEAFHOPPER VECTOR, GRAMINELLA NIGRIFRONS
(HOMOPTERA: CICADELLIDAE)
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Joao Roberto Spotti Lopes, B.S., M.S.
*****
The Ohio State University
1993
Dissertation Committee:
R.E. Gingery
C.W. Hoy Approved by
L.V. Madden
L.R. Nault
P.L. Phelan Department of Entomology This dissertation is dedicated with love to my wife Mate ACKNOWLEDGEMENTS
I take this opportunity to acknowledge those who have helped me to achieve this major goal of my graduate education. First of all, I express my sincere gratitude to
Dr. Lowell R. Nault, my advisor, for giving me the opportunity to work in his program, for his excellent guidance throughout my research, and for his enthusiasm, friendship and support when I it needed most.
I thank the members of my dissertation committee: Dr.
P. L. Phelan, Dr. R. E. Gingery, Dr. L. V. Madden, and Dr.
C. W. Hoy for reviewing the manuscripts and providing suggestions that much improved this work. I especially thank Dr. Phelan and Dr. Gingery for providing space and training in their laboratories, and Dr. Madden for the statistical advice.
Special thanks go to William (Bill) Styer, Thomas
Lanker and Robert Whitmoyer for the valuable assistance in the technical aspects of my research, and to Bert L.
Bishop (Stat Lab) and Ed Zaborski for the very helpful statistical advice.
I would like to thank all the graduate students and post-docs in the Department of Entomology at Wooster for
iii the friendship and support, especially to Mercedes Ebbert for the encouragement during my first and difficult months of course-work, and for the ideas during my research.
Many thanks go to the support staff of the
Departments of Entomology and Plant Pathology, Statistics and Photography Laboratories, and Library of the Ohio
Agricultural Research and Development Center (OARDC) for their cordial assistance.
I wish to acknowledge R. W. Van Keuren (Department of
Agronomy/OARDC) for the identification of grass species, and R. C. Pratt (Department of Agronomy/OARDC), S.
Linscombe (Louisiana State University Agricultural Center,
Crowley, LA), and R. Hawes (Wilderness Center, Wilmot, OH) for providing seeds or stolons of grasses.
I especially acknowledge my sponsor, the Conselho
Nacional de Desenvolvimento Cientifico e Tecnologico
(Brasilia, DF, Brazil) for my scholarship, and the
Universidade de Sao Paulo (Sao Paulo, Brazil) for giving me the opportunity to pursue the Ph.D.
Finally, I thank my wife, Maria Tereza (Mate), for the continuous support and love that helped me to overcome all the difficult moments of pursuing the Ph.D., my son
Andre for understanding my short time available to play, and my parents and siblings for the frequent letters of encouragement. VITA
July 15, 1964 ...... Born - S. J. do Rio Preto, Sao Paulo (SP), Brazil.
1985 ••«Be«B**aa B . S., Agronomic Engineering Universidade de Sao Paulo Piracicaba, SP - Brazil
1988 aaaaaaaaaaa M. S., Entomology Departamento de Entomologia Universidade de Sao Paulo Piracicaba, SP - Brazil
1989-present ...... Assistant Professor Departamento de Entomologia Universidade de Sao Paulo Piracicaba, SP - Brazil
PUBLICATIONS
Lopes, J.R.S., Parra, J.R.P., Justi Junior, J., and Oliveira, N.H. 1989. Metodologia para infestacao artificial de ovos de Diatraea saccharalis (Fabr., 1794) em cana-de-acucar, visando estudos com Trichoqramma. Anais da Escola Superior de Agricultura "Luiz de Queiroz" 46:375-390.
Parra, J.R.P., Lopes, J.R.S., Biral, E., and Goveia, P.C.R. 1989. Numero ideal de ovos de Anaqasta kuehniella (Zeller, 1879) por caixa de criacao para pesquisas com Trichoqramma. Anais da Sociedade Entomologica do Brasil 18:391-402.
Parra, J.R.P., Lopes, J.R.S., Serra, H.J.P. and Sales Junior, 0. 1989. Metodologia de criacao de Anaqasta kuehniella (Zeller, 1879) para producao massal de Trichoqramma spp. Anais da Sociedade Entomologica do Brasil 18:403-415.
v Lopes, J.R.S. and Parra, J.R.P. 1991. Efeito da idade de ovos do hospedelro natural e alternative no desenvolvimento e parasitismo de duas especies de Trichoqramma. Revista de Agricultura 66:221-224.
FIELDS OF STUDY
Major Field: Entomology BIOLOGY OF MAIZE CHLOROTIC DWARF VIRUS AND MOBILITY OF ITS LEAFHOPPER VECTOR, GRAMINELLA NIGRIFRONS (HOMOPTERA: CICADELLIDAE)
By Joao Roberto Spotti Lopes, Ph.D.
The Ohio State University, 1993
Professor Lowell R. Nault, Advisor
The biological properties of maize chlorotic dwarf virus (MCDV) strains and flight behavior of Graminella niarifrons were studied in the laboratory. MCDV-T and MCDV-
M1 strains were compared as to leafhopper transmission, vector specificity, and host plant range. Among nine
Deltocephalinae leafhopper species tested, no differential vector was found, but in general MCDV-T was transmitted at higher rates than MCDV-M1 by efficient vectors. Rates of loss of inoculativity of MCDV-T and MCDV-M1 by G. niarifrons were similar, but slower at 15°C than at 30°C.
Nineteen grass species, mostly panicoids and andropogonoids, of 46 tested were susceptible to each strain. Two differential hosts were found. Tertiary vein clearing in host leaves induced by MCDV-T was always more pronounced than that caused by MCDV-M1. The periodicity of takeoff from a maize seedling canopy (vertical movement) by
G. niarifrons was dependent on light intensity and
vii photoperiod, and varied according to the sex and age of leafhoppers. Males left the canopy only after light was reduced to simulate dusk. Unlike males, most females left the canopy before dusk. Vertical movement of females was not observed under constant light or darkness, suggesting that a circadian rhythym was not involved. Fewer young males and females (3 d old) moved vertically compared to older ones (1-2 wks old). Males showed a higher rate of interplant movement during daylight hours than did females.
Interplant movement by males was reduced during the scotophase. Migratory potential of G. niarifrons was analyzed under simulated summer (long day, higher temperature) and spring (short day, lower temperature) conditions and compared to the migrant, Macrosteles guadrilineatus. Fewer G. niarifrons made long flights in a vertical flight chamber compared to M. guadrilineatus.
Flight propensity of both species was enhanced by rearing under spring conditions, but was not affected by 2-d starvation. Peak period of takeoff from the plant canopy by both species was advanced from early evening under summer conditions to late afternoon under spring conditions.
Weight gain under spring conditions was lower in G. niarifrons than in M. guadrilineatus. Spring forms of G. niarifrons had darker wings than M. ouadri1ineatus.
Possible implications of seasonal polyphenism on flight propensity and overwintering strategies are discussed.
viii TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... iii
VITA ...... v
DISSERTATION ABSTRACT ...... vii
LIST OF TABLES ...... xi
LIST OF F I G U R E S ...... xii
LIST OF PLATES ...... xvi
INTRODUCTION ...... 1
CHAPTER PAGE
I. LEAFHOPPER TRANSMISSION, HOST PLANT RANGE, AND SPECULATION ON THE ORIGIN OF MAIZE CHLOROTIC DWARF VIRUS STRAINS ...... 6
Introduction ...... 6 Materials and Methods ...... 8 Results ...... 19 Discussion...... 33
II. DIAL ACTIVITY PERIODICITY OF GRAMINELLA NIGRIFRONS (HOMOPTERA: CICADELLIDAE) AND IMPLICATIONS FOR LEAFHOPPER DISPERSAL ...... 43
Introduction ...... 43 Materials and Methods ...... 45 R e s u l t s ...... 50 Discussion...... 61
III. COMPARATIVE FLIGHT AND MIGRATORY POTENTIAL OF GRAMINELLA NIGRIFRONS AND MACROSTELES OUADRILINEATUS (HOMOPTERA: CICADELLIDAE) . . . 70
Introduction ...... 70 Materials and Methods ...... 73 R e s u l t s ...... 83 Discussion...... 113
ix EPILOGUE ...... 126
LIST OF REFERENCES ...... 132
X LIST OF TABLES
TABLE PAGE
1 . Transmission efficiency of MCDV-M1 and MCDV-T by nine leafhopper species from the subfamily Deltocephalinae ...... 21
2 . Regression slopes and half-lives of inoculativity of MCDV-M1 and MCDV-T by Graminella niarifrons. at 15 and 30°C .... 26
3. Susceptibility of Gramineae species to MCDV-T and MCDV-M1, based on symptomatology and detection assays ...... 27
4. Susceptibility of grasses to MCDV-M1 and MCDV-T according to their taxonomic status within the G r a m i n e a e ...... 32
5. Cyclic light intensity regime used to simulate dawn, photophase, dusk and scotophase, to study activity periodicity of G. niarifrons...... 48
6 . Analysis of variance statistics obtained for the effects of rearing conditions, sex and starvation on flight duration, number of bouts and mean bout duration by M. quadri1ineatus in a vertical flight tunnel ...... 85
Statistics obtained from the linear logit analysis for the effects of rearing condition, adult starvation, and sex on the proportion of G. niarifrons that fly in a vertical flight t u n n e l ...... 95
xi LIST OF FIGURES
FIGURES PAGE
1. Observed mean (points) and predicted log- transformed transmission rates [log(p)] (lines) of MCDV-M1 and MCDV-T by Graminella niarifrons over time elapsed after virus acquisition (t), at 15 and 30°C. Regression equations and associated coefficients of determination (R2) obtained for MCDV-M1 at 15 and 30°C were, respectively, log(p) = -0.584-0.024t, R2 = 87.1%, and log(p) = -0.376-0.063t, R2 = 97.0%. For MCDV-T at 15 and 30°C, regression equations and associated R2s were log(p) = -0.328-0.039t? R2 = 89.1%, and log(p) = -0.183-0.088t, R2 = 92.7%, respectively, p = transmission rate for single insects, =1-(1-J)1/k, in which I is the proportion of infected plants and k is the number of insects placed per test plant (Swallow, 1985)...... 23
2. A) Number of G. niarifrons virgin males (1-2 wk old), and B) virgin females (1-2 wk old), observed within and above the maize canopy in a Plexiglas™ chamber, at half-hour or one-hour intervals during simulated dawn, photophase, dusk and scotophase. Bars at the top of the graph represent periods of scotophase (solid), dawn or dusk (hatched), and photophase (open). Values correspond to means (+ s.e.) of three t r i a l s ...... 51
3. Number of G. niarifrons virgin males and females with different ages observed above the maize canopy in a Plexiglas™ chamber, from 2 h before the onset of the scotophase to 0.5 h after the onset of the photophase. Values represent means (+ s.e.) of three trials for: A) 3 d old and 1-2 wk old males, and B) 3 d old and 1-2 wk old females. Bars at the top of the graph indicate periods of photophase (open), dusk or dawn (hatched) , and scotophase (solid)...... 54
xii 4. Log-transformed number of flights of G. niarifrons males and females (1-2 wk. old) within the maize canopy per 10-min. observation periods. Observations were done at half-hour or one-hour intervals during simulated dawn, photophase, dusk and scotophase. Bars at the top of the graph indicate periods of photophase (open), dusk or dawn (hatched), and scotophase (solid). Values on the curve represent means (+ s.e.) of three trials ...... 56
5. Number (mean + s.e.) of G. niarifrons virgin females (1-2 wk old) observed above the maize canopy at one-hour intervals, during: A) 6 d at 14:10 (L:D) h (entrainment photoperiod)? B) 4 d at 14:10 (L:D) h, followed by 2 d under continuous darkness (0.3 h E/t&2/ s ) ; and C) 4 d at 14:10 (L:D) h, followed by 2 days under constant light (61 ME/m2/s) . Bars at the top of the graph indicate periods of light (open) and darkness (hatched)? subjective nights during the regimes of constant darkness or light are indicated by two dots. Values on the curves represent means of three t r i a l s ...... 59
6. A) Total flight duration (mean + s.e.), B) number of flight bouts (mean + s.e.), and C) bout duration (mean + s.e.) of M. quadrilineatus males and females in a vertical flight chamber when reared under summer or spring conditions, and maintained continuously on maize seedlings as adults (non-starved) or starved for 2 d. Fifteen individuals were tested for each c o n d i t i o n ...... 87
7. A) Total flight duration (mean + s.e.), B) number of flight bouts (mean + s.e.), and C) bout duration (mean + s.e.) of spring-reared M. guadrilineatus males and females in a vertical flight chamber when maintained continuously on maize seedlings as adults (non-starved) or starved for 2 or 4 d before the test. Fifteen individuals were tested for each condition . . 90
i i * Xlll 8. Proportion of M. guadrilineatus adults that flew longer than X (flight duration [min]) in a vertical flight chamber as a function of; A) rearing conditions (summer or spring-reared), B) adult starvation (adults kept continuously on maize seedlings or starved for 2 d) and C) sex. x = mean (± s.e.) flight duration for the sample of leafhoppers (n = 60) tested in each condition. D = maximum difference in proportion of fliers observed between two samples. An asterisk after the D-value indicates significant difference in distribution between the two samples (a = 0.05; Kolmogorov-Smirnov two sample test); n.s. = difference not significant ...... 92
9. Proportion (+ s.e.) of A) G. niarifrons males and females; and B) female M. guadrilineatus and G. niarifrons that initiate flights lasting >30 sec in a vertical flight chamber when reared under summer or spring conditions, and fed continuously on maize seedlings as adults (non- starved) or starved for 2 d. Fifteen leafhoppers was tested for each condition...... 97
10. Proportion of G. niarifrons males and females that flew longer than X (flight duration [min]) in a vertical flight chamber when reared under A) summer or B) spring conditions. X = mean (± s.e.) flight duration for the sample of leafhoppers (n = 30) tested in each condition. D = maximum difference in proportion of fliers observed between two samples. An asterisk after the D-value indicates significant difference in distribution between the two samples (a = 0.05; Kolmogorov-Smirnov two sample test); n.s. = difference not significant. One data point corresponding to flight duration of 113 min was not included in B ...... 100
xiv 11. Proportion of M. quadri1ineatus and G. niarifrons females that flew longer than X (flight duration [min]) in a vertical flight chamber when reared under A) spring or B) summer conditions. X = mean (± s.e.) flight duration for the sample of leafhoppers (n = 30) tested in each condition. D = maximum difference in proportion of fliers observed between two samples. An asterisk after the D-value indicates significant difference in distribution between the two samples (a = 0.05? Kolmogorov-Smirnov two sample test); n.s. = difference not significant. Data points corresponding to flight durations above 80 min were not included in A ...... 102
12. Number of G. niarifrons and M. quadrilineatus females observed above the maize canopy in rearing cages (A), at one-hour intervals, under simulated summer conditions with photoperiod of 14:10 (L:D) h and fluctuating temperatures (B). Solid bars indicate scotophase? hatched bar, photophase. Values and bars in A represent mean numbers of leafhoppers and s.e., respectively, of four replications of the experiment .... 105
13. Number of G. niarifrons and M. quadrilineatus females observed above the maize canopy in rearing cages (A), at one-hour intervals, under simulated spring conditions with photoperiod of 12:12 (L:D) h and fluctuating temperatures (B). Solid bars indicate scotophase? hatched bar, photophase. Values and bars in A represent mean numbers of leafhoppers and s.e., respectively, of four replications of the experiment .... 108
14. A) Wing cell length (mean + s.e.), B) dry body weight (mean + s.e.), and C) wing cell color intensity (mean + s.e.) of G. niarifrons and M. quadrilineatus females when reared under summer or spring conditions. For color intensity, a higher light intensity value indicates lighter wing coloration, whereas a lower value indicates darker coloration. Means followed by the same lowercase letter are not significantly different (P = 0.05? LSD test preceded by two-way ANOVA using untransformed data)...... Ill
xv LIST OF PLATES
PLATE PAGE
I. Plexiglas™ chamber used for observations of activity periodicity of G. niarifrons. with fixture of fluorescent and incandescent bulbs located above ...... 47
xv i INTRODUCTION
Maize chlorotic dwarf virus (MCDV) (a new genus name, "waikavirus", is awaiting approval by the
International Committee of Taxonomy of Viruses) is the most frequent pathogen associated with corn stunt disease in the United States (Gordon & Nault 1977). Recently, it was found that severe stunting of maize (Zea mays L.) observed in the field can result from a synergistic interaction between the type isolate of MCDV (MCDV-T) and a second isolate, MCDV-M1 (Gingery & Nault 1990). Either
MCDV-M1 or MCDV-T alone causes no stunting or only mild stunting and vein clearing (Gingery & Nault 1990), which is probably of little economic importance. These two isolates were designated as separate strains based on differences found in serology and molecular weight of coat proteins (Gingery & Nault 1990). However, a recent study revealed little nucleic acid homology between MCDV-
M1 and MCDV-T (Ngazimbi 1993), which argues for considering them as distinct virus species.
MCDV-T is a foregut-borne virus transmitted semipersistently by several leafhoppers in the subfamily
Deltocephalinae, but one species, Graminella niarifrons
1 2
(Forbes), is the principal field vector (Nault et al.
1973; Nault & Madden 1988). This virus has not been transmitted mechanically or by seed (Knoke & Louie 1981), and is retained in the vector for just 2-4 d (Nault
1977). Because of these transmission characteristics,
MCDV depends on a perennial host plant to survive through the winter. A host plant range study with MCDV-T indicated that the perennial weed, johnsongrass [Sorghum halepense (L.) Pers.), is the principal and perhaps the only overwintering reservoir for this virus in the US
(Nault et al. 1976). This appears to explain why MCDV is endemic to the region of overlap between the distributions of johnsongrass and G. niarifrons. which extends from eastern Texas to the Atlantic Coast and from the Gulf of Mexico northwards to Pennsylvania, southern
Ohio, and areas bordering the Ohio River Valley (Gordon &
Nault 1977). It is noteworthy that MCDV has not been observed in areas distant from the presence of johnsongrass, such as northern Ohio, despite the presence of the vector. This suggests that MCDV spread is mostly restricted to short distances from the overwintering inoculum sources.
A major goal of this dissertation was to determine what factors besides johnsongrass distribution are possibly contributing to the observed pattern of incidence of MCD disease in the US and to the apparent 3 lack of long-range spread of MCDV. According to Berger &
Ferriss (1989), virus retention times, vector behavior associated with long and intermediate-range movement, and weather conditions are critical factors determining between-field spread of plant viruses. Applying these criteria to the MCD system where two virus strains (MCDV-
T and MCDV-M1) are involved in the induction of severe stunting, I hypothesized that the absence of long-range spread may be related to one or a combination of the following factors: a) efficiency of leafhoppers to separately acquire and transmit both strains; b) retention of each strain during long-range flight; c) propensity of G. niarifrons for long-distance flight; d) vector behavior associated with long-range transport
(with or without frequent feeding stops); and e) chances of landing on a maize crop in the early growth stages, when plants are more susceptible to virus infection and more suitable for vector feeding and reproduction.
To examine whether any of the above conditions is likely to explain the lack of MCD disease incidence in northern areas of the Corn Belt, and to approach other relevant questions on the biology of MCDV strains and flight behavior of G. niarifrons. I conducted three studies presented in the following chapters.
In Chapter I, I tested the hypothesis that the observed differences in serology and electrophoretic 4 mobility of coat proteins between MCDV-M1 and MCDV-T
(Gingery & Nault 1990) may translate to significant differences in their biological traits. Thus, MCDV-M1 and
MCDV-T were compared on the basis of vector specificity, retention of inoculativity in G. niarifrons. and host plant range. The biological properties of MCDV-T, but not of MCDV-M1, have been studied before. This information is basic to understanding the spread of both MCDV strains and might be helpful in establishing relationships between them.
Chapters II and III are laboratory studies done to better understand the flight behavior of G. niarifrons and to examine the migratory potential of this leafhopper. In Chapter II, I investigated the influence of light intensity, photoperiod, sex and age of adults on the periodicity of G. niarifrons dispersal from the plant canopy (vertical movement). Also, I tested the hypothesis that a circadian rhythm is involved in this photoperiodic response.
In Chapter III, the propensity of G. niarifrons to participate in prolonged flight was analyzed in comparison with the aster leafhopper, Macrosteles guadrilineatus (=fascifrons) Forbes, which is thought to be a regular migrant in the north-central and midwestern states (Drake & Chapman 1965; Hoy et al. 1992). In addition, I investigated the effects of adult starvation and environmental conditions (temperature and photoperiod) during egg and nymphal development on flight activity of adults. I also described how periodicity of vertical movement changes as a function of temperature and photoperiodic conditions, and discussed the implications of these changes on probability of wind- assisted migration. Finally, I quantified seasonal variations in body size and pigmentation of the leafhoppers as an attempt to understand differences in flight propensity between the two species and speculate on possible overwintering strategies. CHAPTER I
LEAFHOPPER TRANSMISSION, HOST PLANT RANGE AND SPECULATION
ON THE ORIGIN OP MAIZE CHLOROTIC DWARF VIRUS STRAINS
Introduction
Since the discovery of maize chlorotic dwarf virus
(MCDV) (Bradfute et al. 1972; Pirone et al. 1972), various isolates have been described that differ in severity of tertiary veinbanding and stunting produced on maize (Zea mays L.) (Nault et al. 1976; Hunt et al. 1988; Gingery &
Nault 1990). Recently, it was found that severe stunting observed in the field can result from a synergistic interaction between the type isolate (MCDV-T) and a second isolate, MCDV-M1 (Gingery & Nault 1990). With single infections, either isolate causes no stunting or only mild stunting and veinbanding in maize.
Two of the three coat proteins of MCDV-M1 (CP2 and
CP3) are larger than the corresponding ones of MCDV-T
(Gingery & Nault 1990). The CPIs of MCDV-M1 and MCDV-T have similar molecular weights, but are not serologically related (Gingery & Nault 1990). A further distinction is that crystals of virus-like particles (VLP) found in the
6 7 vacuoles of cells from MCDV-M1-infected leaves contain a high proportion of "empty” or "partially empty" particles
(Ammar et al. 1993). In cells of plants infected with type-like isolates, mainly "full" VLP are observed.
Because MCDV-M1 and MCDV-T can be differentiated based on electrophoretic mobility and serology of coat proteins, but are morphologically similar and serologically related, MCDV-M1 was designated as a new
MCDV strain (Gingery & Nault 1990). However, the low frequency of nucleic acid homology between these two strains suggests that they may be ultimately designated as distinct viruses (Ngazimbi 1993). To better understand the relationships between them, other criteria such as biological properties should be examined. Work on leafhopper transmission (Nault et al. 1973; Nault 1977;
Nault & Madden 1988) and host plant range (Nault et al.
1976; Nault et al. 1982) of MCDV-T has been conducted, yet there is no comparable information on the biological properties of MCDV-M1.
Coat proteins have been shown to affect biological properties of plant viruses such as vector specificity
(Gera et al. 1979; Atreya et al. 1990; Chen & Francki
1990; Gal-on et al. 1990) and symptom development (Sarkar
1986; Saito et al. 1989). Because of the considerable differences in coat proteins between the two MCDV strains,
I anticipated that their biological traits might also 8 differ. This study tests this hypothesis by comparing the two strains with respect to host plant range, vector range and retention (persistence) of inoculativity. Based on a previous vector range study for MCDV-T (Nault & Madden
1988), five vector species, including the principal field vector, Graminella niorifrons (Forbes), and four nonvector leafhopper species in the subfamily Deltocephalinae were selected for comparison. A collection of grass species representing the major Gramineae assemblages was used to compare host plant range, including most grasses found to be susceptible to MCDV-T in a previous study (Nault et al.
1976). This paper reports for the first time the vector and host plant ranges of MCDV-M1 and the effect of temperature on the rate of loss of inoculativity of MCDV by G. niarifrons. A discussion of the origin of MCDV is presented.
Materials and Methods
MCDV strains and leafhopper rearing. Isolates of
MCDV-T and MCDV-M1 were obtained from johnsongrass rhizomes collected near Portsmouth, OH, in 1972 (Nault et al. 1973) and 1988 (Gingery & Nault 1990), respectively.
Both strains were maintained in maize (Zea mavs L., 'Early
Sunglow' sweet corn) by serial leafhopper transmission.
The differences in severity of tertiary veinbanding caused by MCDV-M1 and MCDV-T on maize were used to distinguish the strains. In addition to G. niarifrons. the vector used in studies of host plant range and retention of inoculativity, eight Deltocephalinae species that use maize as a developmental host (Madden & Nault 1988) were chosen for the vector range experiment; three from the tribe Deltocephalini rAmblvsellus qrex (Oman), G. sonora
(Ball) and Planiceohalus flavicostatus (Van Duzee)]; three from Euscelini (Euscelidius varieoatus (Kirshbaum),
Ollarianus strictus (Ball) and Stirellus bicolor (Van
Duzee)]; and two from Macrostelini (Dalbulus maidis
(DeLong & Wolcott) and Macrosteles quadrilineatus
(=fascifrons) Forbes]. Voucher specimens for most species, with information on collectors, locality and date of collection of leafhoppers used to establish the laboratory colonies, are deposited in The Ohio State University
Collection of Insects and Spiders (see Nault & Madden
1988). Voucher specimens were not selected for P. flavicostatus and S. bicolor, which were collected in
Wooster, OH, in September/1990; however, specimens of the populations used here were identical to those from populations used by Nault & Madden (1988), for which vouchers were deposited.
Cages and tubes used in the leafhopper rearing and transmission studies were described previously (D'Arcy and
Nault 1982). Oviposition and development hosts used to rear A. qrex. G. niqrifrons. G. sonora. M. quadrilineatus 10 and P. flavicostatus were oats (Avena sativa L.) and maize
('Aristogold Evergreen Bantam' or 'Early Sunglow' sweet corn). E. varieqatus and S. bicolor were reared on rye
(Secale cereale L.), and D. maidis and 0. strictus. only on maize. The colonies were kept in a growth room at 26 ±
2°C and photoperiod of 14:10 (L:D) h.
Virus acquisition and inoculation. Adults used in experiments were 1-3 wks old. Leafhoppers were placed within rearing cages for a 48-h acquisition access period
(AAP) on maize source plants, which had been inoculated with either MCDV-M1 or MCDV-T 10-14 days before the test
(Hunt et al. 1988). Fifty to 80 insects were used per source plant. To maximize acquisition, non-symptomatic leaves from the source plants were removed (Nault & Madden
1988). Viruliferous leafhoppers were then transferred to test plants for a 48-h inoculation access period (IAP), either in tube cages or in rearing cages. Unless otherwise stated, both AAPs and IAPs were carried out in a walk-in chamber adjusted to 28 ± 2°C (day) and 20 ± 2 °C (night), and photoperiod of 14:10 (L:D) h. After the IAP, the leafhoppers were manually removed and the test plants were sprayed with a pyrethroid insecticide to eliminate residual insects. Test plants were then placed in the greenhouse and rated for symptoms at 14 and 21 days after inoculation, unless specified otherwise. 11
Vector range study. Transmission efficiencies of
MCDV-M1 and MCDV-T by each leafhopper species were
evaluated in three to six trials. In each trial, 20 maize
seedlings (inbred 'VA-35') at the two- to three-leaf stage
were inoculated by confining five insects per test plant
within tube cages. For G. niarifrons. whose transmission
rate is higher than the other species (Nault & Madden
1988), only three individuals per plant were used. To
detect transmission rates as low as 1%, a minimum of 300
insects were tested in all trials for each vector-virus
combination (Zeyen & Berger 1990). Controls using G.
niqrifrons were included in all trials to assure that
experimenta1 conditions were adequate for transmission.
Estimated transmission rates for single leafhoppers
(p) were calculated as described by Swallow (1985). Values
of p obtained for each vector-virus combination were
transformed to arcsin/p and submitted to a two-way
analysis of variance using the General Linear Model (GLM)
procedure of Minitab (Minitab Inc., State College, PA
16801; 1989). Data obtained for nonvectors were not
included in the statistical analysis. Least significant
difference (LSD) (a = 0.05) was calculated and used for multiple comparisons of the means. Because sample size varied, the LSD was estimated based on the harmonic mean
(fi) of the sample sizes of the treatments (Steel & Torrie
1980). 12
Rate of loss of inoculativity. The rate of loss of inoculativity of MCDV-M1 and MCDV-T by G. niarifrons was assessed at 15 and 30°C in growth chambers. Only adult females were used because they are more efficient vectors than males when confined on test plants (Choudhury &
Rosenkranz 1983). Maize seedlings ('Early Sunglow' sweet corn) at the two- to three-leaf stage were placed individually in tube cages for inoculation. In each treatment trial, 20 groups of three viruliferous females were serially transferred to test seedlings for successive
4-h IAPs, up to 24 h after the AAP. Thus, transmission rates for each strain and temperature were evaluated at the intervals of 0-4, 4-8, 8-12, 12-16, 16-20 and 20-24 h following virus acquisition. Two groups of insects were used to obtain six time intervals without running the experiment continuously for 24 h. A first group was transferred in the morning and was tested for transmission between 0 and 12 h after the AAP. In the evening of the same day, a second group of leafhoppers was removed from source plants, placed on a group of 20 healthy "holding plants" and left for 12 h under the same experimental temperatures. The next morning, this second group was transferred for inoculation of test plants during the intervals of 12-16, 16-20 and 20-24 h after the AAP.
Because inoculativity of plant viruses is usually lost by insect vectors at a logarithmic rate over time 13
(Yarwood & Sylvester 1959; Frazier & Sylvester 1960;
Berger et al. 1987), transmission rates for single leafhoppers (p) estimated for each time interval (4-h IAP) were submitted to logarithmic transformation [=log(p)].
Least-squares linear regression was used to fit the exponential model to the transmission data obtained for each strain and temperature. Transmission rates used in the analyses were means of three and four trials at 30 and
15°C, respectively. Midpoints of each time interval (4-h
IAP) were used as independent variable. Slopes of the regression equations were compared by a t test (P = 0.05) to detect differences in the rate of loss of inoculativity between the treatments. Based on the regression slopes, retention half-lives (t1/2) for each strain at the two temperatures were estimated by using the formula, t1/2 = log(0.5)/slope. This formula also was used to calculate
95% confidence intervals (95% CIs) for the half-lives, from 95% CIs estimated for the slopes.
Host plant range study. Forty-six species in thirty- one genera of grasses from the main groups of Gramineae, including oryzoids, festucoids, chloridoids, panicoids and andropogonoids, were tested for susceptibility to MCDV-M1 and MCDV-T. Most species were obtained from the Southern and Western Regional Plant Introduction Stations, and from commercial seed companies. Others were collected by the authors and identified by R. W. Van Keuren, Department of 14
Agronomy, Ohio Agricultural Research and Development
Center (OARDC), Wooster, OH. A few grasses were provided by R. Hawes, Wilderness Center, Wilmot, OH; J. H. Tsai,
Fort Lauderdale Research and Education Center, Fort
Lauderdale, FL; and S. Linscombe, Lousiana State
University Agricultural Center, Crowley, LA. Except for
Andropoqon virqinicus L., Leersia hexandra Sw., L. orvzoides (L.) Sw., Muhlenbergia sobolifera (Muhl.) Trin.,
Schizachvrium scooarium (Michx.) Nash, Spartina pectinata
Link and Tripsacum dactvloides (L.) L., which were propagated vegetatively from rhizomes or stolons, remaining grasses were grown from seed.
Test plants were grown in 10-cm diam. plastic pots and thinned to one to three plants per pot after germination. At the three- to five-leaf stage, ten plants of each grass species were confined with 130-150 viruliferous leafhoppers within a rearing cage for a 48-h
IAP. Another five to ten plants not exposed to viruliferous leafhoppers were used as a negative control for each species. As a positive control for the detection assays and to check for the inoculativity of the leafhoppers used in each trial, ten healthy maize seedlings ('Early Sunglow' sweet corn) were simultaneously exposed to a sample of these insects. In all trials, transmission to maize was 80-100%. 15
Test and control plants were placed in the greenhouse and evaluated for diagnostic MCDV symptoms (tertiary vein clearing) at 3 and 4 wks after inoculation, by comparing leaves of inoculated and non-inoculated plants of the same species. After symptom evaluation, leaf tissue of test and control plants was sampled for detection assays. Dot blot hybridization (DB), which tests samples for the presence of viral nucleic acid, was the main assay used because of the large number of samples to be tested and availability of cDNA probes specific for each strain. Many samples were also tested for the presence of viral coat proteins by electrophoresis followed by electro-blot immunoassay
(western blots) (WB), to confirm results obtained with DB.
All grass species in which any of the strains could replicate and reach levels detectable by either DB or WB, were considered experimental hosts regardless of the presence or absence of visible symptoms.
Samples consisted of 5 g of leaf tissue collected from the whole plant. Inoculated test plants showing symptoms were sampled individually, while symptomless ones were pooled in groups of five and a single sample was taken from each group. A pooled sample was also taken from three to five non-inoculated plants of each grass species
(negative control) and from maize plants inoculated with each strain (positive control). Each sample was extracted in 20 ml 0.3 M potassium phosphate buffer, pH 7.0, containing 0.5% 2-mercaptoethanol. The extract was filtered through two layers of cheesecloth and clarified by emulsification with 1/5 volume CHC13, followed by low- speed centrifugation (5,900 g for 10 min.). The supernatant was recovered and centrifuged at high speed
(105,000 g for 2 h) to pellet the virus. The pellet was then resuspended overnight in 0.25 ml 0.3 M sodium phosphate buffer, pH 7.0, with gentle agitation at 4°C.
From this volume, 0.1 ml was used for WB and 0.15 ml for
DB.
Dot blot hybridization. Viral nucleic acid was extracted using a mixture of TE (10 mM Tris-HCl, 1 mM
EDTA, pH 7.9)-saturated phenol (AMRESCO Inc., Solon, OH
44139) and chloroform-isoamyl alcohol (25:24:1) according to Sambrook et al. (1989), except that samples were first mixed with 25 /xl 10% SDS and 300 /xl TE-saturated phenol, and centrifuged at 16,000 g for 3 min. Purified nucleic acid was precipitated with 600 /xl of cold 95% ethanol and
0.2 M NaCl at -20°C, for at least 24 h. The precipitated nucleic acid was pelleted by centrifugation at 16,000 g for 15 min., washed with 70% ethanol, and repelleted by another 15-min. centrifugation (Sambrook et al. 1989). The pellet was resuspended in 0.1 ml distilled water and the samples were applied (10 /xl per well) to polyvinylidene difluoride (PVDF) membranes (Imobilon-N) (Milipore Corp.,
Bedford, MA) using a dot blot apparatus (Bio Dot™) (Bio- 17
Rad Laboratories, Richmond, CA). PVDF membranes had been previously wet in methanol, rinsed in water and stabilized in 2OX SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) transfer buffer for 15 min. Each group of test samples, including positive and negative controls, was blotted in duplicate to two separate membranes: one to be tested for
MCDV-M1, the other for MCDV-T. After blotting, membranes were baked at 85°C under vacuum for 2 h for fixation of the nucleic acid. Dry membranes were wet in methanol, rinsed with water and incubated for at least 3 h in a prehybridization buffer [1 ml denatured salmon sperm DNA
(250 jug/ml), 5X SSC, 1.5X Denhardt's, 37.5 mM Tris, pH
8.0, 0.15% sodium dodecyl sulfate (SDS), 7.5 mM EDTA], in heat-sealed plastic bags. A heat-denatured 32P-labeled cDNA probe was then added and hybridization was carried out for 18 h. Both prehybridization and hybrization were done at 65°C, under gentle agitation. Duplicate membranes to be tested for MCDV-M1 and MCDV-T, were kept in separate bags. After hybridization, the membranes were washed five times in 2X SSC, 0.1% SDS at 65°C for 5 min. each wash, followed by another five washes in 0.2X SSC, 0.1% SDS, to eliminate excess probe. Autoradiography of the membranes was done at -70°C for 3-7 days, using an x-ray film (Kodak
X-Omat™ AR, Eastman Kodak Company, Rochester, NY) and intensifying screen. 18
Probes were radiolabeled by random priming using the
procedure of Feinberg & Vogelstein (1983).
Oligodeoxyribonucleotides used were pd(N)6 (hexamers,
Catalog No. 272166-01) purchased from Pharmacia LKB
Biotechnology, Piscataway, NJ. The amount of cDNA probe
used in each preparation was 50-100 ng (5 /xl; 10-20
ng//xl). Polymerization was carried out for 2 h at room
temperature, and stopped with 5 /xl EDTA. Unincorporated
32P-labelled nucleotides were eliminated by filtering the
probe mix in a Nick column® (Pharmacia LKB Biotechnology,
Piscataway, NJ). cDNA clones used to prepare specific
MCDV-T and MCDV-M1 probes were MC-23 and #48,
respectively. Probe MC-23 was provided by M. McMullen
(USDA Agricultural Research Service, Wooster, OH), and is
from the coding region for CPI, in the MCDV-T genome (M.
McMullen, personal communication). Probe #48 was prepared
by C. Mzira, Department of Plant Pathology, Ohio
Agricultural Research and Development Center, Wooster, OH.
Its location on the MCDV-M1 genome is unknown.
Western blots. Each sample (0.1 ml) was mixed with
an equal volume of loading buffer (0.13 M Tris-HCl, pH
6.8, 4% SDS, 20% glycerol, 10% mercaptoethanol, 0.5%
sucrose, 10 ppm bromophenol blue) and boiled for 4 min.
Viral coat proteins were then separated by electrophoresis
on 12.5% vertical slab polyacrylamide gels, using the
Laemmli system (Laemmli 1970). Electrophoresis was done at 200 V for 50 min. in a Mini-Protean II dual slice slab cell (Bio-Rad Laboratories, Richmond, CA). Besides samples of inoculated grasses, each gel contained negative and positive control samples and pre-stained standards. After electrophoresis, proteins were transferred from polyacrylamide gels onto PVDF membranes (Imobilon-P)
(Milipore Corp., Bedford, MA) at 30 V for 18 h, as described previously (Gingery & Nault 1990). Following the transfer, viral coat proteins were visualized by using the immunostaining procedure detailed by Gingery & Nault
(1990), except that total protein staining was not used.
For each membrane, two immunostaining series were done to detect the two strains. In the first, the blot was probed with the primary antibody specific for CPI of MCDV-M1.
Soon after development with the nitro blue tetrazolium/5- bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) solution, the membrane was photographed, reblocked, and probed with the primary antibody specific for CP2 of MCDV-T.
Results
Vector Range. No differential vectors for MCDV-M1 and
MCDV-T were found. However, ANOVA indicated differences in transmission efficiency (p) among vector species
(F = 44.5; df = 5,62; P < 0.001) and between strains
(F - 9.6; df = 1,62; P < 0.001). G. niarifrons. which was tested simultaneously with all the other species as a control, efficiently transmitted both strains in all 20 trials and always at a higher rate than other vector species (Table 1). A. qrex was also an efficient vector, but transmitted MCDV-M1 at a lower rate than MCDV-T. Two other species, P. flavicostatus and S. bicolor, were relatively efficient vectors of MCDV-T, but poor vectors of MCDV-M1. G. sonora and D. maidis were poor vectors of both strains, whereas E. variegatus, 0. strictus and M. fascifrons were non-vectors (Table 1).
There was a statistical interaction between vector species and strains (F = 2.9; df = 5,62; P = 0.019), which reflects the differential effect of vector species on the transmission efficiency of the two strains. Efficient and relatively efficient vectors (A. qrex. P. flavicostatus and S. bicolor), except G. niqrifrons. transmitted MCDV-T at higher rates than MCDV-M1 (Table 1). Inefficient vectors (D. maidis and G. sonora), however, transmitted both strains at equally low rates.
Rate of loss of inoculativity of MCDV strains.
Transmission efficiencies of MCDV-M1 and MCDV-T by G. niqrifrons at 15 and 30°C decreased at a logarithmic rate with time after virus acquisition (Fig. 1). Regression equations obtained for the mean transmission rates of the two strains at 15 and 30°C were all statistically significant (P < 0.05) ; R2 values ranged from 87.1 to 97%
(Fig. 1). Comparisons by t test (P = 0.05) indicated no significant differences between the regression slopes Table 1. Transmission efficiency of MCDV-M1 and MCDV-T by nine leafhopper species from the subfamily Deltocephalinae
Transmission byv Transmission byv G. niqrifrons G. niqrifrons check Leafhopper MCDV species strain N p N
Amblvsellus MCDV-M1 530 0,067(0.011)bw 294 0.184(0.021) qrex MCDV-T 520 0.175(0.037)a 285 0.250(0.025) Dalbulus MCDV-M1 625 0.017(0.009)c 378 0.192(0.031) maidis MCDV-T 625 0.008(0.006)c 372 0.273(0.042) Euscelidius MCDV-M1 295 0 189 0.141(0.015) varieqatusx MCDV-T 305 0 189 0.171(0.013) Graminella MCDV-M1 780 0.204(0.022)a NAy NA niqrifrons MCDV-T 771 0.270(0.031)a NA NA G. MCDV-M1 360 0.025(0.003)bc 231 0.176(0.030) sonora MCDV-T 370 0.012(0.007)c 228 0.207(0.023) Macrosteles MCDV-M1 520 0 294 0.227(0.033) cuadrilineatus* MCDV-T 500 0 285 0.295(0.038) 01 lari anus MCDV-M1 450 0 252 0.190(0.011) strictus* MCDV-T 445 0 246 0.293(0.008) PIaniceohalus MCDV-M1 475 0.010(0.007)c 294 0.155(0.015) flavicostatus MCDV-T 475 0.079(0.025)b 291 0.201(0.028) Table 1 (Continued) Stirellus MCDV-M1 400 0.012(0.008)c 234 0.230(0.054) bicolor MCDV-T 340 0.059(0.016)b 234 0.340(0.083) vTransmission of MCDV strains by each species was tested in three to six trials. Insects were allowed a 48-h AAP on virus source plants and subsequently placed five per plant on maize test plants for a 48-h IAP. Unlike other species, G. niqrifrons was placed three per plant on test plants. G. niqrifrons was included in all trials to check if source plants and experimental conditions were adequate to transmission. N = total number of leafhoppers tested in all trials; p = transmission rates for single insects, =1-(1-7)1/k, in which I is the proportion of infected plants and k is the number of insects placed per plant (Swallow, 1985). Number in parenthesis is the standard error of p. “Mean transmission rates followed by the same lower case letter are not significantly different IP = 0.05; LSD test preceded by a two-way analysis of variance using transformed transmission rates (arcsin/p)]. xNon-vectors were not included in the statistical analysis. yNot applicable. Fig. 1. Observed mean (points) and predicted log- transformed transmission rates [log(p)] (lines) of MCDV-
M1 and MCDV-T by Graminella niqrifrons over time elapsed
after virus acquisition (t), at 15 and 30°C. Regression
equations and associated coefficients of determination
(R2) obtained for MCDV-M1 at 15 and 30°C were,
respectively, log(p) = -0.584-0.024t, R2 = 87.1%, and
log(p) = -0.376-0.063t, R2 = 97.0%. For MCDV-T at 15 and
30°C, regression equations and associated R2s were
log(p) = -o.328-0.039t; R2 = 89.1%, and
log(p) = -0.183-0.088t, R2 = 92.7%, respectively, p = transmission rate for single insects, =1-(1-J)1/k, in which I is the proportion of infected plants and k is the
number of insects placed per test plant (Swallow, 1985).
23 Log (p) 3 2 0 1 A — — - -- --Q A ie fe vrs custo (hours) acquisition virus after Time MCDV—M1 -T V D C M -T V D C M MCDV-M1 8 Figure 1 Figure 12 16 20 24 24 25
obtained for MCDV-M1 and MCDV-T, either at 15 or 30°C
(Table 2). This result suggests that, at the same
temperature, the two strains have similar rates of loss of
inoculativity. However, slopes were affected by
temperature. At 30°C, slopes of both strains were
significantly steeper than at 15°C (Table 2),
demonstrating that virus inoculativity by G. niqrifrons
declines faster at higher temperatures. As a result,
estimated half-lives for MCDV-M1 and MCDV-T were shorter
at 30°C than 15°C (Table 2).
Host Plant Range. Nineteen grass species were
susceptible to each strain based on symptomatology and
detection assays (Tables 3 and 4). Among these, 18 species
were common hosts to both strains. Two differential hosts were found and both were symptomless: Sorahastrum nutans
was susceptible to MCDV-T, but not to MCDV-M1, whereas the
reverse was observed for S. scoparium (Table 3). S. nutans
was confirmed as susceptible to MCDV-T by dot blot
hybridization (DB) and western blots (WB). S. scoparium.
however, tested positive to MCDV-M1 by DB, but not by WB
(Table 3); thus, its susceptibi1ity to MCDV-M1 is
uncertain. MCDV strains differed with respect to symptom
expression. The diagnostic veinbanding caused by MCDV-T on
susceptible grasses was always more pronounced than that
induced by MCDV-M1. Furthermore, three susceptible grasses
(Ischaemum rugosum. Panicum miliaceum and Setaria qlauca) 26
Table 2. Regression slopes and half-lives of inoculativity of MCDV-M1 and MCDV-T by Graminella niqrifrons. at 15 and 30°C
Half-live (hours)" Temperature MCDV Regression (°C) strain slopev Mean 95% Cl 15 MCDV-M1 -0.024(0.005)as 12.5 [7.9, 30.0] 15 MCDV-T -0.039(0.007)a 7.7 [5.1, 15.0] 30 MCDV-M1 -0.063(0.006)b 4.8 [3.9, 6.1] 30 MCDV-T -0.088(0.012)b 3.4 [2.5, 5.5]
“Slopes estimated by least-squares linear regression of log- transformed transmission rates (means of three and four trials at 30 and 15°C, respectively) over time elapsed after virus acquisition. Number in parenthesis is the standard error of the slope. "Mean half-life (t1/2) estimated based on the slope, ti/2 = log(0.5)/slope. 95% confidence interval (Cl) for mean half-life was estimated from the 95% Cl for the slope, by using the same formula. "Regression slopes followed by the same lower case letter are not significantly different (P = 0.05; t test). Table 3. Susceptibility of Gramineae species to MCDV-T and MCDV-M1, based on symptomatology and detection assays
Growth MCDV-T MCDV-M1 Major groups of Gramineae® habitb ------Species Symptoms0 DBd WBe Symptoms DB WB
ORYZOIDS
Leersia hexandra Sw. P - - Nf - - N
L. orvzoides (L.l Sw. P - N - - N
:ESTUCOIDS
AaroDvron reoens (L.l Beauv. P - - - - -
Avena sativa L. A - - - --
Bromus secalinus L. A -- - --
Dactvlis alomerata L. P - --- - Hordeum vulaare L. A - - --- Lolium Derenne L. P - N - - N Muhlenberaia sobolifera P - + + - + + (Muhl.) Trin.3 Phalaris arundinacea L. P ------Table 3 (continued)
Poa pratensis L. Secale cereale L. Triticum aestivum L. + N + N
CHLORIDOIDS Eleusine coracana (L.) Gaertn. + N + + N
E. indica (L.) Gaertn. Eraarotis cilianensis + N + N (All.) E. Mosher9 Spartina pectinata Link
ARUNDINOID-DANTHONOIDS Danthonia pilosa R. Br.
PANICOIDS Diaitaria decumbens Stent D. ischaemum + N + + N (Schreb.) Schreb. ex Muhl.3 to 03 Table 3 (continued)
D. sanauinalis (L.) Scop. A + + N + + N
Echinochloa crusaalli A + + N + + + (L.) Beauv. var. crusaall Panicum capillare L. A P. miliaceum L. A + P. viroatum L. P Paspatum notatum Fluegge P Pennisetum americanum (L. Leeke A + + N + + N Setaria faberi Herrm. A + + N + + N
S. alauca (L.) Beauv. A + + + + +
S. maana Griseb A + + + + + + S. viridis (L.) Beauv. A + + N + + N
ANDR0P0G0N0IDS Andropoaon aerardii Vitm. P A. ternarius Michx. P N
A. virqinicus L.9 P + to VD Table 3 (continued)
HeteroDoaon contortus P ------(L.) Beauv. ex Roem. & Schult.
Ischaemum ruaosum Salisb9 A + + + - + + Rottboelia exaltata L. f.9 A + + N + + N
Schizachvrium condensatum P - - N - - N (Kunth) Nees
_ Schizachyrium scoDarium P - - - + - (Michx.) Nash
Sorahastrum nutans (L.l Nash9 P - + + - - - Sorahum sudanense (Pioerl StaDf A + + + + + + S. bicolor fL.l Moench A + + N + + N
TriDsacum dactvloides (L.l L. P - - N - - N
T. lanceolatum Ruor. ex Fourn. P ------
Zea Derennis P ------(Hitchc.) Reeves & Mangelsd.
Z. diDloDerennis P ------litis, Doebley & Guzman
“Taxonomic arrangement and species names according to Watson & Gibbs (1974) and Edward E. Terrel (A Checklist of Names for 3,000 Vascular Plants of Economic Importance, Agri Handb. 505, 1977), respectively. bP = perennial growth habit; A = annual growth habit. ‘Symptoms: + = present; - = absent. dDot blot hybridization: + = tested positive; - = tested negative. “Western blots: + = tested positive; - = tested negative. fNot carried out. 9New, previously unreported host of MCDV-T. Table 4. Susceptibility of grasses to MCDV-M1 and MCDV-T according to their taxonomic status within the Gramineae
Major Susceptible speciesb Susceptible hastsc Susceptible generad Groups of Gramineae* MCDV-M1 MCDV-T MCDV-M1 MCDV-T MCDV-M1 MCDV-T Oryzoids 0/2 0/2 NAe NA 0/1 o/i Festucoids 2/11 2/11 0/2 0/2 2/11 2/11 Chloridoids 2/4 2/4 1/2 1/2 2/3 2/3 Arundinoid- 0/1 0/1 NANA o/i o/i Danthonoids Panicoids 9/13 9/13 7/9 9/9 5/6 5/6 Andropogonoids 6/15 6/15 3/6 4/6 5/9 5/9 Total 19/46 19/46 11/19 14/19 14/31 14/31
“Taxonomic arrangement based on Watson & Gibbs (1974). bNumber of susceptible species over total tested. cNumber of species showing symptoms over total susceptible. dNumber of susceptible genera over total tested. “Not applicable. 33 that showed veinbanding when infected with MCDV-T, were symptomless when infected with MCDV-M1 (Table 3).
Most species and genera susceptible to MCDV strains were panicoids and andropogonoids (Table 4). Of 11 festucoid grasses tested, only two (M. sobolifera and
Triticum aestivum) were susceptible. While most panicoid and andropogonoid hosts showed clear tertiary veinbanding, these two festucoid hosts were symptomless (Table 4). The majority of susceptible species were annuals. Sixteen of
21 annual grasses tested were susceptible, with 14 and 11 species showing symptoms when infected by MCDV-T and MCDV-
Ml, respectively. In contrast, only four of 24 perennial grasses evaluated were susceptible to any of the strains, and all were symptomless (Table 3).
Discussion
The rationale for this study was to test the hypothesis that the differences in serology and molecular weight of coat proteins between MCDV-M1 and MCDV-T
(Gingery & Nault 1990) might translate to substantial differences in their biological properties. The results revealed that the two strains are rather similar concerning host plant range, vector range and retention of inoculativity by the vector. However, it was shown that the strains can be distinguished from one another with respect to symptomato1ogy as well as transmission rate by some vector species. In addition, two differential host 34 plants were identified.
It is possible that the more pronounced symptoms induced by MCDV-T and its higher transmission rates by efficient vectors compared to MCDV-M1 result from the differences in coat proteins between the two strains, but there is no evidence available to infer such a causal relationship. Evidence for the role of coat proteins on vector specificity between virus strains has been found for cucumber mosaic cucumovirus (Gera et al. 1979), zucchini yellow mosaic potyvirus (Gal-on et al. 1990) and tobacco vein mottling potyvirus (Atreya et al. 1990).
Induction of symptoms by tobacco mosaic tobamovirus (TMV) also has been shown to be correlated with the presence of specific coding sequences in its coat protein gene (Dawson et al. 1988? Saito et al. 1989). Recently, Ammar et al.
(1993) found by electron microscopy that cells of maize leaves infected with MCDV-M1 show a high proportion of
"empty" (presumably without RNA) and "partially empty" virus particles, whereas cells of plants infected with isolates of MCDV-T contain mainly "full" particles. Thus, another explanation for the milder symptoms and lower transmission efficiency of MCDV-M1 might be a lower titer of infective virions of this strain in plants compared to
MCDV-T.
Similarities in the rates of loss of inoculativity of
MCDV-T and MCDV-M1 by G. niarifrons with time after virus 35 acquisition, suggest that the variations in coat proteins between these strains have no significant effect on their retention in the vector. As has been observed for aphid- transmitted potyviruses and caulimoviruses (Harrison &
Murant 1984? Pirone & Thornbury 1984), there is evidence that transmission of MCDV by leafhoppers is dependent on nonstructural viral-coded proteins known as "helper components" (HC) (Hunt et al. 1988? Creamer et al. 1993), which presumably assist in the binding of virions to retention sites in the vector's foregut (Berger & Pirone
1986; Murant et al. 1988; Ammar & Nault 1991). Because a previous study on the putative HC-mediated transmission of
MCDV showed that HCs of MCDV-M1 and MCDV-T can be used interchangeably (Creamer et al. 1993), it seems reasonable to assume that the two strains have similar HCs. This may explain why these strains have comparable retention half- lives in the vector.
The higher rates of decay of infectivity of MCDV-M1 and MCDV-T at a higher temperature observed here are consistent with previous findings that retention times of
MCDV-T in G. niarifrons are prolonged at lower temperatures (Nault 1977). A similar effect of temperature on retention times has been reported for other foregut- borne viruses transmitted by aphids (Kassanis 1941;
Bradley 1953? Sylvester 1954; Heinze 1959) and leafhoppers
(Ling & Tiongco 1979), including rice tungro spherical 36 virus, the type member of the proposed waikavirus group.
Results of the vector range study of MCDV strains confirm observations of Nault & Madden (1988) that the potential to transmit MCDV among leafhoppers that use maize as a breeding host is correlated with the phylogenetic relatedness of the species. Among the leafhoppers tested here, only species from the
Deltocephalini (G. nigrifrons. A. arex and P. flavicostatus) and recent Euscelini (S. bicolor) were efficient vectors of MCDV-T. MCDV-M1 was efficiently transmitted only by two Deltocephalini species (G. nigrifrons and A. grex).
D. maidis was an inefficient vector of both MCDV strains. Previous reports list D. maidis as a non-vector
(Nault et al. 1973; Nault & Madden 1988; Wayadande 1991).
E- maidis is an important vector of two maize mollicutes fSpiroolasma kunkelii Whitcomb et al. and maize bushy stunt mycoplasma) and a propagative virus (maize rayado fino marafivirus) in the Southern US, Mexico, and Central and South America (Nault 1980; Nault et al. 1980), but has never before been reported as a vector of a foregut-borne, semiperstently-transmitted virus. The smaller number of insects tested in previous studies (100-300) may be why the low levels of MCDV transmission by D. maidis were not detected before. With 300 insects, the probability of transmission could still be 0.01 when all observed insects 37 failed to transmit.
Recently, Ammar & Nault (1991) showed that D. maidis acquires and retains MCDV in the foregut, a prerequisite for transmission of this virus. However, for successful inoculation to occur it is assumed that virions bound to the foregut's cuticle must detach and then be expelled through the stylets back to the plant by extravasation
(McLean & Kinsey 1984; Ammar & Nault 1991). Studies on
MCDV transmission coupled with monitoring of leafhopper feeding provided evidence that extravasation in G. nigrifrons is a behavior associated with recorded X- waveforms when leafhopper probe the phloem (Wayadande &
Nault 1993). Moreover, they observed that X-waveforms of
G. nigrifrons and other leafhopper vectors are qualitatively similar and are more complex and distinct from those of non-vector species, including D. maidis.
With this in mind, they proposed that D. maidis fails to transmit MCDV because extravasation in this species either is qualitatively or quantitatively distinct from those of vector species, or is missing entirely. The discovery that
D. maidis is an infrequent vector of both MCDV strains suggests that this leafhopper does extravasate to some extent during phloem probing. Whether D. maidis is a poor vector because extravasation is rare or is distinct from efficient vector species, remains to be resolved.
The host-plant-range comparison between MCDV-M1 and 38
MCDV-T in this study confirms previous observations of
Nault et al. (1976) that most MCDV hosts are non-festucoid grasses, especially panicoids and andropogonoids. This makes sense because MCDV is more prevalent in warm regions in the southeastern states, where non-festucoid grasses are more abundant, and where the main vector, G. nigrifrons. may have evolved (Whitcomb et al. 1987). In fact, the few grasses in which MCDV has been found in nature are either panicoids or andropogonoids (Nault et al
1973; Pirone et al. 1972). This supports the view that a greater number of susceptible hosts is more likely to be found in groups containing plant species that the virus infects naturally (Dawson & Hilf 1992).
Eight grasses were identified as new experimental hosts of MCDV-T in this research: four andropogonoids (A. virqinicus. I. ruaosum. Rottboelia exaltata and S. nutans); two chloridoids (Eleusine coracana and Eraqrotis cilianensis); one festucoid (M. sobolifera) and one panicoid (Digitaria ischaemum). Among 11 species not tested to MCDV-T before, six (all perennials) were found as not susceptible: three andropogono ids (Andropogon ternarius, Heteroooqon contortus and Schizachvrium condensatum), one panicoid (Digitaria decumbens), one chloridoid (S. pectinata) and one oryzoid (L. orvzoides).
Three species originally found to be immune to MCDV-T by
Nault et al. (1976) (A. virqinicus. E. cilianensis and I. 39 rugosum), are reported here as susceptible to this strain.
The reverse was observed for two other species, Eleusine indica and Panicum capillare. Differences in sensitivity between the detection assays used in this study (DB and
WB) and by Nault et al. (1976) (back-inoculation by leafhopper transmission, rate-zonal sucrose density gradient centrifugation and transmission electron microscopy) may be the reason for these conflicting results. Other possibilities include escape (an inadequate number of test plants and/or inoculative leafhoppers used), misidentification of grass species and use of different varieties of a species.
Among the new experimental hosts of MCDV, A. virqinicus. M. sobolifera, S. nutans and S. scoparium are the first native perennial grasses reported to be susceptible to MCDV that occur naturally in areas of MCDV incidence. Previously, the only such species was johnsongrass [Sorghum halepense (L.) Pers.] (Nault et al.
1976), an andropogonoid grass introduced from the
Mediterranean region early in the 19th century (McWhorter
1971). Johnsongrass is considered to be the major overwintering host of MCDV in the US (Nault et al. 1976;
Gordon et al. 1981). Winter wheat (T. aestivum), which is a symptomless host of both strains, was also suggested by
Nault et al. (1976) as a possible overwintering host of
MCDV. However, wheat is not planted extensively where MCDV 40 is found. Thus johnsongrass is still considered the most important overwintering host.
It is possible that MCDV has been introduced into the
US from the Mediterranean region. However, MCDV is not seed-transmitted (johnsongrass seed, not rhizomes was introduced to the US) and there are no reports of any similar diseases of maize outside the US, thus suggesting that MCDV originated in North America (L. R. Nault, unpublished). The discovery of perennial hosts native to the US supports the hypothesis that MCDV is an indigenous virus. It remains to be learned whether any of these experimental perennial hosts are natural overwintering hosts of MCDV.
Although only 12 genera of native perennial grasses have been surveyed for susceptibility to MCDV here and by
Nault et al. (1976), I speculate that MCDV strains evolved in an andropogonoid host, because four (including johnsongrass) of the five perennial grasses found to be susceptible to MCDV are andropogonoids. It is interesting, however, that one of the experimental perennial hosts (M. sobolifera) was found among the festucoid grasses, where the concentration of susceptible species is very low, and all hosts are symptomless. To make more precise inferences about the evolution and overwintering biology of MCDV in the US, a more extensive survey of the susceptibility of native grass species to this virus is necessary. 41
The small number of symptomless perennial MCDV hosts found so far reflects the marked effects of grass growth habit on the susceptibility of Gramineae to MCDV. Among all grasses tested in this study and by Nault et al.
(1976), a higher percentage of annuals (60%) compared to perennials (13.5%) was susceptible to MCDV strains. This trend of a lower susceptibility of perennials compared to annuals also was observed for maize dwarf mosaic potyvirus
(strains A and B) and sugarcane mosaic potyvirus
(Rosenkranz 1987). It has been proposed that perennials are less susceptible because they are exposed to viruses for longer periods of time than annuals and thus subjected to a higher selection pressure for development of resistance (Nault et al. 1982; Rosenkranz 1987). Unlike perennials, annuals more easily escape infection
(especially by non-seed-transraitted viruses) by developing large populations, growing fast and reseeding every year
(Rosenkranz 1987). In the case of MCDV, another explanation may apply as well. According to Dawson & Hilf
(1992), many viral host ranges that are known today may be responses to human manipulation of the environment.
Although wild virus populations usually comprise a range of strains and variants, changes in the conditions of host plants probably has produced host plant range specialization and greater population uniformity in some viruses (Dawson & Hilf 1992). Thus, with the massive 42 change in the composition of Neartic grasslands from native perennial grasses to predominantly annual crop plants and introduced weedy grasses during the European colonization (Whitcomb et al. 1987), it is possible that the original populations of MCDV have become more homogenous and specialized to prevalent introduced annual and perennial grasses, such as maize and johnsongrass. As a result, they may have lost the ability to infect many of their original native perennial hosts.
In summary, this study shows that MCDV-M1 and MCDV-T share very similar biological properties but can be clearly distinguished from one another based on severity of symptoms and transmission efficiency by some of the vector species. Overall, these results support the conclusions of Gingery & Nault (1990) that MCDV-M1 should be considered a new strain of MCDV rather than a distinct virus. Nevertheless, additional information on genome and coat protein structure of MCDV-M1 and MCDV-T, and on the genetic basis for the observed phenotypic variations, is still necessary for a confirmation of the taxonomic status of MCDV-M1. CHAPTER II
DIEL ACTIVITY PERIODICITY OF GRAMINELLA NIGRIFRONS
(HOMOPTERA: CICADELLIDAE) AND IMPLICATIONS FOR
LEAFHOPPER DISPERSAL
Introduction
The blackfaced leafhopper, Graminella nigrifrons
(Forbes), is the main field vector of the semipersistently transmitted, foregut-borne, maize chlorotic dwarf virus
(MCDV) (Nault et al. 1973; Nault & Madden 1988). The
incidence of this virus in maize crops in the United
States is restricted to areas where johnsongrass fSorghum haleoense (L.) Pers.], the primary overwintering host and
source of inoculum, occurs nearby (Gordon & Nault, 1977).
In fact, epidemiological studies have indicated that the
spread of MCDV by G. nigrifrons within maize plots is
limited to a few meters from inoculum sources (Madden et
al. 1990; Rodriguez et al. 1993; Hunt et al. 1993). This
is probably due to the short persistence of MCDV in the vector (Chapter I ; Nault et al. 1973; Nault 1977) and,
perhaps, to the low rate of movement of G. nigrifrons
observed in maize fields (0.5-4.0 m/day) (Alverson et al.
43 44
1980? Madden et al. 1990).
Although G. nigrifrons is one of the most abundant leafhoppers on maize and other grasses in the eastern
United States (Kramer 1967? Pitre & Hepner 1967), there is not much information about its flight behavior and movement. In a laboratory study of mate-location behavior by G. nigrifrons. Hunt (1988) noted that most males and virgin females, but not mated females, take off from plants and fly above the canopy when dusk is simulated. He suggested that this behavior in nature could lead to short-range dispersal or, possibly, migration. A recent field study of G. nigrifrons flight periodicity with
Johnson-Taylor suction traps revealed that flight activity is indeed maximum at dusk and early evening (Rodriguez et al. 1992). Whether leafhoppers trapped at sunset were engaged in local or migratory flights was unknown, but it was speculated that spread of MCDV far from the sources could happen during evening flights. Like G. nigrifrons. many other leafhoppers and planthoppers, including migratory species, show a crepuscular (twilight) pattern of flight activity with major peaks around dusk (Lawson et al. 1951? Lewis & Taylor 1964? Perfect & Cook 1982? Taylor
& Reling 1986).
To better understand dispersal of G. nigrifrons and the implications of movement on spread of MCDV, further information on the flight behavior of this leafhopper is necessary, particularly on the crepuscular exodus of males and virgin females from the plant canopy (here termed
"vertical movement"), which was previously observed by
Hunt (1988). This paper describes the activity periodicity of G. nigrifrons on maize seedlings under laboratory conditions, during simulated dawn, photophase (light hours), dusk and scotophase (dark hours). One study was carried out to investigate the effects of light intensity on the vertical distribution and interplant movement of specific aged male and female adults. In a second study, the role of photoperiod in maintaining the periodicity of vertical movement by virgin females was evaluated, and possible timing mechanisms involved in this photoperiodic response are discussed. Finally, the prospects for G. nigrifrons to be transported downwind over long distances are examined based on take-off time and possible atmospheric conditions.
Materials and Methods
Leafhopper rearing and experimental conditions. G. nigrifrons was reared in organdy-covered cages (D'Arcy &
Nault 1982) on a combination of oats (Avena sativa L.) and maize seedlings (Zea mays L.) ('Sunglow', sweetcorn).
Cages were kept at 26 ± 2°C and constant photoperiod of
14:10 (L:D) h, with lights on at 06:00 h. Observations were conducted in a transparent Plexiglas™ chamber with four circular openings: two covered with nylon screen for 46 ventilation, and two with removable lids for introduction of plants and insects (Plate I). Temperature and photoperiodic conditions were similar to those at which the insects were reared and entrained, except that a cyclic light intensity regime was used to simulate dawn, photophase, dusk and scotophase (Table 5). Light intensities were measured inside the chamber, at the top of the plants (31 cm), with a photometer (LI-185B) (LI-COR
Inc., Lincoln, NE 68504). Fluorescent bulbs located 35 cm above the observation chamber (Plate I) and in the ceiling of the room were used for illumination during dawn, photophase and dusk. A minimum illumination for observations during the scotophase was provided by two incandescent red bulbs (25 W) located above the chamber
(Plate I). A flashlight with a red filter was used to observe insects on plants.
Activity periodicity study. To assess the effect of light intensity, sex and age on periodicity of activity of
G. nigrifrons. four treatments were studied: a) 3-d old virgin males; b) 1-2 wk old virgin males; c) 3-d old virgin females; and d) 1-2 wk old virgin females. Because
Hunt (1988) observed no vertical movement by mated females, only virgin females were tested here. For each treatment and replication, 45 leafhoppers were introduced in the observation chamber containing nine potted maize seedlings in the four-leaf stage; plants were spaced 15 cm 47
PLATE I. Plexiglas™ chamber (89x65x49 cm) used for observations of activity periodicity of G. nigrifrons. with fixtures of fluorescent and incandescent bulbs located 35 cm above top of the chamber.
■■■ .■ •■■■
- . I 48
Table 5. Cyclic light intensity regime used to simulate dawn, photophase, dusk and scotophase, to study activity periodicity of G. nigrifrons.
Period Time Light intensity3 (ME/m2/s)
Dawn I 5:00 - 5:30 h 7.5
Dawn II 5:30 - 6:00 h 31.0
Photophase 6:00 - 20:00 h 61.0
Dusk I 20:00 - 20:30 h 31.0
Dusk II 20:30 - 21:00 h 7.5
Scotophase 21:00 - 5:00 h 0.3 aLight intensity measured at a height of 31 cm inside the
Plexiglas™ chamber. apart. After an 18-30 h acclimatization period, the
vertical distribution of the leafhoppers in the chamber
and the number of flights per 10-min within the plant
canopy were evaluated every half hour, for 24 h. In the
period between 01:00 and 05:00 h (scotophase), 10-min
observations were spaced by 1-h intervals. For vertical
distribution, individuals were recorded as those: a)
within the maize canopy (on plants, ground and walls up to
40 cm from the chamber floor); and b) those above the
maize canopy (on ceiling and walls above 40 cm from
chamber floor). Observations were not done continuously
for 24 h, but divided in two periods: a) from 05:30 to
20:00; and b) from 19:00 to 6:30 h. The two observation
periods were carried out on different days, using
different groups of insects. For presentation, the results
of the two periods were pooled. Observations of 3-d old virgin male and females were done only during the period
between 19:00 and 06:30 h. Each treatment was repeated
three times.
Persistence of vertical movement under constant
darkness or light. To test whether the periodicity of vertical movement of virgin females (Fig. 2B) involved a
circadian rhythm, its persistence was evaluated under
continuous darkness or light. One day before the onset of
observations, 75 1-2 wk old virgin females were introduced
in the observation chamber, which contained 15 maize 50 seedlings, each with three expanded leaves. Plants were spaced 15 cm from each other. After 4 days at 14:10 (L:D) h (Table 1), a regime of either continuous photophase (61 jitE/m2/s) or continuous scotophase (0.3 jiE/m2/s) was imposed for another 2 days. As a control, the periodicity of vertical movement was also evaluated for 6 days under the entrainment photoperiod [14:10 (L:D) h].
Except during the first 18 h, which represented the acclimatization period, the ceiling and upper walls of the chamber were recorded continuously by using a RCA video camera (TC1005/U9) connected to a Panasonic VCR (NV-8950) and a RCA (TC1112) video monitor. The number of individuals located above the plant canopy was then counted at 1-hour intervals by playing back the recordings in the video monitor. Each treatment was replicated three times. To check if survival was affected by continuous light or darkness, the total number of leafhoppers alive in the chamber was counted at the end of the observation periods.
Results
Activity periodicity. Vertical distribution of G. nigrifrons virgin males and females was affected by light intensity. During the entire photophase, a large majority of males was observed within the maize canopy (Fig. 2A).
At the onset of dusk (20:00 h) about 60% of the males left the plants and moved above the plant canopy (Fig. 2A). Fig. 2. A) Number of G. nigrifrons virgin males (1-2 wk old), and B) virgin females (1-2 wk old), observed within and above the maize canopy in a Plexiglas™ chamber, at half-hour or one-hour intervals during simulated dawn, photophase, dusk and scotophase. Bars at the top of the graph represent periods of scotophase (solid), dawn or dusk (hatched), and photophase (open). Values correspond to means (+ s.e.) of three trials.
51 Number of females Number of m ales 20 30 40 50 20 30 40 50 10 10 O o - 7 1 3 5 7 9 1 3 5 3 1 3 2 21 19 17 15 13 11 9 7 5 ----- . . I i I 1 I I I i 1 I I . I . I vl. wti canopy within „ r i—i— i—i— i— i—i— i— i—i— i— bv canopy above ie (hours) Time Figure 2 Figure
__ —1 I 1— I— __ —I i— -- 1 -- —I 1— -- —1 1— - A: L. 53
Similarly, most virgin females stayed within the plant canopy during the photophase and moved in large numbers above the canopy towards the scotophase (Fig. 2B).
However, females started this upward vertical movement earlier than males (around 16;00 h) and most left the plant canopy before the onset of dusk (Fig. 2B). Males and females that flew to the ceiling of the chamber stayed for a few hours during the scotophase before returning to plants (Fig. 2 A,B). Return was gradual, starting 1 h after the onset of the scotophase. During the morning and early afternoon (8:00-15:00 h), fewer females compared to males were located above the canopy.
Age had a marked effect on vertical movement of both sexes. Fewer young males and females ( 3 d old) were observed above the maize canopy during dusk and scotophase compared to older ones (1-2 wk old) (Fig. 3A,B).
Movement within the canopy was affected by gender and light intensity. Males showed a high rate of interplant movement during the photophase (Fig. 4). However, with the decrease in light intensity at dusk, the number of flights performed by males within the canopy decreased abruptly and remained low throughout the scotophase. Compared to males, rate of movement by females within the canopy was low throughout the light and dark periods (Fig. 4).
Although not quantified, the small peak observed for females late in the photophase (17:00-20:00 h) consisted Fig. 3. Number of G. nigrifrons virgin males and females with different ages observed above the maize canopy in a
Plexiglas™ chamber, from 2 h before the onset of the scotophase to 0.5 h after the onset of the photophase.
Values represent means (+ s.e.) of three trials for: A) 3 d old and 1-2 wk old males, and B) 3 d old and 1-2 wk old females. Bars at the top of the graph indicate periods of photophase (open), dusk or dawn (hatched), and scotophase
(solid).
54 ft of females above canopy # of males above canopy 40 24 32 32 40 24 16 16 8 8 —i —> —i —> —i —i —i —i —i —■ r ■— i— i— i— i— i— i— i— i— i— i— i— >— i— i— i— >— i— i— i— — i - ® - w old wk 1-2 —®— 9 1 3 5 7 5 3 1 3 2 21 19 _ o d old d 3 ___o— V cr T . I I I I I I I I I I L- I I I I I I I I 1 I I I I I I I I I I I I . I ______- A _ l l i a — 2 w old wk -2 1 d old d 3 / i© (hours) Tim©
I _ _ Figure 3 Figure I i I ■ _ _ I _ _ i _ I _ _ xi i _ _ I _ _ XL i _ _ --1 I- - X - - i. 1 - - 1 - - —i-i— A B L. 55 Fig. 4. Log-transformed number of flights of G. niqrifrons males and females (1-2 wk. old) within the maize canopy per 10-min. observation periods.
Observations were done at half-hour or one-hour intervals during simulated dawn, photophase, dusk and scotophase.
Bars at the top of the graph indicate periods of photophase (open), dusk or dawn (hatched), and scotophase
(solid). Values on the curve represent means (+ s.e.) of three trials.
56 Log [<# of flights/10 min.) 2.40 + 0.00 0.60 3.00 1.20 1.80 5 i i i i i i ■
? i V ign e ales fem Virgin ign ales m Virgin t 7
1 ---- 9 1 ----
1 ---- 11 j 1 ---- i i V 4 i i
( ---- ie (hours) Time 13 1 ---- Figure 4 Figure
1 ---- 15 1 ----
1 ___ ---- 17 i —i i i— i— 1 ----
1 ---- 19 r
21 ■
\
23 a /
1 i
V . i. 3
5 57 58 mostly of flights by individuals leaving the plant canopy.
Persistence of vertical movement under constant darkness or light. Under the entrainment photoperiod
[14:10 (L:D) h], the periodicity of vertical movement by virgin females in the observation chamber was maintained unchanged for at least 6 d (Fig. 5A). This periodicity was characterized by cycles of higher numbers of females on the ceiling during late photophase and scotophase (with peaks around dusk), and lower numbers during most of the photophase (6:00-15:00 h) .
By exposing leafhoppers to either continuous photophase or scotophase after 4 d at 14:10 (L:D) h, the periodicity clearly was disrupted (Fig. 56,0). When constant scotophase was imposed, the cyclic response stopped and relatively high numbers of females were continuously observed above the plant canopy, until the end of the observation period (Fig. 5B).
When the continuous-light treatment was imposed on the fourth day (Fig. 5C), females started leaving the canopy at the expected time, because they were still experiencing a normal photoperiod, with photophase preceding scotophase. With the persistence of light after
20:00 h when otherwise there would be the onset of scotophase, females started returning to the plant canopy earlier and faster than observed under the 14:10 (L:D) h photoperiod (Fig. 5C). On the final 2 d of continuous Fig. 5. Number (mean + s.e.) of G. nicrrifrons virgin females (1-2 wk old) observed above the maize canopy at one-hour intervals, during: A) 6 d at 14:10 (L:D) h
(entrainment photoperiod); B) 4 d at 14:10 (L:D) h, followed by 2 d under continuous darkness (0.3 /iE/m2/s) ; and C) 4 d at 14:10 (L:D) h, followed by 2 days under constant light (61 juE/m2/s) . Bars at the top of the graph indicate periods of light (open) and darkness (hatched); subjective nights during the regimes of constant darkness or light are indicated by two dots. Values on the curves represent means of three trials.
59 No. of females above the canopy 12 16 12 16 12 16 6 818 18 6 J __ —L I— 6 ie (hours)Time Figure 5 Figure 18 6 18 6 ij 18 1...1 6 18 6 61 light, vertical movement was depressed and cyclic movement was no longer apparent.
Leafhopper survival was little affected by the regime of continuous photophase, and an average of 95.6% of the initial number of individuals was still alive at the end of the observation period (Day 6, Fig. 50). However, only
76.4% of the leafhoppers were alive after the period of constant scotophase (Day 6, Fig. 5B), suggesting that extended darkness affected leafhopper survival directly or indirectly, perhaps, by decline in host plant quality.
Discussion
Flight periodicity in most insects is mediated by cyclic changes in environmental factors. Time of flight is usually determined by light intensity, while amplitude is mainly influenced by temperature (Lewis & Taylor 1964).
Recently, Rodriguez et al. (1992) showed that flight activity of G. niorifrons in the laboratory is strongly crepuscular, being highest at intermediate levels of light intensity used to simulate dawn and dusk. They further demonstrated that response to light intensity is modified by temperature, being reduced or suppressed at dawn under laboratory or field conditions, when temperature drops to
18°C or less.
This study provides additional evidence that light intensity is an influential factor on activity periodicity of G. niarifrons and shows that response to light 62 intensity also varies with sex and age of adult leafhoppers. The effect of light intensity on virgin males is illustrated by the drastic decrease in the rate of interplant movement, followed by take-off from the plant canopy when dusk is simulated. The response to changes in light intensity was less obvious for virgin females.
Unlike males, females showed a very low rate of interplant movement at all times, and started leaving the plant canopy in greater numbers late in the afternoon, before the onset of low light at dusk.
A higher rate of interplant movement by G. niarifrons males compared to virgin females during the photophase was reported previously (Hunt & Nault 1991), and was shown to be a function of mate-location behavior. G. niarifrons males are active during the day and use a "call-fly" strategy to locate virgin females. Virgin females are sedentary and are found on the upper half of the plant canopy. The decrease in interplant movement by virgin males during the scotophase may be due to an inhibition of mate-location behavior, which in turn is mediated by light
(Hunt & Nault 1991).
That fewer young (3 d old) virgin males and females leave the plant canopy at the end of the photophase and dusk compared to older ones (1-2 wk old) suggests that there might be a teneral period for flight to occur, as observed for many other insects. During this period, the 63 process of cuticle deposition, hardening and darkening is completed, and the enzyme-substrate system necessary for sustained flight is established (Johnson 1974).
Alternatively, the observed effect of age on the propensity of 6. niarifrons adults to leave the plant canopy may be related to mating success. As suggested by
Hunt (1988), the tendency for virgin females to initiate dispersal may depend on how long they remain unmated, and for males, may depend on the success in locating virgin females. Mate deprivation was shown to increase flight propensity of the milkweed bug, Oncooeltus fasciatus
(Dallas) (Dingle 1985).
Because vertical movement of virgin females consistently started »4 h before the simulation of dusk, I considered that this behavior in females could be regulated by a circadian rhythm. However, the cessation of vertical movement when females were exposed to constant darkness or light, after being entrained at 14:10 (L:D) h
(Fig. 5B,C), suggests that this periodicity is not mediated by a circadian clock; rather it appears to rely on exogenous time cues experienced on a daily basis, such as the transition from light to dark (dusk) or vice-versa
(dawn), and possibly night or day length. If G. niarifrons females use such time cues to coordinate onset of vertical flights, then they probably have a mechanism to measure time that has passed since the last dawn or dusk. 64
Alternatively, this behavior may be a response to photoperiodic changes in the host plant. Since a circadian oscillator does not appear to account for the observed periodicity, it is possible that the timing of vertical movement may be associated with a non-rhythmic timer or
"hourglass" clock (Beck 1980).
Strong evidence for the involvement of a nonoscillatory hourglass mechanism in photoperiodic time measurement has been found in the aphid Megoura viciae
(Lees 1966, 1973), in the moth Ostrinia nubilalis (Hvibner)
(Bowen & Shopik 1976) and in the butterfly Pieris brassicae (Veerman et al 1988). In these three cases, scotophase is the critical period measured. According to the hourglass hypothesis, time measurement is dependent on a biochemical reaction, in which a threshold amount of a metabolic product is accumulated at a critical day or night length (Lees 1973). The basic difference between the hourglass and the oscillator clocks is that the former executes a single act of measurement in each dark/light cycle, even if the night period is extended (Veerman et al. 1988). If night is the period measured, a subsequent period of light is required to reset the clock ("to turn over" the hourglass) for another act of measurement. In contrast, the oscillator clock resets itself during prolonged nights, and repeats itself with circadian periodicity (Veerman et al. 1988). As a result, it can 65 make more than one act of measurement during extended dark periods.
Positive evidence for the hourglass model is obtained when photoperiodic response is shown to be related in a predictable way to the duration of the dark and light periods in the cycle (Lees 1973). Induction of pupal diapause in P. brassicae. for instance, requires a night length of 16 h. When larvae are reared at 8:16 (L:D) h or
16:16 (L:D) h, the incidence of diapause is 100%. In contrast, no pupal diapause is observed when larvae are reared at 8:8 (L:D) h or 16:8 (L:D) h (Veerman et al.
1988). At this point, it seems premature to speculate on the involvement of an hourglass clock in the timing of vertical movement of G. niarifrons. because it is still unknown whether lights-on or lights-off initiates time measurement, and whether night or day length is measured in this leafhopper species.
Another relevant question concerns the ultimate reason for upward vertical movement of G. niarifrons around dusk. Hunt (1988) suggested that this behavior in nature could be associated with short-range dispersal or perhaps, migration. The possibility of migration is particularly relevant, because it could result in transport of MCDV far from inoculum sources at night, as speculated by Rodriguez et al. (1992). During the day, most G. niarifrons males and females stay within the plant 66 canopy (Fig. 2A,B), and MCDV spread is probably restricted to short distances from sources.
But, what are the prospects for long-distance flights by G. niarifrons after dusk take-offs? Long distance movement of small insects such as aphids and leafhoppers is thought to be entirely dependent on wind (Taylor 1989).
To be transported downwind, these insects must fly above the boundary layer interface, where wind speeds exceed insect flight speeds (Taylor 1958), which are usually <1 m/s (Farrow 1986). The act of crossing the flight boundary layer is considered migratory and presumably reguires particular behaviors that maximize the chance of lift of the insect (Taylor 1974; Teraguchi 1986). Aphids, for example, enhance their chance of lift by initiating flight during the day, when upward air transport (thermal convection and turbulence) is usually available (Lewis &
Taylor 1964; Taylor 1974) and the boundary layer for small insects (<1.5 cm) is rarely deeper than a few meters
(Farrow 1986).
The specific hypothesis here is that the movement of
G. niarifrons above the canopy observed in the laboratory at dusk is a behavior that results in the transport of leafhoppers to higher altitudes where chances of downwind migration are enhanced. The problem is that the atmosphere is usually very stable near the surface at dusk, and there is little or no atmospheric lift (convection) to assist in 67 the upward movement of these insects (Taylor 1974), except in the zones of convergence of passing cold fronts (Farrow
1986; Taylor & Reling 1986). In fact, dusk is thought to be an ideal time for non-migratory species to disperse within the habitat, because the chances of being displaced downwind are the lowest (Taylor 1974). To engage in downwind migration at dusk or early in the evening, small insects are thought to actively ascend several meters before they can reach a significant wind flow (Farrow
1986). This appears to be the case for the brown planthopper, Nilaoarvata lugens Stal. In 1 h after take off at dusk or late afternoon, large numbers of this migratory planthopper were observed to reach heights of
800 m or more in the absence of updrafts (Riley et al.
1991). This indicates that they were actively climbing at a rate of 0.2 m/s. The potato leafhopper, Empoasca fabae
(Harris), also shows crepuscular flight activity and initiates migratory flight around dusk. But its upward displacement appears to be favored by special weather conditions associated with the passage of cold fronts late in the summer (Taylor & Reling 1986). These frontal systems cause early dusk and presumably stimulate leafhoppers to take off earlier in the day, when conditions likely to provide lift such as thermal convection and frontal conveyors occur. 68
In this study, a few G. niarifrons males and females were observed above the plant canopy in mid afternoon and may take advantage of available thermal lift to be carried above the boundary layer. However, the majority of the individuals took off from the plants late in the afternoon or at dusk, and are less likely to experience updrafts in normal weather. Therefore, their chances of achieving a significant upward displacement should depend mostly on their own power of flight. If flights of G. niarifrons that leave the plant canopy around dusk are not persistent enough to reach altitudes where there is a significant wind flow, most movements of these leafhoppers probably will be restricted to the flight boundary layer and limited to short-range dispersal.
In summary, this study indicates that periodic patterns of movement of G. niarifrons virgin adults are dependent on light intensity and photoperiod, and vary according to the sex and age of the leafhoppers. That G. niarifrons virgin females were observed to leave the plant canopy earlier in the afternoon than males suggests that chances of downwind dispersal are higher for females. This may represent an adaptation of virgin females to minimize chances of interbreeding with sibling males. To speculate further on the potential of G. niarifrons to engage in downwind migration at night and to spread MCDV far from the sources, we must learn more about the propensity of 69 this leafhopper to perform long upward flights. Flight propensity of G. niarifrons in a vertical flight tunnel is the subject of study in Chapter III. CHAPTER III
COMPARATIVE FLIGHT AMD MIGRATORY POTENTIAL OF
GRAMIMELLA NIGRIFRONS AND MACROSTELES OPADRILINEATP8
(HOMOPTERA: CICADELLIDAE)
Introduction
The leafhopper, Graminella niarifrons (Forbes), is the principal vector of the maize chlorotic dwarf virus
(MCDV), a semipersistently transmitted, foregut-borne virus (Nault & Madden 1988). This virus has been observed in maize (Zea mays L.) only in areas of the Unite States where johnsongrass [Sorghum haleoense (L.) Pers.], its only known overwintering reservoir, occurs nearby (Nault et al. 1976; Gordon & Nault 1977). Although this is probably due to the short retention of the virus in the vector (Chapter I; Nault et al. 1973), it also may be related to vector mobility. For example, epidemics of maize dwarf mosaic potyvirus, which is transmitted nonpersistently by aphids, were reported to occur in areas far north of the southern US and Ohio River Valley, where this virus is endemic (Gordon et al. 1981; Zeyen et al.
1987). These northerly epidemics are likely due to the
70 71 long-distance transport by migratory aphids (Zeyen et al.
1987) .
Aphids are known to migrate from southern breeding sites to northern states in the spring (Medler 1962).
Similarly, two leafhoppers that coexist with G. niqrifrons in the southern US, Macrosteles auadrilineatus
(=fascifrgns) Forbes and Emooasca fabae (Harris) (Pitre &
Hepner 1967), are considered to be regular spring migrants, arriving in the north-central and midwestern states from southern states (Pienkowski & Medler 1964;
Drake & Chapman 1965; Taylor 1989; Hoy et al. 1992).
Northward migration in the spring also is suspected for G. niqrifrons (Sedlacek & Freytag 1986), but this has not been investigated extensively. Recently, it was found that
G. niqrifrons can overwinter as eggs, but not as adults or nymphs, in Ohio (Anderson et al. 1991). The very low number of G. niqrifrons adults observed in northern Ohio in May or early June (Knoke & Louie 1981; Teraguchi 1986;
Anderson et al. 1991) when migrant leafhoppers usually arrive, and the lack of spread of MCDV to northern regions of the Corn Belt (Gordon & Nault 1977), raise questions about the potential of G. niqrifrons to migrate to areas north of the Ohio River Valley.
Migratory behavior in many insects is initiated by a decline in response to directional cues from habitat resources and orientation to sky light (Kennedy 1985), which results in movement of the insect into high-speed winds above the boundary layer where horizontal transport occurs (Taylor 1965). Previous laboratory studies have shown that many G. niarifrons virgin male and female adults take off from plants and move above the canopy late in the photophase and around dusk, a behavior that in nature is possibly associated with either local movements or migration (Chapter II; Hunt 1988). A recent field study showed that flight activity of G. niarifrons above the plant canopy is indeed maximum at dusk and early evening
(Rodriguez et al. 1992). Yet, it is questionable whether these dusk flights can result in wind-assisted migration, because atmospheric lift rarely occurs at dusk (Taylor
1974) and leafhoppers must actively ascend several meters to reach a significant wind flow where downwind migration is possible (Farrow 1986).
This research was designed to examine the potential of G. niarifrons to engage in downwind migration and to investigate possible environmental factors enhancing migratory propensity. The known migrant, M. quadrilineatus. was used for comparison in all studies. In the first study, the propensities of G. niarifrons and M. quadrilineatus to exhibit persistent phototactic flights in a vertical flight chamber were evaluated as a function of temperature and photoperiodic conditions experienced by immatures during development, and of adult starvation. To 73 this end, insects were reared under temperatures and photoperiods that simulated spring or summer, and after adult eclosion maintained continuously on maize seedlings
(non-starved) or starved for a few days.
Besides flight propensity, the ability of small insects to cross the boundary layer is dependent on a temporal coincidence of flight activity and appropriate air currents (Taylor 1958). Flight periodicity of some crepuscular insects was observed to be affected by seasonal variations in temperature and photoperiod (Lewis
& Taylor 1964). Therefore, a second study was carried out in growth chambers to evaluate the diel periodicity of take-offs from the plant canopy ("vertical movement")
(Chapter II) of G. niqrifrons and M. quadrilineatus females under simulated summer and spring conditions.
Finally, to understand the differences in flight propensity between the two leafhoppers and to speculate about possible overwintering strategies, variations in body size and wing pigmentation between "summer" and
"spring" forms (seasonal polyphenism) of each species were quantified.
Materials and Methods
Leafhopper rearing. Colonies of G. niqrifrons and M. quadrilineatus originated from adults collected, respectively, on grasses on the Ohio Agricultural Research and Development Center, Wooster, OH, in September/1990, 74 and on vegetable crops in Celeryville, OH, in July/1992.
Leafhoppers were reared in growth chambers under two different regimes of temperature and photoperiod designed to simulate: a) mid summer (July) conditions in central
Ohio; and b) spring (March/April) conditions in the putative source areas of the migrant M. quadrilineatus
(western Arkansas, northwestern Louisiana, northeastern
Texas, and eastern Oklahoma) (Chiykowski & Chapman 1965;
Hoy et al. 1992). Mean temperature and daylength in central Ohio in July are «23°C and 14.7 h, respectively.
Average March and April temperatures in the southern states listed above range from 10 to 19.5°C; daylength is ca. 12-13 h (National Oceanic and Atmospheric
Administration 1980; Hanson 1990). Based on this information, the two sets of experimental conditions used to rear G. niqrifrons and M. quadrilineatus were: a)
26.7°C/23.5°C (L:D) temperature cycle and 14:10 (L:D) h photoperiod, with lights on at 6:00 h ("summer" conditions); and b) 23°C/17°C (L:D) temperature cycle and
12:12 (L:D) h photoperiod, with lights on at 8:00 h
("spring" conditions). Relative humidity was 60-90% in both conditions. I did not select lower temperatures to simulate the spring because this would elongate considerably the egg-to-adult developmental time (see
Larsen et al. 1990). 75
In both summer and spring conditions, leafhoppers were maintained on potted maize seedlings ('Early Sunglow' sweet corn) at the four- to five-leaf stage in rearing cages (19.5 x 38.5 x 39 cm) (D'Arcy and Nault 1982). In each condition, about 150 adults («1:1 male:female ratio) were placed on 12 seedlings per cage for an oviposition period of 5 d. Adult eclosion started around 30 and 40 d later under summer and spring conditions, respectively.
Nymphs in the last stadium were sexed and placed on maize seedlings within 30.5 x 7.5 (dia) cm tubes cages (D'Arcy &
Nault 1982). Eclosed adults were kept under these conditions until the age to be tested. Virgin adults were used in all studies, except for the evaluation of seasonal polyphenism.
General procedures for evaluation of flight propensity. Experiments were done in a black-walled vertical flight chamber modified from Kennedy and Booth
(1963). Construction and functioning details are described by Blackmer & Phelan (1991). A mercury-vapor lamp
(Philips, 175 W) with emission peaks at 400, 430, 550 and
580 nm was used as the light source. Leafhoppers were flown at a height of 20-40 cm below the light window, where the light intensity was wl.O /j,E/m2/s. Air speed was maintained constant from the top to the bottom of the chamber by adjusting the two moveable side walls perpendicularly to the chamber floor (Blackmer & Phelan 76
1991). Temperature inside the chamber was kept at 25 ±
2°C.
Since flight activity of G. niarifrons is highest at dusk under summer conditions (Chapter II? Rodriguez et al.
1992), flight evaluations were conducted in the period between 1.5 h before, to 2.5 h after the onset of the scotophase. Leafhoppers to be tested were placed individually in 2.5 (height) x 2.5 (base dia) cm jelly plastic cups. This release container had the top and bottom covered with parafilm, and was equipped with two lateral openings: one screened for ventilation, and the other corked for introduction of the insects. Leafhoppers were held on the chamber floor for a preconditioning period of 0.5 h, after which the plastic cups were placed on a flat-black platform 15 cm above chamber floor. The top parafilm membrane was then removed and each leafhopper was allowed 10 min to take off. Leafhoppers were tested individually. If a 1ight-oriented, persistent flight bout lasting at least 30 sec was not started within 10 min after take-off, the test was terminated. Free-flight behavior of G. niarifrons and M. quadrilineatus in the vertical wind tunnel is characterized by a series of short phototactic bouts (1-3 min, mean duration) spaced by brief landings on the side walls. All bouts and landed periods were timed by a stopwatch. Results of time measurements were dictated and recorded on cassette tapes for latter 77 review. Observations were ceased if, after performing one or more bouts, the leafhoppers failed to re-initiate flight (>30 sec) within 1.5 min after landing. Total duration of phototactic flight of a leafhopper was considered as being the sum of durations of all bouts performed, excluding the time periods spent landed. Each individual was tested once.
Flight propensity experiments. Two experiments were carried out using the procedures described above. A randomized complete block design with 15 blocks and one replicate/treatment/block, was used in both experiments.
Each replication consisted of a single leafhopper. Each observation period (evening) represented one block. In each block, treatments were tested in a random order.
Leafhoppers tested were 8-d old adults.
In experiment 1, the influence of rearing conditions
(summer or spring-reared) and adult starvation (kept continuously on maize seedlings, or maintained on water for 2 or 4 d before tested) on flight propensity of M. guadrilineatus males and females were assessed. Variables evaluated were means of total flight duration, number of bouts and bout duration, as well as frequency distribution of total duration of flights.
For the 2-d and 4-d starvation treatments, respectively, 6-d and 4-d old adults were transferred from maize seedlings to starvation chambers modified from 78
Larsen (1991), in each rearing condition. Each chamber consisted of a 15.5 x 3.5 (dia) cm tube cage (D'Arcy &
Nault 1990) placed on a 1.4 x 5.3 (dia) cm petri dish
(open side up) filled with white sand. A waffled piece of
3 x 2 cm paper towel was placed on the sand inside the tube as a substrate and shelter for the insects. The sand and paper were kept moist by adding distilled water on a daily basis.
Preliminary tests indicated a high mortality of M. quadrilineatus males when leafhoppers are starved longer than 3 d under summer conditions. Thus, the 4-d starvation treatment was tested only under spring conditions. Because of the unbalanced design, experiment 1 was subdivided in two overlapping experiments for purposes of analysis of variance: la and lb. Factors and levels (in parenthesis) analyzed in experiment la were: rearing conditions (summer or spring-reared); sex (male or female); and adult starvation (non-starved or 2-d starved). Experiment lb included only spring-reared insects, and the only factors tested were sex and starvation (non-starved, 2-d or 4-d starved). In both experiments, data of flight duration, number of bouts and bout duration were transformed to log(x+l), where x is a response, and submitted to analysis of variance (ANOVA). In the case of bout duration, the
General Linear Model (GLM) procedure of MINITAB (Minitab
Inc., State College, PA 16801; 1989) was used for analysis 79 of variance because the sample size varied. After ANOVA, the three levels of starvation in experiment lb were compared by orthogonal contrasts calculated according to
Hicks (1973). For analysis of distribution of flight duration, proportions of fliers as a function of flight duration were calculated for the levels of each factor studied (except starvation for 4 d). Maximum differences in cumulative distributions of proportion of fliers between two levels of each factor were then tested for significance by the Kolmogorov-Smirnov two sample test (a
= 0.05), as described by Sokal and Rohlf (1981).
Experiment 2 had two purposes: a) to evaluate flight propensity of G. niarifrons males and females as a function of rearing conditions (summer or spring-reared) and adult starvation (non-starved or 2-d starved); and b) to compare the flight propensity of G. niarifrons and M. quadrilineatus females. Variables studied were proportion of leafhoppers that initiated flights >30 sec, and frequency distribution of flight duration. Means of flight duration and number of bouts were not analyzed because a very low proportion of G. niarifrons flew >30 sec in some treatments, and the data failed to meet ANOVA's assumption of homogeneity of variances (Steel & Torrie 1980).
Proportions of fliers were submitted to linear logit analysis using the CATMOD procedure of SAS (SAS Institute
Inc., Cary, NC 27512; 1987). Because males of G. 80 niqrifrons. but not of M. quadrilineatus. were tested in experiment 2, the results were divided in two overlapping data sets (experiments) for statistical analysis: 2a and
2b. Experiment 2a included only G. niqrifrons males and females, and factors studied were sex, rearing conditions, and adult starvation. Experiment 2b included females of G. niqrifrons and M. quadrilineatus. with species, rearing conditions, and adult starvation as main factors.
Frequency distributions of flight duration of G. niqrifrons males and females were compared when reared under summer or spring conditions by the Kolmogorov-
Smirnov two sample test (a = 0.05), as described for experiment 1. Comparisons between the distributions of flight duration of G. niqrifrons and M. quadrilineatus females were done for summer or spring rearing conditions, using the same procedure. In all cases, data from non- starved and 2-d starved treatments were combined.
Seasonal periodicity of vertical movement. The periodicities of vertical movement of G. niqrifrons and M. quadrilineatus females were studied under "summer" and
"spring" conditions simulated by the two regimes of photoperiod and temperature described before.
Insects tested under summer and spring conditions were 1-2 wk old virgin females reared from egg to adult under the simulated summer and spring regimes, respectively. Observations were carried out in the same 81 walk-in growth chambers where the insects were reared, within organdy-covered cages similar to that described by
D'Arcy and Nault (1982), but larger (30 x 30 x 102 cm).
Light was provided by fluorescent and incandescent bulbs.
Light intensities during the photophase and scotophase, as measured at the height of 50 cm within the observation cages, were 132 and 0 ixE/m2/s, respectively. Observations during the scotophase were done with a flashlight equipped with a red filter.
For each treatment (season/species) and replication,
45 females were introduced in one observation cage containing nine potted maize seedlings (1 plant/pot)
('Early Sunglow' sweet corn) at the four-leaf stage, which were spaced «10 cm from one another. Leafhoppers were introduced at 18:00 h and evaluations started at 5:00 h on the next day. For every hour until 5:00 h on the following day, vertical movement was monitored by counting the number of individuals located above the maize canopy (on the ceiling and walls of the cages above 50 cm from the cage floor). The experiment was repeated four times; each time with a new group of insects. All treatments were tested simultaneously in each trial.
Seasonal polyphenism. To examine seasonal variations in morphology and coloration of G. niarifrons and M. cruadrilineatus. an assessment of body weight, and wing length and pigmentation, was carried out on females of 82 both species reared under summer and spring conditions.
Wing cell length and dry body weight were evaluated according to Larsen et al. (1990). Two to three wk old adults were frozen and stored at -20°C. After thawing, leafhoppers were sexed and females were separated for each analysis. Measurements of the second median cell in the right forewing were done on ten randomly selected females of each species and rearing condition under a dissecting microscope. For evaluation of dry body weight, females were dried in a convection oven at 80°C and weighed in groups of ten on a balance to the nearest 10'5 g. Ten replicates of ten females were weighed per treatment
(species/rearing condition).
Differences in color intensity of wing pigmentation between summer and spring-reared females were assessed with a method modified from Larsen (1991), in which light intensity transmitted through the second median cell in the forewing of the leafhoppers was measured. The right forewing of ten females from each treatment were removed and placed individually on 5 x 5 cm squares of transparent acetate sheet (0.5-mm thick). A piece of transparent tape was used to fix the wings on the acetate squares. Wings were then projected on a white screen by a slide projector on high power at a distance of 9 m in a dark room. Light intensity proj ected through the wings was measured using a photometer with a light sensor (LI-185B) (Li-COR Inc., 83
Lincoln, NE 68504). The sensor was positioned in front of the viewing screen, facing the projector, and within the projected image of the second median cell.
Untransformed means of each variable studied were submitted to two-way ANOVA, with species and rearing conditions as main effects. Least significant difference
(LSD) (a = 0.05) was calculated and used for multiple comparison of the means.
Results
Flight propensity-preliminary observations. To establish criteria for the flight propensity studies, the minimum times required for 1-2 wk old M. quadrilineatus adults to leave the release container and then to start persistent, light-oriented flight bouts in the vertical flight chamber were measured. All individuals tested (16 males and 13 females) took off from the container in less than 10 min after the plastic wrap was removed, and most males (75%) and females (83%) started phototactic flight bouts within 10 min after taking off. Persistent bouts (>2 min) were observed only for females that performed initial bouts of at least 30 sec. FIight bouts lasting 15 sec or less were usually very erratic and indicated low photokinetic and phototactic responses of the insects.
Additional tests were done to assess the effect of the type of light source and leafhopper age on the propensity of M. quadrilineatus to perform flight bouts > 84
30 sec within 10 min after take-off. A higher proportion of leafhoppers was observed to fly under the clear mercury lamp than under a high pressure sodium lamp (Philips
400 W; major emissions at 490, 565, 580, 590 and 615 nm).
Flight propensity was also affected by age. A reduced percentage (20%) of 1-2 d old individuals flew compared to older ones 7-10 d old adults (80%).
Flight propensity experiments. Experiment 1 . ANOVA revealed that (transformed) means of flight duration and number of bouts performed by M. auadrilineatus were affected by rearing conditions (Table 6, experiment la), being higher for spring-reared leafhoppers than for summer-reared ones (Fig. 6A,B). Mean bout duration was not significantly affected by rearing conditions (Table 6, exp. la; Fig. 6C), suggesting that the prolonged flight duration observed for spring-reared insects (Fig 6A) is due more to the larger number of bouts performed (Fig. 6B) than to an increase in duration of these bouts (Fig. 6C).
In fact, a positive linear relationship was found between total flight duration and number of bouts by spring-reared leafhoppers (n = 60; all treatments combined), as indicated by the Pearson's correlation coefficient
(r = 0.81; P < 0.001) (Devore & Peck 1986). Conversely, a linear relationship between flight duration and bout duration was less evident (r = 0.40; P = 0.004). Some rather long flight bouts (>20 min) were recorded for Table 6. Analysis of variance statistics obtained for the effects of rearing conditions, sex and starvation on flight duration, number of bouts and mean bout duration by M. ouadrilineatus in a vertical flight tunnel.
Flight duration* No. of bouts* Bout duration0 Effect* F df PF df PF df P Experiment la (Sorina- and Summer-reared insects! Block 0.95 14, 98 0.51 1.38 14, 98 0.18 0.82 14, 73 0.64 Rearing 13.08 1, 98 <0.001 13.01 1, 98 <0.001 0.74 1, 73 0.39 conditions (R) Starvation 0.65 1, 98 0.42 0.15 1, 98 0.70 0.70 1, 73 0.41 Sex 0.59 1, 98 0.45 0.39 1, 98 0.54 0.45 1, 73 0.50 R x Sex 0.03 1, 98 0.85 0.08 1, 98 0.78 0.06 1, 73 0.81 R x Starv. 0.01 1, 98 0.93 0.10 1, 98 0.75 <0.01 1, 73 0.98 Sex x Starv. 0.04 1, 98 0.84 0.04 1, 98 0.84 0.85 1, 73 0.36
R x Sex x Starv. 1.49 VO 00 0.23 1.27 1, 98 0.26 0.21 1, 73 0.65
Experiment lb (onlv Snrina-reared insects! B1 ock 0.81 14, 70 0.65 0.73 14, 70 0.73 1.07 14, 53 0.41
Sex 1.04 1, 70 0.31 1.55 70 0.22 0.03 1, 53 0.87 03 1, “Factors and levels studied in experiment la were rearing conditions (Spring or Summer-reared), starvation (non-starved or 2-d starved), and sex (male or female). Experiment lb was carried out only with Spring-reared leafhoppers, and three levels of starvation were studied: 0, 2 or 4 days. bData of flight duration and number of bouts per leafhopper (n = 15) were transformed to log(Y+l) for ANOVA. cData of mean bout duration per leafhopper were transformed to log(Y+l) and submitted to ANOVA using the General Linear Model procedure of Minitab (Minitab, Inc., State College, PA 16801; 1989). Analysis of orthogonal contrasts revealed no significant differences between the average effect of starvation for 0 and 2 days and starvation for 4 days on flight duration (F = 3.11; df = 1, 70; P - 0.082), number of bouts (F = 1.92; df = 1, 70; P = 0.17), and bout duration (F = 3.19; df = 1, 53; P = 0.080). Also, no significant differences were found between the effects of starvation for 0 and 2 days on flight duration (F = 0.31; df = 1, 70; P = 0.59), number of bouts OO (F = 0.18; df = 1, 70; P = 0.67), and bout duration (F = 3.19; df = 1, 53; P = 0.44). ^ Fig. 6. A) Total flight duration (mean + s.e.), B) number of flight bouts (mean + s.e.), and C) bout duration (mean + s.e.) of M. quadrilineatus males and females in a vertical flight chamber when reared under summer or spring conditions, and maintained continuously on maize seedlings as adults (non-starved) or starved for 2 d. Fifteen individuals were tested for each condition. 87 Flight duration (min) Bout duration (min) No. of flight bouts f? 3 (D3 $D H- (D“T (Qu I fl>(D a(D J I I i I I L oo 00 89 spring-reared individuals, but they were infrequent and had a small impact on the means. Mean flight duration, number of bouts and bout duration were not significantly affected by 2-d starvation or by sex of the leafhoppers, and no interaction between main effects was detected (Table 6, exp. la). Also, no significant effects of starvation on these three variables were detected in the overlapping experiment lb, where spring-reared males and females were submitted to three levels of adult starvation (non-starved, or starved for 2 or 4 d) (Table 6, exp. lb). However, there was a noticeable reduction in flight propensity in 4-d starved leafhoppers (Fig. 7A,B,C). Orthogonal contrasts revealed no significant decline in flight propensity of 4-d starved leafhoppers in relation to those starved for 0 or 2 d (Table 6, exp. lb, footnotes), but the low P values (<0.10) obtained in such analyses suggest that negative effects of 4-d starvation could have been detected had a larger sample size been used. Frequency distributions of flight duration of summer and spring-reared leafhoppers differed significantly by the Kolmogorov-Smirnov two sample test (a = 0.05). Both summer and spring-reared groups of leafhoppers showed a large proportion (>60%) of individuals that flew less than 4 min (Fig. 8A). However, no summer-reared leafhoppers flew longer than 18 min, while at least a fourth of the Fig. 7. A) Total flight duration (mean + s.e.), B) number of flight bouts (mean + s.e.), and C) bout duration (mean + s.e.) of spring-reared M. cruadrilineatus males and females in a vertical flight chamber when maintained continuously on maize seedlings as adults (non-starved) or starved for 2 or 4 d before the test. Fifteen individuals were tested for each condition. 90 VD H Flight duration (min.) No of bouts Bout duration (min.) Figure t Fig. 8. Proportion of M. ouadrilineatus adults that flew longer than X (flight duration [min]) in a vertical flight chamber as a function of: A) rearing conditions (summer or spring-reared), B) adult starvation (adults kept continuously on maize seedlings or starved for 2 d) and C) sex. x = mean (± s.e.) flight duration for the sample of leafhoppers (n = 60) tested in each condition. D = maximum difference in proportion of fliers observed between two samples. An asterisk after the D-value indicates significant difference in distribution between the two samples (a = 0.05; Kolmogorov-Smirnov two sample test); n.s. = difference not significant. 92 93 0.90 l“T“» i r-|-r-r-r "TT-rr-i I 1‘ i ’T i | i "i "i i | i i-ri | i i it X A 0.75 £ A Stmmer-reared : x = 3.1 ±0.54 A 0) c 0.60 a Spring-reared; y = 12.5±22 *4— 0.45 D = 0.28* s 0.30 o Q. 0 0.15 I X tN 0. A *4 A 0.00 I. I I I..I. l l a ' i l M i A A - ' A 1 A ^ . 1— J-A I A 2 1 1 1 1 1 1 1 1 1 A 1 C 10 20 30 40 50 60 70 SO 0.90 X A 0.75 ® Non-starved; x = a s ± 1.8 B ? 0.60 o Starved; x = 7.0 ± 1.7 > M- 0.45 D = 0.17as. c o *-> 0.30 «• o “Of? & 0.15 R> o? n V. 00, ” « «B Ol °f °,oo. ,Q>, fa 18'- 10 20 30 40 50 60 70 80 0.90 X 0.75 11 A M a les; 5? = 7.1 ±1.5 O) c 0.60 > Fem ales; y = 8.5±2.0 H- 0.45 0 .1 7as c o t 0.30 S o 0.15 a 0.00 0 10 20 30 40 50 60 70 8 0 Flight duration (min) Figure 8 94 spring-reared leafhoppers showed a total flight duration longer than 20 min (Fig. 8A). No differences in distributions of flight duration were observed by comparing non-starved with 2-d starved leafhoppers (Fig. 8B), or males with females (Fig. 8C); this is consistent with the ANOVA results for means of flight duration (Table 6, exp. la). Experiment 2. Logit generalized linear model showed a significant effect of rearing conditions on the proportion of G. nictrifrons that initiated upward flights >30 sec, but no effects of starvation (Table 7, exp. 2a). The effect of rearing conditions was dependent on sex, as indicated by the significant statistical interaction observed between these two factors (Table 7, exp. 2a). The proportion of fliers among females reared under spring conditions was higher than that observed under summer conditions, while the proportion of fliers among males was equally low in both rearing conditions (Fig. 9A). This statistical interaction explains why the overall effect of sex on propensity to fly was not clearly significant (P = 0.063) (Table 7, exp. 2a). Females showed a higher propensity to initiate flight than males when both were spring-reared, but summer-reared males and females did not differ in this regard (Fig. 9A). This is further demonstrated by the significant differences in frequency distribution of flight duration observed between the two 95 Table 7. Statistics obtained from the linear logit analysis for the effects of rearing condition, adult starvation, and sex on the proportion of G. niqrifrons and M. quadrilineatus adults that fly in a vertical flight tunnel8 Experiment Effects" X2 df P 2a. G. niarifrons Rearing 10.58 1 0.001 (males and females) conditions (R) Starvation (Starv.) 0.22 1 0.64 Sex 3.45 1 0.063 R x Sex 7.77 1 0.005 R x Starv. 1.94 1 0.16 Sex x Starv. 0.86 1 0.35 R x Sex x Starv. 0.86 1 0.35 2b. G. niarifrons and R 6.98 1 0.008 M. auadrilineatus (females only) Species 55.44 1 <0.001 Starv. 2.83 1 0.093 R x Species 12.98 1 <0.001 R x Starv. 4.67 1 0.031 Species x Starv. 0.06 1 0.81 R x Species x Starv. 0.06 1 0.81 ‘Proportion of fliers represent the number of leafhoppers, over the total tested for each treatment (n = 15), that performed upward flights > 30 sec. Proportions were submitted to linear logit analysis using the CATMOD procedure of SAS (SAS Institute Inc., Cary, NC 27512; 1987). 96 bRearing conditions: spring-reared or summer-reared; starvation: non starved or starved for 2 d; species: 6. niarifrons or M. auadrilineatus. Fig. 9. Proportion (+ s.e.) of A) G. niarifrons males and females; and B) female M. quadrilineatus and G. niarifrons that initiate flights lasting >30 sec in a vertical flight chamber when reared under summer or spring conditions, and fed continuously on maize seedlings as adults (non-starved) or starved for 2 d. Fifteen leafhoppers was tested for each condition. 97 M ales F em ales G. n igrifron s 0)1.° M. quadriIin e a tu s c ^ 0 . 8 §0.6 +-> Q o .4 c l o CL 0.2 o non-starv. starved non-starv. starved Summer—reared Spring-reared Figure 9 99 sexes when reared under spring conditions (Fig. 10B), but not under summer conditions (Fig. 10A). In the comparison of species, linear logit analysis results indicated significant overall effects of species and rearing conditions on the proportion of leafhoppers that flew >30 sec (Table 7, exp. 2b). M. quadrilineatus showed a higher proportion of fliers than G. niarifrons in all conditions tested (Fig. 9B). The enhanced propensity of G. niarifrons to fly when reared under spring conditions, in contrast with the lack of influence of rearing conditions on proportion of fliers among M. quadrilineatus females (Fig. 9B), accounts for the significant interaction observed between rearing conditions and species (Table 7, exp. 2b). The” significant interaction between rearing conditions and starvation (Table 7, exp. 2b) suggests that starvation under spring conditions, but not under summer conditions, tend to increase the propensity of both G. niarifrons and M. quadrilineatus for flight (Fig. 9B). Comparison of distributions of flight duration of female G. niarifrons and M. quadrilineatus revealed marked differences between the two species regardless of rearing conditions (a = 0.05; Kolmogorov-Smirnov two sample test) (Fig 11A,B). Under spring conditions, the proportion of females flying longer than 10 min was 15-fold higher for M. quadrilineatus compared to G. niarifrons (Fig. 11A). Under summer conditions, less than 5% of G. niarifrons Fig. 10. Proportion of G. niarifrons males and females that flew longer than X (flight duration [min]) in a vertical flight chamber when reared under A) summer or B) spring conditions, x = mean (± s.e.) flight duration for the sample of leafhoppers (n = 30) tested in each condition. D = maximum difference in proportion of fliers observed between two samples. An asterisk after the D-value indicates significant difference in distribution between the two samples (a = 0.05; Kolmogorov-Smirnov two sample test); n.s. = difference not significant. One data point corresponding to flight duration of 113 min was not included in B. 100 Proportion flying > X Proportion flying > X O p o o o 0 o 0.60 o k) to 01 cn o ^ m (j ^ in o o o o o o o o o o o o o o 0 1 Tj H* K) o co K> o 01 Fig. 11. Proportion of M. quadrilineatus and G. niarifrons females that flew longer than X (flight duration [min]) in a vertical flight chamber when reared under A) spring or B) summer conditions, x = mean (± s.e.) flight duration for the sample of leafhoppers (n = 30) tested in each condition. D = maximum difference in proportion of fliers observed between two samples. An asterisk after the D-value indicates significant difference in distribution between the two samples (a = 0.05; Kolmogorov-Smirnov two sample test); n.s. = difference not significant. Data points corresponding to flight durations above 80 min were not included in A. 102 X Proportion flying > X Proportion flying 0.75 A 0.75 0.15 0.00 0.15 0.30 0.45 0.60 0.90 0.30 0.45 0.60 0.90 0.00 1 2 3 4 5 6 7 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 QO 70 60 50 40 30 20 10 0 ~0 -O \ & I ■1 I l IL l I I I ■1 I I I J G ni fons n 'fro n ig n G. A © -o o o G ni fons n 'fro n ig n G. A o o .. , . | r , . .|...r . ii a us tu ea ri/in d a u q M. \ s u t a e n h i r d a u q . M MdkA ; A-A A- ■ 1 T-.i— A Figure 11 Figure = 433-x- 3 3 .4 0 = D ■o- 1 : - i ; i 1 1 1 I i j ____ = x = = x - —I 1— = 18. .6 5 ± .0 8 1 = x _ __ -- ______1 __ I I —I I I “ I— I— I | I 1 3.1 1 __ 1 I ±0. 7 .3 0 ± __ 1 __ 3.5+0.98 1 __ 1 __ I __ 1 __ I I ' I | »' I— I 1 I 1 1— I __ 1 __ -- —1 1 1— 1— 1 : A i — •e - A- -- T— 103 104 females flew longer than 30 sec, in contrast to about 30% of M. quadrilineatus females (Fig. 11B). Mean total flight durations of M. quadrilineatus females were around 6-7 times longer than those observed for G. niarifrons in both conditions (Fig. 11A,B). Despite the differences in frequency of fliers, persistent flights were observed for both species. Longest flight durations observed for M. quadrilineatus and G. niarifrons females were 133 and 113 min, respectively (data points not shown in Fig. 11A). Maximum bout durations for these species were 64 and 68 min, respectively. Both sets of values were observed for 2-d starved, spring-reared females. Seasonal periodicity of vertical movement. Under simulated summer conditions (long day and warm night temperature) (Fig. 12B), M. quadrilineatus females showed a bimodal diel periodicity of exodus from the plant canopy with a peak early in the morning, 1-2 h after lights were turned on, and a second peak early in the evening (Fig. 12A). Similarly, G. niarifrons females showed a peak of vertical movement early in the scotophase under summer conditions, but no morning peak was observed (Fig. 12A). In the early morning peak of M. quadrilineatus. females started taking off from the plants soon after lights were turned on. In the evening peaks, both M. quadrilineatus and G. niarifrons started moving in greater numbers above the canopy late in the afternoon, around 2-3 h before Fig. 12. Number of G. niarifrons and M. quadrilineatus females observed above the maize canopy in rearing cages (A), at one-hour intervals, under simulated summer conditions with photoperiod of 14:10 (L:D) h and fluctuating temperatures (B). Solid bars indicate scotophase; hatched bar, photophase. Values and bars in A represent mean numbers of leafhoppers and s.e., respectively, of four replications of the experiment. 105 2 5 -i— i— i— i— i— i— i— i— i— i— '— ;— i— i— i i r M. quadri/ineatus A 20 G. nigrifrons 15 10 5 0 3 0 2 7 2 4 21 18 15 5 7 9 11 13 15 17 19 21 23 1 3 5 Figure 12 107 lights off (Fig. 12A). Under spring conditions, with a shorter day and lower night temperature (Fig. 13B), the evening peaks of vertical movement of both species were shifted «4 h ahead to mid or late afternoon, around 2-4 h before scotophase (Fig 13A). During the hour that preceded the onset of the scotophase and the decline in temperature, most females located above the canopy returned to the plants, and only a low number of insects were observed above the canopy throughout the dark and cold period (Fig. 13B). G. niarifrons females, which consistently initiated vertical movement late in the photophase under summer conditions, started leaving the plants in greater numbers around noon under spring conditions, and the resulting curve of vertical movement became skewed to the left (Fig. 13A). Due to these changes in periodicity, a larger number of females of both species were observed above the canopy in the afternoon under spring conditions (Fig. 13B), than under summer conditions (Fig. 12B). Seasonal polyphenism. Wing cell length and dry body weight were affected by rearing conditions (F[wing] = 32 and ^ [weight] =72' df = 1 >35'' p < 0.001) and species ( F [wjng] = 271 and F [wei.ght] =93; df = 1,36; P < 0.001) . For both species, means of wing cell length and dry body weight were greater for females reared under spring conditions than for those reared under summer conditions Fig. 13. Number of G. niarifrons and M. ouadrilineatus females observed above the maize canopy in rearing cages (A), at one-hour intervals, under simulated spring conditions with photoperiod of 12:12 (L:D) h and fluctuating temperatures (B). Solid bars indicate scotophase; hatched bar, photophase. Values and bars in A represent mean numbers of leafhoppers and s.e., respectively, of four replications of the experiment. 108 Temperature { °C) No. of females above canopy 5 2 30 20 27 21 4 2 15 10 15 18 0 5 T - - - . quadrilineatus M. girns igrifron n G iue 13 Figure -» T T T hours 5 109 110 (P < 0.05; LSD test) (Fig. 14A,B). For each rearing condition, G. niarifrons females were heavier and had shorter wing cells than M. guadrilineatus females (Fig. 14A,B), suggesting a lower wing loading in the latter species. For dry body weight, there was a significant interaction between species and rearing conditions (F = 6.8; df = 1,36; P = 0.013), indicating that differences in body weight between summer and spring forms were larger for M. guadrilineatus than for G. niarifrons females. Spring forms of M. quadri1ineatus were 26.7% heavier than summer forms, whereas in G. niarifrons weight gain in spring conditions was only 9.3%. Light intensity readings were used as an indirect measure of wing pigmentation. Darker, more pigmented wings were indicated by lower light intensity readings, whereas lighter colored wings were indicated by higher light intensity values. A significant statistical interaction was found between rearing conditions and species (F = 8.9; df = 1,36; P = 0.005). Forewing coloration, as measured by light intensity transmitted through the second median cell, was darker in spring-reared G. niarifrons females than in summer-reared females of the same species (P < 0.05) (Fig. 14C). In contrast, there was no significant difference in wing pigmentation between summer and spring-reared M. guadrilineatus females (Fig. 14C). Fig. 14. A) Wing cell length (mean + s.e.), B) dry body weight (mean + s.e.), and C) wing cell color intensity (mean + s.e.) of G. niarifrons and M. guadrilineatus females when reared under summer or spring conditions. For color intensity, a higher light intensity value indicates lighter wing coloration, whereas a lower value indicates darker coloration. Means followed by the same lowercase letter are not significantly different (P = 0.05; LSD test preceded by two-way ANOVA using untransformed data). I l l 2.00 Y//\ Summer-reared A F 1BO £ Spring—reared 4-’ x: 1.60 ye o 1.40 o O) 1.20 £ 1.00 G. nigrifrons M. qu a d n iin ea tu s H-s ± 9 r (« O V/A Summer-reared 8 ; O T~ I888881 Spring-reared H- 7 - u Ts) £ to ; +-> -i= O! 5 ; o * 4 - ■O o CO 3 G. nigrifrons M. qu ad n iin ea tu s 2.00 \//\ Summer-reared c ^ 1.80 Spring-reared UJ 1.60 a i 1-40 § c 1.20 +-> O) 1.00 _ l 0.80 G. nigrifrons M. qu a d n iin ea tu s Figure 14 113 Discussion Compared to holometabolous insects, leafhoppers are weak fliers with erratic flight behavior characterized by repeated bouts of short duration spaced by brief landings (Taylor et al. 1992). This study confirms that M. quadrilineatus and G. niarifrons are not exceptions. The majority of fliers of both species performed mostly short flight bouts which lasted 1-3 min on average. Thus, most of the long total flight durations calculated for M. guadrilineatus and G. niarifrons were due to an increase in frequency, but not in duration, of bouts except for a few individuals of each species that flew for relatively long periods of time without landing. Similarly, Rose (1972a) observed that tethered flights of Cicadulina leafhoppers were mostly of short duration. Unlike leafhoppers, aphids have been shown to fly continuously for long periods in a vertical flight chamber. Work done with Aphis fabae Scop, showed that first flight bouts lasted an average of 80 min, and represented about 75% of total flight duration (Kennedy & Booth 1963). One goal of this study was to investigate environmental conditions that increase flight propensity of M. guadrilineatus and G. niarifrons. Temperature, photoperiod, and food level or quality, are factors often associated with induction of migration and diapause in insects (Dingle 1985; Rankin et al. 1986). Raising rearing 114 temperature from 23 to 27°C reduced the proportion of Oncooeltus fasciatus (Dallas) females performing persistent tethered flights (>30 min) from «30% to less than 8% (Dingle 1968). Short daylength induces reproductive diapause in 0. fasciatus in the fall and lengthens the age interval that adults perform long flights (Dingle 1978). In the aphid A. fabae. alate forms produced under short days (autumn form, gynoparae) differ from those reared under long days (summer form, virginoparae) with respect to flight behavior in a flight chamber (David & Hardie 1988). Gynoparae appeared to be more migratory than virginoparae in the sense that they were faster upward fliers and less responsive to green targets presented for 5 sec at 1-min intervals. In this study, the combination of low temperature and short days (spring conditions) clearly increased total flight duration of M. guadrilineatus males and females, and the proportion of individuals performing phototactic flights among G. nigrifrons females. These results are consistent with migration of M. guadrilineatus to the north-central states in the spring (Drake & Chapman 1965; Hoy et al. 1992) and suggest that seasonal cues such as temperature and photoperiod may induce development of migrant adults in these species. Whether the effect of temperature and/or photoperiod on the insect is direct, or indirect through changes in host plant quality, remains to be resolved. 115 Unlike observed with the lygaeid, O. fasciatus (Dingle 1968), and the nitidulid, Carpophilus hemipterus (L.) (Blackmer & Phelan 1991), starvation did not enhance flight propensity of G. nigrifrons and M. guadrilineatus. The apparent decline in flight duration with starvation for 4 d suggests that this condition was severe and possibly affected the reserves necessary for sustained flight. In fact, preliminary tests indicated poor survival of males after 4-d starvation under simulated spring conditions. Low host quality is a less severe condition that is likely to be experienced by leafhoppers in the field, and which could be tested in future studies. A previous study showed no dramatic effects of rearing host age on periodic flight activity of G. nigrifrons (Rodriguez et al. 1992), but effects on flight duration were not examined. Suboptimal food is thought to induce migration in the beet leafhopper, Circulifer tenellus (Baker) (Lawson et al. 1951), and was shown to increase flight readiness in Cicadulina spp. (Rose 1972a). The comparison of flight propensity between the two leafhopper species provides evidence that M. guadrilineatus has a greater migratory tendency than G. nigrifrons. Long phototactic flights (>20 min) were observed for both species, but were rather infrequent for G. nigrifrons. Overall, the proportion of fliers and mean flight duration were higher in M. guadrilineatus than in 116 G. niarifrons. These results are consistent with observations of flight activity of G. niarifrons and M. guadrilineatus in northern Ohio (Teraguchi 1986). Although she considered both species as highly migratory, her data show that numbers of M. guadrilineatus trapped well above the flight boundary layer (at 9.14 m) were about five fold greater than for G. niarifrons. especially in late September when return migration to southern areas may occur. Comparisons between sexes indicated that G. niarifrons females are more likely to start persistent phototactic flights and engage in downwind migration than males under spring conditions. This may result in a lower male:female ratio among migrants, as observed for Empoasca fabae (Harris) (Taylor 1989). In contrast, M. guadrilineatus males and females appeared to be equally apt to perform long upward flights. Thus, the considerably low male:female ratio observed in populations of immigrant M. guadrilineatus populations in Wisconsin (Drake & Chapman 1965) is probably due to factors other than propensity to fly, perhaps differential survival during the migratory flight. For E. fabae. the low tolerance of males to extremes of temperature and relative humidity (Kouskolekas & Decker 1966; Decker & Cunningham 1967) was indicated as a likely reason for the low proportion of males observed among migrants (Taylor 1989). 117 Although G. niarifrons showed a relatively low propensity to initiate phototactic flight in this study, its potential for long-range movement cannot be ruled out. The increase in proportion of fliers among females in response to development under simulated spring conditions is consistent with the hypothesis of northward migration in the spring (Sedlaceck & Freytag 1986). Because only a small fraction of G. niarifrons fly longer than 10 min in the vertical flight chamber, one question that arises is how long persistent phototactic flights should be for leafhoppers to reach altitudes where downwind transport is possible. According to the flight boundary layer concept (Taylor 1958, 1974), this depends on take-off time and atmospheric conditions. The study on seasonal periodicity of vertical movement provides valuable information on periods of the day that leafhoppers are most likely to leave the plant canopy and start phototactic flights. Under summer conditions, most G. niarifrons females were observed to take off late in the afternoon (1-2 h before scotophase) and early evening. In general, atmospheric stability near the surface is maximum at dusk and there is no convective mixing (thermal convection) to aid in the upward movement of flying leafhoppers to higher-speed winds (Taylor 1974 ? Farrow 1986), except in zones of convergence of passing cold fronts (Farrow 1986; Taylor & Reling 1986). Thus, the 118 success of leafhoppers in reaching a significant wind flow at dusk, which may be as high as 50 m (Farrow 1986), may depend mostly on their own power of flight. Considering that over 95% of summer-reared G. niarifrons females flew 30 sec or less in the vertical flight chamber, it is likely that most G. niarifrons movement in the summer is limited to low altitudes and, perhaps, restricted to short-range dispersal. However, there may be opportunities for intermediate or long-range movement of G. niarifrons near the surface. M. guadrilineatus. for example, is thought to migrate considerable distances on strong surface («2 m above ground) winds that often move in a north or northeasterly direction from the Gulf Coast area during the spring (Chiykowski & Chapman 1965; Hoy et al. 1992). In the summer, the prevailing direction of surface winds is similar (U.S. Department of Commerce 1983). It should also be cautioned that the results of the vertical flight chamber study might represent an unde re s t imate of flight propensity by G. niarifrons under summer conditions. Other conditions that may stimulate upward flight of G. niarifrons in the summer, such as suboptimal food quality, were not tested here. Chances of long-range transport are apparently higher under spring conditions. Movement of G. nigrifrons females to above the plant canopy was advanced about 4 h and most individuals left the plants in mid afternoon (3-4 h before 119 scotophase), when the flight boundary layer for small insects in the field is only a few meters deep (Farrow 1986) and atmospheric lift is usually available (Lewis & Taylor 1964? Taylor 1974). Moreover, at least 20% of spring-reared G. nigrifrons females performed phototactic flights longer than 4 min. Rate of climb of the leafhoppers in the vertical flight chamber was not determined in this study, but it is known to be around 0.2 m/s for migratory aphids (David & Hardie 1988) and planthoppers (Riley et al. 1991). Assuming a conservative rate of climb of 0.1 m/s for G. niarifrons. this leafhopper could potentially reach heights of 24 m within 4 min after take-off. This added to the available lift provided by convection might result in a vertical displacement of the leafhoppers of tens or hundreds of meters, to altitudes where low-level jet (LLJ) winds are observed commonly in the lower Mississippi Valley regions during mid and late spring (Sedlacek & Freytag 1986? Taylor 1986? Zeyen et al. 1987). This speculation is supported by the capture of G. niarifrons along with M. ouadri1 ineatus and other leafhoppers at the altitude of 610 m by airplane over Texas (Glick & Noble 1961). Once in a LLJ stream, insects might travel long distances without providing an horizontal component to their flight, although some wing beating is necessary to compensate for their terminal velocity and keep then aloft (L. R. Taylor 1 2 0 1986; R. A. J. Taylor 1989). Considering the conditions described above, the potential for northward spring migration of M. quadrilineatus is obvious. Take-off of females from the plant canopy also was concentrated in the afternoon under spring conditions, and at least 25% of spring-reared individuals showed a total flight duration greater than 20 min. Actually, the potential of M. cruadrif ineatus to engage in long-range transport is also high under summer conditions, because a reasonable proportion of summer- reared females (1 0 %) showed total flight duration longer than 10 min in the vertical flight chamber. Moreover, under simulated summer conditions many M. guadrilineatus females were observed to leave the plant canopy in the morning when updrafts in the field become more frequent. Seasonal changes in flight periodicity similar to those observed for G. nigrifrons and M. guadrilineatus were reported for other crepuscular-flying insects and are thought to be determined by temperature and time of sunrise or sunset (Lewis & Taylor 1964). In general, these insects show peaks of flight at dawn and dusk in the summer, and tend to advance the dusk peak to mid or late afternoon on cold days in the fall or winter when evening temperatures fall below their thresholds for flight. This trend has been documented for the brown planthopper Nilaparvata lugens Stal (Ohkubo & Kisimoto 1971; Riley et 1 2 1 al. 1991) and for the leafhoppers C. tennelus (Lawson et al. 1951), Cicadulina spp. (Rose 1972b) and Dalbulus spp. (Taylor et al., in press). Temperature was already known to alter the flight periodicity of G. nigrifrons. In a laboratory study, Rodriguez et al. (1992) observed that nocturnal flight activity is significantly reduced and a greater number of adults fly late in the day when night temperature is lowered from 26.6 to 18.3°C. The periodicity of vertical movement of G. nigrifrons virgin females observed under simulated summer conditions and fluctuating temperature [23.5/26.7°C (L:D)] in this study was similar to that observed at constant 26 ± 2°C in Chapter II (Fig. 2B). The analysis of seasonal variation in body size and wing pigmentation for G. nigrifrons and M. quadrilineatus revealed differences between the two species that may explain the superior flight propensity of M. guadrilineatus. The larger gain of weight under spring conditions in M. guadrilineatus (26.7%) compared to G. nigrifrons (9.3%), for example, suggests a greater accumulation of reserves for flight in the former species. The fat content in C. tenellus was observed to decrease 75% after a 300-mile flight (Fulton & Romney 1940; DeLong 1971), indicating that lipid reserves are important in leafhopper migration. It would be interesting to learn whether the differences in body weight between summer and 1 2 2 spring forms of M. guadrilineatus and G. nigrifrons are due to an increase in fat content. In other insects, the relationship between body size and flight duration is controversial. Roff (1991) predicted that migrant insects will tend to be larger than nonmigrants because larger body size allows migrants to fly longer without refueling. However, Rankin & Burchsted (1992) found no differences in average body size of migrants and nonmigrants individuals in migratory species such as 0 . fasciatus. Melanoolus sanguinipes (Fabr.), Hippodamia convergens Guerin- Meneville, and Anthonomus grandis grandis Boheman. Melanization is widespread in insects and in many cases can be a thermoregulatory adaptation to seasonally varying thermal conditions, as shown in Colias and Pieris butterflies (Watt 1969; Kingsolver & Wiernasz 1991). Darker forms usually develop under colder temperatures and more efficiently absorb heat than light forms. Photoperiod appears to be the most common environmental switch in this polyphenism. In Colias butterflies (Watt 1969) and in the leafhoppers Eucelius incisus (Muller 1979) and Stirellus bicolor (Whitcomb et al. 1972), dark forms are induced by short daylength. It is likely that the short daylength (12 h) used to simulate spring conditions in this study was the factor inducing wing darkening in G. nigrifrons. By rearing G. nigrifrons at low temperature (18°C) , but long daylength (16 h), Larsen et al. (1990) observed a general 123 increase in body size and weight of adults, but reported no differences in coloration. In the leafhopper Euotervx uticae (Fab.) and in Dalbulus species, development of dark pigmentation was associated with distribution at high altitudes and low temperature (Stewart 1981; Larsen 1991). Therefore, the darker wing coloration observed under simulated spring conditions for G. niarifrons compared to M. guadrilineatus may be an indication that the former species is better adapted to endure cold weather in the adult stage, and possibly rely on overwintering in the northern areas as an alternative to southward migration in the fall. For day-flying insects in temperate climates, darker coloration might also be advantageous for migration, because it can aid insects to raise body temperature above the ambient temperature and the minimum levels required for flight (Lewis & Taylor 1964). It is noteworthy that M. quadrilineatus populations in Wooster, OH, usually decrease faster in September and October than populations of G. niarifrons (R. J. Anderson, personal communication). Moreover, fewer G. niarifrons adults compared to M. guadrilineatus were observed above the boundary layer (at 9.14 m) in northern Ohio in late Septempber (Teraguchi 1986), suggesting that the latter species displays a higher migratory activity at this time. These facts added to the observation that only a small 124 proportion of G. niarifrons performed long phototactic flights when reared under simulated spring conditions suggest that this leafhopper may have a dual overwintering strategy, in which part of the population overwinters in northern states and the rest returns to the southern areas in the fall. There is evidence that M. guadrilineatus and G. nigrifrons can overwinter in northern regions of US in the egg stage, but not as nymphs or adults (Drake & Chapman 1965; Saini 1967; Sedlacek et al. 1990; Anderson et al. 1991). In fact, populations of M. guadrilineatus in New York and eastern Washington are thought to originate primarily from local overwintering eggs (Hervey & Schroeder 1947; Hagel et al. 1973). In north-central and midwestern states, however, first adults collected in late April or early May were considered migrants from southern areas, because they appeared several weeks before the overwintering population could develop from eggs to adults (Drake & Chapman 1965; Teraguchi 1986; Hoy et al. 1992). Similarly, the first G. nigrifrons adults in Kentucky were observed in early May, before sufficient degree-days had been accumulated for complete egg-adult development of the overwintering population (Sedlaceck & Freytag 1986). The coincidence of this early appearance of G. nigrifrons with the occurrence of surface and LLJ streams blowing from the south and southwest, prompted Sedlacek & Freytag (1986) to 125 hypothesize that adults collected in May were immigrants from southern areas. It is interesting that the occurrence of G. nigrifrons in northern Ohio is considerably delayed compared to Kentucky and southern Ohio. In northern Ohio first adults have been observed in low numbers only in late May, and the first peak has not been recorded before late June or July (Knoke & Louie 1981; Teraguchi 1986; Anderson et al. 1991; Rodriguez et al. 1992). In contrast, the first peak of G. nigrifrons in southern Ohio and Kentucky has been observed as early as late May/early June (Knoke & Louie 1981; Sedlacek & Freytag 1986). These observations indicate that G. nigrifrons populations in northern Ohio may be mostly of local origin, as previously suggested by Anderson et al. (1991). Alternatively, it is possible that colonization of northern Ohio is by populations that originate from first generation adults from Kentucky and southern Ohio. In any case, if regular northward migration of G. nigrifrons in the spring indeed takes place as suspected by Sedlacek & Freytag (1986), most migrant leafhoppers may not reach northern Ohio when maize plantings are in the early growth stages. This might be another reason why MOD disease epidemics are not observed in northern areas of the Corn Belt (Gordon & Nault 1977), in addition to the absence of johnsongrass and to the short retention time of MCDV in G. nigrifrons. EPILOGUE The research presented in this dissertation improves our understanding of the biology of MCDV strains (MCDV-T and MCDV-M1), the flight behavior of Graminella niarifrons (Forbes) and Macrosteles guadrilineatus Forbes, and the patterns of incidence of MOD disease in the US. This body of information suggests that the synergism between MCDV-T and MCDV-M1, half-life of both strains in G. niarifrons. leafhopper behavior associated with long distance movement, and possibly the delayed colonization of northern areas of the US by migrant G. nigrifrons. are likely factors limiting the spread of MCDV to northern regions of the Corn Belt where this virus is not endemic. When examining the possibility of long-range spread of MCDV by leafhoppers, it should be considered that both MCDV-T and MCDV-M1 are necessary for full expression of MCD disease symptoms (Gingery & Nault 1990). This requires that both strains be carried by the same vector during long-range movement, otherwise the chances of co inoculation of a plant distant from the source are remote. Among the six leafhoppers confirmed as vectors in 126 127 Chapter I, only G. niarifrons transmitted both strains efficiently. Thus, it appears to be the only species likely to carry both strains over long distances. Besides G. niarifrons. the only other vector species that could be of importance for spread of MCDV is Amblvsellus arex (Oman). This species ranked second in transmission efficiency of both strains. As noted by Nault & Madden (1988), A. arex would be of special concern if MCDV was introduced in the western US, because along with the main overwintering host of MCDV, johnsongrass [Sorghum halepense (L.) Pers.], this leafhopper is abundant in California and other western states (Kramer 1971; Nault & Madden 1988). Other two relatively efficient vectors of MCDV-T, Planiceohalus flavicostatus (Van Duzee) and Stirellus bicolor (Van Duzee), were poor vectors of MCDV- M1 and are considerably less abundant than G. niarifrons in maize and nearby grasses (Pitre & Hepner 1967; Nault & Madden 1988). Based on the short retention half-lives of MCDV-M1 and MCDV-T in G. niarifrons (3.8-12.5 h) (Chapter I), I predict that co-inoculation of these strains after movements lasting longer than a day will occur at very low rates. It is possible that the rate of loss of virus in G. niarifrons is reduced under continuous flight when leafhoppers are deprived of food, as observed in the case of aphid transmission of MDMV (Berger et al. 1987). 128 However, the sequence of short flight bouts and frequent landings that characterized the flight behavior of leafhoppers in the vertical flight chamber (Chapter III) suggests that feeding stops may not be uncommon during migration. In fact, feeding stops are thought to occur in migratory flights of the beet leafhopper, Circulifer tenellus (Baker) (Lawson et al. 1951), and M. guadrilineatus (Chapman 1973). The results of flight propensity and periodicity of take off (vertical movement) of G. niarifrons (Chapter III) does not argue against the hypothesis of northward spring migration by this leafhopper (Sedlacek & Freytag 1986). However, it appears that the bulk of this migratory movement does not extend to areas north of the Ohio River Valley, such as northern Ohio, because very low numbers of G. niarifrons are observed in these areas in May and early June (Knoke & Louie 1981? Teraguchi 1986? Anderson et al. 1991), when spring migrants normally arrive. Colonization of northern areas of Ohio by second generation migrants from Kentucky and southern Ohio may occur in July, but at this time field corn is beyond the phenological stage that is most susceptible to virus infection and adequate for leafhopper feeding and reproduction. Moreover, since rates of loss of inoculativity of both strains are faster at higher temperatures (Chapter I), fewer leafhoppers are likely to retain MCDV during long-distance movement in 129 mid-summer when the average temperature is higher. Besides the problem of long-range spread of MCDV, other important questions were addressed in this research. The comparison of biological properties between MCDV-M1 and MCDV-T revealed that these two strains share very similar host plant and vector ranges, as well as transmission characteristics (Chapter I). Should these two viruses be considered distinct species based on differences in nucleic acid sequence (Ngazimbi 1993), the similarities in biological properties suggest that they are probably closely related. The discovery of native perennial plants susceptible to MCDV-M1 and MCDV-T supports the hypothesis that MCDV originated in North America (L. R. Nault, unpubl.). The only perennial host shown to be susceptible in a previous study was johnsongrass (Nault et al. 1976) , which was introduced in the US from the Mediterranean region early in the 19th century (McWhorter 1971). The new experimental perennial hosts are wild native grasses that occur in old fields or woods (Hitchcock 1950). If any of these grasses are natural hosts of MCDV in the US, they are unlikely to be important reservoirs for this virus because, unlike johnsongrass, they are not abundant nearby maize fields. The absence of MCDV in northern Ohio where these new hosts occur (Hitchcock 1950), supports this view. The discovery that Dalbulus maidis (DeLong & Wolcott) is an infrequent 130 vector of both MCDV strains contributes to our understanding of MCDV vector specificity by indicating that this leafhopper does extravasate to some extent during phloem probing, despite the differences in X- waveform behavior observed between D. maidis and efficient vectors (Wayadande & Nault 1993). In Chapter II, I demonstrated the role of photoperiod and light intensity on the maintenance of the periodicity of vertical movement of G. niarifrons. and how this response varies according to the age and sex of the leafhoppers. To better understand the mechanism involved in photoperiodic time measurement of this periodicity, a different experimental set up should be used in which only the insect, not the plants, are exposed to the variable photoperiod. An important contribution of Chapter III, besides determining the relative flight propensities of G. niarifrons and M. ouadrilineatus. was to indicate that photoperiod and/or temperature are possibly involved in induction of migratory forms in these leafhoppers. I believe that seasonal variations in body size and coloration of G. niarifrons and M. guadrilineatus have implications on their flight propensity and overwintering strategies, and deserve further attention. 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