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INTERACTIONS AMONG Dalbulus SPECIES (HOMOFTERA: CICADELLIDAE) AND THEIR ASSOCIATED ORGANISMS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Gustavo Moya-Raygoza, B.S., M.S.
*****
The Ohio State University 1997
Dissertation Committee:
L.R. Nault, Adviser Approved by
D.J. Horn
B.R. Stinner Adviser ' Department of Entomology D.T. Gordon UMI Number: 9813318
UMI Microform 9813318 Copyright 1998, by UMI Company. All rights reserved.
This microform edition Is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeh Road Ann Arbor, MI 48103 ABSTRACT
Effects of temperature and vector age on the transmission of maize bushy smnt phytoplasma (MBSP) isolates by the com leafhopper Dalbulus maidis were studied. Mean latent period in the leafhopper and transmission rate of the Tlaltizapan MBSP isolate had quadratic relation with temperature. Mean latent period was longer at low (15 °C) than at high (25 “ and 30 °C) temperatures, whereas transmission rate was low at 15 °C and high at 25 °C. The Tlaltizapan isolate was transmitted better by nymphs than adults when maintained at 15, 20, and 25 °C.
The transmission biology of three (Tlaltizapan, El Batan, and Poza Rica) MBSP isolates was investigated with nymphs ofD- maidis at 15 ° and 25 °C. Differences among the three isolates were found only at 25 °C. At this temperature, the Poza Rica isolate had longer mean latent period and higher transmission rate than the El Batan and Tlaltizapan isolates. Moreover, the Poza Rica isolate had longer incubation period in the plant host and produced more basal tillering in infected maize than the other two isolates. Based upon biological findings in this study, as well as molecular smdies reported elsewhere, I propose that MBSP has a polyphyletic origin and evolved from the aster yellows phytoplasma.
The myrmecophile leafhopper Dalbulus quinquenotatus has an obligatory association with ants. Greenhouse experiments showed that JD. quinquenotams. but not the
ii non myrmecophile Dalbulus gelbus. kills its gamagrass host plant. However, when D.
quinquenotatus is attended by ants they regulate leafhopper numbers and maintain host
plant health. Furthermore, results in the field showed that D. quinquenotatus and ants do
not have negative effect on the gamagrass. Food supplements supplied to ants alter their
response to leafhoppers. Ants attended D. quinquenotatus best when they received only
protein, but not when they received honey and protein or no food supplements. Ants
reduced D. quinquenotams populations in all treatments compared to controls.
Other benefits among D. quinquenotams. ant species and gamagrass were smdied.
Ants received honeydew and selectively prey on D- quinquenotams to supplement protein
requirements. The leafhopper and gamagrass benefit from the ants. Ants remove
honeydew, benefitting bothD. quinquenotams and the gamagrass host plant. Furthermore,
ants expel non myrmecophile Dalbulus when gamagrass is small in size. Finally, in the
field ants protect _Q. quinquenotams from several taxa of spider predators; moreover,
protection occurs in different gamagrass species and in two distinct habitats. Greenhouse experiments confirmed the protection of D- quinquenotams against the predator Nabis
americoferus by the ant Formica subsericea.
in ACKNOWLEDGMENTS
This dissertation and my doctoral program have been possible by the help of many persons. First, I am very grateful to my major adviser Dr. Lowell (Skip) Nault for his friendship, advice, orientation, and support during my most difficult moments. Also I wish to thank my dissertation committee members Drs. David Horn and Ben Stinner, not only for reviewing the manuscript and making suggestions that improved this study, but also for their guidance throughout my doctoral program.
I am especially grateful to William (Bill) Styer for his friendship and assistance at anytime, for anything, and anywhere. Thanks to Drs. Larry Madden, Casey Hoy, and
Graham Head for their statistical advice. Thanks to all the Entomology graduate students from Wooster for their help with any difficulty. Thanks to Le Ann Beanland and Todd
Blackledge for the identification of spiders and David Smith (Systematic Entomology
Laboratory, Agricultural Research Service, USDA) for the identification of ants.
I want to acknowledge my two sponsors, the Consejo Nacional de Ciencia y
Tecnologia (CONACYT) for giving me part of my scholarship, and the Universidad de
Guadalajara for the other part of my scholarship. Both Mexican institutions supported all my doctoral education even at a time when Mexico had serious economic problems.
IV Finally I am thankful for the support of my family. Especially to my wife Martha and son Gustavito (Gustalbulitus) for sharing with me interesting and often times challenging situations. Thanks to my mother Nene for all her help in Mexico and to ray sisters Celina and Gena. VITA
August 31, 1963...... Bom- San Agustin, Jalisco, México.
1985...... B.S., Biology, Universidad de Guadalajara, Guadalajara, Jalisco
1986-1990...... Research Assistant, Institute Manandan, Universidad de Guadalajara
1990...... M.S., Entomology, Colegio de Postgraduados, Montecillo, Edo. de Mexico
1990-1994...... Researcher in Entomology and Coordinator of Agroecology Program Institute Manandân, Universidad de Guadalajara
PUBLICATIONS
Moya-Raygoza, G., E. Santana C., and P.Plaza. 1988. Bùsqueda de resistencia en Zea diploperennis (Gramineae) para disminuir el dano per plagas del suelo en mafz. In: M.A. Moron and C. Deloya Eds., Tercera Mesa Redonda Sobre Plagas del Suelo. 179-196 pp., Sociedad Mexicana de Entomologia, México.
Moya-Raygoza, G., V. Bedoy V., and E. Santana C. 1990. Seasonal patterns of insect abundance in natural patches of Zea diploperennis. Maydica 35: 177-182.
VI Larsen, K.J., F.E. Vega, G. Moya-Raygoza, and L.R. Nault. 1991. Ants (Hymenoptera; Formicidae) associated with the leafhopper Dalbulus quinquenotatus (Homoptera; Cicadellidae) on gamagrasses in Mexico. Ann. Entomol. Soc. Am. 84: 498-501.
Hernandez-Vazquez, S., G. Moya-Raygoza, K.J. Larsen, and L.R. Nault. 1992. Densidad estacionai de Dalbulus maidis (Homoptera: Cicadellidae) en maiz CZea mays') y en el teosinte perenne Zea diploperennis. Folia Entomol. Mex. 86: 15-24.
Larsen, K.J., LJl. Nault, and G. Moya-Raygoza. 1992. Overwintering biology of Dalbulus leafhopper (Homoptera: Cicadellidae): Field population dynamics and drought hardiness. Environ. Entomol. Soc. Am. 21: 566-577.
Moya-Raygoza, G. 1993. Dinamica pobalcionai de Dalbulus spp. (Homoptera: Cicadellidae) en mafz fZea mavsl(Gramineae) y sus parientes cercanos. Folia Entomol. Mex. 87: 21-29.
Moya-Raygoza, G. 1993. Plagas subterraneas del mafz (Zea mays L.) cultivado bajo agricultura de montaha. In: M.A. Moron Ed., Diversidad y raanejo de plagas subterraneas. 105-112 pp., Sociedad Mexicana de Entomologia, Mexico.
Moya-Raygoza, G. 1993. Diagnostfco de las plagas insectiles en mafz cultivado bajo agricultura de subsistencia. Agrociencia 4: 153-163.
Moya-Raygoza, G., and J. Trujillo-Arriaga. 1993. Dryinid (Hymenoptera: Dryinidae) parasitoids of Dalbulus leafhopper (Homoptera: Cicadellidae) in Mexico. Entomophaga 38:41-49.
Moya-Raygoza, G., and J. TmjUlo-Arriaga. 1993. Evolutionary relationships between Dalbulus leafhopper (Homoptera: Cicadellidae), and its dryinid (Hymenoptera: Dryinidae) parasitoids. J. Kans. Entomol. Soc. 66: 41-50.
Moya-Raygoza, G., V. Bedoy V., E. Santana C. 1993. Patrones estacionales de la abundancia de insectos en manchones naturales de Zea diploperennis. In: B.F. Benz Ed., Biologfa, ecologfa y conservaciôn del género Zea. 165-177 pp., Universidad de Guadalajara, Mexico.
Moya-Raygoza, G. 1994. Diversity of leafhoppers and their hymenopetrous parasitoids in maize, teosinte, and gamagrass related ecosystems. Maydica 39: 225-230.
Moya-Raygoza, G. 1995. Fire effects on insects associated with the gamagrass Tripsacum dactvloides in Mexico. Ann. Entomol. Soc. Am 88: 434-440.
vu FIELD OF STUDY
Major Field: Entomology
vui TABLE OF CONTENTS Page
ACKNOWLEDGMENTS...... iv
VTTA...... vi
LIST OF OF TABLES...... x
LIST OF FIGURES...... xii
LIST OF PLATES...... xvii
PROLOGUE...... I
CHAPTERS:
1. TRANSMISSION BIOLOGY OF THREE MAIZE BUSHY STUNT PHYTOPLASMA ISOLATES BY THE CORN LEAFHOPPER Dalbulus maidis (HOMOPTERA: CICADELLIDAE)...... 4 Introduction ...... 4 Materials and Methods ...... 8 Results...... 13 Discussion ...... 26
2. MUTUALISTIC ASSOCIATION BETWEEN THE MYRMECOPHILE LEAFHOPPER Dalbulus quinquenotatus (HOMOPTERA: CICADELLIDAE) AND ITS TENDING ANTS.. 38 Introduction ...... 38 Materials and Methods ...... 40 Results...... 48 Discussion ...... 72
LIST OF REFERENCES...... 88
ix LIST OF TABLES
Table Page
1. Effect of temperature on transmission rate of maize bushy stunt phytoplasma (isolate Tlaltizapan) by nymphs or adults of Dalbulus maidis (biotype Tlaltizapan). Data zure means ± SE from three replicates, each with 24 individuals (n=72). Data are expressed as percentages. Means flanked by the same letter are not signifrcantly different (LSD Test) ...... 15
2. Transmission rate of three isolates of maize bushy stunt phytoplasma by three biotypes of Dalbulus maidis nvmphs at 25 °C. Data are means ± SE from three replicates, each with 24 individuals (n=72). Data are expressed as percentages. Means flanked by the same letter are not significantly different (LSD Test) ...... 21
3. Percentage of plants with basal tillering produced by three maize bushy stunt phytoplasma isolates when transmitted by three Dalbulus maidis biotypes. Results were based on means of two replicates, each with 48 plants (n=96). Values in parenthesis are the total number of plants with symptoms ...... 23
4. Weibull scale (b) and shape (c) parameters of the survival for Dalbulus maidis (biotype Tlaltizapan) nymphs and adults when exposed to maize bushy stunt phytoplasma at four different temperatures. Values are the average of three replicates, each with 24 individuals (n=72). b is in units of time (days) and c is a unitless shape parameter, when c=l, the Weibull model reduces to the exponential model ...... 25 5. Comparison of Dalbulus quinquenotatus adults and nymphs when attended by Formica subsericea colonies. Number of adults and nymphs are averages from 9 replicates (n=9) for attended and 3 replicates (n=3) for unattended leafhoppers. Ants attended nymphs and adults during 16 days under 3 treatments. The first was when ants did not receive food supplement; the second when received protein; and the third when received honey and protein...... 57
6. Percentage of surface covered with honeydew produced by Dalbulus quinquenotatus on Tripsacum dactvloides when attended by Formica subsericea and unattended. Data were taken 16 days after ant colonies attended Dalbulus quinquenotatus. During this experiment ants received protein as food supplement ...... 59
XI LIST OF FIGURES
Figure Page
1. Effect of temperature on mean latent period of maize bushy stunt phytoplasma (isolate Tlaltizapan) when acquired by nymphs or adults of Dalbulus maidis (biotype Tlaltizapan). Data points are means from three replicates, each with 24 individuals (n=72). Bars are standard errors ...... 14
2. Effect of temperature on mean latent period of maize bushy stunt phytoplasma (isolate Tlaltizapan, El Batan, and Poza Rica) when acquired by nymphs of Dalbulus maidis (biotype Tlaltizapan, El Batan, and Poza Rica). Data points are means from three replicates, each with 18 individuals (n=54). Bars are standard errors ...... 17
3. Effect of temperature on transmission rate of maize bushy stunt phytoplasma (isolate Tlaltizapan, El Batan, and Poza Rica) when acquired by nymphs of Dalbulus maidis (biotype Tlaltizapan, El Batan, and Poza Rica). Data points are means from three replicates, each with 18 individuals (n=54). Bars are standard errors ...... 19
4. Transmission rate over time of maize bushy stunt phytoplasma (isolate Tlaltizapan, El Batan, and Poza Rica) when acquired by nymphs of Dalbulus maidis (biotype Tlaltizapan, El Batan, and Poza Rica) and maintained at 25 °C. Data points are means from three replicates, each with 18 individuals (n=54) ...... 20
5. Survival of Dalbulus maidis bio type Tlaltizapan when nymphs or adults exposed to Tlaltizapan maize bushy stunt phytoplasma isolate. Nymphs and adults were maintained at four different temperatures. Data points are means from three replicates, each with 24 individuals (n=72) ...... 24
XU 6. Survival of inoculative and non-inoculative Dalbulus maidis (biotype Tlaltizapan) when nymphs and maize bushy stunt phytoplasma isolate Tlaltizapan were maintained at 15 and 25 °C. Data points are means of three replicates, each with 18 individuals (n=54) ...... 27
7. Survival of inoculative and non-inoculative Dalbulus maidis (biotype Poza Rica) when nymphs and maize bushy stunt phytoplasma isolate Poza Rica were maintained at 15 and 25 °C. Data points are means of three replicates, each with 18 individuals (n=54) ...... 28
8. Survival of inoculative and non-inoculative Dalbulus maidis (biotype El Batan) when nymphs and maize bushy stimt phytoplasma isolate El Batan were maintained at 15 and 25 °C. Data points are means of three replicates, each with 18 individuals (n=54) ...... 29
9. Total precipitation (mm) every two weeks from April to July 1996 in three stations from Central Mexico in Jalisco state. Those stations are Zapopan, Agua Caliente, and San Agustin. Data were provided by the Mexican National Water Commission 41
10. Average number of Dalbulus quinquenotatus and Dalbulus gelbus adults and nymphs on Tripsacum dactvloides. Bars indicate standard error. Each data point is the mean of five replicates (n=5). Data were taken every four days during 76 days. Stars from days 64 to 76 is when T. dactvloides with D. quinquenotatus died ...... 49
11. Average number of stems per Tripsacum dactvloides clone under three treatments: Tripsacum with unattended Dalbulus quinquenotatus: Tripsacum with Dalbulus gelbus: and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment was repeated five times (n=5) ...... 51
12. Average number of leaves per Tripsacum dactvloides clone under three treatments: Tripsacum with Dalbulus quinquenotatus: Tripsacum with Dalbulus gelbus: and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment was repeated five times (n=5). Data were taken every four days during 76 days. Stars from days 64 to 76 is when T. dactvloides with D. quinquenotatus died ...... 52
xin 13. Average height of Tripsacum dactvloides stems under three treatments; Tripsacum with unattended Dalbulus quinquenotatus: Tripsacum with Dalbulus gelbus: and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment was repeated five times (n=5). Data were taken every four days during 76 days. Stars from days 64 to 76 is when T. dactvloides with D. quinquenotatus died ...... 53
14. Average number of leaves per Tripsacum pilosum clone in Zapopan, Jalisco, Mexico in two treatments: Tripsacum with Dalbulus quinquenotatus attended by ants; and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment had four replicates (n=4). Data were taken from May to July, 1996 ...... 54
15. Average number of leaves per Tripsacum dactvloides clone in Agua Caliente, Jalisco, Mexico in two treatments: Tripsacum with Dalbulus quinquenotatus attended by ants; and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment had four replicates (n=4). Data were taken from May to July, 1996 ..... 55
16. Average number of leaves per Tripsacum dactvloides clone in San Agustin, Jalisco, Mexico in two treatments: Tripsacum with Dalbulus quinquenotatus attended by ants; and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment had four replicates (n=4). Data were taken from May to July, 1996 ..... 56
17. Average number of Dalbulus gelbus and Dalbulus guTmani adults on Tripsacum pilosum in Zapopan, Jalisco, Mexico. These leafhoppers were found on Tripsacum under two treatments: without Dalbulus quinquenotatus + ants; and with D- quinquenotatus + ants. Bars indicate standard error. Each treatment had four replicates (n=4). Data were taken from May to July, 1996 ...... 60
18. Average number of Dalbulus gelbus and Dalbulus quinquenotatus adults on large and small Tripsacum dactvloides. Bars indicate standard error and each data point represents four replicates (n=4). Data were taken every 30 m during the first six hours and once at 24 hours after Formica subsericea started to attend Dalbulus quinquenotatus...... 61
XIV 19. Average number of Dalbulus quinquenotatus and attending ants on Tripsacum pilosum in Zapopan, Jalisco, Mexico. Bars indicate standard error and each treatment had four replicates (n=4). Data were taken every four days from May to July, 1996 ...... 63
20. Average number of Dalbulus quinquenotatus and attending ants on Tripsacum dactvloides in San Agustin, Jalisco, Mexico. Bars indicate standard error and each treatment had four replicates (n=4). Data were taken every four days from May to July, 1996 ...... 64
21. Average number of Dalbulus quinquenotatus and attending ants on Tripsacum dactvloides in Agua Caliente, Jalisco, Mexico. Bars indicate standard error and each treatment had four replicates (n=4). Data were taken every four days from May to July, 1996 ...... 65
22. Number total of spiders found on unattended Dalbulus quinquenotatus. Spiders from Zapopan were collected on Tripsacum pilosum. Spiders from Agua Caliente and San Agustin were collected on Tripsacum dactvloides. Data were taken every four days from May to July, 1996 ...... 66
23. Average number ofNabis americoferus adults and nymphs when predator was maintained in two conditions: the first was when Dalbulus quinquenotatus was attended by Formica subsericea: and the second when Dalbulus quinquenotatus was unattended. Bars indicate standard error. Each treatment had seven replicates (n=7). Data were taken every four days during 60 days ...... 69
24. A verse number of Dalbulus quinquenotatus adults when maintained with the predator Nabis americoferus under three conditions: the first was with large ant colony (n=4); the second with small ant colony (n=3); and the third was with unattended Dalbulus quinquenotatus (n=7). Bars indicate standard error. Data were taken every four days during 60 days ...... 70
25. Average number of Dalbulus quinquenotatus nymphs when maintained with the predator Nabis americoferus under three conditions: the first was with large ant colony (n=4); the second with small ant colony (n=3); and the third was with unattended Dalbulus quinquenotatus (n=7). Bars indicate standard error. Data were taken every four days during 60 days ...... 71
XV 26. Average number of Dalbulus quinquenotatus adults when ants were maintained in three dietary conditions: in the first ants received no food supplement; in the second ants received protein; in the third ants received honey and protein. Bars indicate standard error. Each treatment had nine replicates (n=9). Data were taken every four days during 16 days ...... 73
27. Average number of Dalbulus quinquenotatus nymphs when ants were maintained in three dietary conditions: in the first ants received no food supplement; in the second ants received protein; in the third ants received honey and protein. Bars indicate standard error. Each treatment had nine replicates (n=9). Data were taken every four days during 16 days ...... 74
28. Average number of Formica subsericea found in screen cages with Dalbulus quinquenotatus when ants maintained under three dietary conditions: in the first ants received no food supplement; in the second ants received protein; in the third ants received honey and protein. Bars indicate standard error. Each treatment had nine replicates (n=9). Data were taken every four days ...... 75
29. Beneficial effects of the Dalbulus quinquenotatus/ants relationships 86
XVI LIST OF PLATES
Plate Page
I. Symptoms in maize of three maize bushy stunt phytoplasma isolates: maize infected with El Batan isolate is more bushy and stunted; maize infected with Tlaltizapan isolate shows more reddening, and; maize infected with Poza Rica isolate shows intermediate symptoms.... 6
xvu Prologue
Despite that the leafhopper genus Dalbulus originated in Mesoamerica, it has been
studied broadly by scientists in Ohio at The Ohio State University. These studies began with
the first species descriptions by Dwight M. DeLong in 1923 and 1950, and continue to
present studies conduced by L.R. Nault and his colleagues and students. During the past 24
years they have generated so much information on this group of leafhoppers that Dalbulus
is considered one of the best known genera of insects in the world based on what is now
known on behavior, biogeography, biology, ecology, evolution, pathogen interactions, and
systematics. Therefore, I am proud to present the results of this dissertation which continues
to increase the knowledge of these leafhoppers and their associated organisms.
The genus Dalbulus is composed of 13 described species and grouped into the
subfamily Deltocephaline, tribe Macrostelini. Species are distributed from the United States
to Argentina. Throughout this geographic area Dalbulus uses maize fZea mays), its wild
relatives, the teosintes (Zea species) or the gamagrasses (Tripsacum species) as host plants.
Some species such as the com leafhopper Dalbulus maidis are specialists on maize and
teosintes, others such as D. gelbus are found on maize and Tripsacum. while others such as D- quinquenotatus are Tripsacum specialists. This thesis focuses on two species, D. maidis and Q. quinquenotatus. The com leafhopper first specialized on maize possibly 8-10,000 years ago, when maize was domesticated from the annual teosinte Zea mavs ssp. parviglumis. Since then D. maidis and three pathogens it transmits, the maize bushy stunt phytoplasma (MBSP), com stunt spiroplasma and maize rayado fino marafivirus have adapted to different environmental conditions throughout Neotropic and different D. maidis biotypes and pathogens isolates have originated. The com leafhopper has been found in Florida, California, and Texas in the United States, throughout Mexico, the Caribbean Islands, Central America, and South
America. There the com leafhopper transmits MBSP. This phytoplasma from different regions of Mexico varies in its symptoms in maize and variation among MBSP isolates has been found in their DNA. Few studies have showed the relation between genetics and biological function. Results presented in chapter I show that there is a link between the genetics of MBSP with their transmission biology (biological function). Here I worked with the interactions of three MBSP isolates and their respective vector biotypes from Central
Mexico. I showed that the MBSP isolates differ with respect to mean latent period, mean transmission rate, pattem of transmission, and mean incubation period in maize when transmitted by nymph or adult leafhoppers at several temperatures.
The fivespotted gamagrass leafhopper Dalbulus quinquenotatus. which is found in
Central Mexico and Costa Rica, differs from other Dalbulus species in color, size, behavior, production of honeydew, and microhabitat. The adults are brownish orange, while the adults of the other Dalbulus species are generally yellow. The last instar nymph of the fivespotted gamagrass leafhopper has four black stripes that extend the length of the body, while its
9 sister species do not have these stripes. In addition, the fivespotted gamagrass leafhopper persists on its host plant even during the Mexican dry season. The association of this leafhopper with ants is favored because of its gregarious and sedentary behavior and because of its high and firequent production of honeydew.
Only 13 species of leafhoppers, including the deltocephaline D. quinquenotatus. have been reported in association with ants (K.J. Larsen, unpublished). These species belong to seven subfamilies; Agalliinae, Deltocephalinae, Hecalinae, lassinae, Idiocerinae, Ledrinae, and Macropsinae. No studies have tested the mutual benefits between any of these myrmecophile leafhoppers and ants. Several mutual benefits have been reported separately for other Homopterans and ants; however, no studies have tested the many benefits enjoyed by a single myrmecophile Homoptera species by its association with ants. In chapter 2 the mutual benefits between D. quinquenotatus. the only known deltocephaline myrmecophile, and ants is demonstrated. 1 tested the hypotheses as to whether D. quinquenotatus and ants have an obligatory association, if ants protect the fivespotted gamagrass leafhopper firom predators, regulate the population of this leafhopper, and expel other Dalbulus species that try to use the same plants occupied by D. quinquenotatus. Interesting results not only show the mutual benefits between D. quinquenotatus and ants but also describe how food supplements influence the ants attendance with the fivesppoted gamagrass leafhopper. CHAPTER 1
TRANSMISSION BIOLOGY OF THREE MAIZE BUSHY STUNT PHYTOPLASMA ISOLATES BY THE CORN LEAFHOPPER Dalbulus maidis (HOMOPTERA: CICADELLIDAE)
INTRODUCTION
The com leafhopper Dalbulus maidis (DeLong & Wolcott) (Homoptera; Cicadellidae) is the most important leafhopper pest of maize (Zea mavs L.) (Gramineae) in Latin America
(Nault, 1990). The direct damage caused by feeding injury is rarely important; however, serious economic loss is caused by pathogens transmitted by the leafhopper; the com stunt spiroplasma (CSS), the maize bushy stunt phytoplasma (MBSP), and the maize rayado fino marafi virus (MRFV) (Nault, 1980; Nault et al., 1981).
The com leafhopper is distributed throughout the Neotropics from the southem United
States of America to Argentina as well as the Caribbean Islands (Nault, 1983). Furthermore,
D. maidis is found at a wide range of elevations on its maize host from sea level to as high as 3,200 m in the Pemvian Andes (Nault et al., 1979). The distribution of the leafhopper coincides with the distribution of the pathogens it transmits. Specifically, MBSP, the focus of this study, was thought to be primarily found at high elevations in Mexico (Davis, 1973:
Davis, 1977) but appears to be found at all elevations elsewhere in the Neotropics (Nault et al., 1979, 1981).
Based on analysis of pathogen 16S rDNA for classification of mycoplasmalike organisms, MBSP has been assigned to the 16Srl-B cluster which includes the aster yellows and periwinkle yellows phytoplasmas (Lee et al., 1993). Isolates of MBSP collected from
Costa Rica, Mexico and the United States have been differentiated genetically (Harrison et al., 1996). Similarly, isolates from three geographic regions in Mexico, El Batan in the state of Mexico, Tlaltizapan in Morelos and Poza Rica in Veracruz, have been differentiated based on total DNA (N. A. Harrison, pers. comm.). Moreover, these Mexican isolates from field-collected Dalbulus leafhoppers, produce distinctly different symptoms in maize (L.R.
Nault, unpublished) (Plate I).
These differences prompted me to ask whether the three isolates behave differently when transmitted by their leafhopper vector. For example, Murral et al. (1996) have shown that two strains of aster yellows differ in relationship with their aster leafhopper vector
Macrosteles quadrilineatus Forbes. Acquisition and infection rate, latent period, and effects on vector survival are just a few biological traits that can be genetically regulated by the phytoplasma genome. I report on the effect of phytoplasma isolates, D. maidis biotypes, and temperature on 1) latent period, 2) transmission rate, 3) pattem of transmission over time,
4 ) mean retention time and pattem of transmission and, 5) survival of adults. In addition, I discuss the evolutionary relationships between MBSP and aster yellows, its sister phytoplasma, and speculate on the evolution of the three MBSP isolates. Plate I. Symptoms in maize of three maize bushy stunt phytoplasma isolates: maize infected with El Batan isolate is more bushy and stunted; maize infected with Tlaltizapan isolate shows more reddening, and; maize infected with Poza Rica isolate shows intermediate symptoms. Plate I MATERIALS AND METHODS
EXPERIMENTAL MATERIAL. Three biotypes of D. maidis and isolates of
MBS? each, were collected in 1992 from Central Mexico at the three experiment stations of
Centro Intemacional de Mejoramiento de Maiz y Trigo (CIMMYT) by M.A. Ebbert and L.R.
Nault. The Tlaltizapan D. maidis biotype and MBS? isolate were obtained from Tlaltizapan in the state of Morelos. Tlaltizapan is at 940 m above sea level. The dry season is from
November to April with a mean temperature of 21 °C, while the wet season, from May to
October, has a mean temperature of 25 “C. The El Batan Q. maidis biotype and MBS? isolate were collected at El Batan, near Texcoco in the state o f Mexico. El Batan is at 2,249 m above sea level. The dry season is from November to March with a mean temperature of 13 °C, while the wet season is from April to October with a mean temperature of 16 ''C. The Poza
Rica D. maidis biotype and MBS? isolate were collected in the state of Veracruz. Poza Rica is 60 m above sea level, with a dry season from December to March and a mean temperature of 21 °C; the wet season is from April to November with a mean temperature of 27 ^C. The climate data cited are based on a ten year average (1973-1983) and provided by CIMMYT.
The three leafliopper biotypes and MBS? isolates were maintained in com plants
(Early Sunglow sweet com). The biotypes were reared in growth chambers at 25 "C, 50 % relative humidity, while the MBSP isolates were kept in greenhouse rooms at 20-35 °C . All work on transmission biology was performed using Early Sunglow sweet com. MEAN LATENT PERIOD, TRANSMISSION RATE, MEAN RETENTION PERIOD,
AND PATTERN OF TRANSMISSION. Isolate and biotype Tlaltizapan. The Tlaltizapan
D. maidis biotype was reared to obtain two groups of individuals of different ages. One
group was composed of second instar nymphs, while the other was composed of one week
old adults. To obtain these two insect groups, females of Tlaltizapan D. maidis biotype were given an oviposition access period (GAP) of two days to get individuals of similar age. The
GAP was on maize plants confined to rearing cages, which were maintained in a walk-in plant growth chamber kept at 25 °C, 50 % relative humidity, and a 14:10 (L;D) cycle. Cages were kept in the walk-in chamber until the two insect groups reached their nymphal and adult stages, respectively.
Test insects were given a 48-hour acquisition access period (AAP) on maize plants infected with Tlaltizapan MBSP isolate. This AAP was performed at the same temperature, relative humidity, light cycle and rearing cages as in the GAP. After AAP, each nymph and adult group was divided into four subgroups of nymphs and adults, respectively. Each subgroup was placed in rearing cages supplied with 5 healthy, 4-leaf stage maize plants for an eight day holding period. Each nymph and adult subgroup was held at one of the following temperatures: 15,20,25, or 30 °C in bench-top growth chambers at 50 % relative humidity and 14:10 (L:D) cycle.
Following the 8-day holding period, individual leafhoppers were caged on a single plant in a numbered butyrate tube. Insects were transferred to new test plants every 48 or 72 hrs. The environmental conditions maintained during all the transfers were the same as in the holding period using the same bench-top growth chambers. If any insect died during the
9 first three transfers, it was replaced with individuals in reserve which had been reared under the same conditions. The transfers were repeated 24 times in each one of the eight treatments until all insects had died. Each treatment was repeated three times.
The ordinary runs analysis was used to test for the pattern of transmission for nymphs of the Tlaltizapan biotype maintained at 20 °C. Low numbers of transmissions at 15,25, and
30 °C limited the analysis of pattern of transmission at those temperatures. A description of this analysis for pattern of transmission of aster yellows phytoplasma was reported previously (Murral et al., 1996). The null hypothesis is that the sequence of infected plants is random (P>0.05) and the alternative hypothesis is that the sequence is clustered (P<0.05)
(gladden et al., 1982). Only leafhoppers that transmitted at least once and later survived nine more transfers were included in the analysis.
Isolates and biotypes Tlaltizapan, El Batan, and Poza Rica. Each of the three D. maidis biotypes were reared to obtain second instar nymphs of the same age. Each colony was started with females, given a 2-day GAP. The GAP was on maize plants in rearing cages, maintained in a walk-in plant growth chamber at 25 °C, 50 % relative humidity and
14:10 (L:D) cycle.
When nymphs reached the second instar, each colony was confined to MBSP infected plants firom the same locality as the vector e.g. El Batan biotype exposed to El Batan N'IBSP isolate. Gnce leafhoppers completed a 48-hr AAP, insects were held for eight days. In this step each biotype colony was divided into two groups, one at 15 °C and the other at 25 °C.
Insects from the six treatments were maintained in bench-top growth chambers at 50 % relative humidity and 14:10 (L:D) cycle. When the holding period ended, test insects were
10 transferred in the same way as previously described. Transfers were repeated 18 times for
each treatment and each treatment was replicated three times. The ordinary runs analysis was
applied to the pattern of transmission for nymphs of Tlaltizapan, El Batan, and Poza Rica
biotypes maintained at 15 °C. Low numbers of transmissions at 25 °C limited the analysis
of vector transmission over time at this temperature.
Transmission rate of each MBS? isolate by each D. maidis biotype. Second instar
nymphs were obtained for each biotype. Nymphs of each biotype were exposed for a 48 hrs
AAP to each MBSP isolate, e.g. El Batan biotype was exposed separately to El Batan, Poza
Rica, or Tlaltizapan MBSP isolates. Once the AAP was completed, nymphs were transferred
to holding maize plants for 35 days. The GAP, AAP and holding period were conduced at
25 “C using the same procedure as in the previous experiment.
Following the holding period, insect transmission rate was determined by placing
leafhoppers one per plant into individually numbered butyrate tubes. Each insect was transferred sequentially to two healthy maize plants for 48 hrs each. During the transfers, the leafhoppers were maintained in bench-top growth chambers at the same environmental conditions as in holding period. A test insect was scored as inoculative if at least one of the two tested plants became infected with MBSP. Each treatment was replicated three times, each with 24 individuals.
Test plants were placed in the greenhouse, sprayed with 1% Resmethrin, and held for four weeks to allow symptoms expression.
11 MEAN INCUBATION PERIOD AND SYMPTOMS ON MAIZE. The mean incubation
period of MBSP in the plant was determined for each strain by exposing inoculative
leafhoppers to test plants for 48 or 72 hrs prior to transfer to the greenhouse. Greenhouse
temperature averaged 27 °C. Plants were watered daily. Supplemental light was from a
1,000 watt metal halide lamp for 16 hrs/day 145 cm above the plant bench. Greenhouse
plants were examined daily to determine when phytoplasma symptoms first became visible
(incubation period). Observations continued 35 days post incubation period to follow
symptom development. The mean incubation period was obtained from two replicates, with
each replicate composed of 36 plants, and the symptoms (tillering) on maize were obtained
from two replicates, with each replicate composed of 48 plants.
SURVIVAL OF VECTOR LEAFHOPPERS. Survival was determined for nymphs and
adults of Tlaltizapan D. maidis biotype exposed to the Tlaltizapan MBSP isolate. Survival
was evaluated at 15, 20, 25, and 30 °C beginning 14 days after AAP to when all the
leafhoppers had died. The experiment was replicated three times for each treatment with 24
leafhoppers per treatment. The Weibull model was used to test survival of leafhoppers. This
model has two parameters: the scale parameter (b), which is inversely related to rate of
population decline and is expressed in units of time, and the shape parameter (c), which
allows the model to take many forms (i.e. when c=l, the Weibull model is reduced to
exponential) (Madden and Nault, 1983; Madden et al., 1984).
In addition, survival of the three biotypes was recorded when they acquired their
respective phytoplasma as nymphs at 15° and 25 °C. Survival was checked from 14 days after the second instar nymph acquired the phytoplasma until each insect had died. Three
12 replicates were run, each with 18 individuals. Survival between inoculative and non- inoculative leafhoppers was compared by using the Gehan’s Wilcoxon test.
RESULTS
MEAN LATENT PERIOD, TRANSMISSION RATE, MEAN RETENTION
PERIOD AND PATTERN OF TRANSMISSION. Isolate and biotype Tlaltizapan.
Mean latent period of Tlaltizapan MBSP isolate was similar for nymphs and adults of
Tlaltizapan D. maidis biotype at four different temperatures (Figure 1). The analysis of variance (ANCVA) shows that the mean effect for stages (nymphs and adults) was not significant (P=0.787; df=l); however, the mean effect of temperature was significant
(P<0.001; df=l). In addition, the ANOVA value indicates that the interaction term stage by temperature was not significant for mean latent period (P=0.777; df=l). The longest mean latent period of MBSP for nymphs and adults occurred when they were maintained at 15 °C, whereas the shortest mean latent period was at 25 °C. Mean latent period did not continue to decline from 25“ to 30 “C; rather it increased for nymphs and adults. However, mean latent period at 30 “C was calculated from only two nymphs and two adults each that transmitted MBSP at that temperature. Mean latent period for nymphs and adults had a quadratic relationship with temperature for both stages.
Transmission rate of Tlaltizapan MBSP strain was different for nymphs and adults
(Table 1). The ANOVA indicates that the mean effect temperature (P<0.001 ; df=I) and stage
13 nymphs O) CO
■O o
CL C o « c CO CD
Temperature
Figure 1. Effect of temperature on mean latent period of maize bushy stunt phytoplasma (isolate Tlaltizapan) when acquired by nymphs or adults of Dalbulus maidis (biotype Tlaltizapan). Data points are means from three replicates, each with 24 individuals (n=72). Bars are standard errors.
14 Temnerature °C Leafhopper stage 15 _ 20 25 30 a b b c nymphs 31.6 ± 1.3 73.6 ± 1.3 73.7 ± 11.0 2.8 i 2.8
c a a c adults 6.6 ± 1.3 34.7 ± 6.1 30.3 ± 1.3 2.8 ± 2.8
Table 1. Effect of temperature on transmission rate of maize bushy stunt phytoplasma (isolate Tlaltizapan) by nymphs or adults of Dalbulus maidis (biotype Tlaltizapan). Data are means ± SE from three replicates, each with 24 individuals (n=72). Data are expressed as percentages. Means flanked by the same letter are not significantly different (LSD Test).
15 (P<0.001; df=l) were significant as well as the interaction terms temperature by stage
(P=0.077; df=l). Nymphs transmitted better than adults at 15, 20, and 25 °C. As for mean
latent period, transmission rate for nymphs and adults had a quadratic relationship with
temperature for both stages.
Nymphs retained the phytoplasma longer than adults. Mean retention period of
Tlaltizapan MBSP isolate was 48.0 and 37.0 days for nymphs at 20° and 25 °C, respectively.
For adults it was 40.0 and 29.5 days at 20° and 25 °C, respectively. The mean effect stage
(P=0.005; df=l) and temperature (P<0.001; df=l) were significant; however, the interaction
term stage by temperature was not significant (P=0.920; df=l).
Of 72 leafhoppers, 57 transmitted the Tlaltizapan MBSP isolate when nymphs were
maintained at 20 °C. Of these 57 leafhoppers, 50 were tested by the ordinary runs analysis
(those that survived nine serial transfers). Of these 50 leafhoppers 33 had a random pattern
and 17 had clustered pattern of transmission.
Isolates and biotypes Tlaltizapan, El Batan, and Poza Rica. Mean latent period of the three isolates in their respective biotypes was longer at 15 °C than at 25 °C (Figure
2). The ANOVA indicates that the mean effect temperature (P<0.001; df=l) and the interaction terms temperature by biotype (P=0.085; df=2) were significant. The mean effect of bio type was not significant (P=0.153; df=2); thus, the mean latent period of Tlaltizapan,
El Batan, and Poza Rica isolates is similar when they acquired by their respective biotype at 15° and 25 °C. However, at 25 °C the mean latent period of Poza Rica isolate was longer than for the Tlaltizapan and El Batan isolates (P=0.010; df=2).
16 60
^ Tlaltizapan c/3 50 CO □ El Batan
0 Poza Rica XJ 40 o © a. 30 c © © 20 c m © « © 10 M
1 5 25
Temperature C
Figure 2. Effect of temperature on mean latent period of maize bushy stunt phytoplasma (isolate Tlaltizapan, El Batan, and Poza Rica) when acquired by nymphs of Dalbulus maidis (biotype Tlaltizapan, El Batan, and Poza Rica). Data points are means from three replicates, each with 18 individuals (n=54). Bars are standard errors.
17 Transmission rate of the three MBSP isolates acquired by their respective biotypes was higher at 25 °C than at 15 °C (Figure 3). The ANOVA indicates that not only was the mean effect of temperature (?<0.001; df=I) significant but also the mean effect of biotype
(?=0.041; df=2). Poza Rica isolate was transmitted at a higher rate than the other two isolates (P=0.030; df=2).
Leafhoppers did not transmit MBSP isolates consistently through time (Figure 4).
The transmission rate was similar during the first several days once transmission began; however, transmission rate declined during the last several days of transmission.
Only 14 Tlaltizapan leafhoppers, 21 El Batan leafhoppers, and 25 Poza Rica leafhoppers survived nine serial transfers subsequent to their first transmission and were analyzed for pattem of transmission. There was no difference in cluster (P=0.862; df= 2,4) or randomness (P=0.859; df=2,4) pattem of transmission among the three MBSP isolates as transmitted by their respective biotypes at 15 °C. For the Tlaltizapan biotype 10 leafhoppers transmitted randomly and 4 with a clustered pattem; for the El Batan biotype 16 leafhoppers transmitted randomly and 5 with a clustered pattem, and; for the Poza Rica biotype 17 leafhoppers transmitted randomly and 8 with clustered pattem.
Transmission rate of MBSP isolates by leafhopper biotypes. Each of the three leafhopper biotypes transmitted each of the three isolates (Table 2). The biotypes Tlaltizapan and El Batan transmitted isolates from other localities better than the isolate from their own locality. For instance, the transmission rate was higher when Tlaltizapan bio type transmitted the isolates El Batan and Poza Rica than when it transmitted the isolate Tlaltizapan.
Similarly the El Batan biotype transmitted the Poza Rica isolate better than the El Batan
18 100
S Tlaltizapan
□ El Batan 80 - o M Poza Rica (0 oc 60 - C o T tô .2 40 -
(0 c ea H 20
1 5 Temperature C
Figure 3. Effect of temperature on transmission rate of maize bushy stunt phytoplasma (isolate Tlaltizapan, El Batan, and Poza Rica) when acquired by nymphs of Dalbulus maidis (biotype Tlaltizapan, El Batan, and Poza Rica). Data points are means from three replicates, each with 18 individuals (n=54). Bars are standard errors.
19 100
Tlaltizapan
80 El Batan
0) Poza Rica (0 OC c o w en 40 E 21 24 26 28 31 33 35 38 40 42 45 47 Time (days) Figure 4. Transmission rate over time of maize bushy stunt phytoplasma (isolate (Tlaltizapan, El Batan, and Poza Rica) when acquired by nymphs of Dalbulus maidis (biotype Tlaltizapan, El Batan, and Poza Rica) and maintained at 25 °C. Data points are means from three replicates, each with 18 individuals (n=54). 2 0 Biotvpe Isolate Tlaltizapan El Batan Poza Rica a b a Tlaltizapan 44.3 ± 5.1 63.9 ± 8.4 55.4 ± 8.5 b b a El Batan 65.1 ± 12.0 66.7 ± 8.3 50.0 ± 6.3 c c c Poza Rica 80.6 ± 1.4 86.1 ± 3.6 72.2 ± 9.0 Table 2. Transmission rate of three isolates of maize bushy stunt phytoplasma by three biotypes of Dalbulus maidis nymphs at 25°C. Data are means±SE from three replicates, each with 24 individuals (n=72). Data are expressed as percentages. Means flanked by the same letter are not significantly different (LSD Test). 21 isolate. The mean effect of biotype was not significant (P=0.I19; dfi=2); however, the mean effect isolate was significant (P<0.001 ; dfi=2), with all three leafhopper biotypes transmitting the Poza Rica isolate best. MEAN INCUBATION PERIOD AND SYMPTOMS ON MAIZE. Mean incubation period among Tlaltizapan, El Batan, and Poza Rica isolates was different (P<0.001; df=2). The longest incubation period occurred for the Poza Rica isolate, with an average of 21.4 days, while a shorter mean incubation period occurred for the Tlaltizapan and El Batan isolates, with 18.3 and 17.0 days, respectively. The symptoms produced fay the three isolates on maize leaves were similar in coloration. Maize infected with all three isolates first showed reddening in the margins of the old leaves, generally fi-om the middle to the tip of leaves. Red color was followed by chlorosis 3 or 4 days latter followed by senescence of leaves. Each leaf that developed after the onset of disease followed this progression of symptoms. Simultaneously the plant becomes stunted, and often developed basal tillers. Amount of basal tillering in symptomatic plants was different (P<0.05; df=4) among the three isolates with Poza Rica isolate producing a higher proportion of plants with basal tillering compared to the Tlaltizapan and El Batan isolates (Table 3). SURVIVAL. Biotype Tlaltizapan. Temperature influenced the survival of Tlaltizapan D. maidis biotype nymphs and adults exposed to the Tlaltizapan MBSP isolate (Figure 5). The survival of nymphs and adults exposed to those four temperatures was fitted to Weibull model (Table 4). ANOVA showed that temperature had significant effect on the Weibull scale (b) (P<0.001; df=3) but not on shape (c)(P=0.170; df=3) parameters. The b (P<0.001; df=l) and c (P<0.001; df=l) parameters were different between nymphs and ~ ) i Biotvpe Isolate Tlaltizapan El Batan Poza Rica Tlaltizapan 5.1 (39) 0.0 (58) 1.8 (51) El Batan 1.8 (54) 12.6 (56) 7.5 (40) Poza Rica 19.1 (68) 53.8 (78) 45.4 (66) Table 3. Percentage of plants with basal tillering produced by three maize bushy stunt phytoplasma isolates when transmitted by three Dalbulus maidis biotypes. Results were based on means of two replicates, each with 48 plants (n=96). Values in parenthesis are the total number of plants with symptoms. 23 100 NYMPHS 80 - ce > > 60 - C CD 40 - ü 20 - 0 2 4 6 8 1 0 . 1 2 . 1 4 . 1 6 .1 8 100 ADULTS 80 - ce > > \— 60 - 3 co c o 40 - o CD CL 20 - 0 2 4 . 6 8 . 1 0 . 1 2 . 1 4 . 1 6 . 1 8 Time (weeks) Figure 5. Survival of Dalbulus maidis biotype Tlaltizapan when nymphs or adults exposed to Tlaltizapan maize bushy stunt phytoplasma isolate. Nymphs and adults were maintained at four different temperatures. Data points are means from three replicates, each with 24 individuals (n=72). 24 Nymphs Adults Temperature °C b c b c. 15 81.1 2.0 51.6 1.6 20 72.6 3.7 48.3 1.5 25 52.1 3.1 21.9 1.4 30 19.0 1.7 10.8 1.3 Table 4. Weibull scale (b) and shape (c) parameters of the survival for Dalbulus maidis (biotype Tlaltizapan) nymphs and adults when exposed to maize bushy stunt phytoplasma at four different temperatures. Values are the average of three replicates, each with 24 individuals (n=72). b is in units of time (days) and c is a unitless shape parameter, when 0 = 1, the Weibull model reduces to the exponential model. 25 adults. In addition the interaction terms temperature by stage were not significant for b CP=0.308; df=3) and c (P=0.233; df=3) parameters. Survival for the three biotypes. Inoculative individuals of the three biotypes lived longer than non-inoculative individuals when leafhoppers were maintained at 15 °C (Figures 6,7,8). The Gehan’s Wilcoxon test shows that inoculative leafhoppers from Tlaltizapan lived longer than non-inoculative leafhoppers at 15 °C (P=0.037), but not at 25 °C (P=0.195). In addition, leafhoppers inoculative with the Poza Rica and El Batan isolates lived longer at 15 °C (?<0.001; P<0.001, respectively), but not at 25 °C (P=0.347; P=0.197, respectively). DISCUSSION The influence of temperature on the mean latent period of phytoplasmas in their nymph and adult vector has not been well studied. Here it was found that latent period did not differ between nymphs and adults transmitting the MBSP Tlaltizapan isolate at four temperatures. Murral et al. (1996) reported similar results for nymphs and adults of the aster leafhopper M. quadrilineatus transmitting a strain of the aster yellows phytoplasma. Mean latent period of MBSP in both leafhoppers stages is inversely related to temperature from 15° to 25 °C. Jensen (1972) reported similar results for the Western X phytoplasma transmitted by the leafliopper Colladonus montanus (Van Duzee). This phytoplasma had the longest (125 days) median incubation (latent) period (1P50) at 15 °C and the shortest (26 days) median incubation period at 25 °C. 26 100 80 - inoculative CO > non-inoculative > 3 60 - CO c Ü03 40 - 03 o_ 20 - 0 2 4 6 8 10 . 12 . 14 . 16 . 18 100 25 °C 80 - ■ inoculative CO > non-inoculative > 60 - 3 C/3 c 03 40 - O 03 CL 20 - 0 2 6 8 10 . 12 . 14 . 16 . 184 Time (weeks) Figure 6. Survival of inoculative and non-inoculative Dalbulus maidis (biotype Tlaltizapan) when nymphs and maize bushy stunt phytoplasma isolate Tlaltizapan weremaintained at 15 and 25 °C. Data points are means o f three replicates, each with 18 individuals (n=54). 27 100 inoculative non-inoculative 80 - (C > > 3 60 - CO c CD 40 - Ü 0 0_ 20 - 0 2 4 6 8 10 . 12 . 14 . 16 . 18 100 inoculative non-inoculative 80 - cc 25 °C > > 60 - 3 CO c 0 40 - O 0 CL 20 - 0 2 4 6 . 8 1 0 . 1 2 . 1 4 . 1 6 .1 8 Time (weeks) Figure 7. Survival of inoculative and non-inoculative Dalbulus maidis (biotype Poza Rica) when nymphs and maize bushy stunt phytoplasma isolate Poza Rica weremaintained at 15 and 25 °C. Data points are means of three replicates, each with 18 individuals (n=54). 28 100 inoculative non-inoculative 80 - cC > > 60 - 3 CO c (D 40 - Ü 0 2 4 6 . 8 1 0 . 1 2 . 1 4 . 16.18 100 inoculative non-inoculative 80 - oJ > 25 °C > 60 - c oCD 40 - CD Q_ 20 - 0 24 6 8 1 0 . 1 2 . 1 4 . 16.18 Time (weeks) Figure 8. Survival of inoculative and non-inoculative Dalbulus maidis (biotype El Batan) when nymphs and maize bushy stunt phytoplasma isolate El Batan weremaintained at 15 and 25 °C. Data points are means of three replicates, each with 18 individuals (n=54). 29 Although mean latent period is temperature dependent, the relationship between these two variables is not linear over the entire range of temperatures. Results showed that mean latent period for Tlaltizapan MBSP isolate has quadratic relation with temperature. At the highest temperature tested, 30 °C, mean latent period was longer than at 25 °C. A similar quadratic relations between mean latent period and temperature have been reported in other phytoplasmas. For example, similar mean latent periods were found for two aster yellows strains transmitted by M. quadrilineatus at 25° and 30 °C (Murral et al., 1996). In addition, the mean latent period for Western X phytoplasma was 26 days at 25 °C; while at 30 °C it was 38 days (Jensen, 1972). The mean latent period of the three MBSP isolates, when transmitted by their respective D. maidis biotypes (e.g. biotype-isolate; El Batan-El Batan), also is temperature dependent Moreover, the three isolates had similar mean latent periods in their respective biotypes at 15 °C but not at 25 °C. At that temperature the Poza Rica isolate had a longer mean latent period than the Tlaltizapan and El Batan isolates. Differences in latent period among strains of phytoplasmas have been reported for other phytoplasmas. Gold and Sylvester (1982), comparing Western X phytoplasma strains, found that the LP 50 (latent period) of the Dixon strain was 35 days while the LP 50 of the Berkeley strain was 28 days when both strains were maintained in their vector at 25 °C. Granados and Chapman (1968) found differences among strains of aster yellows as well. They reported a mean latent period of 21.1 days for the Western strain compared with the mean latent period of 14.7 days for the Eastern strain when both strains were maintained in M- quadrilineatus at 25 °C. In addition, Murral et al. (1996) found that the aster yellows bolt and severe strains had 30 different mean latent periods in their M- quadrilineatus vector at 15° and 25 °C. Transmission rate of the TIaltizapan MBSP isolate by the Tlaltizapan D. maidis biotype also had a quadratic relation with temperature. Transmission rate of MBSP for D. maidis nymphs and adults was higher at 20° and 25 °C, than at 15° and 30 °C. Similar quadratic relation between transmission rate and temperature has been reported for the aster yellows and Western X phytoplasma. Nymphs and adults of M- quadrilineatus transmitted two aster yellows strains better at 20° and 25 °C than at 15° and 30 °C (Murral et al., 1996). In addition, a higher transmission rate of Western X phytoplasma by Q. montanus was reported at 20° and 25 °C than at 15° and 30 °C . High temperatures may reduce the reproduction of phytoplasmas and explain the reduced rates of transmission and longer latent period of MBSP when leafhoppers were held at 30 °C. For instance, temperatures of 38° and 41 °C during a period of 7 to 20 days inactivated the Western X phytoplasma or reduced its concentration in the vector, C. montanus (Jensen, 1968). Furthermore, Kunkel (1937) found that Cicadula sexnotata (Fall.) lost its ability to transmit the aster yellows within 24 hours after being maintained at 30 °C and never regained the ability to transmit when maintained at that temperature. Nymphs of planthoppers (Delphacidae) are generally more efBcient vectors of persistently transmitted, propagative viruses than are adults (Nault, 1994). Phytoplasmas have similar relationships with their vectors as seen in this study; at all temperature tested, except 30 °C, nymphs were more efBcient vectors. Similar to this study, Murral et al. (1996) foimd that nymphs of M. quadrilineatus transmitted two aster yellows strains better than adults at four temperatures. However, other studies have not found these differences in 31 transmission rate between nymphs and adults. For example, Sinha and Chikowski (1967) reported no differences in transmission by nymphs and adults when they acquired aster yellows between 22° and 28 °C. Swenson (1971), on the other hand, reported that M- quadrilineatus adults transmitted better than nymphs when both stages acquired the aster yellows between 18° and 25 °C. Reasons for these differing reports are not known but could be explained by different phytoplasma strains and vector biotypes used by these investigators. The Poza Rica MBSP isolate not only was different in its latent period when acquired at 25 °C but also different in its transmission rate when acquired by nymphs at that temperature. The Poza Rica isolate was transmitted at a higher rate than the El Batan and Tlaltizapan isolates. Difference in transmission rate of phytoplasma strains by leafhopper vectors has been documented in aster yellows strains. For example, Murral et al. (1996) found that nymphs of M. quadrilineatus transmitted the bolt strain better than the severe strain at 15° and 25 °C. Chiykowski (1997), working with the same vector, reported significant differences in the transmission rate of two aster yellows strains at 24 °C . This leafhopper transmitted the B strain better than the A strain. In addition, Granados and Chapman (1968) reported that the aster yellows Eastern strain was transmitted better than the Western strain by M- quadrilineatus. Differences in transmission rate not only has been found in aster yellows strains but also in the Western X phytoplasma. Gold and Sylvester (1982) found that the Berkeley strain had a higher transmission rate (33%) than the Dixon strain (25%) when transmitted by Ç. montanus. 32 The Poza Rica MBSP isolate not only is transmitted better by the Poza Rica D. maidis biotype but also by the other two biotypes. On the other hand, the three leafhopper biotypes do not differ among each other in transmitting the three N'IBSP isolates. I expected that each D. maidis biotype would be best coadapted to the MBSP from its locality and transmit the isolate at a higher rate. This was not the case, that is if transmission rate is a measure of coadaptation, it was not detected here. The pattern of transmission tested by ordinary runs analysis is similar among the three isolates. Preferentially the three isolates are transmitted by D. maidis in a random fashion. Therefore, the three isolates share the common characteristic that they were not transmitted to every test plant in every transfer period. Survival of nymphs and adults of the Tlaltizapan D. maidis bio type exposed to Tlaltizapan MBSP isolate is temperature dependent. A similar pattern in nymphal survival was reported by Murral et al. (1996) when M. quadrilineatus acquired aster yellows at different temperatures. They found that this leafhopper lived more than 18 weeks at 15 °C, 14 weeks at 20 °C, 12 weeks at 25 °C, and 7 weeks at 30 °C. Madden et al. (1986) determined the survival of mollicute-free adults of D- maidis and similar pattern in survival was found for moUicute-exposed adults. Exposed and mollicute-free adults of D. maidis live longer as temperature decreases. Survival of inoculative and non-inoculative D. maidis differed at 15 °C but not 25 °C. Here it was found that at 15 °C inoculative individuals of all three D. maidis bio types survived longer than non-inoculative ones. A similar finding was reported by Ebbert and Nault (1994), but with a spiroplasma. They reported that CSS improved the survival of D- 33 maidis at low temperature (10-20 °C) when the vector was maintained on moist sand, oats or maize. Most likely, both pathogens produce a positive effect on D. maidis at low temperatures. Low temperatures (between 13° and 21 °C) occur during the dry season in the three sites where the MBSP isolates were collected. Consequently, MBSP may help to increase the survival of the vector during the dry season, especially when the com leafhopper’s maize host plant is not present. On the other hand, high temperatures (25 °C) do not produce positive or negative effects in the survival of inoculative or non-inoculative individuals. No effect in vector survival has been reported in several studies when the molhcute is maintained with its vector leafhopper at high temperatures. For example. Madden and Nault (1983) and Madden et al. ( 1984) found that Q. maidis survival was not affected when nymphs acquired the CSS at 26 °C. Alivizatos and Markham (1986) working with the same vector and spiroplasma found no effect in the survival of D. maidis when it was injected with CSS and maintained at 29 °C. Similar results were reported by Hsu and Banttari (1979) in the survival of M. quadrilineatus when it acquired the aster yellows by feeding at 24 °C. In addition, Chiykowski (1991) found that the longevity of inoculative, Paraphlepsius irroratus (Say) leafhoppers, was not different from non-inoculative ones when nymphs acquired the clover phyllody phytoplasma at 24 °C. The mean incubation period of the Poza Rica MBSP isolate in maize was significantly longer than for the Tlaltizapan and El Batan isolates. Similarly, latent period of the Poza Rica MBSP isolate was longer in the vector than were the Tlaltizapan and El Batan isolates. This similarity in difference of mean incubation period and mean latent 34 period aise has been reported for aster yellows. Chiykowski (1977) found that aster yellows A strain had a mean incubation period of 12.6 days in the plant and a mean latent period of 20. 8 days in the vector, while the aster yellows B strain had a mean incubation period of 13.4 days and a mean latent period of 21.8 days. Mean incubation period and mean latent period of phytoplasmas reflect in part, the rate of multiplication of these bacteria in their hosts. These rates, regardless of insect or plant host, apparently are consistent and stable biological traits. The traditional criteria applied to separate phytoplasma strains has been the type of symptoms produced in the host plant. In this case each MBSP isolate produces distinct symptoms in maize. Those infected with the Poza Rica MBSP isolate had more basal tillering and were less stunted than the other two isolates. Possibly the Poza Rica isolate stimulates the production of hormones (auxins), which increase tillering. Kuske and Kirkpatrick (1992) indicated that plant pathogenic bacteria stimulate auxins production that are responsible for increasing tillering in the host plant. 1 hypothesize that MBSP evolved from aster yellows as isolates and/or strains that specialized in their D. maidis vector (and host) and Zea plant hosts. Moreover I propose that N'IBSP has a polyphyletic origin, evolving more than once from aster yellows ancestors. Lee et al. (1993) previously established that the MBSP type strain from Texas is grouped with the 168 rDNA-I-subgroup B with the aster yellows phytoplasmas. More recently, N.A. Harrison (pers. comm.) indicated that the Poza Rica isolate is distinct from the El Batan and Tlaltizapan isolates by their reaction to two aster yellows probes, FPV-A44 (8.0-kb) and FPV-A 7 (1.0-kb). This is consistent with data presented here showing that the leafhopper 35 transmission characteristics and symptoms (tillering) in maize of the Poza Rica isolate differ significantly fi'om the other two isolates. This data suggests that the Poza Rica isolate should be considered as a strain distinct firom a second strain represented by the Tlaltizapan and El Batan isolates. Aster yellows, unlike MBSP, has a wide distribution, primarily Nearctic, infects a broad range of plant hosts and is a M- quadrilineatus specialist. The distribution of aster yellows strains in the United States and Canada has been reported by Chapman (1973). Five strains have been reported occur in Japan (Okuda et al., 1997). Aster yellows has been reported firom 350 different plant species (Peterson, 1973). Most host are dicots (Kunkel, 1953), but eight monocots have been reported as hosts (Banttari, 1966). Attempts to infect maize with two Ohio strains of aster yellows (^lurral et al., 1996) proved unsuccessful ( L. R. Nault pers. comm.). Failure was not a result of inability of M. quadrilineatus to feed on maize. Indeed, the leafhopper has host plants firom 54 different families (Errampalli et al., 1991) including maize which has been used at times in our laboratory to rear this species. Nault (pers. comm.) did succeed in transmitting the type isolate fi'om Texas but not the three isolates used in study firom maize to maize with M. quadrilineatus. The evolution of MBSP most likely happened when AY-infected M- quadrilineatus carried the phytoplasma to susceptible maize. This likely is a rare event Resident D. maidis feeding on the maize became infected and perpetuated the disease in maize. That such an event happened at least twice is suggested by the genetic affinity of the Poza Rica isolate to a different aster yellows strain than that for the Tlaltizapan and El Batan isolates. Moreover the latter two are high elevation isolates with similar climatic habitats compared to the Poza 36 Rica isolate which is found at low elevation with a very different climate. Furthermore, weather patterns in Mexico would allow for transport of Q. maidis to and from El Batan and Tlaltizapan but not Poza Rica (Taylor et al., 1993). Further work is needed on additional MBSP isolates, not only those from Mexico but elsewhere in Latin America, to test the hypothesis that MBSP has a polyphyletic origin. 37 CHAPTER 2 MUTUALISTIC ASSOCIATION BETWEEN THE MYRMECOPHILE LEAFHOPPER Palbulus quinquenotatus fHOMOPTERA: CICADELLIDAE) AND ITS TENDING ANTS INTRODUCTION The fivespotted gamagrass leafhopper. Dalbulus quinquenotatus DeLong & Nault differs from its congeners in many respects. It is a gregarious species forming dense colonies of nymphs and adults at the bases of its host plants, which are species of Mexican gamagrasses (Tripsacum species) (Nault et al., 1983). Also, unlike other Dalbulus species, Û- quinquenotatus is sedentary, rarely responding to mechanical stimuli (Heady and Nault, 1985). Moreover, D quinquenotams produces greater quantities of honeydew at higher rates than other Dalbulus (Larsen et al., 1992). All of these behaviors are common to homopterans that are attended by ants, otherwise known as myrmecophiles. Indeed, colonies of D. quinquenotatus are rarely found in the field without the presence of attendent ants. Larsen et al. (1991) reported 18 species of ants from four subfamilies associated with D. quinquenotatus in Mexico. 38 Beneficial effects between myrmecophile Homoptera and ants have been reported for aphids (Vinson and Scarborough, 1991), coccids (Hanks and Sadof, 1990; Bach, 1991), psyllids (Novak, 1994), pseudococcids (Way, 1963), and membracids (Messina, 1981; Fritz,, 1982; De assis Dansa and Duarte Rocha, 1992). Homopterans excrete honeydew which is used as food by ants. Moreover, at times, ants also prey upon myrmecophile Homoptera to supplement their requirements for protein. In doing so, ants regulate populations of attended myrmecophiles preventing overpopulation and exhaustion of the host plant (Way, 1963; Fritz, 1983). When ants remove honeydew they perform a sanitary function that benefits both the homopteran and the host plant (Bartlett, 1961; Washburn et al., 1985). In addition, ants expel herbivores that compete with myrmecophilous Homoptera for their host plant (Messina, 1981; Fritz, 1983; Seibert, 1992). Furthermore and perhaps most importantly, ants protect the myrmecophile homopterans from natural enemies (Way, 1963; Banks and Macaulay, 1967; Bristow, 1984). None of the above mentioned benefits have been reported for ant-attended _D. quinquenotams. or for that matter any myrmecophilous leafhopper, rather these benefits have been assumed. Previously, Larsen et al. (1991) considered JD. quinquenotams to be a facultative myrmecophile. Later, Larsen et al. (1992) suggested that the relationship could be obligatory. Here I show that the leafhopper is an obligatory myrmecophile. In the absence of ants and predators the leafhopper kills its hosts. If ants are absent and predators are present, leafhopper populations are extinguished. Ants free plants from damaging honeydew, regulate leafhopper populations and protect leafhoppers from predators and competing herbivores. 39 MATERIALS AND METHODS Field Studies. This study was conducted in the field at three locations, Zapopan, Agua Caliente, and San Agustin, in the state of Jalisco, Central Mexico in 1996. Two seasons, dry and wet, occur at those sites. In Zapopan and Agua Caliente the wet season began the third week of May, while in San Agustin the wet season started the first week of May (Figure 9). At Zapopan, jQ- quinquenotatus is attended by Brachvmyrmex obscurior Forel, Camponotus sp., and Pheidole sp. on the gamagrass Tripsacum pilosum Scriber and Merrill. Furthermore, on this host the nonmyrmecophile leafhoppers Dalbulus gelbus DeLong, and D. guzmani DeLong & Nault are found. In Agua Caliente and San Agustin D. quinquenotatus is attended by£. obscurior on Tripsacum dactyloides L.. Tripsacum dactyloides patches were burned in Agua Caliente two months before this study was begun. Tripsacum had few clones with green stems and leaves at the three sites, when the study begun. At that time, I . pilosum from Zapopan had an average of 4.4 leaves; T. dactvloides from San Agustin had an average of 4.5 leaves, while T. dactyloides from Agua Caliente had an average of 4.1 leaves. The leaves averaged 30, 26 and 8 cm long in Zapopan, San Agustin, and Agua Caliente, respectively. Obligatory association between D. quinquenotatus and ants. Three treatments were established in the greenhouse at GARDC-GSU in Wooster, OH. The first was JT. dactyloides with D. quinquenotams. the second J . dactyloides with D- gelbus. and the third I . dactyloides alone. Each treatment had five replicates and each replicate included a single gamagrass clone. At the start of the experiment, the gamagrass clones had 3 stems, between 3 and 4.5 leaves per stem and were 8 and 11 cm in height. Eight females 4 0 Figure 9. Total precipitation (mm) every two weeks from April to July 1996 in three stations from Central Mexico in Jalisco state. Those stations are Zapopan, Agua Caliente, and San Agustin. Data were provided by the Mexican National Water Commission. 41 o \o Precipitation (mm) Precipitation (mm) Precipitation (mm) m 00 t04^cncx)oro4^cn ê o o s o o o o o o o o o _* > •O t\3 0 3 4^ I -* 00 4^ - c no 3 (D w 4^ ho C 0 0 4^ T 1------1------1------r and eight males of D. quinquenotatus were released per clone in the first treatment. The same number of females and males of D- gelbus was released in the second treatment. Each gamagrass clone with and without leafhoppers was maintained in screen cages 100 X 30 cm during the experiment in the greenhouse, with a light-dark period of 10:14 h, and the average temperature was 24 °C. Plants were watered every two days. Every four days the number of nymphs and adults per clone in the first and second treatments was determined, and in the three treatments the number of stems, number of green leaves and height of T.dactyloides was recorded. The day on which the gamagrass clone with D. quinquenotatus died was registered. Clones withD- gelbus or no leafhoppers did not die. The number of adults of Q. quinquenotatus and D . gelbus was compared by using the r- test. The same test was applied to compare nymphs of both species. The analysis of variance (ANOVA) was used to compare the number of stems, number of leaves, and height of gamagrass in the three treatments. Two treatments were conducted under natural conditions at each of the three sites previously mentioned. One treatment was Tripsacum withD- quinquenotams and ants, and the other was Tripsacum free of insects. Resifiim tridente (Cipermetrina and diclorvos) insecticide was applied every month to Tripsacum in this last treatment to eliminate aU insects. Four gamagrass clones per treatment were chosen at each location. Every two weeks from May to July 1996 the number of leaves in the chosen clones was registered and the number of leaves for Tripsacum with and without D- quinquenotams and ants was compared by using the r-test. 43 Regulation of D. quinquenotatus population, removal of honeydew, and attendance by ants. Three treatments were conducted under greenhouse conditions to determine possible benefits from ants. In the first treatment Formica subsericea Say attended D- quinquenotatus without food supplements; in the second, f . subsericea attended D- quinquenotatus with supplements of honey and insect protein (dead yellow jackets Vespula maculifrons (Buysson)), and in the third, f . subsericea attended D quinquenotatus with supplement of insect protein alone. Three large colonies of F. subsericea (2,851, 2, 055, and 1,915 workers respectively), which were collected from Wooster, Ohio, were used in the three experiments. Ant colonies were maintained in bushel baskets filled 3/4 with soil. Each of these colonies was connected by tygon tubing to three T. dactyloides clones with D. quinquenotatus adults. In addition, three J". dactvloides clones with D. quinquenotatus adults but no ants were used as control. Eight females and eight males were released on eachX- dactvloides clone, ten days prior to ants’ being connected to the clones. Honey and insect protein were supplemented in the second treatment and insect protein was supplemented in the third treatment at the same time that the ant colony was connected to D- quinquenotatus. Supplements were added three times per week. Each gamagrass clone with its D quinquenotatus colony was maintained in screen cages 100 x 30 cm. Formica subsericea had free access to attend J3. quinquenotams in these screen cages. Once connected to the ants, the number of J3. quinquenotams nymphs and adults in ant treated and untreated colonies was counted randomly every four days from 900 to 1300 hr. During each experiment, plants were watered every other day and the ant colonies every day. The temperature average in the greenhouse was 26 °C, 44 and the light-dark period was 14:10 h. The effect of ants onD- quinquenotatus was evaluated by comparing the numbers of attended and unattended leafhoppers. Both adults and nymphs were compared by using the f-test in each of the three treatments. Leaf surface of Tripsacum covered with honeydew produced by D. quinquenotatus was compared 16 days after ants were connected to D- quinquenotatus colonies. Each sheath and leaf of J . dactvloides was examined to estimate the percent of surface covered with honeydew. Attendance of JQ. quinquenotatus by ants was evaluated in the three treatments. The number of attended nymphs and adults of D. quinquenotatus was compared when ants did not receive food supplement versus when they received protein or honey and protein. In addition, the number of ants found in screen cages was determined. The number of attended D- quinquenotatus among the three treatments was compared by using the ANOVA test. This was made for nymphs and adults. Moreover, ANOVA was used to compare the number of ants found inside the screen cages in the three treatments. Reaction of nonmyrmecophile Dalbulus to ants. The numbers of D. gelbus and D. guzmani were determined in two treatments at Zapopan. These species were counted on I . pilosum with and without D. quinquenotatus and ants. Four clones per treatment were chosen and adults of D gelbus and D. guzmani were determined every four days, between 900 and 1300 hrs, from May to July, 1996. The effect of ants on the number of D. gelbus was tested in the greenhouse with small and large Tripsacum plants. The small plants were an average of 24.7 cm in height, while the large plants averaged 91.5 cm in height. One large (2,055 workers) colony of 45 F. subsericea that received protein as supplement was given access to one small and one large plant. Twenty four hours before access, 16 adult leafhoppers of each species were deposited on each small plant and another 16 adults of each species were deposited on each large plant. Once ants were given access the number of adult leafhoppers was counted every 30 m during the first 6 h and once at 24 h. Leafhoppers had opportimity to leave plants if disturbed by ants. This experiment was repeated four times. Protection of D. quinquenotatus by ants against predators. Exclusion experiments were conducted in Zapopan, Agua Caliente, and San Agustin from May to July 1996. Ten days before the experiment started the number of D. quinquenotatus per gamagrass clone was determined at those three locations in order to start with similar numbers of leafhoppers in the two treatments (ant attended and unattended D. quinquenotatus). One treatment was Tripsacum with D. quinquenotams attended by ants and the other was Tripsacum with only D quinquenotams. Four gamagrass clones with D. quinquenotams and ants were selected randomly at each site. In addition, four other gamagrass clones with green stems and leaves but without quinquenotams and ants were selected at each site. This was because colonies of D quinquenotams are never found in the absence of attending ants (Larsen et al., 1991). At each of these last four clones, five adults and 15 nymphs of D quinquenotams were placed on plants. Ants were excluded from these plants by isolating them with a plastic pot with the bottom removed. The side of the pot was buried in the soil approximately five cm. In addition, the pot was covered with Fluon (Northern Products, Inc) from the middle to the top. Fluon was applied on each pot every 25 days during the smdy. 46 Arthropod density was evaluated at each location. For each clone the number of quinquenotatus. number of ants, number of spiders, and number of D. Q uinquenotflm s nymphs parasited by the dryinid Anteon ciudadi Olmi was cotmted. This parasitoid is distinctive from other dryinids in that larva produce a “black-bag” in the thorax o f^ . quinquenotatus nymphs (Moya-Raygoza and Trujillo-Arriaga, 1993). Consequently, every D. quinquenotams nymph with a black bag in the thorax was assumed to have been parasited by A. ciudadi. Arthropods were counted directly on the plant without removal between 900 and 1300 hr every four days at each site. The order in which the plants were counted was randomized each day. The number of JD. quinquenotams from the two treatments at each site was compared by using the f-test. In addition ant protection against D. quinquenotams predators was tested by using £. subsericea and the generalist nabid predator, Nabis americoferus Carayon, in the greenhouse. Preliminary observations confirmed that this hemipteran preys upon D. quinquenotatus adults and nymphs. The predator catches the leafhopper with its forelegs and pierces it with its stylets. Once the stylets are introduced, the predator feeds on the leafhopper approximately 10 minutes and then discards the empty exoskeleton. The£l. americoferus used in this smdy were collected from an alfalfa field in Wayne County, Ohio. Eight female and eight male quinquenotams were deposited on each of 14 J . dactvloides clones. Each clone with its 16 adults was maintained in a screen cage 100 x 30 cm in the greenhouse. Ten days after release, four of the clones were connected to a large (2,851 workers) ant colony o ff. subsericea. three clones were connected to a small 47 (887 workers) ant colony o f the same ant species, and the other seven clones received no attendance by ants. Three (two females and one male) americoferus per clone were released two days after ants were given access to leafhopper colonies. Nabis americoferus and D. quinquenotatus numbers were counted between 900 and 1300 hr every four days in each clone for 64 days. Gamagrass had an average of 4.4 leaves when the experiment started. Gamagrass clones were watered every other day and the ant colonies were watered daily. The average temperature in the greenhouse was 25.5 °C and the light-dark period was 14:10 h. The j^. americoferus treatments were compared by using the f-test. RESULTS Obligatory association between D. quinquenotatus and ants. Tripsacum dactvloides infested with D quinquenotatus died in a period between 64 and 72 days, whereas I . dactvloides with D gelbus did not die. Four days before the gamagrass died, D. quinquenotatus had higher number of nymphs (P=0.002; df=4) and adults (P=0.003; df= 6) than D gelbus (Figure 10). Adults of D gelbus. but not adults of quinquenotatus. decreased in number during the first 32 days on%. dactvloides: however new adults of D- gelbus appeared at day 36, while new adults of quinquenotams appeared four days later. Nymphs of both leafhoppers started to appear 16 days after adults were released. Number of nymphs from the first generation decreased sooner for D gelbus than D. quinquenotams. 48 J _ J I I I I I I I I I I I I I 1 I I I L D. quinquenotatus i2 °— D. gelbus ■a CO g œ CL CL O “cô o 5 E 3 C di < t I I t I I I I t t r I t I t t r I t t en D. quinquenotatus XI Q. E o— D. gelbus >» c en 03 Q. 200 - O(3 . 15 CD 150 - 03 100 - E 3 C en > < Figure 10. Average number of Dalbulus quinquenotatus and Dalbulus gelbus adults and nymphs on Tripsacum dactvloides. Bars indicate standard error. Each data point is the mean of five replicates (n=5). Data were taken every four days during 76 days. Stars firom days 64 to 76 is when T. dactvloides with D. quinquenotatus died. 49 Dalbulus quinquenotatus not only killed Tripsacum but also affected its growth. Stems of I . dactyloides with D quinquenotatus started to decrease 44 days after the experiment began (Figure 11). At that time, stems started to senesce and die, while stems of this gamagrass with D. gelbus and no leafhoppers increased in number. When number of stems decreased in plants with D- quinquenotams. the number of green leaves decreased as well (Figure 12). Tripsacum withD- quinquenotams had fewer leaves than with D gelbus or without leafhoppers (P <0.001; df=2,282). Although Tripsacum with D. quinquenotams had fewer leaves, their stems reached similar height than stems from control plants (P=0.16; df=144) (Figure 13). Tripsacum with D. gelbus had fewer leaves than control plants (P=0.024; df=99) (Figure 12). Furthermore, Tripsacum with D. gelbus was shorter than those Tripsacum with D. quinquenotams. or without leafhoppers (P<0.001; df=2,237) (Figime 13). Tripsacum infested Dalbulus quinquenotams and attended by ants in the field did not show visible signs of stress and no plants died. Tripsacum pilosum in Zapopan (Figure 14), and T. dactvloides in Agua Caliente (Figure 15) and San Agustin (Figure 16) had similar number of leaves when infested withD- quinquenotams and ants as those plants without leafhoppers (P=0.14; df=43, P=0.75; df=44, P=0.94; df=44, respectively). Regulation of quinquenotatus population and removal of honeydew by ants. Depending on how ants were maintained, they had effect on JQ. quinquenotams populations. Formica subsericea preyed on fivespotted gamagrass leafhopper adults and nymphs under three dietary conditions (Table 5). Adults of D. quinquenotams were 50 D. Quinquenotatus □ D. gelbus control CO E 03 03 03 E ZJ c C) < Figure 11. Average number of stems per Tripsacum dactvloides clone under three treatments: Tripsacum with unattended Dalbulus quinquenotatus: Tripsacum with Dalbulus gelbus: and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment was repeated five times (n=5). 51 9 D. quinquenotatus 8 D. gelbus 7 œntrol OJ q) 6 > CO CD 5 0 jQ E 4 3 3 G) > < 2 1 0 0 8 1 6 24 . 32 . 40 . 48 56 64 76 Days Figure 12. Average number of leaves per Tripsacum dactvloides clone under three treatments: Tripsacum with Dalbulus quinquenotatus: Tripsacum with Dalbulus gelbus: and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment was repeated five times (n=5). Data were taken every four days during 76 days. Stars from days 64 to 76 is when T. dactvloides with D. quinquenotatus died. 52 40 D. quinquenotatus D. gelbus control E o S G) 0) SI o > < 0 8 1 6 2432 40 48 56 64 72 Days Figure 13. Average height of Tripsacum dactvloides stems under three treatments: Tripsacum with Dalbulus quinquenotatus: Tripsacum with Dalbulus gelbus: and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment was repeated five times (n=5). Data were taken every four days during 76 days. Stars firom days 64 to 76 is when T. dactvloides with D. quinquenotatus died. 53 ZAPOPAN attended D. quinquenotatus c/3 □ control CD > ce CD CD E Z3 C < Figure 14. Average number of leaves per Tripsacum pilosum clone in Zapopan, Jalisco, Mexico in two treatments: Tripsacum with Dalbulus quinquenotatus attended by ants; and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment had four replicates (n=4). Data were taken from May to July, 1996. 54 AGUA CALIENTE attended D. quinquenotatus □ control Figure 15. Average number of leaves per Tripsacum dactvloides clone in Agua Caliente, Jalisco, Mexico in two treatments: Tripsacum with Dalbulus quinquenotatus attended by ants; and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment had four replicates (n=4). Data were taken j&om May to July, 1996. 55 SAN AGUSTIN attended D. quinquenotatus ^ w □ control (D CD JD £ 3 C d) > < May Figure 16. Average number of leaves per Tripsacum dactvloides clone in San Agustin. Jalisco, Mexico in two treatments: Tripsacum with Dalbulus quinquenotatus attended by ants; and Tripsacum without leafhoppers (control). Bars indicate standard error. Each treatment had four replicates (n=4). Data were taken from May to July, 1996. 56 No food supplement Adults Nymphs Davs attended unattended attended unattended 0 9.5 9.6 0 0 4 0.2 8.6 0 0 8 0 7.3 0 20.0 12 0 6.3 4.3 40.7 16 0 5.0 2.6 59.7 Protein 0 13.7 14.3 0 0 4 7.9 13.0 0 0 8 7.1 12.3 11.6 31.0 12 6.4 12.3 65.9 90.0 16 5.4 11.6 129.0 164.3 Honey and Protein 0 11.4 12.3 0 0 4 5.8 12.0 0 0 8 4.5 12.0 25.8 62.8 12 3.1 12.0 44.2 110.0 16 2.1 11.0 56.8 110.0 Table 5. Comparison of Dalbulus quinquenotatus adults and nymphs when attended by Formica subsericea colonies. Number of adults and nymphs are averages from 9 replicates (n=9) for attended and 3 replicates (n=3) for unattended leafhoppers. Ants attended nymphs and adults during 16 days under 3 treatments. The first was when ants did not receive food supplement; the second when received protein; and the third when received honey and protein. 57 predated significantly by F. subsericea when ants did not receive any food supplement (P < 0.001; df=72), received protein (P < 0.001; df=98) or honey and protein (P<0.001; df=85). Several times I observed ants carrying JD. quinquenotams adults into the nest under the three dietary conditions. Formica subsericea removed the honeydew produced by D. quinquenotatus (Table 6). No honeydew was found on the microhabitat (upper leaf base) o f^ . quinquenotatus when attended by F. subsericea. However Tripsacum with unattended D. quinquenotams had their surface covered with honeydew in the microhabitat of this leafhopper. Honeydew was found inside sheath of the two first leaves of Tripsacum when D- quinquenotams was attended. There nymphs produced honeydew which could not be removed by subsericea workers. Reaction of nonmvrmecophile Dalbulus to ants. Dalbulus gelbus and D. guTmani adults were observed in the field primarily on Tripsacum not infested with D. quinquenotams and ants (P< 0.001; df=110) (Figure 17). Furthermore, these two nonmyrmecophile leafhoppers were collected on I. pilosum occupied by D- quinquenotams and ants only when plants were large In size. Experiments in the greenhouse showed that on small Tripsacum. £. subsericea expeledD- gelbus adults one hour after given access to D- quinquenotams adults (Figure 18). No J3. gelbus adults were found after 24 hours on these small plants but they were found on large plants with quinquenotams and ants. Predation on D- gelbus by F. subsericea was not observed on small and large gamagrass. 58 Attended Unattended % leaf surface area covered % leaf surface area covered Leaf with honevdew with honevdew Upper leaf base I 0 50.0 2 0 56.0 3 0 49.7 4 0 16.0 Inside sheath I 2.4 0 2 9.9 0 3 0 0 4 0 0 Table 6. Percentage of surface covered with honeydew produced by Dalbulus quinquenotatus on Tripsacum dactvloides when attended by Formica subsericea and unattended. Data were taken 16 days after ant colonies attended Dalbulus quinquenotatus. During this experiment ants received protein as food supplement. 59 60 without D.qulnquenotatus +ants 50 with D. quinquenotatus + ants O) 40 30 20 D) 18 26 3 . 11 . 1 9 . 27 . 5 . 1 3 21 29 May June July Figure 17. Average number of Dalbulus gelbus and Dalbulus guTmani adults on Tripsacum pilosum in Zapopan, Jalisco, Mexico. These leafhoppers were found on Tripsacum under two treatments: without Dalbulus quinquenotatus + ants; and with D. quinquenotatus + ants. Bars indicate standard error. Each treatment had four replicates (n=4). Data were taken from May to July, 1996. 60 20 large Tripsacum small Tripsacum w ZJ 14 - (D 05 d i 03 E o c d) < ■Q" — §— g— -5— $— ^ 2 - 0 1 2 3 4 5 6 24 large Tripsacum cn 3 small Tripsacum o o c 0) 3 O" c '3 O' 10 - Ql JD £ 3 c 0 3 < 0 1 . 2 3 5 6 244 time (hr) Figure 18. A v erse number of Dalbulus gelbus and Dalbulus quinquenotatus adults on large and small Tripsacum dactvloides. Bars indicate standard error and each data point represents four replicates (n=4). Data were taken every 30 m during the first six hours and once at 24 hours after Formica subsericea started to attend Dalbulus quinquenotams. 61 Protection of D. quinquenotatus by ants against predators. Many more_D. quinquenotatus were found when they were attended than not attended by ants in Zapopan (P < 0.001; df=79) (Figure 19), San Agustin (P < 0.001; df=78) (Figure 20), and Agua Caliente (P< 0.001; df=80) (Figure 21). Twenty-four days after the experiment began, unattended D. quinquenotatus from the three localities almost or completely disappeared. However, a few nymphs ofD- quinquenotatus were observed in the three localities. Large numbers of D. quinquenotatus always were observed in the three localities when attended by ants. Dalbulus quinquenotatus was attended by several ant species through time in Zapopan (Figure 19). There this leafhopper was attended by three ant species on two of the four clones. The first of the two clones was occupied by JB. obscurior. latter by Camponotus sp. and finally by Pheidole sp. The second clone was occupied by B. obscurior and latter by Pheidole sp. in Zapopan. The leafhopper was attended only by the diminutive (1.5-2 mm long) B. obscurior in San Agustin (Figure 20) and Agua Caliente (Figure 21). Although small in size, this ant species occurred in high number in the latter two sites. In Agua Caliente the total mean of JS. obscurior was higher than the total mean of ants (three species) collected in Zapopan (P<0.001; df=19). In addition, B- obscurior from San Agustin was higher in number than those collected in Zapopan (P=0.033; df=35). Spiders were collected and sometimes observed preying on unattended quinquenotatus in Zapopan, Agua Caliente, and San Agustin (Figure 22). Spiders were not collected or observed in the microhabitat of ant attended J3- quinquenotatus. Spiders 62 200 ZAPOPAN 180 - attended D. quinquenotatus 160 - unattended D. quinquenotatus ants 140 - 120 - 100 - O E 80 - 3 C 60 - O) > < 40 - 20 - 1 8 26 3 1 1 1 9 27 5 13 . 21 . 29 May June July Figure 19. Average number of Dalbulus quinquenotatus and attending ants on Tripsacum pilosum in Zapopan, Jalisco, Mexico. Bars indicate standard error and each treatment had four replicates (n=4). Data were taken every four days from May to July, 1996. 63 220 SAN AGUSTIN 200 attended D. quinquenotatus 180 unattended D. quinquenotatus M 160 ü ants I 140 ~ 120 0 100 tt" C 80 d) > < 20 ■ 28 13 . 21 29 15 . 23 31 May June July Figure 20. Average number of Dalbulus quinquenotatus and attending ants on Tripsacum dactvloides in San Agustin, Jalisco, Mexico. Bars indicate standard error and each treatment had fom- replicates (n=4). Data were taken every four days from May to July, 1996. 64 800 AGUA CALIENTE 700 - attended D. quinquenotatus unattended D. quinquenotatus w 600 - oÜ ants (n c 500 - O XI 400 - E ZJ c 300 - C35 > < 200 - 100 - 1 9 27 4 12 20 28 6 1 4 22 30 May June July Figure 21. Average number of Dalbulus quinquenotatus and attending ants on Tripsacum dactvloides in Agua Caliente, Jalisco, Mexico. Bars indicate standard error and each treatment had four replicates (n=4). Data were taken every four days fiom May to July, 1996. 65 Figure 22. Number total of spiders found on unattended Dalbulus quinquenotatus. Spiders from Zapopan were collected on Tripsacum pilosum. Spiders from Agua Caliente and San Agustin were collected on Tripsacum dactvloides. Data were taken every four days from Mav to Julv. 1996. 66 ►n fto to Total No of Spiders Total No of Spiders Total No of Spiders O ro CO o> o o cn CD o o CD o (/) to 00 ro CD o\ ■o cCL. 3 CD (Û CD 4 • o> tn c cn w ro CO CO to collected in Zapopan and San Agustin belong to two different taxa, the family Anyphaenidae (genus Anyphaena) and from the family Philodromidae. Spiders collected in Agua Caliente belonged to four taxa; two taxa from the family Thomisidae and two others from the Araneidae. Zapopan had the highest number of spiders, while San Agustin had the fewest. Dalbulus quinquenotams nymphs were parasited by the dryinid A- ciudadi when attended by obscurior in Agua Caliente and San Agustin but not in Zapopan. Parasited nymphs were observed in the last days from July, and maximum parasitism of leafhoppers was three percent in Agua Caliente and nine percent in San Agustin. Adults (P< 0.001; df = 175) and nymphs (P< 0.001; df=98) of the predator j^. americoferus were higher when confined with D- quinquenotams alone than with JD- quinquenotatus and ants (Figure 23). Young nymphs of this predator were observed 22 and 55 days after predator adults were released in cage w i t h quinquenotams but no ants. On the other hand, N. americoferus adults and nymphs were completely eliminated b y f. subsericea during the first 16 days once released in cages with ant attended J^. quinquenotams. Unattended D- quinquenotams adults were preyed upon and eliminated by americoferus 16 days after the predator was released (Figure 24), while D. quinquenotams nymphs were eliminated in 24 days (Figure 25). In addition, quinquenotams attended by large ant colony had fewer individuals that reached the adult stage and no nymphs from a second generation were found. In this last treatment ants were observed carrying to their nest not only americoferus but also D quinquenotatus. On the other hand, adults of_D. 68 6 leafhoppers attended 3 5 "O leafhoppers not attended (0 o 4 TJ CD 3 JQCD E 3 2 C d) > < 1 0 0 8 1 6 24 32 40 48 56 I I I J I I L ' ' ' ' ' leafhoppers attended cn leafhoppers not attended Q. E >» c o cS T3 CD CD E 3 C d) < Days Figure 23. Average number of Nabis americoferus adults and nymphs when predator was maintained in two conditions; the first was when Dalbulus quinquenotatus was attended by Formica subsericea: and the second when Dalbulus quinquenotatus was unattended. Bars indicate standard error. Each treatment had seven replicates (n=7). Data were taken every four days during 60 days. 69 cn large ant colony 3 T 3 (0 small ant colony cn 3 unattended 00 o c CD 3 a c ■3 o 10 - > < 0 8 1 6 24 3 2 40 48 56 Days Figure 24. Average number of Dalbulus quinquenotatus adults when maintained with the predator Nabis americoferus under three conditions: the first was with large ant colony (n=4); the second with small ant colony (n=3); and the third was unattended Dalbulus quinquenotatus (n=7). Bars indicate standard error. Data were taken every four days during 60 days. 70 40 large ant colony Q. E small ant colony >» c OT 30 - unattended 3 3 O c CB 3 O' Ç 20 - 3 C Q o S3 10 - S 3 C o> < 8 1 6 244032 48 5 60 Days Figure 25. Average number of Dalbulus quinquenotatus nymphs when maintained with the predator Nabis americoferus under three conditions: the first was with large ant colony (n=4); the second with small ant colony (n=3); and the third was with unattended Dalbulus quinquenotatus (n=7). Bars indicate standard error. Data were taken every four days during 60 days. 71 quinquenotatus attended by the smaller ant colony completed a generation and a second generation of nymphs was observed. Attendance of j). quinquenotatus by ants. Dalbulus quinquenotatus was not attended and protected by F. subsericea when these ants did not receive food supplements (Figure 26 and 27). Adults and nymphs of D. quinquenotams were consumed by subsericea in less than eight days. However D- quinquenotams adults and nymphs were attended by large F. subsericea colonies when ants received protein or honey and protein as supplements. Higher numbers of Formica subsericea were counted with D. quinquenotams when ants received a protein diet than when they received honey and protein or did not receive a food supplement (P<0.001; df=2,105) (Figure 28). These ants that received protein only built a gallery in the groimd at the base of Tripsacum. DISCUSSION When populations of D. quinquenotams maintained under experimental conditions in the greenhouse were allowed to develop without attending ants, they quickly overwhelmed and killed their Tripsacum host. Once leafhopper populations reached high numbers on leaves and stems, plants began to senesce and die. Similar drastic effects to host plants have been reported for other mymecophilous homopterans when ants were not present. High densities of the scale Pulvinariella mesembryanthemi (Vallot) stopped 72 LJ no foocj supplement 3 ■a cn protein CO 3 honey and protein m o c æ 3 cO' '3 CT (U E 3 c d) < Days Figure26. Average number of Dalbulus quinquenotatus adults when ants were maintained in three dietary conditions: in the first ants received no food supplement; in the second ants received protein; in the third ants received honey and protein. Bars indicate standard error. Each treatment had nine replicates (n=9). Data were taken every four days during 16 days. 73 180 n no food supplement .C Q. 160 E ^ protein >* c 140 I honey and protein cn 3 E 120 o c eu 3 100 a c '3cr 80 60 CD E 3 40 c D) > 20 < 0 D ays Figure 27. Average number of Dalbulus quinquenotatus nymphs when ants were maintained in three dietary conditions: in the first ants received no food supplement; in the second ants received protein; in the third ants received honey and protein. Bars indicate standard error. Each treatment had nine replicates (n=9). Data were taken every four days during 16 days. 74 40 ■ no food supplement □ protein S honey and protein « 30 - C < o XJ E 3 20 - 0 3 > < X i a i 1 2 1 6 D ays Figure 28. Average number of Formica subsericea found in screen cages with Dalbulus quinquenotatus when ants maintained under three dietary conditions: in the first ants received no food supplement; in the second ants received protein; in the third ants received hooey and protein. Bars indicate standard error. Each treatment had nine replicates (n=9). Data were taken every four days. 75 production of new leaves and shoots of the host plant when ants were absent (Washbum et al., 1985). In addition, the scale Coccus viridis (Green) caused significant leaf death to its host plant Pluchea indica (L.) compared to plants when the scale was unattended by Pheidole meeacephala (Fabr.) (Bach, 1991). Dalbulus gelbus affected Tripsacum growth but did not kill plants. Dalbulus gelbus colonies reduced the number of leaves and height of plants. These are the most common signs of plant stress reported for nonmyrmecophile Homoptera. For example, the scale Eriococcus coriaceus Maskell reduces the leaf area and height of its host. Eucalyptus blakelyi Maiden (Vranjic and GuUan, 1990). Similar results were found for planthopper Prokelisia dolus Wilson. This homopteran reduced the leaf production and tiller elongation of its host Spartina altemiflora Loisel (Olmstead et al., 1997). In some cases the height of the plant has been reported as the only negative effect produced by nonmyrmecophile Homoptera. This has been found in the aphid Schizaphis graminum (Rondani) feeding on A vena sativa L. (Gellner and Kieckhefer, 1992), in the leafhopper Empoasca fabae (Harris) feeding on soybean (Yeargan et al., 1994), and in the scale£ . coriaceus feeding on£. blakelyi (Vranjic and Gullan, 1990). Formica subsericea regulated D. quinquenotams populations to levels that were not harmful to its Tripsacum host. Similar results have been found with other myrmecophüe homopterans when attended by ants. For instance, the aphid Aphthargelia symphoricarpi was preyed upon by Formica fusca L. which reduced aphid numbers by 64 % (Andersen, 1991). The coccid Saissetia sp. was attacked, killed and removed by Oecophvlla longinoda (Latreille) when this coccid reached high densities (Way, 1963). By regulating 76 populations of attended homopterans, ants ensure that honeydew resources are sustained. Formica subsericea removed the honeydew produced bv D. quinquenotams on the upper surfaces of basal leaves of Tripsacum. The fivespotted gamagrass leafhopper prefers to oviposit in clusters on the upper surface of midribs of basal leaves of its host (Nault et al., 1983; Heady et al., 1985). Therefore, when honeydew is removed by ants, death of leafhopper eggs by suffocation with accumulated honeydew and sooty mold is prevented. Also, the presence of sooty mold reduces plant photosynthesis (Way, 1963). Other myrmecophile Homoptera experience similar benefits when ants remove honeydew. Such is the case with aphids attended by Lasius. coccids attended by Acropvga. and pseudococcids attended by Crematogaster (Way, 1963). On the other hand, when myrmecophile Homoptera are not attended by ants accumulation of honeydew occurs that directly affects the plant. For example, when the ant P. megacephala is excluded, the host plant P. indica has higher degrees of honeydew accumulation with resultant sooty mold growth and greater rates of leaf death (Bach, 1991). Formica subsericea. which is similar in size to Pheidole sp. observed in the field, did not remove the honeydew produced by D- quinquenotams nymphs on the inside sheath of Tripsacum basal leaves. This part of the leaf is tightly appressed to the stem and ants species with large heads cannot harvest the honeydew. However, B. obscurior. the small ant that attends the fivesppoted gamagrass leafhopper in the field, would not have problems harvesting the honeydew produced by nymphs in these .small microhabitats. Ants attending D. quinquenotatus in the field expelled D. gelbus and D. guzmani. Adults of these two species respond readily to mechanical stimuli (Heady and Nault, 1985) 77 and can avoid capture and predation by ants, especially on large plants. Large plants with more foliage can provide food and refuge to visiting herbivores high in the plant canopy. For example, damage by visitor herbivores occurred but was less on meristems where the membracid Aconophora teligera (Germar) was attended by ants than on meristems where the membracid and ants were absent (De assis Dansa and Duarte Rocha, 1992). Protection of the host plant by ants against herbivores has been observed in other membracids. For instance, the ants Formica spp. attacked adults of two species the chrysomelid beetles Trirhabda when they tried to feed on plants where the membracid Publilia concava (Say) is attended (Messina, 1981). Similar results have been reported with the membracid Vanduzea arquata Say, attended by f . subsericea. Here the host plant is protected against the locust leaf-mining beetle Odontota dorsalis (Thunb.)(Fritz, 1983). The complete protection of Tripsacum from D. gelbus by ants occurred under namral and controlled conditions when plants were small in size. Ants spend less energy to expel D. gelbus when Tripsacum is small, whereas ants must travel longer distances from D. quinquenotatus colonies at the plant base on large plants and spend more energy to expel this herbivore. Tripsacum is small during the dry season when plants can suffer severe damage if consumed by herbivores during this time. I found that ants not only expelled D. gelbus from small Tripsacum but also removed some D. quinquenotatus from overpopulated plants. This would reduce the impact ofD- quinquenotatus on plants dining the dry season which is critical to plant survival. Results obtained in this study support the hypothesis that ants protect quinquenotatus from arthropod predators. Exclusion experiments in Central Mexico 78 showed that ants protected the fivespotted gamagrass leafhopper in two Tripsacum habitats. Ant protection occurred in Agua Caliente where J . dactvloides burned during the dry season and in San Agustin where J . dactvloides had not burned. Although in both places n . quinquenotams were foimd in high number when attended and protected by £. obscurior. the fivespotted gamagrass leafhopper number was higher in the burned Tripsacum habitat. New stems and leaves from burned Tripsacum were greenest during the first days in May. Possibly, this was because ash accumulated on the soil surface and released nutrients. These new stems and leaves are unexploited resource for herbivores. Therefore, D quinquenotatus colonized and exploited these resources more effectively and had higher number on burned compared with imbumed T[. dactvloides. Similar results were reported by Moya-Raygoza (1995) where nymphs and adults ofD- quinquenotams were always more abundant in burned than unbumed T. dactvloides in Central Mexico. Nitrogen increases the number of D maidis (Power, 1989), and likely burned gamagrass could provide more nitrogen which increases the number ofD- quinquenotams as well. Not only ants protected D. quinquenotatus in different Tripsacum habitats but also in different Tripsacum species. Dalbulus quinquenotams was attended by ants on T. dactvloides in San Agustin and J . pilosum in Zapopan. Morphological and possibly biochemical differences between these two Tripsacum species are not obstacles to J2- quinquenotams colonization and their attendance by ants. Tripsacum pilosum is larger than I . dactyloides and the sheath, the oviposition site o f£ . quinquenotams (Heady et al., 1985), is heavily covered with trichomes (Randolph, 1970). Even with these differences between host plants, D- quinquenotams is attended by ants. Ants have been observed 79 attending D quinquenotatus on all Tripsacum species it uses as a host (L. R. Nault, unpublished). The fivespotted gamagrass leafhopper has been found on T. dactvloides. T. pilosum. I . zopilotense Hernandez and Randolph, J . lanceolatum Ruprecht ex Fournier, T. latifolium Hitchcock, I . laxum Nash, and J . manisuroides de Wet and Harlan (Triplehom and Nault, 1985). Dalbulus quinquenotams is protected by a single or several ant species over time in the field. When this leafhopper is attended by several ant species, displacement among them occurs. Displacement among ant species suggests competition fbrD- quinquenotams as a source of food. Observations suggest that interspecific competition between ant species involves chemical warfare. Pheidole sp. workers were observed in Zapopan raising their abdomen and producing droplets firom the anus to repel Camponoms sp. Those droplets likely contain allomone produced in the pygidial gland of Pheidole when they compete for territory or food (Holldobler and Wilson, 1990). Interspecific ant competition likely results because honeydew produced by myrmecophile Homoptera is a rich source of sugars (fructose, glucose, saccharose, and trehalose), amino acids, vitamin B, lipids, and minerals (Way, 1963; Strong, 1965; Holldobler and Wilson, 1990; Douglas, 1993). Barchymyrmex obscurior was the only ant species that attended D. quinquenotams in Agua Caliente and San Agustin. This small ant species occurred in high numbers in those sites. Theoretically fi. obscurior needs less honeydew than larger species to feed its colony. Dreising (1988) concluded that the ingestion rate of honeydew by ants increases with the body size of the ant, and the ingestion rate decreased when ants forage in high numbers. 80 Ants protected D . quinquenotatus against spiders in the field. The rapid appearance of spiders in unattended D quinquenotatus may be that honeydew attracts not only ants but also spiders. The spider Hibana velox (Becker) was observed feeding on extrafloral nectaries in the base of Hibiscus tiliaceus L. leaf at night (Taylor and Foster, 1996). These nectaries, like honeydew, provide water, sugars, amino acids, lipids and vitamins that attract visitors. Four spider taxa were found with unattended D- quinquenotatus on burned Tripsacum (Agua Caliente). This new open gamagrass habitat may favor the colonization of spiders firom different groups. A similar pattern has been reported in other burned perennial habitats. For instance, in the burned wetland dominated by Spartina (Poaceae), a greater species richness of spiders was reported than in unbumed wetlands. These burned plants are more productive and have more sap feeding insects, which are controlled by those spiders (Johnson, 1995). Spiders are generalist arthropods predators and the six spider taxa collected in this smdy must compete with ants to obtain quinquenotams. Such competition between spiders and ants for aphids has been reported (Halaj et al., 1997). Spiders observed in this study are not ant mimics. Myrmecomorphy is a convergent mechanism of evolution, which is common in spiders that attack myrmecophilous Homoptera and facilitate spider attack on Homoptera without detection by ants. For example, the ant-like spider Micaria sp. is a common predator of myrmecophile aphids on sagebrush. This spider steals aphids attended by Formica (Mclver and Stonedahl, 1993). Formica subsericea protected D. quinquenotatus firom the predator N. americoferus in greenhouse studies. This predator not only was expelled by ants when they tried to approach D- quinquenotams but also were captured and transported to the ant nest and 81 presumably used as food. In other studies predators avoid but are not consumed by ants when they try to attack myrmecophile Homoptera. For example. Banks and Macaulay (1967) found that the predator coccinellid Adalia bipunctata (L.) attacked the aphid Aphis fabae Scop, at low rates because of protection by the ant Lasius niger L.. In addition. Way (1963) mentioned that L- niger quickly repelled and carried off predators from aphid aggregations. Possibly, H- americoferus was captured and consumed by £ . subsericea in this study because the experiment was conducted in closed cages where americoferus could not escape from ants. Several studies have reported that ants require animal protein to complete their nutritional requirements (HoUdobler and Wilson, 1990). Predation by F. subsericea not only was found against M- americoferus but also against D. quinquenotams. This occurred only when large f . subsericea colonies, but not small colonies, were used in greenhouse smdies. Results from this study showed that large colonies of F. subsericea destroyedJD. quinquenotams population (nymphs and adults) when not supplied with protein. Perhaps large colonies of ants must obtain more protein to maintain their association with j). quinquenotams. Large colonies of Formica exsectoides Forel consume more protein and less honey compared with small colonies, which consume less protein and more honey (Ayre, 1966). Independent of ant colony size, ants need a balanced diet with essential amino acids. Ants obtain limited amino acids from honeydew. These amino acids, glutamic acid, glutamine, asparagine, and serine are nonessentials (Douglas, 1993), and represent less than 0.7% of concentration in the honeydew (Gray, 1952). Therefore, if no other source of protein is available, ants will consume attended Homoptera to meet this 82 requirement. No studies have explored the protein requirements for ants attending myrmecophilous Homoptera. However, several studies have found that a supplement of protein has direct effect in ant colony growth. For example, a Formica colony becomes larger when fed protein (Ayre, 1966). A similar effect in colony growth was found in Solenopsis invicta Buren. Its colony grew larger and maintained a larger biomass with increasing cricket (protein) availability (Porter, 1989; Macom and Porter, 1995). In addition, protein deficiency affects the production of reproductive castes. Male larvae of Iridomyrmex humilis (Mayr) fail to pupate when deprived of protein (Passera et al., 1988). Protein deficiency not only affects colony growth but also may alter the behavior of workers. Buschinger and Pfeifer (1988) suggest that Harpagoxenus sublaevis (Nyl.) and Leptothorax acervorum (Fabr.) intensify their search for food when protein deficiency occurs. Sugar supplements in the form of honey dramatically altered the response ofJF. subsericea to D. quinquenotams. When supplied with protein and honey, ants made few visitations to leafhopper host plants. In contrast, when ants were supplied only with protein, the numbers of ant visitations to host plants was significantly higher. Moreover, visitations and attendance benefitted populations of leafhopper adults and nymphs. However in smdies with other Homopterous, sugar supplements do not alter ant attendance. For example, the membracid Guavaquila was attended bv Camponoms spp. even when an alternative sugar source was on the host plant Didymopanax and it did not interfere with attendance by ants (Del-Claro and Oliveira, 1993). Similarly extrafloral 83 nectaries did not affect ant attendance of myrmecophiles as reported by Fiala (1990). Consequently, ants may obtain sugar from other sources such as extrafloral nectaries and still attend D- quinquenotams in natural conditions. Some plant species reported with extrafloral nectaries in Panamanian forest such as Acacia and Ipomoea (Schupp and Feener, 1991) occur in the same habitat where Tripsacum grows (Moya-Raygoza and Trujillo-Arriaga, 1993). Formica subsericea attended quinquenotatus in high numbers when the ant colonies only received protein but no honeydew. This number found under laboratory conditions was similar to ant numbers that attended D. quinquenotams in Zapopan. Ants likely increased their attending numbers because the honeydew produced by the fivespotted gamagrass leafhopper was their only carbohydrates resource. Carbohydrate resources are important to workers because they provide fuel for activity (Deslippe and Savolainen, 1994; Macom and Porter, 1995). Formica subsericea. when supplied with protein, built galleries in the ground at the base of Tripsacum. Those galleries likely are used by ants to avoid the heat of the day as found in other Formica species by Seibert (1992), who observed Formica obscuripes Forel building galleries in the ground at the base of the plant where the aphid Lachnus allegheniensis McCook was attended. Larsen et al. (1991) concluded from field observations that D. quinquenotams has a facultative association with ants. The leafhopper can be maintained on seedling maize for many generations over several years in the laboratory. Later, following intensive smdies of interactions with ants and leafhoppers in the laboratory, Larsen et al. (1992) suggested that the relationships could be obligatory. From my smdies, I conclude that J3. 84 quinquenotatus requires ants for its survival and that the leafhopper has an obligatory relationship with ants. In controlled experiments in the greenhouse, colonies of quinquenotatus eventually overpopulated and killed their gamagrass host plants. When ants are present this does not happen. Ants remove contaminating honeydew from leaf surfaces and regulate leafhopper populations to levels that do not cause serious damage to the gamagrass host. In the field, exclusion of ants from gamagrass leaves D. quinquenotams vulnerable to attack from predators that extinguish leafhopper populations. It would seem that D. quinquenotams is totally dependent on ants for its existence. A schematic diagram (Figure 29) summarizes the interactions among D- quinquenotams. gamagrass and the ant species associated with the gamagrass habitat. 85 Figure 29. Beneficial effects o f the Dalbulus quinquenotatus/ ants relationships. 86 *n f GAMAGRASS food (honeydew + prey) LEAFHOPPER ______\ A N T S (Dalbulus quiuquenolatus) ^ remove honeydew (planl) (many species) predator protection expel herbivores population regulation other habitats and food sources LIST OF REFERENCES Alivizatos, A.S., and P.O. Markham. 1986. Acquisition and transmission of com stunt spiroplasma by its leafhopper vector Dalbulus maidis. Ann. appl. Biol. 108: 535-544. Andersen, M. 1991. An ant-aphid interaction: Formica fusca and Aphthargelia svmphoricarpi on Mount st. Helens. Am. Midi. Nat. 125: 29-36. Ayre, G.L. 1966. Colony size and food consumption of three species of Formica. Ent. exp. & appl. 9: 461-467. Bach, G.E. 1991. Direct and indirect interactions between ants fPheidoIe megacephaial. scales rCoccus viridis) and plants fPIuchea indical. Oecoiogia 87: 233-239. Banks, C.J., and E.D.M. Macaulay. 1967. EGects of Aphis fabae Scop, and of its attendant ants and insect predators on yields of field beans (Vicia faba L.). Ann. app. Biol. 60:445- 453. Banttari, E.E. 1966. Grass host of aster yellows virus. Plant Dis. Rep. 50: 17-21. Bartlett, B.R. 1961. The influence o f ants upon parasites, predators, and scale insects. Ann. Entomol. Soc. Am. 54: 543-551. Bristow, C. 1984. Differential benefits firom ant attendance to two tropical species of homopterans of New York ironweed. J. of Animal Ecology 53: 715-726. Buschinger, A., and E. Pfeifer. 1988. Effects of nutrition on brood production and slavery in ants (Hymenoptera, Formicidae). Insectes Sociaux 35: 61-69. Chapman, R.K. 1973. Fruit and vegetable insects. Proc. North Cent. Branch Entomol. Soc. Am. 28: 38. Chiykowski, L.N. 1977. Reduction in the transmissibility of a greenhouse-maintained isolate of aster yellows agent. Can. J. Bot. 55: 1783-1786. 88 Chiykowski, L.N. 1991. Vector-pathogen-host plant relationships of clover phyllody mycoplasmalike organism and the vector leafhopper Paraphlepsius irroratus. Can. J. of Plant Pathol. 13: 11-18. Davis, R.E. 1973. Occurrence of a spiroplasma in com stunt-infected plants in Mexico. Plant Dis. Rep. 57: 333-337. Davis, R.E. 1977. Spiroplasma: Role in the diagnosis of com stunt disease. In: L. E. Williams, D.T. Gordon and L.R. Nault, eds., Proc. Maize Virus Dis. Coloq. and Workshop. Ohio Agric. Res. and Dev. Center, Wooster, Ohio. De assis Dansa, C.V., and C.F. Duarte Rocha. 1992. An ant-membracid-plant interaction in a cerrado area of Brazil. J. of Tropical Ecology 8:339-348. Del-Claro, K., and P.S. Oliveira. 1993. Ant-Homoptera interaction: do altemative sugar sources distract tending ants ?. Oikos 68: 202-206. Deslippe, R.J., and R. Savolainen. 1994. Role of food supply in structuring a population o f Formica ants. J. of Animal Ecology 63: 756-764. Douglas, A.E. 1993. The nutritional quality of phloem sap utilized by natural aphid populations. Ecological Entomology 18: 31-38. Dreising, H. 1988. Foraging rate of ants collecting honeydew or extrafloral nectar, and some possible constraints. Ecological Entomology 13: 143-154. Ebbert, M.A., and L.R. Nault. 1994. Improved overwintering ability in Dalbulus maidis (Homoptera: Cicadellidae) vectors infected with Spiroplasma kunkelii (Mycoplasmatales: Spiroplasmataceae). Environ. Entomol. 23: 634-644. Errampalli, D., J. Fletcher, and P.L. Claypool. 1991. Incidence of yellows in carrot and lettuce and characterization of mycoplasmalike organism isolates in Oklahoma. Plant Dis. 75: 579-584. Fiala, B. 1990. Extrafloral nectaries vs ant-Homoptera mutualisms: a comment on Becerra and Venable. Oikos 59: 281-282. Fritz, R.S. 1982. An ant-treehopper mutualism: effects of Formica subsericea on the survival of Vanduzea arquata. Ecological Entomology 7: 267-276. Fritz, R.S. 1983. Ant protection of a host plant’s defoliator: consequence of an ant- membracid mutualism. Ecology 64: 789-797. 89 Gellner, J.L., and R.W. Kieckhefer. 1992. Response in oat to infestation at the seedling growth stage by two species of cereal aphids (Homoptera: Aphididae). J. Kansas Entomol. Soc. 65: 141-145. G old, R.E., and E.S. Sylvester. 1982. Pathogen strains and leafhopper species as factors in the transmission of Wester X-disease agent under varying light and temperature conditions. Hilgardia 50: 1-43. Granados, R.R., and R.K. Chapman. 1968. Identification of some new aster yellows virus strains and their transmission by the aster leafhopper, Macrosteles fascifrons. Phytopathology 58: 1685-1692. Gray, R.A. 1952. Composition of honeydew excreted by pineapple mealybugs. Science 115: 129-133. Halaj, J., D.W. Ross, and A.R. Moldenke. 1997. Negative effects of ant foraging on spiders in douglas-fir canopies. Oecoiogia 109: 313-322. Hanks, L.M., and C.S. Sadof. 1990. Host tree selection by the neotropical ant Paraponera clavata (Hymenoptera: Formicidae). Biotropica 22: 213-214. Harrison, N.A., P.A. Richardson, J.H. Tsai, M.A. Ebbert, and J.B. Kramer. 1996. PCR assay for detection of the phytoplasma associated with maize bushy stunt disease. Plant. Dis. 80: 263-269. Heady, S.E., and L.R. Nault. 1985. Escape behavior of Dalbulus and Baldulus leafhoppers (Homoptera: Cicadellidae). Environ. Entomol. 14: 154-158. Heady, S.E., L.V. Madden, and L.R. Nault. 1985. Oviposition behavior of Dalbulus leafhoppers (Homoptera: Cicadellidae). Ann. Entomol. Soc. Am. 78: 723-727. HoUdobler, B., and E.O. Wilson. 1990. The ants. Bemap. Harvard University Press, Cambridge, Massachusetts. Hsu, T. P., and E.E. Banttari. 1979. Dual transmission of the aster yellows mycoplasmalike organism and the oat blue dwarf virus and its effect on longevity and fecundity of the aster leafhopper vector. Phytopathology 69: 843-845. Jensen, D.D. 1968. Influence of high temperature on the pathogenicity and survival of Western X-disease virus in leafhoppers. Virology 36: 662-667. Jensen, D.D. 1972. Temperature and transmission of the Western X-disease agent by Colladonus montanus. Phytopathology 62: 452-456. 90 Johnson, S.R. 1995. Spider communities in the canopies of annually burned and long-term unbumed Spartina pectinata wetlands. Environ Entomol. 24: 832-834. Kunkel, L.O. 1937. Effect of heat on ability of Cicadula sexnotata (Fall.) to transmit aster yellows. Am. J. Bot. 24: 316-327. Kunkel, L.O. 1953. Aster yellows. In Plant Diseases. U.S. Dept, of Agr., 1953 Yearbook of Agriculture. Kuske, C.R., and B.C. Kirkpatrich. 1992. Distribution and multiplication of western aster yellows mycoplasmalike organisms in Catharanthus roseus as determined by DNA hybridization analysis. Phytopathology 82:457-462. Larsen, K.J., F.£. Vega, G. Moya-Raygoza, and L.R. Nault. 1991. Ants (Hymenoptera: Formicidae) associated with the leafhopper Dalbulus quinquenotatus (Homoptera: Cicadellidae) on gamagrasses in Mexico. Ann. Entomol. Soc. Am. 84: 498-501. Larsen, K.J., S.E. Heady, and L.R. Nault 1992. Influence of ants (Hymenoptera: Formicidae) on honeydew excretion and escape behaviors in a myrmecophile, Dalbulus quinquenotatus (Homoptera: Cicadellidae), and its congeners. J. of Insect Behavior 5: 109-122. Lee, I.-M., R.W. Hammond, R.E. Davis, and D.E. Gundersen. 1993. Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms. Phytopathology 83: 834-842. Macom, T.E., and S.D. Porter. 1995. Food and energy requirements of laboratory fire ant colonies (Hymenoptera: Formicidae). Environ. Entomol. 24: 387-391. Madden, L.V., and L.R. Nault. 1983. Differential pathogenicity of com stunting moUicutes to leafhopper vectors in Dalbulus and Baldulus species. Phytopathology 73: 1608- 1614. Madden, L.V., R. Louie, J.J. Abt, and J.K. Knoke. 1982. Evaluation of tests for randomness of infected plants. Phytopathology 72: 195-198. Madden, L.V., L.R. Nault, S.E. Heady, and W.E. Styer. 1984. Effect of maize stunting moUicutes on survival and fecundity of Dalbulus leafhopper vectors. Ann. appl. Biol. 105: 431-441. Madden, L.V., L.R. Nault, S.E. Heady, and W.E. Styer. 1986. Effect of temperature on the population dynamics of three Dalbulus leafhopper species. Ann. appl. Biol. 108: 475- 485. 91 Mclver, J.D., and G. Stonedahl. 1993. Myrmecomorphy: Morphological and behavioral mimicry of ants. Annu. Rev. Entomol. 38: 351-379. Messina, F.J. 1981. Plant protection as a consequence of an ant-membracid mutualism: interactions on goldenrod fSolidago sp.). Ecology 62: 1433-1440. Moya-Raygoza, G. 1995. Fire effects on insects associated with the gamagrass Tripsacum dactvloides in Mexico. Ann. Entomol. Soc. Am. 88: 434-440. Moya-Raygoza, G., and J. Trujillo-Arriaga. 1993. Evolutionary relationships between Dalbulus leafhopper (Homoptera: Cicadellidae) and its dryinid (Hymenoptera: Dryinidae) parasitoids. J. Kansas Entomol. Soc. 66: 41-50. Murral, D.J., L.R. Nault, C.W. Hoy, L.V. Madden, and S.A. Miller. 1996. Effects of temperature and vector age on transmission of two Ohio strains of aster yellows phytoplasma by the aster leafhopper (Homoptera: Cicadellidae). J. Econ. Entomol. 89: 1223-1232. Nault, L.R. 1980. Maize bushy stunt and com stunt: A comparison of disease symptoms, pathogen host ranges, and vectors. Phytopathology 70: 659-662. Nault, L.R. 1983. Origins in Mesoamerica of maize viruses and mycoplasmas and their leafhopper vectors. In: R.T. Plumb and J.M. Thresh, eds.. Plant virus epidemiology: The spread and control of insect-bome viruses., Blackwell Scientific Pub., Ltd., Oxford, England. Nault, L.R. 1990. Evolution of an insect pest: Maize and the com leafhopper, a case study. Maydica 35: 165-175. Nault, L.R. 1994. Transmission biology, vector specificity, and evolution of planthopper- transmitted plant viruses. In: R.F. Denno and T.J. Perfect, eds., Planthoppers; their ecology and management. Chapman & Hall, New York. Nault, L.R., D.T. Gordon, R.E. Gingery, O.E. Bradfiite, and J. Castillo Loayza. 1979. Identification of maize viruses and moUicutes and their potential insect vectors in Pern. Phytophatology 69: 824-828. Nault, L.R., D.T. Gordon, and J. Castillo Loayza. 1981. Maize virus and mycoplasma diseases in Pern. Trop. Pest. Manag. 27: 363-396. 92 Nault, L.R., DM . DeLong, B.W. Triplehorn, W.E. Styer, and J.F. Doebiey. 1983. More on the association of Daibujus (Homoptera: Cicadellidae) with mexican Tripsacum (Poaceae), including the description of two new species of leafhoppers. Ann. Entomol. Soc. Am. 76: 305-309. Novak, H. 1994. The influence of ant attendance on larval parasitism in hawthorn psyllids (Homoptera; Psyllidae). Oecoiogia 99: 72-78. Okuda, S., J.P. Prince, R.E. Davis, E.L. Dally, I.-M. Lee, B. Mogen, and S. Kato. 1997. Two groups of phytoplasmas from Japan distinguished on the basis of amplification and restriction analysis of 16S rDNA. Plant Dis. 81: 301-305. Olmstead, K.L., R.F. Denno, T.C. Morton, and J.T. Romeo. 1997. Influence of Prokelisia planthoppers on amino acid composition and growth of Spartina altemiflora. J. of Chemical Ecology 23: 303-321. Passera, L., L. Keller, and J.P. Suzzoni. 1988. Control of brood male production in the argentine ant Iridomvrmex humilis (Mayr). Insects Sociaux 35: 19-33. Peterson, A.G. 1973. Host plant and aster leafhopper relationships. Proc. North Cent. Branch Entomol. Soc. Am. 28: 66-70. Porter, S.D. 1989. Effects of diet on the growth of laboratory fire ant colonies (Hymenoptera: Formicidae). J. Kansas Entomol. Soc. 62: 288-291. Power, A.G. 1989. Influence of plant spacing and nitrogen fertilization in maize on Dalbulus maidis (Homoptera: Cicadellidae), vector of com stimt. Environ. Entomol. 18: 494- 498. Randolph, L.F. 1970. Variation among Tripsacum populations of Mexico and Guatemala. Brittonia 22: 305-337. Seibert, T.F. 1992. Mutualistic interactions of the aphid Lachnus allegheniensis (Homoptera: Aphididae) and its tending ant Formica obscuripes fHvmenoptera: Formicidae). Ann. Entomol. Soc. Am. 85: 173-178. Schupp, E.W., and D.H. Feener. 1991. Phylogeny, lifeform, and habitat dependence of ant- defended plants in a Panamanian forest. In: C.R. Huxley and D.F. Cutler, eds., Ant-Plant interactions, Joshua Associates Ltd, Oxford, England. Sinha, R.C., and L.N. Chiykowski. 1967. Initial and subsequent sites of aster yellows virus infection in a leafhopper vector. Virology 33: 702-708. 93 Strong, F.E. 1965. Detection of lipids in the honeydew of an aphid. Nature 205; 1242. Swenson, K.G. 1971. Relation of age, sex, and mating of Macrosteles fascifrons to transmission of aster yellows. Phytophatology 61: 657-659. Taylor, R.AJ., L.R. Nault, and W.E. Styer. 1993. Experimental analysis of flight activity of three Dalbulus leafhoppers (Homoptera: Auchenorrhyncha) in relation to migration. Ann. Entomol. Soc. Am. 86: 655-667. Taylor, RAl., and W.A. Foster. 1996. Spider nectarivory. American Entomologist 42: 82-86. Triplehom, B.W., and L.R. Nault. 1985. Phylogenetic classification of the genus Dalbulus (Homoptera: Cicadellidae), and notes on the phylogeny of the Macrostelini. Ann. Entomol. Soc. Am. 78: 291-315. Vinson, S.B., and T.A. Scarborough. 1991. Interactions between Solenopsis invicta (Hymenoptera: Formicidae), Rhopalosiphum maidis (Homoptera: Aphididae), and the parasitoid Lvsiphlebus testaceipes Cresson (Hymenoptera: Aphidiidael. Ann. Entomol. Soc. Am. 84: 158-164. Vranjic, J.A., and P.J. Gullan. 1990. The effect of a sap-sucking herbivore, Eriococcus coriaceus (Homoptera: Eriococcidae), on seedling growth and architecture in Eucalvptus blakelvi. Oikos 59; 157-162. Washburn, J.O., G.W. Frankie, and J.K Grace. 1985. Effects of density on survival, development, and fecundity of the soft scale, Pulvinariella mesembrvanthemi (Homoptera: Coccidae), and its host plant. Environ. Entomol. 14: 755-761. Way, M.J. 1963. Mutualism between ants and honeydew-producing homoptera. Ann. Rev. Entomol. 8: 307-344. Yeargan, K.V., S.K. Braman, and W.E. Barney. 1994. Effects of potato leafhoppers on soybean plant growth and yield. J. Kansas Entomol. Soc. 67: 29-36. 94