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Diapause and nutritional influences on reproduction and diapause induction in the elm leaf beetle,Xanthogaleruca luteola (Muller) (Coleoptera:Chrysomelidae)
Young, Curtis Eugene, Ph.D.
The Ohio State University, 1991
UMI 300 N. Zeeb R& Ann Arbor, MI 48106
DIAPAUSE AND NUTRITIONAL INFLUENCES ON REPRODUCTION AND
DIAPAUSE INDUCTION IN THE ELM LEAF BEETLE,
XANTHOGALERUCA LUTEOLA (MULLER)
(COLEOPTERA: CHRYSOMELIDAE)
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Curtis Eugene Young, B.S., M.S.
* * * * *
The Ohio State University
1991
Dissertation Committee: Approved by D. L. Denlinger iU lq R. W. Hall Adviser Department of Entomology D. J. Horn
C. A. Triplehorn Copyright by Curtis Eugene Young 1991 To my wife, Linda Mull Young, B.A., M.S., Ph.D. and my daughter, Kaitlin Christine Young.
ii ACKNOWLEDGMENTS
I would like to extend my most sincere gratitude to my advisor, Dr. Richard W. Hall for his guidance through my studies and research work at The Ohio State University. I also wish to thank him for his patience in waiting for the completion of this dissertation. Additional thanks are extended to Dr. Hall for the use of his laboratory space, equipment and supplies and periods of financial support.
I wish to express my sincere appreciation to my general examination committee, Drs. Ralph E. Boerner, David
L. Denlinger, R.W. Hall and David J. Horn, and to my final oral examination committee, who also reviewed the dissertation manuscript, Drs. D.L. Denlinger, R.W. Hall,
D.J. Horn and Charles A. Triplehorn. The financial support from The Ohio State University through graduate teaching and research associateships and a Dean's graduate research fellowship is gratefully acknowledged.
I wish to thank my friends and co-workers, Michael R.
Hamerski and Brian A. Giles for their support and help while we shared the same laboratory space, equipment and supplies and office space at The Ohio State University.
iii Finally, to my wife, Linda, I offer my sincerest gratitude for her encouragement and patience while I fretted over the completion of this dissertation.. Also, for her unwavering love, I am eternally grateful. VITA
June 6, 1959 ...... Born - Pittsburgh, Pennsylvania
1981...... B.S., Edinboro State College of Pennsylvania, Edinboro, Pennsylvania Biology
1985...... M.S., The Ohio State University, Columbus, Ohio Entomology
1985 - 1988 ...... Research and Teaching Associate, Department of Entomology, The Ohio State University, Columbus, Ohio
1988 ...... Control Entomologist, Ohio Department of Health - Vector Borne Disease Unit, Columbus, Ohio
1989 ...... Ornamentals Extension Entomologist, Ohio Cooperative Extension Service, The Ohio State University, Columbus, Ohio
1988 - Present ...... Instructor of Biology, Department of Biological Sciences, Ohio Northern University, Ada, Ohio
v PUBLICATIONS
Hall, R. W. , D. G. Nielsen, C. E. Young, and M. R. Hamerski. 1988. Mortality of elm leaf beetle (Xanthoaaleruca luteola)(Coleoptera: Chrysomelidae) larvae exposed to insecticide bands applied to elm bark. J. Econ. Entomol. 81: 877-879.
Hall, R. W . , D. G. Nielsen, C. E. Young, and M. R. Hamerski. 1990. Insecticidal bark bands for reduction of defoliation by elm leaf beetle (Coleoptera: Chrysomelidae). J. Environ. Hort 8: 61-63.
Hall, R. W . , and C. E. Young. 1986. Suitability of three Asiatic elms to elm leaf beetle (Xanthoaaleruca luteola)(Coleoptera: Chrysomelidae). J. Environ. Hort. 4: 44-46.
Young, C. E., and R. W. Hall. 1986. Factors influencing suitability of elms for elm leaf beetle, Xanthoaaleruca luteola (Muller) (Coleoptera: Chrysomelidae). Environ. Entomol. 15: 843-849.
Young, C. E. 1985. Suitability of elms for elm leaf beetle, Xanthoaaleruca luteola (Muller) (Coleoptera: Chrysomelidae). M.S. Thesis, The Ohio State University, Columbus.
FIELDS OF STUDY
Major Field: Entomology
Insect/plant interactions - Dr. R. W. Hall TABLE OF CONTENTS
DEDICATION ...... ii
ACKNOWLEDGMENTS...... iii
VITA ...... v
LIST OF TABLES ...... ix
LIST OF FIGURES ...... xii
INTRODUCTION ...... 1
CHAPTER PAGE
I. DIAPAUSE IN THE ELM LEAF BEETLE, XANTHOGALERUCA LUTEOLA (MULLER) (COLEOPTERA: CHRYSOMELIDAE) WITH REFERENCE TO SEASONAL ANATOMICAL CHANGES . 16
Introduction ...... 16 Materials and Methods ...... 19 Results and Discussion ...... 25 List of References ...... 45
II. LARVAL HOST INFLUENCE ON OVARIOLE NUMBER AND FECUNDITY OF THE ELM LEAF BEETLE, XANTHOGALERUCA LUTEOLA (COLEOPTERA: CHRYSOMELIDAE) ...... 63
Introduction ...... 63 Materials and Methods ...... 65 R e s u l t s ...... 68 D i s c u s s i o n ...... 70 List of References ...... 73
vii III. ADULT HOST INFLUENCE ON DIAPAUSE INDUCTION IN THE ELM LEAF BEETLE, XANTHOGALERUCA LUTEOLA (MULLER) (COLEOPTERA: CHRYSOMELIDAE) ...... 91
Introduction ...... 91 Materials and Methods ...... 94 Results and Discussion ...... 101 List of References ...... Ill
CONCLUSION ...... 129
APPENDIX ...... 134
LIST OF REFERENCES ...... 143
viii LIST OF TABLES
TABLE PAGE
1. Variation in the accessory gland development in overwintered male and ovarian development and mated condition of overwintered female X. luteola adults collected from U. pumila. April and May 1988...... 48
2. Mean percent lipid content of dry weight of reproductively-active and diapause-destined male and female X. luteola adults of different ages reared under long day (15:9 L:D), short day (12:12 L;D) at 25 C or ambient field conditions, autumn 1986 ...... 49
3. Results of one-tailed paired t' tests comparing mean percent lipid contents of dry weight of reproductively-active and diapause- destined male and female X. luteola adults sampled at 1, 2, and 3 weeks of ag e ...... 50
4. Number of ovarioles in female X* luteola reared on different elm species as larvae and fed U. pumila as adults, February 1987...... 75
5. Oviposition of X» luteola reared on different elm species as larvae and fed U. pumila as adults, February 1987 ...... 76
6. Adult X- luteola standard synthetic diet ingredients...... 114
7. Number of diapausing and non-diapausing adult X. luteola fed lush-green actively- growing ("high" quality) or tough-bronzed senescing ("low" quality) U. pumila foliage for 14 days after eclosion under 15:9 L:D at 25°C...... 115
ix 8. Number of diapausing and non-diapausing adult X* luteola fed U. pumila foliage that was actively-growing, senescing from the field or senescing from the greenhouse for 14 days after eclosion under 15:9 L:D at 25°C...... 116
9. Percent leaf water content, leaf protein content and soluble leaf carbohydrate content of JJ. pumila foliage of different conditions...... 117
10. Percent leaf water content and leaf protein content of 'Urban' elms under different fertilization treatments...... 118
11. Number of diapausing and non-diapausing adult X* luteola reared under 15:9 L:D at 25°C and fed for 14 days after eclosion on foliage from 'Urban' elms conditioned with different fertilization treatments...... 119
12. Percent diapause induction in male and female X* luteola reared under 15:9 L:D at 25°C and starved for 0, 1, 2, 3, 4 or 5 days after eclosion then fed U. pumila foliage . . . 120
13. Percent diapause induction in male and female X« luteola reared under 15:9 L:D at 25°C and starved for 1, 2, 3, 4, 5 or 6 days after eclosion then fed 30% protein diet or were alternatively starved and fed 30% protein diet over a 2 week period ...... 121
14. Mean percent diapause induction in female and male X* luteola fed foliage or artificial diets with different levels of protein supplements (%) and flavonols (g) and exposed to 15:9 L:D or 12:12 L:D at 25°C. . . . 122
15. Percent mortality in X* luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) U. pumila and exposed to different photoperiods at 25°C, July 1986 ...... 135
x 16. Percent diapause induction in £. luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) U. pumila and exposed to different photoperiods at 25°C, July 1986 . . . 136
17. Percent mortality in X. luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) ' Urban' elms and exposed to different photoperiods at 25t>C, August 1986 . . 139
18. Percent diapause induction in X. luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) 'Urban' elms and exposed to different photoperiods at 25°C, August 1986 . . 140
xi LIST OF FIGURES
FIGURE PAGE
1. Mean percent lipid content of dry weight of reproductively-active and diapause-destined male and female X* luteola adults of different ages reared under long day (15:9 L:D), short day (12:12 L:D) at 25°C or ambient field conditions, autumn 1986. Bars within clusters, designated by the same letter are not significantly different (P = 0.05; SNK preceded by a one-way ANOVA)...... 51
2 . Percentage diapause induction in groups of X« luteola which were transferred to alternate photoperiods (upper panel, 15:9 L:D -> 12:12 L:D & lower panel, 12:12 L:D -> 15:9 L:D) on designated days following pupation and eclosion at 25°C. Bars at each end of figure represent groups which were held under one photoperiod throughout the experiment ...... 53
3. Replication 2 percentage diapause induction in groups of X* luteola which were transferred to alternate photoperiods (upper panel, 15:9 L:D -> 12:12 L:D & lower panel, 12:12 L:D -> 15:9 L:D) on designated days following eclosion at 25°C. Bars at each end of figure represent groups which were held under one photoperiod throughout the experiment...... 55
Replication 3 percentage diapause induction in groups of X. luteola which were transferred to alternate photoperiods (upper panel, 15:9 L:D -> 12:12 L:D & lower panel, 12:12 L:D -> 15:9 L:D) on designated days following eclosion at 25°C. Bars at each end of figure represent groups which were held under one photoperiod throughout the experiment...... 57
xii 5. Laboratory photoperiodic response curve for diapause induction in £. luteola from Columbus, Ohio...... 59
6. Field photoperiodic response curve for diapause induction (*) in luteola from Columbus, Ohio, July and August 1986. The dates correspond to the day of eclosion and placement in the field. The hours of light/day values (a) correspond to the midweek daylength experienced by the sample of adults placed in the field at each date. . . 61
7. Frequency distribution of ovariole numbers per ovary in X. luteola reared as larvae on U. stilssniana# 'Urban elm, or a. pumila. February 1987 ...... 77
8. Relationship between the ovary with the maximum number of ovarioles and the ovary with the minimum number of ovarioles within female X* luteola reared on U. wilsoniana as larvae and fed U. pumila as adults. The r value is statistically significant at the 5% level...... 79
9. Relationship between the ovary with the maximum number of ovarioles and the ovary with the minimum number of ovarioles within female luteola reared on 'Urban' elm as larvae and fed U. pumila as adults. The r value is statistically significant at the 5% level...... 81
10. Relationship between the ovary with the maximum number of ovarioles and the ovary with the minimum number of ovarioles within female X. luteola reared on H. pumila as larvae and fed U. pumila as adults. The r value is statistically significant at the 5% level...... 83
11. Frequency distribution of ovariole numbers per female in luteola reared as larvae on U. wilsonianaf /Urban/ elm, or U. pumila. February 1987 ...... 85
xiii 1 2 . Relationship between the number of eggs deposited in the first 7 days of oviposition and the number of ovarioles/female X. luteola. The r value is statistically significant at the 5% level ...... 87
13. Relationship between the number of eggs deposited in the first 14 days after adult eclosion and the number of ovarioles/female X. luteola. The r value is statistically significant at the 5% level ...... 89
14. Percent diapause induction in female and male X. luteola fed artificial diets with three levels of protein supplements and added flavonols or foliage for 14 days after adult eclosion under 15:9 L:D at 25°C and 2 levels of protein supplement and flavonols or foliage under 12:12 L:D at 25°C...... 123
15. Percent diapause induction in female X* luteola fed artificial diets with three levels of protein supplements and added flavonols or foliage for 14 days after adult eclosion under 15:9 L:D at 25°C and 2 levels of protein supplement and flavonols or foliage under 12:12 L:D at 2 5 ° C ...... 125
16. Percent diapause induction in male X* luteola fed artificial diets with three levels of protein supplements and added flavonols or foliage for 14 days after adult eclosion under 15:9 L:D at 25°C and 2 levels of protein supplement and flavonols or foliage under 12:12 L:D at 2 5 ° C ...... 127
17. Percent mortality and percent diapause induction in X* luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) U. pumila and exposed to photoperiods of (1) 15:9 L:D, (2) 14.25:9.75 L:D, (3) 13.75:10.25 L:D or (4) 12:12 L:D at 25°C, July 1986 .... 137
xiv 18. Percent mortality and percent diapause Induction in X. luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) /Urban/ elms and exposed to photoperiods of (1) 15:9 L:D, (2) 14.75:9.25 L:D/ (3) 14.5:9.5 L:D or (4) 14.25:9.75 L:D at 25°C, August 1986 . . . . 141
xv INTRODUCTION
A REVIEW OF THE STUDY OF XANTHOGALERUCA LUTEOLA
Xanthoaaleruca luteola (Muller), the elm leaf beetle,
was given several binomial names by different authors in
the late 1700's and mid-1800's. The first known, published
binomial given to the elm leaf beetle was chrysomela
luteola by F. Muller in 1766 (Wilcox 1971). After Muller,
several other authors published binomials (synonyms) for
the elm leaf beetle: Fabricius in 1775, Crioceris
calmariensis: Schrank in 1781, Chrvsomela xanthomelaena:
Geoffroy in 1785, Galerica ulmi; Fabricius in 1801,
Galleruca aelatinariae; and Joannis in 1866, G. crataeai
(Wilcox 1971). Of the synonyms published, Schrank's
species name, xanthomelaena. in the genera Galleruca.
Galeruca and Galerucella appeared most frequently in the
literature and persisted for an extensive period of time.
It was still in use as late as 1961 (e.g., Robertson 1961).
Muller's species name for the elm leaf beetle was
eventually placed into the genus Galerucella by Bedel in
1897 (Wilcox 1971). G. luteola (Muller) was most commonly
used by authors from the early 1900's until 1963 when a
revised name for the elm leaf beetle appeared in the literature, Pyrrhalta luteola (Muller) (Gressitt and Kimoto
1963). This binomial was dominant in the literature into the 1980's and is the binomial used in the most recent edition of the Entomological Society of America's book,
Common Names of Insects & Related Organisms 1989 (Stoetzel
1989). However, a revised binomial for the elm leaf beetle proposed by Bechyn'e in 1961, Xanthoaaleruca luteola
(Muller) was revived by Silfverberg (1974). According to
Wilcox (personal communication), Silfverberg presented convincing evidence to justify the revival of the genus
Xanthoaaleruca for the elm leaf beetle and one other beetle, subcoerulescens Weise (Silfverberg 1974). Thus, in all publications since 1985, investigators, including myself, at The Ohio State University have used this up-to- date binomial for the elm leaf beetle and recommend that all others do the same.
luteola is an Old World insect that was accidentally imported into the United States from Europe in the early 1800's (Glover 1867). In Europe, £. luteola was known to cause "great mischief", especially in Italy,
Austria, and southern portions of France and Germany (Riley
1883, Britton 1907). As early as 1837, 1838 and 1839, X. luteola was reportedly causing significant levels of defoliation to elm trees in Baltimore, Md., the probable port of entry for £. luteola (Glover 1867, Britton 1907).
Since then, X. luteola has spread throughout most of the 3 conterminous states from the East Coast to the West Coast.
luteola has been in Ohio since the early 1900's (Houser
1918).
The spread of luteola was probably facilitated by the natural geographical distribution and abundance of native, although not highly preferred hosts of X. luteola
(e.g., Ulmus americana L.) and by the extensive planting of highly desirable hosts of £. luteola (e.g., U. glabra
Hudson and IJ. procera Salisbury) in urban landscapes, in shelter belts and as windbreaks (see Rosendahl 1955 and
Preston 1976 for geographical distributions for some of these elms). In some areas where these highly desirable hosts of )(. luteola are used in urban landscapes (e.g., southern California), luteola can defoliate elm trees twice a year and poses serious economic and aesthetic problems (Luck and Scriven 1976). X. luteola is now recognized as an important pest of elms in most regions of the United States where it is found (Kielbaso and Kennedy
1983, Nielsen et al. 1985). Pest status of X. luteola results from a combination of several factors: (1) defoliation of elms reduces shade and aesthetic value of trees; (2) large numbers of larvae synchronously descend from the trees and pupate on front lawns, patios, porches and in homes; (3) high density populations produce large quantities of frass that falls on people and their properties; and (4) adults tend to overwinter in homes and other structures (Wene et al. 1968, Hall 1986).
X- luteola overwinters as an adult away from the host plant in protected places such as sheds, attics, woodpiles, under siding, etc. (Fernald 1901). Adults leave overwintering sites in the spring and fly to trees around the time new foliage begins to expand. After a period of feeding, females deposit egg-masses on the lower surfaces of leaves. The resulting larvae also feed on the elm foliage. Larvae pass through three instars. Mature third- instar larvae abandon the foliage and descend the bole of the tree to pupation sites. The majority of the larvae reach the soil surface before pupation. Adults eclose, spend a short period of time in the pupation site, and then fly or crawl back into the tree and begin feeding. A thorough description of the developmental stages of X« luteola is given by Riley (1883). £. luteola can be multivoltine and is reported to have 1-4 generations per year (Britton 1932, Eikenbary and Raney 1968, Wene 1968,
King et al. 1985). In central Ohio, X. luteola normally has 2 generations per year and occasionally a partial third. The first occurs from May through June and the second from June into August. In those years when a third generation occurs, it starts in late July and ends in
September. Most beetles of the second generation and probably all of the beetles of the third generation overwinter. X« luteola larvae and adults consume the foliage of many elms fUlmus spp.) and certain Zelkova spp. Many of the hosts of X* luteola are exotic elm species that were introduced into the United States for shade and ornamental purposes. Early observers saw that X* luteola fed upon most varieties and species of elms; however, the beetle showed a distinct preference for European elm species over other Asiatic and American elm species (Riley 1883, Felt
1907, Houser 1918). Recently, U. pumila L., an Asiatic elm species, was also found to be a highly preferred host susceptible to attack by X* luteola (Weber and Thompson
1976, Luck and Scriven 1979, Mittempergher and Ferrini
1984, Hall 1986). European elm species and U. pumila are often cited as the elms that are the most heavily damaged by X* luteola in any given year (Riley 1883, Fernald 1901,
Houser 1918, Luck and Scriven 1979, Hall and Young 1986).
The adult choice of host plant is also influenced by the proximity of the tree to favored overwintering sites
(e.g., man-made structures)(USDA 1964, Hall 1986). Foci of
X- luteola spring activity in urban and suburban areas are observed on trees that are nearest to buildings. As the season progresses, the beetles radiate out from these foci to infest other nearby trees. Once the adults are on the host, it appears that much of their activity centers on the south and east sides of the tree (Riley 1883, Weber and
Thompson 1976). This behavior of aggregating on certain 6 trees and in certain areas within those trees may facilitate mate location when X» luteola populations are low. The warmth of the south and east sides of the trees may also help accelerate X- luteola development in the spring. Thus, it is apparent from observations made on X- luteola in the field that patterns of infestation of elm trees are influenced by host species and location.
Numerous reports on chemical control of X- luteola appear throughout the literature (e.g., Riley 1883, Fernald
1901, Britton 1932, Knowlton 1952, Wene et al. 1968, Brewer
1973, Hall et al. 1988b). Apparently, because of the relative ease of control of X* luteola with insecticides, little attention was given to X» luteola biology beyond its life history and host plants upon which it feeds. The main means of X* luteola control used in the United States remains foliar applications of insecticides. However, the acceptance of chemical control is declining because of expense and increasing restrictions on insecticide use in urban environments. As a result, an increased interest in alternatives to foliar applications of insecticides for X. luteola control has developed. Alternatives that are being examined include: (1) novel methods of insecticide application (Hall et al. 1988b); (2) introduction and inoculative releases of natural enemies (Hamerski 1988,
Dreistadt and Dahlsten 1990); and (3) breeding and selection of X. luteola resistant elms for urban landscape 7 use (Hall and Townsend 1987). To increase the probability of success in this type of research, a thorough understanding of X* luteola biology and how it is influenced by its environment and host plant is needed.
Early X* luteola literature in the United States dealt mainly with the beetle's life cycle, elm hosts attacked, and control recommendations (e.g., Riley 1883, Fernald
1901, Parks 1936). Until recently, relatively few biological studies had been conducted on X* luteola. Weber and Thompson (1976) found that X* luteola oviposition-site preference was influenced by the distribution of light on a leaf. In the laboratory, X* luteola showed no preference of oviposition-site on uniformly lit leaves. However, in the field, although X- luteola oviposited most frequently on the portions of trees that received the most sunlight, beetles preferred to oviposit on shaded areas of the leaves in those portions.
The duration of X* luteola life stages reared at constant 25.5°C was determined by Wene (1968). King et al.
(1985) examined the influence of 5 separate constant temperatures (15.6, 22.2, 28.8, 32.2 and 36.1°C) under a
16:8 L:D (hours of light and dark in a 24 h period) on developmental time and survival of immature stages of X* luteola. Hall and Young (1986) and Young and Hall (1986) have also followed larval developmental time at constant 25
± 1°C under 16:8 L:D or 15:9 L:D as a bioassay of host 8
suitability of elms for X* luteola. Average larval
developmental time determined in the various investigations
ranged from 14.1 to 20.3 days at 25 ± 1°C. These results
indicate that temperature is only one of possibly several
environmental factors influencing developmental rates of X. luteola- The influence of different elm species and hybrids on
developmental rates, survivorship, fecundity, preference
and feeding behavior of X* luteola has also been
investigated (Luck and Scriven 1979, Hall 1986, Hall and
Young 1986, Young and Hall 1986, Hall et al. 1987, Hall and
Townsend 1987, Giles 1989). Luck and Scriven (1979) found
that English elm (U. procera) and Siberian elm (U. puroila)
were more suitable for X» luteola survivorship, development
and fecundity than were American elm (U. americana L.) and
Chinese elm (U. parvlfolia Jacq.). Hall (1986) examined
the preference for and suitability of three of these elm
species and 'Urban' elm (an elm hybrid) for adult X*
luteola and found the preferred hosts, U. pumila and
'Urban' elm, were also the most suitable elms for adult
survivorship and fecundity. Young and Hall (1986) found
that X* luteola larvae reared on the preferred hosts had higher survivorship and developed faster than larvae reared
on the non-preferred hosts, U. parvifolia and U. wilsoniana
Schneid. Hall et al. (1987) performed a more extensive
survey of the suitability of thirteen elm species for X. luteola which included European, Asian and North American
elm species. Suitability was measured in terms of adult
mortality and fecundity over a two week period. Hall et
al. (1987) found that European elms were better hosts for
X* luteola than American or Asian elm species. From these
studies, it appears that the preference for hosts expressed
by adult X» luteola is related to the suitability of hosts
for growth, development, reproduction and survival of
larvae and adults. Thus, it seems that the patterns of
defoliation caused by X* luteola noted by earlier observers
of X* luteola (e.g., Riley 1883) can be explained in part
by adult preference for hosts of high suitability.
Some of the differences in suitability of elms for X.
luteola appear to be genetically determined (Hall and
Townsend 1987, Hall et al. 1987). Crosses between high
suitability hosts and low suitability hosts produce hybrid offspring of intermediate suitabilities. Because of the range of differences in suitability among the offspring, it was also suggested that suitability was governed by more than one gene (Hall and Townsend 1987). However, significant differences in suitability within elm species and within vegetatively propagated elm hybrids (assumed genetically identical) exist due to influences of environmental factors (Young and Hall 1986, Hall et al.
1988a). Differing levels of fertilization and watering
(Young and Hall 1986) and air pollution (Hall et al. 1988a) 10 have been shown to alter suitability of elm foliage for X- luteola.
Age of foliage and resulting changes in foliage characteristics have also been implicated in altering suitability of elms for X*- luteola (Wene 1968). Wene
(1968) found that survival and development of larvae and adults were lower on older tough foliage than they were on younger succulent foliage of the same tree. Thus, the suitability of an elm tree is most probably influenced by some combination of genetics and environmental factors and can change over time as a result of aging processes.
One aspect of X* luteola biology that has received little attention is X* luteola dormancy. Dormancy serves at least two important functions in insect seasonal cycling: (1) it provides a mechanism to survive prolonged adverse environmental conditions and (2) it synchronizes the life cycles of individuals within a population and the population with the seasonal changes of the environment.
The study of insect seasonal cycles is of great interest to entomologists in several disciplines (e.g., physiology, ecology, insect pest management). Nearly all physical factors in the environment show seasonal cycles.
Changes in environmental factors are major selective forces in the evolution of insect life histories. As a result, many insects use environmental changes as cues to synchronize their life cycles with the seasonal changes in the environment. In the temperate zone, photoperiod, the length of light and dark periods, has a predictable seasonal pattern which is highly correlated with seasonal changes in temperature, moisture, food supply and other factors affecting insect development (Tauber et al. 1986).
Photoperiod is used by many insects as the primary, anticipatory, seasonal cue in the regulation of their seasonal cycling. However, other environmental stimuli
(i.e., food quality and/or availability, temperature and moisture) with relatively predictable seasonal patterns of change may also be used by many insects as a signal of impending change, either in conjunction with or independently of photoperiod (Tauber et al. 1986).
In the temperate zone during colder months, most insects do not remain active outside man-made structures.
Insects without a mechanism to survive the adverse winter conditions will succumb. One of the most common mechanisms used by temperate zone insects to survive inhospitable environmental conditions is diapause (Tauber et al. 1986).
Diapause is a genetically determined, neurohormonally mediated, physiological arrest in the development and/or reproductive activity of an insect. Diapause is induced in advance of the actual event by environmental stimuli which act as 'token' indicators of the coming unsuitable conditions. Diapause can occur in every stage of the insect life cycle, but usually is restricted to one stage 12 of the life cycle of a given species. In response to the token environmental stimulus(i), the insect undergoes specific physiological, behavioral and morphological modifications that prepare the insect for future adverse conditions. These preparatory events occur in a species- specific sequence, and are all part of an overall set of events referred to as the diapause syndrome (Tauber et al.
1986).
Diapause syndrome is broken down into three phases:
(1) prediapause, a period of preparation; (2) diapause, the period of dormancy; and (3) postdiapause, a period between the end of dormancy and the return of favorable environmental conditions. Pre- and postdiapause frequently include seasonal migrations to and from overwintering sites, and seasonal polyphenism, any seasonal pattern of change in color and/or structure of the insect's body or wings. And usually associated with diapause is cold-, heat- or drought-hardiness (Tauber et al. 1986).
In Ohio, adult £. luteola in the field exhibit behavioral and morphological changes that are typically associated with diapause syndrome. luteola adults halt reproductive activity about two months before unfavorable environmental conditions for growth and development prevail. As a part of their preparation for the unfavorable conditions, adults migrate away from host plants to favored overwintering sites. Beetles collected from overwintering sites exhibit a distinct elytral
coloration change from the yellow-green of summer
reproductively-active beetles to a dark olive-green to
black coloration. Only after favorable conditions for
growth and development return the following growing season,
do the adults migrate back to host plants and resume
reproductive activity. At this time, elytra go through a
second color change from the dark olive-green of the
overwintering beetle back to a green-yellow. These
observations suggest that X. luteola overwinters in a state
of adult diapause.
Adult diapause has been studied extensively in
Leptinotarsa decemlineata (Say), the Colorado potato beetle
(see de Kort and Granger 1981, de Wilde 1983, and
references sited therein for reviews and important
contributions to the study of diapause). Because of the
body of information accumulated on adult diapause in h.
decemlineata. it serves as a model of adult diapause.
Diapause in L. decemlineata is mainly induced by
photoperiod. However, temperature and condition of food to which adults are exposed, and the photoperiod to which
late-instar larvae are exposed also influence diapause
induction (de Wilde et al. 1959, de Wilde et al. 1969, de
Kort and Khan 1984). Diapause is induced in the newly-
eclosed adult when it is exposed to short day photoperiods
(e.g., 12:12 L:D). In response to the short day photoperiod, £. decemlineata undergoes distinct physiological and behavioral changes. The beetle stops feeding, halts reproductive activities, suppresses oogenesis, becomes positively geotactic, burrows into the soil, degenerates flight muscles, produces unique classes of proteins into the blood, elevates levels of lipid and glycogen within the body and suppresses its metabolic rate
(Denlinger 1985, and references therein). These physiological and behavioral changes have been shown to be neurohormonally controlled (de Kort and Khan 1984,
Denlinger 1985, and references therein).
The role of the brain and hormone production under its control in adult diapause of L. decemlineata have been well documented (de Kort and Khan 1984, Denlinger 1985, and references therein). Changes in juvenile hormone titer have been shown to be correlated with the physiological and behavioral changes that occur during the prediapause, diapause and postdiapause periods of £. decemlineata.
Juvenile hormone is produced and secreted by the corpora allata. The activity of the corpora allata is apparently stimulated by hormone(s) released from neurosecretory cells of the brain that are carried to the corpora allata in the blood. In diapausing £. decemlineata. neurosecretory cell and corpora allata activity are highly reduced and juvenile hormone titer is very low. Many of the same adult diapause characteristics observed in L. decemlineata may also be exhibited in the adult diapause of X* luteola. However, there may also be many unique characteristics in X* luteola diapause. Thus, because of the important role that diapause plays in insect seasonal cycling, it is important to examine each species for its own characteristic diapause syndrome.
In this dissertation I expand the body of knowledge pertaining to X* luteola biology. Specifically, I (1) determined the incidence and described characteristics of
X. luteola diapause, (2) examined X* luteola response to photoperiod as an environmental token stimulus for diapause induction, and (3) examined the role of the host plant in
X. luteola seasonal cycling as it pertains to diapause induction and aspects of reproduction. CHAPTER I
DIAPAUSE IN THE ELM LEAF BEETLE, XANTHOGALERUCA LUTEOLA
(MULLER) (COLEOPTERA: CHRYSOMELIDAE) WITH REFERENCE TO
SEASONAL ANATOMICAL CHANGES
Since shortly after the arrival of Xanthocraleruca luteola (Muller), the elm leaf beetle, into the United
States, it has concerned many entomologists charged with reporting on and preventing damage to trees in the urban environment. As a result, much of the early literature chronicled X» luteola destructive activity in successive growing seasons, and chemical and physical means of its control (e.g., Riley 1883). Reports on chemical control of
X. luteola also appear in the current literature (e.g.,
Wene et al. 1968, Brewer 1973, Hall et al. 1988). The use of natural enemies to control X* luteola has also been investigated. X- luteola was found to be a host for several species of native and imported predators (Eikenbary and Raney 1968, Luck and Scriven 1976) and parasites (Berry
1938a,b, Luck and Scriven 1976, Graham 1985, Hall and
Johnson 1983, Hamerski and Hall 1988). Yet efforts to control X* luteola with these natural enemies met with limited success (Hamerski 1988, Dreistadt and Dahlsten 17
1990). The limited success was attributed in part to
asynchrony between the life histories of X* luteola and
those of its natural enemies (Berry 1938b, Eikenbary and
Raney 1968, Luck and Scriven 1976).
Details of X» luteola life history were recorded in
the early literature, concentrating mostly on the portion
of the life history observed during the growing season
(e.g., Parks 1936). Recently, additional information
regarding X* luteola biology and factors that influence X,
luteola development have been reported (King et al. 1985,
Hall 1986, Young and Hall 1986, Hall et al. 1987).
However, an important aspect of X* luteola life history
that has not been covered in any detail is X* luteola
dormancy. A few observational details of X* luteola
dormancy have been referred to by many authors. These
included: X* luteola overwinters as an adult (e.g., Riley
1883, Fernald 1901); these adults have a dark olive-green
almost black coloration (e.g., Felt 1907, Britton 1932);
they migrate to "protective" overwintering sites (favored
sites are man-made structures such as homes, sheds, barns,
etc.) (e.g., Fernald 1901, Britton 1907, Felt 1907); and
they often aggregate in large numbers in the overwintering
sites (Britton 1907). Because of the limited attention given to X« luteola dormancy and the important role that it may play in parasite/host synchronization, I felt it
important to investigate this aspect of X. luteola life 18 history more thoroughly.
In Columbus, Ohio, X* luteola produces 2 generations each year resulting in three peak periods of adult activity on host plants. The first peak occurs in mid-April with the return of adults from their overwintering sites. The other 2 peaks coincide with the first and second generation adults and normally occur in June and July/August. In some years, the beetle produces a partial third generation that extends into late August/September. I have observed that most of the second generation adults and probably all of the third generation adults undergo the overwintering dormancy. I suspected that X* luteola enters a state of diapause to overwinter since diapause is a common form of dormancy used by insects to survive through unfavorable environmental conditions in the Temperate Zone (Tauber et al. 1986).
The objectives of this project were: (1) to examine seasonal anatomical changes in X» luteola and (2) to identify and describe anatomical differences that could be used to distinguish between reproductively active (non- diapausing) and overwintering (diapausing) adults.
Preliminary observations indicated that short day photoperiods (e.g., 12:12 L:D) induced diapause in X* luteola. Thus, our objectives also were: (3) to identify the stage most sensitive to diapause-inducing photoperiods,
(4) to determine the period of sensitivity to diapause- 19
inducing photoperiods and (5) to define a critical photoperiod for diapause induction.
MATERIALS AND METHODS
Characterization of diapause. Adult X. luteola were sampled from the field in Columbus, Ohio during late spring, throughout the summer and early autumn from 1985 to
1988. Beetles were captured from host trees during late spring and throughout the summer by searching the foliage and brushing beetles into sealable containers provisioned with excised foliage (usually Ulmus oumila L. or U. procera
Salisbury). In early autumn, beetles were found aggregated in groups (3 - > 200/group with males and females mixed together) in an overwintering site (a woodpile) 10m from host plants. Beetles were placed in petri dishes for transport and storage. Field collected beetles were brought to the laboratory for dissection, observation and comparison with laboratory-reared specimens.
X. luteola used in experiments were collected from the field or from a laboratory colony maintained under artificial lighting (18 hours of light and 6 hours of dark in each 24 hour cycle) throughout the year on potted U. pumila and 'Urban' elm (an elm hybrid) in a greenhouse at
The Ohio State University as wandering-stage third-instar larvae and pupae. In the laboratory, larvae and pupae were placed into separate disposable plastic petri dishes (50- 20
150/dish) and held in environmental chambers during the completion of their development. During this time, neither required food. Larvae and pupae were examined 2-3 times each day for pupation and adult eclosion. Pupae were further separated into additional petri dishes by sex according to characteristics described by Weber (1976).
Adult beetles used in experiments were kept in 250 ml or 500 ml translucent plastic cups with snap on lids (10-30 beetles/cup). Excised foliage of U. pumila was provided throughout adult life. Foliage was replaced at 2-3 day intervals. Two 2.5-3.5 cm strips of bleached white paper towel (Scott C-fold towel) were placed along the side and across the bottom of each cup to absorb moisture released by the excised foliage.
Environmental chambers provided a temperature of 25 +
1°C and various photoperiods, 24 h lightidark cycles (L:D cycles), employed in each experiment. Two frequently used
L:D cycles were 12:12 L:D as the short day photoperiod and
15:9 L:D as the long day photoperiod.
To identify and describe anatomical differences between non-diapausing and diapausing beetles, beetles were dissected under 70% alcohol in wax-bottomed dissecting dishes at 100X magnification. Differences in the condition of the reproductive structures, amount of fat body masses, and presence of spermatozoa in spermathecae and food in gut were recorded. The presence of spermatozoa in testes and 21 spermathecae was determined by examining temporary squash mounts of these structures under a compound microscope.
Diapause characteristics identified in these dissections were used to determine the incidence of diapause in experimentally-manipulated beetles.
A quantification of changes in fat body content was determined by lipid extraction. The lipid content was measured to quantify differences in the amount of fat body between non-diapausing and diapausing £. luteola. and to measure the rate of fat body accumulation. The development of lipid content was followed for the first three weeks of the adult stage in beetles reared at 25 + 1°C under the short day photoperiod, 12:12 L:D, and under the long day photoperiod, 15:9 L:D. A sample of newly-eclosed beetles was taken to represent the starting point for each group and a sample of diapausing beetles (>3 weeks old) collected from their overwintering site on September 29, 1986 was included for comparison. Beetles were separated by sex, placed in 5 ml culture tubes (1 per tube) and frozen until further processing. Seven to 21 beetles of each sex were preserved. Beetles that were feeding were held without foliage for 2 d before freezing to allow most of the food to pass through the gut. Beetles were dried to a consistent weight at 60°C and dry weight of each beetle was recorded. Lipids were extracted with 2 ml of anhydrous ether. After 24 h, the ether was decanted off and replaced 22 with 2 ml fresh ether for an additional 24 h. The beetles were dried at 60°C for 24 h and a lean weight was determined. Percent lipid content was calculated by dividing the difference between dry weight and lean weight by dry weight and multiplying the result by 100.
Statistical analyses were conducted according to methods outlined in Sokal and Rohlf (1981). Data were subjected to an analysis of variance (ANOVA), alpha = 0.05.
Where significant differences existed, means were compared with Student-Newman-Keuls multiple comparison test at the
5% level. Paired data were compared statistically using an approximate £ test, alpha = 0.05 (Ott 1984).
Determination of sensitive stage and period of sensitivity. Reciprocal transfer experiments were conducted to determine the sensitive stage and period of sensitivity for diapause induction. Preliminary observations indicated that the sensitive stage for diapause induction in £. luteola was experienced between the mature third-instar larva and the 2 week old adult.
Thus, the reciprocal transfer experiments were conducted within this period of luteola development. At the beginning of these experiments, large groups of mature third-instar larvae were exposed to one of two photoperiods, then at a specific age or stage of development, a portion of each group was transferred to the alternate photoperiod where it remained until the completion of the experiment. Only individuals that pupated or eclosed during the daylight hours were used in
the transfers to standardize the age of individuals in each group; i.e., all individuals in a group pupated or eclosed within 8 h of one another. One portion of each initial group was held under one photoperiod throughout the experiments to determine base-line diapause incidences.
The alternative photoperiods used were the long day and short day photoperiods. The number of £. luteola in each transfer group ranged from 20 to 80 which was determined by the number of individuals available at the start of the experiment.
Two types of reciprocal-transfer experiments were conducted. Transfers were made daily from day of pupation through the fourth day of adulthood to determine the stage of sensitivity. Also, transfers were made daily from the day of eclosion through the ninth or tenth day of adulthood to determine the length of the period of sensitivity. At the end of each experiment, survivors were preserved and dissected to determine percent diapause induction in each group. In these and other experiments where percent diapause induction was determined by dissection, only those individuals that fully expressed the two most reliable adult diapause characteristics (i.e., underdeveloped reproductive systems and large accumulations of fat body) were scored as being diapause destined. Individuals of 24 uncertain status and those that were obviously reproductively active, were scored as non-diapause.
Critical photoperiod - Laboratory and Field. Samples of X. luteola adults were exposed to different photoperiods at 25 ± 1°C from adult eclosion until two weeks of age.
Photoperiods and numbers of adults exposed to each were:
(1) 15:9, n = 380; (2) 14.75:9.25, n = 88; (3) 14.5:9.5, n
= 97; (4) 14.25:9.75, n = 176; (5) 14:10, n = 42; (6)
13.75:10.25, n = 39; (7) 13:11, n = 112 and (8) 12:12 L:D, n = 335. Beetles were fed excised greenhouse-grown U. pumila foliage. Foliage was replaced every third day.
After 2 weeks exposure, adults were preserved and dissected to determine percent diapause induction.
To determine the critical photoperiod for diapause induction under ambient field conditions, newly eclosed beetles (n = 20 - 50) were caged on U. pumila trees in 15 cm X 40 cm nylon mesh cages. The terminal portion of a branch was placed inside a cage, the beetles were then introduced and the end of the cage was tightly closed around the branch and tied with a string. At the end of one week, the branch with the cage was clipped from the tree and returned to the laboratory. The beetles were allowed to feed for two additional days, and survivors were removed, preserved and dissected to determine the percent diapause induction. Groups of newly eclosed adults were placed in the field cages on the following dates: July 22, 25
25, 29, August 1, 5, 8, 12, and 15, 1986. Period between sunrise and sunset for each of these dates was: 14.57 h,
14.48 h, 14.37 h, 14.28 h, 14.13 h, 14.03 h, 13.88 h, and
13.77 h, respectively.
RESULTS AND DISCUSSION
Characterization of diapause. The coloration of the eiytra of male and female £. luteola varied markedly depending upon the age of and time of year in which the beetles were examined. All newly eclosed beetles had lemon-yellow colored elytra. Beetles that became reproductively active, developed a dull yellow to yellow- brown or yellow-green elytra coloration. Overwintering beetles developed a dark olive-green to dull black coloration. Elytra coloration lightened to a yellow-green when the overwintered beetles returned to the host plant the following year. Similar coloration changes were stimulated by photoperiod in laboratory reared X* luteola.
Beetles reared under 15:9 L:D at 25°C developed the yellow- green or yellow-brown elytra coloration and were reproductively active, while most beetles reared under
12:12 L:D at 25°C developed the olive-green coloration, were not reproductively active and appeared to be diapause- destined. The dark olive-green to black elytra coloration of diapausing beetles from the field and the diapause- destined beetles reared in the laboratory was easily assessed. However it may not be a reliable indicator of diapause in X* luteola reared under various laboratory conditions. King and Price (1986) described reproductively active X* luteola reared under 16:8 L:D at 15.6°C with the same dark olive-green elytra coloration as that associated with diapausing beetles. Thus, it appears that differences in elytra coloration in X- luteola may be stimulated independently of diapause by low temperatures as well as with diapause by photoperiod.
More reliable indicators of diapause in X* luteola were found in internal characteristics. The internal reproductive system of X» luteola is structurally similar to that of many other Coleoptera. The male reproductive system consists of a single testis, a pair of vasa deferentia, a pair of accessory glands, an ejaculatory duct and an aedeagus. The bilobed testes of X* luteola are fused to form a single medial four-lobed testicular structure, a common characteristic of Galerucinae (Mann and
Crowson 1983). The testis is located under tergites II and
III slightly right of the midline. The vasa deferentia are very short and attach to the ventral surface of the testis at the midlines of the left and right paired lobes. The hindgut passes between the vasa deferentia and loops over top of the junction of the vasa deferentia, accessory glands and ejaculatory duct. From this junction, which is located near the posterior of the abdomen, the pair of long, highly convoluted accessory glands loop anteriorly under the tergites, then travel ventrally to loop toward the posterior along the sternites. The apical ends of the accessory glands are slightly bifurcated in many males. It appears that the vasa deferentia and the accessory glands attach separately to or fuse just prior to their attachment to the muscular ejaculatory duct. The musculature of the ejaculatory duct increases in bulk and dilates toward the aedeagus. The sclerotized aedeagus of £• luteola is described by Silfverberg (1974).
The female reproductive system consists of a pair of ovaries, each with 27+2 (mean ± SD; n = 126) ovarioles, a pair of lateral oviducts which join posteriorly to form a common oviduct, a vagina and a spermathecal capsule and gland. Each ovariole consists of a terminal filament, germarium, vitellarium and pedicel. The terminal filaments project forward from the tip of each ovariole to connect to the second thoracic phragma which hold the reproductive system in place. The ovarioles attach to the ventral sides of the lateral oviducts. The lateral oviducts and common oviduct are composed of transparently thin tissue. The common oviduct connects to the muscular vagina which opens to the outside of the body. Attached dorsally to the vagina is a sigmoid shaped, sclerotized spermathecal capsule and a short convoluted spermathecal gland. Female accessory glands were not found in J£. luteola. A lack of 28
accessory glands has been suggested to be common among
chrysomelids (Gupta and Riley 1967).
The entire reproductive systems of both male and
female X* luteola. except for the testes, were under developed in newly-eclosed beetles. Testes appeared to be
fully-matured at the time of eclosion. Males collected in the field from mid-May to late July were reproductively
active. In these males, reproductive systems were fully mature and a very prominent feature within the abdominal cavity. The accessory glands were uniformly dilated throughout their length to ca. 4X their original size. The slender tubes of the vasa deferentia were also expanded to a uniform width of ca. 2X their original size. All glands and ducts were filled with milky-white secretions which caused these structures to stand out against the orange- yellow coloration of the other abdominal organs. The musculature of the ejaculatory duct increased in bulk as well.
In overwintering males, the development of the reproductive organs was suppressed. These males were similar in appearance to newly-eclosed males. The accessory glands and vasa deferentia were undilated or only slightly enlarged in some cases and lacked the milky-white
fluid found in the glands of reproductively-active males.
The testes were active in the overwintering males, contained matured spermatozoa and in the early portion of the overwintering period, showed no apparent size
difference from the testes of reproductively-active males.
Matured spermatozoa are frequently found in the testes of
insects during periods of dormancy even though the insects
are reproductively inactive (Beck 1980). A pair of sac-
like structures present but usually not noticeable in
reproductively-active males was found on the ventral
surface of the testes in overwintering males. Temporary
squashes of these sacs revealed them to be storage areas
for the matured sperm bundles produced prior to or during
the overwintering period.
Weekly collections of overwintered males during April
and May 1988, showed that reproductive maturation was not
completed until male beetles returned to the host plant
(Table 1). In the earliest samples, accessory glands and vasa deferentia of most males were in an involuted state
and lacked secretions or were in the early stages of
activation. Beetles were present and feeding on the host plant ca. 3 weeks before fully matured reproductive organs were found in some males. A month after the first beetle was found on the host plant, most males had reached full
activation (Table 1). Testes of overwintered males were
flattened and shriveled and thus, differed in appearance
from other groups of males' testes. The storage sacs under the testes were packed with matured sperm bundles and were stretched to their largest size. Collections from other 30 years showed similar developmental patterns.
Fully matured reproductive organs were found in females on host plants from mid-May into July. These females were actively maturing oocytes, mating and ovipositing. The germaria of each ovariole swelled to ca.
2X its original size as a result of initiation of primary oocyte maturation (vitellogenesis). The vitellaria of each ovariole conformed to the shapes and number of developing oocytes in them. Females that were rapidly producing eggs had 3 and sometimes 4 oocytes in different stages of maturity in each ovariole. The calyx of each lateral oviduct had excess tissue that folded when the lateral oviduct was empty to give it a popcorn textured appearance.
The folds of tissue allowed each lateral oviduct to expand to accommodate about 15 matured oocytes. Oocytes were also held in the common oviduct. Thus, it was not surprising to find 30-40 eggs in some egg masses oviposited by these females. The overall length and width of each ovary were increased by a factor of 2-3X its original size. The muscular vagina appeared to increase in bulk in these females. Although the spermathecal capsule did not change in size or shape, the spermathecal gland increased in length and width when active. Thus, all the soft-tissued, female reproductive organs were largest in reproductively active X. luteola. 31
Overwintering female X* luteola were similar in appearance to newly-eclosed females. There was a slight increase in the size of reproductive organs in these females. The ovaries remained small and compact, and were articulated by a dense network of tracheae. No sign of oocyte development was observed in overwintering females.
Like the overwintered males, overwintered females did not fully mature and activate their reproductive organs until they returned to the host plant (Table 1). Ovaries of overwintered females caught in the early weeks of the growing season were small and compact or were slightly enlarged. None of these females showed signs of advanced oocyte development (i.e., obvious yolk deposition indicated by the development of an orange coloration in the developing oocytes). By the middle of the fifth week of the season (5/15/88), most overwintered females had completed ovarian development and had many matured oocytes ready for oviposition stored in the lateral and common oviducts (Table 1).
The central feature of adult diapause is the suppression of reproduction. In male insects this is characterized by involuted glands with a lack of secretions in accessory glands. In female insects, there is a total lack of or cessation of oocyte maturation (Denlinger 1985) which may be accompanied by undeveloped ovaries or a reduction in the size of the ovaries, respectively. 32
Overwintering (diapausing) male and female X. luteola
exhibited these typical characteristics of adult diapause
and were conspicuously different in appearance from the reproductively active X« luteola.
Another aspect of reproduction that can be suppressed by adult diapause is copulation. The absence of copulation was indirectly assessed by examining spermathecal capsules
for the presence of spermatozoa. Spermatozoa were found in the spermathecae of most reproductively-active females. No overwintering female was found to have spermatozoa in her spermatheca. Initially upon their return to the host plant, no overwintered female had spermatozoa in her spermatheca. At about the same time that eggs were first found in the field, the number of mated overwintered females began to increase (Table 1). Most females collected on 5/15/88 had fully matured ovaries and were mated. The increase in the appearance of spermatozoa in spermathecae of overwintered females also coincided with the increase in the number of males with fully active reproductive systems (Table 1). The observations indicate that while the male and female reproductive systems were suppressed, copulation was also suppressed in diapausing X. luteola. However, this indicator is not entirely reliable since spermatozoa were not found in spermathecae of all ovipositing females. 33
Dissections of beetles also revealed increased fat
body content and absence of food in the gut of diapausing
X. luteola. Overwintering male and female X« luteola had
large accumulations of fat body that obscured most abdominal organs including reproductive organs of both
sexes. This was sharply in contrast to the fat body content of overwintered beetles and reproductively active beetles, both of which had few and discontinuous masses of
fat body.
Partially digested foliage was observed in the digestive tract of all healthy reproductively-active beetles. Beetles collected from the overwintering site had little or no food in their digestive tracts. The digestive tracts of many diapausing insects are emptied to increase cold-hardiness (Tauber et al. 1986). In the early samples of overwintered beetles collected on foliage, most had initiated feeding. Less than 20% of the beetles in the first three samples did not have foliage in their digestive tract. All beetles collected after 5 May had foliage in their digestive tracts.
X- luteola exhibited several characteristics that could be used as indicators of diapause. The most reliable and rapidly assessable indicators were the suppressed condition of the reproductive organs and the increased fat body content. Unfortunately, the observation of these characteristics requires dissection. Adult X» luteola reared on excised foliage under the
15:9 L:D at 25°C became reproductively active and exhibited behavior and appearance similar to that described for field-collected reproductively active beetles. Most adults reared on excised foliage under the 12:12 L:D at 25°C did not become reproductively active and exhibited behavior and appearance, including the elytra coloration change and increased fat body content, similar to that described for field-collected overwintering (diapausing) beetles.
Laboratory-reared beetles ceased feeding approximately 2 weeks after adult eclosion and aggregated in clusters within the rearing container, usually found under the paper towel strips. Thus, diapause could be induced under laboratory conditions.
The lipid contents of male and female non-diapausing and diapause destined beetles are listed in Table 2 and shown in Fig. 1. Significant differences existed between male and female non-diapausing and diapause destined X. luteola at all 3 sample ages after eclosion (Table 3).
Insects that overwinter for lengthy periods as X. luteola does, sequester large quantities of lipids and other reserves into the fat body (Danks 1987). As a result, the fat body undergoes hypertrophy in diapause destined insects
(Tauber et al. 1986). Therefore, the significant differences in lipid contents between the non-diapausing and diapause destined luteola indicate that the fat body 35 should be hypertrophied In diapause destined beetles and not hypertrophied in non-diapausing beetles. Thus, the visual estimates of differences in the amount of fat body between non-diapausing and diapausing X. luteola were validated.
Male beetles reared under 15;9 L:D did not change in lipid content in the first two weeks of adult life. During the third week, lipid content in the male dropped to 1/2 the original amount. This is significantly less than the first three samples (Table 2 & Fig. 1). The drop in lipid content may have been due to their advancing age. Lipid content in females reared under 15:9 L:D began decreasing within the first week of adulthood and continued to decrease through the second week to its lowest level where it remained through the third week of adulthood. The metabolic demands of reproduction could account for the depletion of lipids in the females.
Reproductive activity was suppressed in diapause- destined beetles. Apparently, energy was diverted to fat body accumulation. Both male and female beetles reared under 12:12 L:D showed significant increases in lipid content over the first 2 weeks of adulthood (Table 2 and
Fig. 1). However, this was followed by a significant decrease in lipid content by the end of the third week.
Even with the decrease during the third week, lipid contents remained much higher in the diapause-destined 36 beetles than in non-diapausing beetles.
The drop in lipids in 3 week old diapause-destined adults may reflect completion of preparations for diapause.
Although patterns of metabolic reserve usage during dormancy are highly species-specific, there are coccinellid beetles that use their reserves extensively in the fall
(Tauber et al. 1986). Insects that enter diapause break down glycogen reserves into sorbitol and glycerol (polyols) which function as cryoprotectants (Tauber et al. 1986).
Likewise, a similar break down of some lipid reserves may occur to produce additional cryoprotectants. Also, diapause-destined beetles in the laboratory usually ceased feeding by the end of the second week of adulthood. Thus, during the week between cessation of feeding and sampling, the beetles would have utilized energy reserves for survival. It would have been during this same time that diapause-destined beetles normally migrate from the host plants to overwintering sites. Thus, metabolic rate would have remained high to keep the flight muscles in readiness, again resulting in a consumption of reserves. Lipid content of the diapausing beetles collected from an overwintering site did not differ significantly from the lipid content of the 3 week old diapause-destined beetles in the laboratory (Table 2 & Fig. 1). This may indicate that a similar consumption of reserves occurs in the field as beetles pass through prediapause development and 37 migration.
Sensitive stage and period. In the first reciprocal transfer experiment, 86.4% (n = 44) of beetles held entirely under short day (12:12 L:D) initiated diapause.
Beetles held under long day (15:9 L:D), all became reproductively active (n = 31). The initial photoperiod and the length of time pupae were exposed to that photoperiod had little effect on their adult fate. Most beetles (99.5%, n = 218) exposed to 12:12 L:D as pupae and
15:9 L:D as adults became reproductively active (Fig. 2 A).
Likewise, most beetles (95.5%, n = 111) exposed to 15:9 L:D as pupae and 12:12 L:D as adults initiated diapause (Fig. 2
B) .
The initial photoperiod did not begin to determine the physiological fate of most individuals until after they were 1 day old adults. The more days beyond the 1 day old adult that a group was exposed to its initial photoperiod, the greater the percentage of individuals that committed to the physiological fate expected under that photoperiod.
This was indicated by the decrease in percent diapause induction in the adults that were transferred from 15:9 to
12:12 L:D on days 2, 3 and 4 of adulthood (Fig. 2 A), and by the increase in percent diapause induction in the adults that were transferred from 12:12 to 15:9 on days 2, 3 and especially day 4 of adulthood (Fig. 2 B).
In the second and third replicates of the reciprocal transfer experiment, few individuals in the groups that were transferred from 15:9 to 12:12 L:D, remained sensitive
to diapause induction beyond the 5th day of adulthood
(Figs. 3 & 4 A). The percent diapause induction in the
adults that were transferred from 12:12 to 15:9 leveled off
after the 6th or 7th day of being exposed to the short day
photoperiod before experiencing the long day photoperiod
(Figs. 3 & 4 B). The observed levels of diapause induction
for these groups were as high as or nearly as high as that
of groups of adults exposed to constant short day in each
of these experiments indicating the end of the sensitive period for diapause aversion (Fig. 2 & 3 B). Thus, it
appears that the newly-eclosed adult is the sensitive stage of X. luteola. and it is most highly sensitive to photoperiod during the period of time between the 1 day old adult and the 1 week old adult (at 25°C).
In the reciprocal transfer experiments, there is some
indication that the commitment to diapause induction does not occur as rapidly as the commitment to reproductive activity (i.e., the extended sensitivity into the 6th and
7th day of adulthood). This slightly protracted period of sensitivity may have biological significance in terms of
fitness. Diapause may reduce an individual's fitness by decreasing its reproductive success. When in diapause, the
individual may be exposed to a greater number of mortality agents or be exposed to a set of mortality agents for a greater period of time resulting in death before reproduction. If an individual survives through diapause, energy that could have been used for reproduction would instead have been used to survive during the event of diapause, potentially resulting in a reduction in reproductive output (Tauber et al. 1986). Also, environmental conditions encountered after diapause are unpredictable and may not be conducive to reproductive activity (e.g., unusually cool temperatures, extended periods of wetness, a late frost, etc.). Thus, it may benefit newly-eclosed beetles to avert diapause when conditions to which they are exposed are perceived to be satisfactory for reproduction. The observed delay in commitment to diapause induction could give luteola the time to assess environmental conditions for reproduction and based on that assessment make the decision to avert or enter diapause. The affect of entering diapause on reproductive success in 2£* luteola needs to be investigated.
Additionally, not all of the results were consistent with the hypothesis that photoperiod is the only factor in determining the physiological fate of newly-eclosed luteola. If photoperiod is the only determining factor, groups of beetles held under the short day (diapause inducing) photoperiod from the mature third-instar larva to the 2 week old adult should all be expected to exhibit high 40 incidences of diapause (i.e., 95 - 100%). This did not occur in all experiments; some groups exhibited much lower diapause incidences than the others (e.g., 65%). From these results, it appears that other environmental factors may influence the diapause decision in X* luteola.
Experiments to examine this possibility are reported in chapter III.
Critical Photoperiod in the Laboratory and Field
Verification. Diapause incidence in adult X* luteola exposed to constant photoperiods increased as daylength decreased from 15 h to 13 h per 24 h cycle (Fig. 5). The mean diapause incidence in groups of adults exposed to photoperiods of 13:11 and 12:12 L:D were equally high, 97.4
% and 96.7 % respectively. From these data, it was determined that the laboratory critical photoperiod for X* luteola occurred between photoperiods with 14.5 and 14.25 h of light in a 24 h cycle. Critical photoperiod is the photoperiod at which 50% of the individuals of a sample respond by entering diapause (Danks 1987). In Columbus,
Ohio (40° north latitude), the critical photoperiods correspond to a photoperiod (sunrise to sunset) occurring between 25 July and 2 August. Thus, populations of X. luteola that eclose in the field after 2 August, would be expected to exhibit high incidences of diapause.
The adults used in the field verification of critical photoperiod experienced at least 7 full L:D cycles in the 41
field. By associating mid-week photoperiods with the
observed diapause incidence in the group of beetles placed
in the field at the beginning of that week, a critical photoperiod for the field was determined. From these data,
the critical photoperiod was determined to have occurred between 5 August and 12 August (Fig. 6). Daylength during this week decreased from 14 h 8 min (14.13 h) to 13 h 53 min (13.88 h ) .
The critical photoperiods for £. luteola determined
in the laboratory and the field were relatively close.
Midpoints for laboratory and field critical photoperiods were 14.4:9.6 L:D and 14:10 L:D, respectively. Daylengths
cited for the field data were defined by time of sunrise
and sunset without consideration of minutes of twilight.
It is not known how much twilight might be interpreted as daylength by £. luteola. Twilight could account for the difference between the laboratory and field critical photoperiods. The influence of varying temperature in the
field verses the constant temperature in the laboratory may also account for the discrepancy.
The critical photoperiods determined in these
experiments might not be valid for all populations of X.
luteola found at different latitudes within its geographic
range. X. luteola remains active later in the year at more
southern latitudes than it does at 40° N latitude
(Eikenbary and Raney 1968, Wene 1968). Daylengths under which X* luteola remains active at more southern latitudes are shorter than the critical photoperiods determined for
X. luteola at 40° N latitude. Geographic differences in several quantitative aspects of diapause, including the length of critical photoperiod have been documented for a number of insect species (see Tauber et. al. 1986 for citations). For many insect species with large geographic ranges, critical photoperiods decline (daylengths get shorter) by about one hour for every 5° increase of latitude (Danks 1987). Thus, it may be necessary to determine critical photoperiods for X» luteola populations found at latitudes outside of 40° N latitude.
On 23 January 1978, a sample of beetles was brought from the field to the laboratory and was divided into four groups containing equal numbers of males and females. One group (n = 30) was provided excised elm foliage and held under 15:9 L:D at 25°C. A second group (n = 10) was also held under 15:9 L:D at 25°C but was not provided with foliage. Two similarly treated groups were held under
12:12 L:D at 25°C. The fed groups of beetles under both photoperiods became reproductively active. However, the mean preoviposition period for females held under 12:12 L:D was significantly longer than the mean preoviposition period for females held under 15:9 L:D, 25.3 days and 18.1 days, respectively (t' = 3.392; df = 14; P < 0.005). The females held under 12:12 L:D also deposited fewer eggs/day 43
after 7 days of oviposition than did the females held under
15:9 L:D, 12.5 (n = 11) and 23.6 (n =» 13), respectively (t'
= -2.738; df = 22; P < 0.01). Thus, it appears that 12:12
L:D influenced the reproductive activity of beetles under
that photoperiod.
The unfed groups of beetles held under each
photoperiod also exhibited differences in their response to
photoperiod. Beetles exposed to 15:9 L:D showed signs of
reproductive activation (e.g., fat body reserves were
depleted, male accessory glands were filled with their
milky-white secretions, and the ovaries were partially
enlarged). However, none of the females was mated.
Consumption of fresh foliage may be a requirement for the
completion of reproductive activation in luteola. The unfed beetles held under 12:12 L:D remained inactive with
no apparent signs of male accessory gland activity or
enlargement of the ovaries. Furthermore, none of the
females was mated. Also, some fat body reserves remained
in these beetles.
Postdiapause development in £. luteola appears to be
influenced by both photoperiod and the availability of
food. This seems to be consistent with observations made on overwintered beetles returning to the host plants in spring. Overwintered beetles return to the host plant in an inactive and unmated state. Completion of reproductive activation and initiation of mating did not occur until after a period of foliage consumption (Table 1). The beetles returned to the host plant on a fairly regular basis in mid-April. Thus, £. luteola may have a critical photoperiod that triggers the return migration and initiation of reproductive activation. This too needs to be investigated more thoroughly. LIST OF REFERENCES
Beck, S. D. 1980. Insect photoperiodism, 2nd ed. Academic Press, New York.
Berry, P. A. 1938a. Laboratory studies on Tetrastichus xanthomelaenae (Rond.) and Tetrastichus sp., two hymenopterous egg parasites of the elm leaf beetle. J. Agric. Res. 57: 859-863.
Berry, P. A. 1938b. Tetrastichus- brevistiama Gahan, a pupal parasite of the elm leaf beetle, pp. 1-11. USDA Cir. No. 485.
Brewer, J. W. 1973. Control of the elm leaf beetle in Colorado. J. Econ. Entomol. 66: 162-164.
Britton, W. E. 1907. The elm leaf beetle, pp. 3-15. Conn. Agr. Exp. Stn. Cir. No. 14.
Britton, W. E. 1932. The elm leaf beetle outbreak, pp. 29-34. Conn. Agr. Exp. Stn. Cir. No. 84.
Danks, H. V. 1987. Insect dormancy: An ecological perspective. Biological Survey of Canada, Ottawa.
Denlinger, D. L. 1985. Hormonal control of diapause, pp. 353-412. IQ G. A. Kerkut and L. I. Gilbert [eds.], Comprehensive insect physiology, biochemistry and pharmacology, vol. 8. Pergamon Press, New York.
Dreistadt, S. H., and D. L. Dahlsten. 1990. Distribution and abundance of Ervnnioosis antennata [Diptera: Tachinidae] and Tetrastichus brevistiama [Hymenoptera: Eulophidae], two introduced elm leaf beetle parasitoids in northern California. Entomophaga 35: 527-536.
Eikenbary, R. D., and H. G. Raney. 1968. Population trends of insect predators of the elm leaf beetle. J. Econ. Entomol. 61: 1336-1339.
Felt, E. P. 1907. White marked tussock moth and the elm leaf beetle, pp. 9-14. N. Y. State Mus. Bull. 109.
45 46
Fernald, H. T. 1901. The imported elm leaf beetle, pp. 3- 8. Mass. Agr. Exp. Stn. Bull. 76.
Graham, M. W. R. De V. 1985. Tetrastichus species (Hymenoptera, Eulophidae), parasitizing the elm-leaf beetle Pvrrhalta luteola (Mull.) and allied hosts. J. Natural History 19: 1059-1071.
Gupta, A. P., and R. C. Riley. 1967. Female reproductive system and histology of the ovariole of the asparagus beetle, Crioceris asparaai (Coleoptera: Chrysomelidae). Ann. Entomol. Soc. Am. 60: 980-988.
Hall, R. W. 1986. Preference and suitability of elms for adult elm leaf beetles, Xanthoqaleruca luteola (Coleoptera: Chrysomelidae). Environ. Entomol. 15: 143-146.
Hall, R. W., and N. F. Johnson. 1983. Recovery of Tetrastichus aallerucae (Hymenoptera: Eulophidae), an introduced egg parasitoid of the elm leaf beetle (Pyrrhalta luteola! (Coleoptera: Chrysomelidae). J. Kansas Entomol. Soc. 56: 297-298.
Hall, R. W., A. M. Townsend, and J. H. Barger. 1987. Suitability of thirteen different host species for elm leaf beetle, Xanthoqaleruca luteola (Coleoptera: Chrysomelidae). J. Environ. Hort. 5: 143-145.
Hall, R. W . , D. G. Nielsen, C. E. Young, and M. R. Hamerski. 1988. Mortality of elm leaf beetle (Coleoptera: Chrysomelidae) larvae exposed to insecticide bands applied to elm bark. J. Econ. Entomol. 81: 877-879.
Hamerski, M. R. 1988. A study of Tetrastichus aallerucae (Fonscolombe) and Tetrastichus brevistiama (Gahan), two parasitoids of the elm leaf beetle (Xanthoqaleruca luteola (Muller)) in central Ohio. Ph. D. Dissertation, The Ohio State University, Columbus.
Hamerski, M. R., and R. W. Hall. 1988. Laboratory rearing of Tetrastichus aallerucae (Hymenoptera: Eulophidae), an egg parasitoid of the elm leaf beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol.. 81: 1503-1505.
King, J. E., and, R. G. Price. 1986. Effects of temperature on fecundity and adult longevity of the elm leaf beetle, Pvrrhalta luteola (Muller). Southwest. Entomol. 11: 51-54. 47
King, J. E., R. G. Price, J. H. Young, L. J. Willson, and K. N. Pinkston. 1985. Influence of temperature on development and survival of immature stages of the elm leaf beetle, Pvrrhalta luteola (Muller)(Coleoptera: Chrysomelidae). Environ. Entomol. 14: 272-274.
Luck, R. F., and G. T. Scriven. 1976. The elm leaf beetle, Pvrrhalta luteola. in southern California: its pattern of increase and its control by introduced parasites. Environ. Entomol. 8: 308-313.
Mann, J. S., and R. A. Crowson. 1983. On the internal male reproductive organs and their taxonomic significance in the leaf beetles (Coleoptera: Chrysomelidae). Entomol. Gener. 9: 75-99.
Ott, L. 1984. An introduction to statistical methods and data analysis, 2nd ed. Duxbury Press, Boston.
Parks, T. H. 1936. Insects on elms, pp. 3-9. Ohio State Univ., Agric. Ext. Serv. Bull. No. 172.
Riley, C. V. 1883. Report of the entomologist, pp. 159- 170. Xn Rep. Commissioner of agriculture for the year 1883. Government Printing Office, Washington, D.C.
Silfverberg, H. 1974. The west palaearctic species of Galerucella Crotch and related genera (Coleoptera, Chrysomelidae) contribution to the study of Galerucinae 6. Notulae Entomol. 54: 1-11.
Sokal, R. R., and F. J. Rohlf. 1981. Biometry: the principles and practice of statistics in biological research, 2nd ed. W. H. Freeman and Co., San Francisco.
Tauber, M. J., C. A. Tauber, and S. Masaki. 1986. Seasonal adaptations of insects. Oxford University Press, New York.
Weber, R. G. 1975. Sexing the elm leaf beetle, Pyrrhalta luteola (Coleoptera: Chrysomelidae). Ann. Entomol. Soc. Am. 69: 217-218.
Wene, G. P., J. N. Roney, and S. Stedman. 1968. Control of the elm leaf beetle in Arizona. J. Econ. Entomol. 61: 1180-118.
Young, C. E., and R. W. Hall. 1986. Factors influencing suitability of elms for elm leaf beetle, Xanthoqaleruca luteola (Coleoptera: Chrysomelidae). Environ. Entomol. 15: 843-849. 48
Table 1.
Variation in accessory gland development in overwintered male and ovarian development and mated condition of overwintered female X* luteola adults collected from H. pumila, April and May 1988.
Accessory Gland Mated Collection Development3 Ovarian Development1b Females Date E 1/4 1/2 3/4 F No D L-Di E-10 YD M0 +
4/20/88 2 4 1 0 0 3 1 1 0 0 5 0
4/25/88 12 14 3 0 0 3 11 7 0 0 21 0
4/30/88 19 13 3 0 0 7 19 3 0 0 28 1
5/5/88 3 6 6 5 2 0 3 16 5 0 24 0
5/10/88 0 2 3 5 5 0 1 12 9 8 18 12
5/15/88 1 0 0 3 10 0 0 2 030 4 28
5/20/88 0 0 0 5 6 0 0 2 6 21 3 26
5/25/88 0 0 0 3 13 0 0 0 326 0 29
aE - no development of glands and glands empty; 1/4 - glands 1/4 full sized; 1/2 - glands 1/2 full sized; 3/4 - glands 3/4 full sized; F - glands full sized and filled with milky-white fluid. bNo D - no development of ovaries; L-D - limited development where ovaries were beginning to expand; E-lO - ovaries expanded to full size and first oocyte present; YD - yolk deposition in developing oocytes; MO - matured oocytes in oviducts. 49
Table 2.
Mean percent lipid content of dry weight of reproductively- active and diapause-destined male and female X- luteola adults of different ages reared under long day (15:9 L:D), short day (12:12 L:D) at 25°C or ambient field conditions, autumn 1986.
Percent Lipid Content(±SE) Of Dry Weight
Age Reproductively active Diapause Destined
Malea Female^ Malec Female**
Newly- Eclosed 10.41(0.32)a 11.10(0.68)3 10.41(0.32)3 11.10(0.68)a
1 Week 10.00(0.57)a 8.40(0.67)b 22.83(1.51)b 24.15(1.20)b
2 Weeks 10.81(0.65)3 4.72(0.31)c 34.11(1.10)c 33.36(0.68)c
3 Weeks 5.06(0.79)b 5.01(0.20)c 25.69(0.66)b 23.72(0.92)b
Field 24.83(0.42)b 23.26(0.43)b Samplee
aMeans followed by the same letter are not significantly different (P = 0.05; SNK preceded by a one-way ANOVA [F = 20.65; df = 3,68; P < 0.01]). bF = 27.76; df = 3,63; P < 0.01.
CF = 90.34; df = 4,99; P < 0.01. dF = 85.27; df = 4,99; P < 0.01. eAdults collected from an overwintering site 29 September, 1986. Table 3.
Results of one-tailed paired t' tests comparing mean percent lipid contents of dry weight of reproductively active and diapause- destined male and female luteola adults sampled at 1, 2 and 3 weeks of age.
Age t'a df P
Males
1 Week 7.945 24 < 0.005
2 Week 18.217 31 < 0.005
3 Week 20.127 33 < 0.005
Females
1 Week 11.446 30 < 0.005
2Week 38.650 25 < 0.005
3 Week 19.799 22 < 0.005
- / \ 2 = 0 a n d 1^ : , ^ > 0 w h e r e ^ x was the mean % lipid content of the diapause- destined adult population and/\2 was the mean % lipid content of the reproductively-active adult population, alpha = 0.05. Figure 1.
Mean percent lipid content of dry weight of reproductively- active and diapause-destined male and female X. luteola adults of different ages reared under long day (15:9 L:D), short day (12:12 L:D) at 25°C or ambient field conditions, autumn 1986. Bars within clusters designated by the same letter are not significantly different (P = 0.05; SNK preceded by a one-way ANOVA).
51 A6E OF BEETLE ■ ■ NEWDT ECLOSED M E E Z 3 1 WEEK A 1 ] 2 WEEKS N E 3 3 WEEKS B S 3 FIELD > 3 WEEKS % Hi
2 1 $ MALE FEMALE MALE FEMALE REPRODUCTIVELY ACTIVE DIAPAUSE DESTINED
Figure 1. Figure 2.
Percentage diapause induction in groups of X* luteola which were transferred to alternate photoperiods (upper panel, 15:9 L:D -> 12:12 L:D & lower panel, 12:12 L:D -> 15:9 L:D) on designated days following pupation and eclosion at 25°C. Bars at each end of figure represent groups which were held under one photoperiod throughout the experiment.
53 16.-9 012346 01234 12:12 PUPAE ADULTS
12:12 L:D — > 16:9 L:D
1222 012346 01234 16:9 PUPAE ADULTS DAY OF TRANSFER
Figure 2. Figure 3.
Replication 2 percentage diapause induction in groups of luteola which were transferred to alternate photoperiods (upper panel, 15:9 L:D -> 12:12 L:D & lower panel, 12:12 L:D -> 15:9 L:D) on designated days following eclosion at 25°C. Bars at each end of figure represent groups which were held under one photoperiod throughout the experiment.
55 - 0 6 % z o — i o c o z - m c o c > - o > 100 Figure 3. Figure 1202 1202 59 1 a 6 7 9 12d2 9 8 7 6 6 4 a a 1 o 15:9 A O TASE ATR ECLOSION AFTER TRANSFER DAY OF 0129466709 69 : — IJ L:D I2J2 —) L:D 16:9 16:9 56 Figure 4.
Replication 3 percentage diapause induction in groups of X. luteola which were transferred to alternate photoperiods (upper panel, 15:9 L:D -> 12:12 L:D & lower panel, 12:12 L:D -> 15:9 L:D) on designated days following eclosion at 25°C. Bars at each end of figure represent groups which were held under one photoperiod throughout the experiment.
57 Z O IOCD2- I71COC>T7> % 100 40 Figure 4. Figure 1112 69 1 a 6 7 9 0 Ilia 10 9 8 7 6 6 4 a a 1 0 16:9 A O TASE ATR ECLOSION AFTER TRANSFER DAY OF 0126466769 100126466769 la L ) 16:9LD —) LD Ilia 15:9 LD —15:9 LD >LD 1112 16:9 58 Figure 5.
Laboratory photoperiodic response curve for diapause induction in X* luteola from Columbus, Ohio.
59 % z o — i o c d z - mcoc>-o> D iue 5. Figure 100 - 0 5 12 H0UR8 OF LIGHT IN A 24 h CYCLE h 24 LIGHTINA OF H0UR8 13 14 15 o C\ Figure 6.
Field photoperiodic response curve for diapause induction (*) in X. luteola from Columbus, Ohio, July and August 1986. The dates correspond to the day of eclosion and placement in the field. The hours of light/day values (o) correspond to the midweek daylength experienced by the sample of adults placed in the field at each date.
61 o\ to
I I I S E P T U U C D D N O A N A 100 60 15 15 17 12
10
8 AVGUST 5
3
-B - MIDWEEK 311
29
27
juiy 25
22 - 14.7 14.6 14.1 13.5 13.3 13.9 Figure 6.
HOPMva Oh U n g t iiH S O ^ h CHAPTER II
LARVAL HOST INFLUENCE ON OVARIOLE NUMBER AND FECUNDITY OF
THE ELM LEAF BEETLE, XANTHOGALERUCA LUTEOLA
Examination of the female reproductive system of the elm leaf beetle, Xanthooaleruca luteola (Muller), reveals differences in the number of ovarioles among individuals and between ovaries within individuals. References to ovariole number in Coleoptera are usually given in short descriptions of the female reproductive system (e.g., Gupta
& Kumar 1963, Varma 1963 and Boiteau et al. 1979). In other investigations, ovariole numbers in Coleoptera have been examined for systematic purposes and to identify a characteristic for measuring differences among populations from different geographic locales (e.g., Boiteau & Drew
1986). Ritcher and Baker (1974) and Robertson (1961) dealt exclusively with ovariole number and variation among and within species of Coleoptera. Ritcher and Baker (1974) performed an extensive survey of ovariole numbers in the
Scarabaeoidea and found little or no variation in ovariole numbers in most genera. However, several species of
Pleocoma showed an exceptional amount of variation in the number of ovarioles in each ovary. However, the source of
63 64 this variation was not determined.
Robertson (1961) compiled an extensive list of ovariole numbers in 329 species representing 45 families of
Coleoptera. Among the species examined was X« luteola
(listed as Galerucella xanthomelaena Schrank), in which
Robertson (1961) found a mean of 27 ovarioles per ovary (n
= 16). In a preliminary study, I examined 26 individuals from a population in Columbus, Ohio and also found a mean
(+ SE) of 27 + 0.29 ovarioles per ovary. However, the range of ovariole numbers per ovary around the means did differ, 25-28 (Robertson 1961) and 22-32 (Young, unpublished data). From the range of ovarioles per ovary of the Columbus population, it can be hypothesized that X. luteola females could have a total number of ovarioles per female as low as 44 or as high as 64. The actual range of ovariole numbers per female was 47-59 (mean = 54.2 + 0.54).
The beetles of the preliminary study were reared on U. pumila. a host plant that has been shown to be a preferred and moderately to highly suitable host for X. luteola growth and development (Luck and Scriven 1979, Hall 1986,
Young and Hall 1986). Other host trees were found to be less preferred and less suitable for X* luteola growth and development (e.g., U. parvifolia and U. wilsoniana).
Larvae reared on less suitable hosts took longer to complete development to the pupal stage and had higher mortality (Young and Hall 1986). Adults fed foliage from 65 these hosts also had fewer females oviposit, took longer to begin ovipositing and oviposited fewer eggs than adults fed on U. pumila (Hall 1986, Hall et al. 1987). Thus, the host plant of X. luteola influences several aspects of its growth, development and reproduction. I suspected that variation in the suitability of hosts plants for X* luteola could also differentially influence the development of the reproductive organs in each female, leading to variation in ovariole number per female. Theoretically, significant differences in ovariole numbers could also produce significant fecundity differences between females.
The purpose of this study was to examine the influence of the larval host on ovariole number and the relationship between ovariole number and fecundity.
MATERIALS AND METHODS
Hosts for X* luteola (a European species) larvae were
Ulmus pumila L. (an Asiatic elm), U. wilsoniana Schneider
(an Asiatic elm), and 'Urban' elm (a hybrid of Asiatic and
European elms). U. pumila was chosen because it is a highly preferred and moderately to highly suitable host for
X. luteola. 'Urban' elm was chosen because it is a moderately preferred and suitable host for X. luteola. U. wilsoniana was chosen because it is non-preferred and is a less suitable host for X» luteola than are the other 2 hosts (Hall 1986, Young and Hall 1986). Trees used were 66 ca. 0.5-1 m high, planted in 12 1 pots in a greenhouse.
Trees were maintained in an active growing condition by watering to soil saturation daily followed by application of 1 liter of a fertilizer solution weekly before and during the experiment. The fertilizer solution consisted of 3.3 g of soluble 20-20-20 N-P-K fertilizer per liter of solution. Trees were grown under a 16:8 L:D using a high pressure sodium lamp as the light source.
Eggs were collected from field-collected
'overwintered' adult X- luteola in Columbus, Ohio. Newly hatched, unfed, first-instar larvae from several egg masses were distributed equally among the trees used in the experiment. Approximately 135 larvae were placed on each tree with 2 trees of each species used.
Larvae were reared in the greenhouse under the 16:8
L:D. Temperature varied in the greenhouse but was 25°C or higher. Mature third (final) instar larvae were collected daily and kept separate according to the tree on which they had fed. Seventy to 80 larvae were collected from each tree and placed in an environmental chamber under a 15:9
L:D at 25°C, where development was completed. Both photoperiods (16:8 and 15:9 L:D) are long day photoperiods for X* luteola. Accidental loss of a group of larvae reared on one U. pumila resulted in a reduction in the sample size (four females and several males survived).
Development of larvae on the other U. pumila was not 67 affected.
Shortly after eclosion, 21 pairs of adults (1 male & 1 female) from each tree (except for the U. pumila noted above) were placed in petri dishes and provided excised foliage from greenhouse-grown U. pumila (treated as described for larval hosts). Petri dishes were placed in plastic bags to reduce drying of the foliage. Dishes were examined daily for the onset of oviposition. Eggs were counted and the leaf on which they were laid was replaced with a fresh leaf. Foliage not removed in this manner was changed as needed (every 2-3 d). The experiment was run for 14 d after adult eclosion or until females had oviposited for a minimum of 7 d. Males that died during the experiment were replaced with males reared as larvae on the same host. Two females resulting from larvae reared on
'Urban' elm died before oviposition started and were replaced.
At the end of the experiment, all surviving beetles were dissected. Male accessory glands were examined to determine that males were sexually mature and capable of mating. Reproductively-active male accessory glands are enlarged and filled with milky-white secretions that cause the glands to stand out against the orange-yellow coloration of the other abdominal organs. Accessory glands of non-reproductively-active males are involuted and do not produce milky-white secretions. The ovaries of each female 68 were removed and the ovarioles were teased apart and counted.
Ovariole counts were sorted to order the observations
from each female so that the lower number of ovarioles was listed first and the higher number of ovarioles listed second. Statistical analyses were conducted according to methods outlined in Sokal & Rohlf (1981). Data were subjected to an analysis of variance (ANOVA), alpha = 0.05.
Where significant differences existed, means were compared with Student-Newman-Keuls multiple comparison test at the
5% level. Correlation analysis was used to examine the relationship between the number of ovarioles per ovary within females and the relationship between fecundity and ovariole number.
RESULTS
The number of ovarioles per ovary ranged from 22 to 32 with a mean(+ SE) of 26.8 ± 0.21 for all beetles combined.
Frequencies with which ovariole numbers per ovary occurred within each group of females reared as larvae on the different hosts are shown in Figure 7. The number of ovarioles in each ovary differed in 86% of the females examined. Means (+ SE) for minimum and maximum ovariole numbers per ovary are given in Table 4. There were
significant relationships between the number of ovarioles
in each ovary within females for females reared on U. 69
wilsoniana. r = 0.41, df = 37, P < 0.01(Fig. 8); on 'Urban'
elm, r = 0.41, df = 36, P = 0.01(Fig. 9); and on U. pumilaf
r = 0.60, df =21, P < 0.01(Fig. 10).
The number of ovarioles per individual differed
significantly among females reared on the different larval
hosts (Table 4). Frequencies with which ovariole numbers
per female occurred within each group of females are shown
in Figure 11. Females reared as larvae on U. pumila had
significantly more ovarioles than females reared as larvae
on U. wilsoniana and 'Urban' elm. The number of ovarioles
in females reared on U. wilsoniana did not differ
significantly from females reared on 'Urban' elm (Table 4).
No adult males (n = 42) reared on U. wilsoniana died
during the experiment and mortality for females was 7%
(n = 43). Mortality for males reared on 'Urban' elm was
5% (n = 43) and for females, 14% (n = 44). For males
reared on U. pumila mortality was 4% (n = 26) and for
females, 4% (n = 25).
Preoviposition period did not differ significantly
among the females reared on the different hosts (Table 5).
There were also no significant differences in the number of
eggs oviposited in the 14 d after eclosion or in the number
of eggs oviposited in the 7 d after the onset of
oviposition (Table 5).
When data were pooled across all treatments, a
significant relationship existed between the number of ovarioles per individual and the number of eggs laid 7d after the onset of oviposition, r = 0.25, df = 98,
P = 0.01(Fig. 12). A significant relationship also existed between ovariole number and the number of eggs laid 14 d after eclosion, r = 0.22, df = 98, P = 0.03(Fig. 13).
Within females reared as larvae on U. wilsoniana. 'Urban' elm or U. pumila. there was no significant relationship between ovariole number and the number of eggs laid 7 d after the onset of oviposition, r = 0.13, df = 37, P =
0.42, r = 0.30, df = 36, P = 0.07, and r = 0.31, df = 21, P
= 0.15, respectively. There also was no relationship detected between ovariole number and the number of eggs laid 14 d after eclosion within females reared as larvae on
U. wilsoniana. r = 0.09; df = 37; P = 0.60, on 'Urban' elm, r = 0.24; df = 36; P = 0.15, or on U. pumila. r = 0.32; df
= 21; P = 0.13.
DISCUSSION
The data indicate that the number of ovarioles in X. luteola was influenced by the larval host. The significant relationship between the number of ovarioles per ovary within females indicates that the ovaries of a female responded similarly to the larval host influence. A similar case of variation in ovariole number due to larval host influence was reported for Encarsia formosa Gahan reared in two species of whiteflies, Trialeurodes 71
vaporariorum (Westwood) and Alevrodes proletella (L.) (van
Vlanen & van Lenteren 1986). Variation in ovariole number
also resulted when J£. formosa were reared in I.
vaporariorum reared on various plant species (van Vianen &
van Lenteren 1986). Wiktelius and Chiverton (1985) also
found that the number of ovarioles in Rhopalosiphum padi
(L.) reared on Prunus padus L. of varying host quality
decreased as host quality decreased.
In phytophagous insects (e.g., many Lepidoptera) that
feed only as larvae or only on nectar as adults, the larval
host plant has a significant influence on the adults that
is evident in fecundity (e.g., Banerjee & Haque 1985, Hough
& Pimentel 1978). In our experiment, no differences in
fecundity occurred when adults were fed U. pumila.
Apparently, any larval host plant influence on the
oviposition of these females was either overcome by the
adult host plant influence or the influence was too subtle
to be detected.
The relationship between fecundity and the number of
ovarioles per female was slight, but statistically
significant. Thus, the number of ovarioles was a
contributing factor in determining fecundity, but not the most important factor in this experiment. Under different conditions (e.g., comparisons of females on a single host that had been reared on hosts with greater differences in
larval suitability than those used in this study), ovariole 72
number could be of greater Importance In determining
differences in fecundity.
As was expected from results of earlier studies (e.g.,
Young and Hall 1986), U. pumila was a highly suitable host
for X* luteola as indicated by high adult survivorship and
fecundity and the highest number of ovarioles was attained
in adults reared as larvae on U. pumila. Hall et al.
(1987) found that X« luteola fed foliage from European
(i.e., coevolved) elm species survived longer and laid more
eggs per female than beetles fed Asiatic and American (non coevolved) elm species. I hypothesize that on a host of higher suitability than U. pumila. a higher mean number of ovarioles could be attained, as well as higher fecundity.
Perhaps the larval host influence on ovariole number and suitability of European elms for X* luteola could in part explain the consistent occurrence of high populations of X.
luteola on these preferred hosts.
Additional research needs to be done to determine the effect of European hosts of X- luteola on ovariole number.
Studies examining factors of the host plant that influence the development of ovarioles in X* luteola and how the larva or pupa is affected physiologically by the factors would also be of interest. Finally, the processes and factors that limit the number of eggs produced per ovariole should be examined. LIST OF REFERENCES
Banerjee, T. C., and N. Haque. 1985. Influence of host plants on development, fecundity and egg hatchability of the arctiid moth Dlacrisia casignetum. Entomol. Exp. Appl. 37: 193-198.
Boiteau, G., and M. E. Drew. 1986. The number of ovarioles of the Colorado potato beetle, Leotinotarsa decemlineata (Say), in four north eastern North American localities. Am. Potato J. 63: 233-236.
Boiteau, G., J. R. Bradley, Jr., and J. W. Van Duyn. 1979. Bean leaf beetle: seasonal anatomical changes and dormancy. Ann. Entomol. Soc. Am. 72: 303-307.
Gupta, A. K. D., and R. Kumar. 1963. Morphology and histology of the female reproductive organs of some beetles (Coleoptera). Indian J. Entomol. 25: 147-160.
Hall, R. W. 1986. Preference for and suitability of elms for adult elm leaf beetle (Xanthogaleruca luteola) (Coleoptera: Chrysomelidae). Environ. Entomol. 15: 143- 146.
Hall, R. W., A. M. Townsend, and J. H. Barger. 1987. Suitability of thirteen different host species for elm leaf beetle, Xanthogaleruca luteola. (Coleoptera: Chrysomelidae). J. Environ. Hort. 5: 143-145.
Hough, J. A., and D. Pimentel. 1978. Influence of host foliage on development, survival and fecundity of gypsy moth. Environ. Entomol. 7: 97-102.
Luck, R. F., and G. T. Scriven. 1979. The elm leaf beetle, Pyrrhalta luteola. in southern California: its host preference and host impact. Environ. Entomol. 8: 307-313.
Ritcher, P. 0., and C. W. Baker. 1974. Ovariole numbers in Scarabaeoidea (Coleoptera: Lucanidae, Passalidae, Scarabaeidae). Proc. Entomol. Soc. Wash. 76: 480-494.
73 74
Robertson, J. G. 1961. Ovariole numbers in Coleoptera. Can. J. Zool. 39: 245-263.
Sokal, R. R., and F. J. Rohlf. 1981. Biometry: the principles and practice of statistics in biological research, 2nd ed. H. H. Freeman and Co., San Francisco. van Vianen, A., and J. C. van Lenteren. 1986. The parasite-host relationship between Encarsia formosa Gahan (Hyra., Aphelinidae) and Trialeurodes vaporariorum (Westwood) (Horn., Aleyrodidae). XIV. Genetic and environmental factors influencing body-size and number of ovarioles of Encarsia formosa. J. Appl. Entomol. 101: 321- 331.
Varma, B. K. 1963. A study on the development and structure of the female genitalia and reproductive organs of Galerucella birmanica Jac. (Chrysomelidae; Coleoptera). Ind. J. Entomol. 25: 224-232.
Wiktelius, S., and A. Chiverton. 1985. Ovariole number and fecundity for the two emigrating generations of the bird cherry-oat aphid (Rhopalosiphum padi) in Sweden. Ecol. Entomol. 10: 349-355.
Young, C. E., and R. W. Hall. 1986. Factors influencing suitability of elms for elm leaf beetle, Xanthogaleruca luteola (Coleoptera: Chrysomelidae). Environ. Entomol. 15: 843-849. 75
Table 4.
Number of ovarioles in female X. luteola reared on different elm species as larvae and fed U. pumila as adults in February 1987.
Number Of Ovarioles (± SE)a Larval Host N Minimum Maximum Total*3
V. wilsoniana 39 25.2 ± 0.23 27.3 ± 0.27 52.5 ± 0.42a 'Urban' Elm 38 25.5 ± 0.24 28.2 + 0.32 53.6 + 0.4 5a
U. pumila 23 26.5 ± 0.41 28.6 + 0.40 55.2 ± 0.73b
aMinimum refers to the mean minimum number of ovarioles per ovary and maximum refers to the mean maximum number of ovarioles per ovary.
^Means in columns followed by the same letter are not significantly different (Student-Newman-Keuls multiple comparison test at the 5% level). F = 6.26; df = 2,97; P < 0.01. 76
Table 5.
Oviposition of X* luteola reared on different elm species as larvae and fed U. pumila as adults in February 1987.
Preovi- No. Eggs Laid No. Eggs Laid Larval Host N position 14 d After 7 d After Period3 Eclosion6 Onset Of Oviposition6
U. wilsoniana 39 5.3 ± 0.16 228.7 ± 12.0 182.3 ± 8.8
'Urban' Elm 38 5.6 ± 0.24 249.6 ± 13.0 209.4 ± 8.1
11. pumiJa 23 5.1 ± 0.23 238.1 ± 15.6 192.4 ± 12.1
aMeans(± SE) were not significantly different according to ANOVA; alpha = 0.05. F = 1.13; df = 2,97; P = 0.32. bF = 0.72; df = 2,97; P = 0.50.
CF = 2.48; df = 2,97; P = 0.09. Figure 7.
Frequency distribution of ovariole numbers per ovary in luteola reared as larvae on U. wilsoniana. 'Urban' elm, or U. pumila. February 1987.
77 II IL wllaanlana ao u
10
at 'Urban' Elm ao
10
10
r at
11. p u m i l a ao ii
10
0 aa ai a* ai ao u OVARIOLE NUMBER/OVARY
7- Figure 8.
Relationship between the ovary with the maximum number of ovarioles and the ovary with the minimum number of ovarioles within female X. luteola reared on U. wilsoniana as larvae and fed U. pumila as adults. The r value is statistically significant at the 5% level.
79 33 32 r2 = 0.17 31 30 29 28 27 26 25 24 23
22 t r t 1------1------1------1------r Z 23 24 25 26 27 28 29 30 31 32 3. MINIMUM NUMBER OF OVARIOLES
e 8 . Figure 9.
Relationship between the ovary with the maximum number of ovarioles and the ovary with the minimum number of ovarioles within female 2. luteola reared on 'Urban' elm as larvae and fed U. pumila as adults. The r value is statistically significant at the 5% level.
81 33 32 31 30 29 28 27 26 25 24 23 22 23 24 25 26 27 28 29 30 31 32 MINIMUM NUMBER OF OVARIOLES Figure 10.
Relationship between the ovary with the maximum number of ovarioles and the ovary with the minimum number of ovarioles within female X. luteola reared on U. pumila as larvae and fed H. pumila as adults. The r value is statistically significant at the 5% level.
83 1215 MAX BER OF OVARIOLES iue 10. Figure 33 28- 25- 29- 30- 31- 32- 27- 22 23- 24- 26- 2 3 4 5 6 7 8 9 0 1 2 33 32 31 30 29 28 27 26 25 24 23 22 - - t
IIU NME O OVARIOLES OF NUMBERMINIMUM Figure 11.
Frequency distribution of ovariole numbers per female in X. luteola reared as larvae on U. wilsoniana. 'Urban7 elm, or U. pumila, February 1987.
85 as u. Mtlaonlaaa ao
IB
10
0 at 'urban Elm
ao
is
10
i
O' as 12. pumila ao
u
10
s
O'
OVARIOLE NUMBER/FEMALE
n . Figure 12.
Relationship between the number of eggs deposited in the first 7 days of oviposition and the number of ovarioles/female £. luteola. The r value is statistically significant at the 5% level.
87 NUMBER OF EGGS 400 250- 350- 200 300- 100 150- iue 12. Figure 50- 550 45 - - * 0.09 r*= UBR F VROE PR FEMALE PER OVARIOLES OF NUMBER 560 55 65 Figure 13.
Relationship between the number of eggs deposited in the first 14 days after adult eclosion and the number of ovarioles/female X. luteola. The r value is statistically significant at the 5% level.
89 NUMBER OF EGGS 200 250- 300- 350- 400 100 150- 50- iue 13. Figure 45 - - 0.05 2 = UBR F VROE PR FEMALE PER OVARIOLES OF NUMBER 50 55 60 65 CHAPTER III
ADULT HOST INFLUENCE ON DIAPAUSE INDUCTION IN THE ELM LEAF
BEETLE, XANTHOGALERUCA LUTEOLA (MULLER)
(COLEOPTERA: CHRYSOMELIDAE)
Host plants of herbivores are more than inert, homogeneous, background substrates upon which the dynamic lives of herbivores are displayed. Host plants too are dynamic entities that play an intricate role in the lives of herbivores which feed upon them (Denno and McClure
1983). A natural succession of change in chemical and physical characteristics takes place within a leaf as it ages. There are usually decreases in total nitrogen and water contents (Feeny 1970, Mattson 1980, Scriber and
Slansky 1981) and increases in defensive compounds (i.e., phenols, flavonoids, etc.) (Feeny 1970, Rhoades and Cates
1976) and leaf toughness due to increases in compounds such as silica, waxes, lignin and fibers (Feeny 1970, Scriber and Slansky 1981). These changes in aging leaves can be accelerated or retarded by several environmental factors, including: soil moisture and fertility (Mattson and Addy
1975, McClure 1980, White 1984), feeding by herbivores
(Wallner and Walton 1979, Schultz and Baldwin 1982,
91 Williams and Myers 1984, Raupp and Denno 1984), adverse environmental conditions (Mason and Baxter 1970, White
1984), infection by disease, exposure to insecticides and fungicides, air pollution, physical damage (White 1984, and references therein), and manipulation of plants for ornamental purposes (Hall and Ehler 1980). This inherent variability of leaf chemical and physical properties alters the suitability of host plants as food sources to support herbivore growth, development and reproduction.
For some insects characterized as "senescence feeders", a decline in the condition of the host plant is of greatest benefit (White 1984). This seems to be especially true for insects that feed on the sap of phloem tissues either by tapping it with a stylet such as in aphids, plant hoppers, scale insects and psyllids or by tunneling as in bark beetles (White 1984, and references therein). These insects tap high concentrations of nutrients being translocated from mature plant parts to growing portions of the plant or storage. There are also some cases where insect defoliators prefer to consume mature and senescing leaves. Such is the case with several species of grasshoppers, a katydid and a geometrid moth larva that feed on creosote bushes (Rhoades and Cates
1976), pine sawflies feeding on jack pine (Ikeda et al.
1977) and a grasshopper feeding on sunflower (Lewis 1979).
In these cases, the herbivores consume senescing foliage 93 and avoid high concentrations of secondary plant compounds
(e.g., creosote resin) found in and on newly flushed and actively growing foliage. Other insects characterized as
"flush feeders", prefer, grow, develop and reproduce more vigorously on newly-flushed and actively-growing succulent foliage (e.g., Feeny 1970, Mason and Baxter 1970, Hall and
Ehler 1980, Maier 1983, Raupp and Denno 1983, and references therein, Raupp and Denno 1984). Xanthoaaleruca luteola. the elm leaf beetle, is also a "flush feeder".
In Columbus, Ohio, X* luteola is active in the field in large numbers from mid-April through mid-August, after which its numbers diminish rapidly. Many of its host trees retain their leaves into October. Thus, X* luteola activity is concentrated on newly-flushed to middle-aged foliage. Laboratory studies have shown that X- luteola survives longer, develops faster and lays greater numbers of eggs on younger succulent foliage than it does on older tough foliage (Wene 1968, Luck and Scriven 1979, Hall and
Young 1986, Young and Hall 1986). Also, in experiments to measure suitability of elms for X* luteola. late season foliage and foliage from trees treated in a manner to reduce their suitability for X* luteola. appeared to have induced diapause in several adults reared under a 15:9 L:D
(a non-diapause-inducing photoperiod) at 25 + l°C (Hall
1986, Hall and Young 1986, Young and Hall unpublished data). The physiological condition (age) of a host plant's 94 foliage has been associated with diapause induction in one other leaf feeding beetle, Leptlnotarsa decerolineata (Say), the Colorado potato beetle (de Wilde et al. 1959, Hare
1983). The host-plant-induced diapause of £. decemlineata appeared to be related to changes in foliar nitrogen, lipid and secondary plant compound contents (Hare 1983, and references therein). Foliar chemical composition may likewise induce diapause in X* luteola.
The objectives of this project were: (1) to examine the influence of the host plant on X* luteola diapause induction and (2) to determine the effect of foliar chemical composition on X* luteola diapause induction.
MATERIALS AND METHODS
X. luteola used in experiments were collected from the field or from a laboratory colony maintained throughout the year on potted U. pumila and 'Urban' elm (an elm hybrid) in a greenhouse at The Ohio State University. Mature third- instar larvae and pupae were collected, placed into disposable plastic petri dishes (50-150/dish) and held in environmental chambers during the completion of their development. Larvae and pupae were examined 2-3 times each day for pupation and adult eclosion. Pupae were further separated by sex according to characteristics described by
Weber (1976). 95
Adult beetles used in most experiments were kept in
250 ml or 500 ml translucent plastic cups with snap on lids
(10-30 beetles/cup). Excised foliage of U. pumila was provided throughout adult life. Foliage was replaced at 2-
3 day intervals. Two 2.5-3.5 cm strips of bleached white paper towel (Scott C-fold towel) were placed along the side and across the bottom of each cup to absorb moisture released by the excised foliage.
Environmental chambers provided a temperature of 25 +
1°C and various photoperiods, 24 h light:dark cycles (L:D cycles), employed in each experiment. The two L:D cycles used were 12:12 L:D as the short day photoperiod and 15:9
L:D as the long day photoperiod.
Statistical analyses were conducted according to methods outlined in Sokal & Rohlf (1981). Data were subjected to an analysis of variance (ANOVA), alpha = 0.05.
Where significant differences existed, means were compared with Student-Newman-Keuls multiple comparison test at the
5% level.
Influence of host foliage condition (age). Greenhouse grown U. pumila trees were selected on the basis of obvious differences in condition of foliage. The "high" quality host had lush-green foliage and was producing new leaves.
The "low" quality host had tough-bronzed foliage and was not producing new leaves. Newly-eclosed, adult X. luteola
(n = 120 with equal numbers of males and females) were fed 96 either the "high" or "low" quality foliage and were placed in the long day environmental chamber. At the end of 2 weeks of feeding, survivors were removed from foliage, preserved and dissected to score incidence of diapause.
A second replication of this experiment was conducted with an additional host (senescing field) and analysis of 3 leaf chemical characteristics: percent leaf water content, leaf protein content and soluble leaf carbohydrate content.
Percent leaf water content (% LWC) was determined by weighing a fresh sample of leaves (wet weight [WN]), drying sample to a consistent weight, weighing dried sample (dry weight [DW]) and placing these values into equation 1:
Equation 1.
WW - DW % LWC ------X 100. WW
The amount of protein present in leaf material was assayed by the Bradford protein-dye binding method
(Bradford 1976). Three 1 g samples were removed and minced from the fresh leaves used to determine percent leaf water content for each tree. To each sample, 12 ml of 0.1 M potassium phosphate buffer (pH 7.0) was added. Leaf samples were ground in a tissue homogenizer. The homogenate was centrifuged at 17,000 x G for 20 min. The supernate was collected and used directly for analysis. 97
The raicrogram protein per gram DW was calculated using the equation 2:
Equation 2. g protein no. g protein 1000 1 12 ml
g DW 50 1 sample of WW 1 ml 1 g WW
1 g WW
% LWC
100
The amount of soluble carbohydrates present in leaf material was assayed by a standard method (Allen 1974).
Groups of newly-eclosed, adult X* luteola (n = 120 with equal numbers of males and females) were fed from each host foliage and were placed in the long day environmental chamber. At the end of 2 weeks of feeding, survivors were removed from foliage, preserved and dissected to score incidence of diapause.
Influence of leaf protein content on diapause induction. To determine the influence of high verses low leaf protein content on diapause induction, potted 'Urban' elms were conditioned with treatments of high water and fertilizer to produce high protein content foliage or high water and no fertilizer to produce low protein content foliage. The fertilized trees were given 1 liter of fertilizer solution biweekly. The solution consisted of
3.3 g of water soluble 20-20-20 N-P-K fertilizer per liter of solution. Treatments were begun at least three weeks 98
before beginning and continued throughout the experiment.
Percent leaf water content and leaf protein content were
determined as described above.
Newly-eclosed, adult X* luteola (n = 70 with equal
numbers of males and females) were fed either the "high" or
"low" quality foliage and were placed in the long day
environmental chamber. At the end of 2 weeks of feeding,
survivors were removed from foliage, preserved and
dissected to score incidence of diapause.
Influence of starvation on diapause induction. To
determine the influence of starvation on diapause induction
in X* luteola. food was withheld from groups of newly-
eclosed adults (n = 40 with equal numbers of males and
females) for 0, 1, 2, 3, 4 or 5 consecutive days (period of diapause induction sensitivity), then provided with
actively-growing U. pumila foliage for the remainder of the
first 14 days of adulthood. In a second replication, food was withheld from beetles (n = 80 with equal numbers of males and females) for 1, 2, 3, 4, 5 or 6 consecutive days, then provided with standard 30% protein diet for the
remainder of the experiment. Experiments were conducted in the long day environmental chamber at 25°C. At the end of
2 weeks, survivors were removed from foliage or diet, preserved and dissected to score incidence of diapause.
The effect of non-consecutive days of starvation was tested in beetles (n = 80 with equal numbers of males and females) using standard 30% protein diet and the following treatments: (1) 1 day off diet then 1 day on diet, (2) 1 day off diet then 2 days on diet, (3) 2 days off diet then
1 day on diet, and (4) 2 days off diet then 2 days on diet.
Treatments were continued for a 2 week period in the long day environmental chamber at 25°C. Beetles (n = 80-160 with equal numbers of males and females) were also fed actively-growing U. pumila foliage in the long day and short day chambers to determine base line incidences of diapause. At the end of 2 weeks, survivors were removed from foliage or diet, preserved and dissected to score incidence of diapause.
Effect of synthetic diet on diapause induction. An synthetic diet was developed for adult X* luteola (Hall and
Young, unpublished data). Ingredients of the standard synthetic diet (protein supplements (equal weights of casein and dried egg white) were 30% of the diet dry weight) are given in Table 6. The dry ingredients of fraction A were combined and thoroughly mixed. Deionized water was added and thoroughly mixed into the mixed dry ingredients. This portion of the diet was then autoclaved for 10-15 min at 121°C and 1.0-3.5 kPa. After autoclaving, mixture was allowed to cool to about 70°C. Dry ingredients of fraction B were added in combination, formalin and olive oil were added singly and thoroughly mixed into the autoclaved fraction. The finished diet was cooled, 100 transferred to petrl dishes and refrigerated until it was fed to beetles. Diets were prepared in an identical manner except that diets were supplemented with 10%, 20% or 30% of dry weight added protein and 0, 0.5 or 1 g of added flavonols (equal weights of quercetin and rutin) in all possible combinations. Diets were maintained at a constant weight of dry ingredients of 44.18 g by adjusting the amount of celufil in the diet. A total of 9 different diets and a foliage control were used.
Thirty newly-eclosed, unfed adult X. luteola (15 male and 15 female with a few exceptions) were placed in a 1 liter plastic container (Dow deli-cup) with a thin layer (3 mm thick X 40 mm diameter) of diet spread on the lid or with excised foliage from actively-growing greenhouse U. pumila. A paper towel (Scott C-fold) was placed around the interior perimeter of the container to absorb condensation.
Three replicates of each diet were made. Rearing containers were placed in an environmental chamber under
15:9 L:D at 25°C. Rearing containers provisioned with foliage, 10% protein/0 g and 1 g flavonols, and 30% protein/0 g and 1 g flavonols were also placed in an environmental chamber under 12:12 L:D at 25°C. Foliage and diets were changed every third day. After 14 days of treatment, surviving beetles were removed from diets, preserved and dissected to score percent diapause induction for each treatment. 101
RESULTS AND DISCUSSION
Influence of host foliage condition (age).
Significantly more newly-eclosed female X- luteola entered diapause under 15:9 L:D at 25°C when fed senescing u. pumila foliage than when fed actively-growing u. pumila
foliage, 16.7% and 2.4% (Fisher's Exact Probability (FEP) <
0.05) (Table 7). A slightly higher percentage of male X*
luteola fed senescing foliage entered diapause than did males fed actively-growing foliage, 8.2% and 2.2%, however, the difference was not significant (FEP = 0.17) (Table 7).
Significant differences in the numbers of individuals entering diapause in groups of newly-eclosed adults fed
foliage of different conditions were also found in the second experiment (Table 8). Significant differences were
found between all groups of females that were fed each type of foliage (females fed actively-growing foliage (AGF) vs.
females fed senescing field foliage (SFF), FEP = 0.05; females fed AGF vs. females fed senescing greenhouse foliage (SGF), FEP < 0.05; and females fed SFF vs. females fed SGF, FEP < 0.05). Significant differences in numbers of diapausing individuals also existed between some groups of males fed the different foliages (males fed AGF vs. males fed SFF, FEP = 1; males fed AGF vs. males fed SGF,
FEP < 0.05; and males fed SFF vs. males fed SGF, FEP < 102
0.05). The highest percentages of diapausing female and male beetles were found in the group of adults fed the senescing foliage from the greenhouse-grown trees, 29.9% and 23.8% (Table 8).
The percent leaf water contents, leaf protein contents and leaf carbohydrate contents of the three foliages of different conditions are shown in Table 9. The actively- growing foliage had a higher percent leaf water content than the two senescing foliages which were similar. The leaf protein contents of the actively-growing foliage and senescing foliage from the field were much higher than that of the greenhouse senescing foliage. The leaf carbohydrate content was lowest in the actively-growing foliage and similar in both senescing foliages.
The data from these laboratory experiments indicate that the condition of the adult host plant consumed during the first 2 weeks of adulthood can in part determine whether the adult may enter diapause. Diapause induction appears to be associated with differences in levels of nutrients present in the foliage. Leaf protein content appears to be a contributing factor to diapause induction in X. luteola but perhaps not the only factor because leaf protein content of the senescing foliage from the field was high yet some diapause induction occurred in beetles fed this foliage. This suggests that other factors are involved in conjunction with leaf protein content, such as 103 percent leaf water content, leaf carbohydrate content and/or factors that were not assayed such as secondary plant compounds (e.g., flavonoids).
Differences in foliar water, protein and carbohydrate contents of maize plants have been associated with differences in larval diapause induction in the maize stem borer, Busseola fusca (Usua 1973). Usua (1973) found that
fewer B. fusca larvae entered diapause when fed young maize plants with high water, high protein and low carbohydrate contents while more larvae entered diapause when fed older maize plants with low water, low protein and high carbohydrate contents. Scheltes (1978) working with stalk borers (Chilo spp.) found that water content and carbohydrate content were not as important as protein content in diapause induction in chilo spp. Decreases in protein (nitrogen) content and increases in lipid content of potato leaves has been associated with diapause induction in the decemlineata (Hare 1983, reference cited therein). The common factor among these experiments and the present investigations is protein (nitrogen) content of the foliage consumed. The following experiment examined the influence of leaf protein content on diapause induction in X. luteola.
Influence of experimentally manipulated leaf protein content. Percent leaf water content and leaf protein content of the fertilized and non-fertilized 'Urban' elms 104 are shown in Table 10. The conditioning of the trees resulted in both fertilized and non-fertilized trees having a similar percent leaf water content. Conditioning also resulted in the fertilized trees having a leaf protein content twice that of the non-fertilized trees.
Of the surviving beetles, no female and 1 male beetle that were fed foliage from the fertilized 'Urban7 elms entered diapause. Four female and 4 male beetles that were fed foliage from the non-fertilized 'Urban' elms entered diapause (Table 11). As was expected, more beetles were induced to enter diapause on the host with the lower leaf protein content. However, the differences in the numbers of beetles that entered diapause on the 2 differently treated hosts were not statistically significant
(comparison between females, FEP = 0,06; comparison between males, FEP = 0.23).
The results of this experiment were more subtle than the results from the experiments that compared actively- growing foliage to senescing foliage. The limited difference in response of the beetles to the fertilized and non-fertilized hosts could be attributed to the following;
The foliages used in the previous experiments were of different ages, whereas the foliages in this experiment were of the same age. The aging process changes many leaf characteristics simultaneously of which some may influence diapause induction in conjunction with protein content of the foliage. It is unknown how the fertilizer treatments influenced levels of other leaf chemical characteristics that were not assayed. The difference between the leaf protein contents was 2 fold; however the lower leaf protein content may not have been low enough to be at or below a
"critical threshold" for diapause induction in X. luteola.
Two additional replications of this experiment were performed, one using U. pumila and one using 'Urban' elm as the fertilized/non-fertilized host plants. Various amounts of diapause induction occurred in all adult X* luteola treatment groups (n = 180/group fed U. pumila and n =
140/group fed 'Urban' elm). However, interpretation of the results was complicated by excessive mortality caused by a microsporidian infection. Depending on the tissues that were infected by the microsporidian (i.e., reproductive tissue, nervous tissue, digestive tract tissue, endocrine tissue, etc.), the infection may have been the direct cause of diapause induction rather than condition or quality of the foliage consumed (see Appendix for data).
Influence of starvation. Reductions in the availability of food have been shown to regulate diapause- induction in a few insect spp. (e.g., the mohave strain of
Chrysopa carnea) (Tauber et al. 1986). Although the example cited is of a predator/prey relationship, it does suggest that starvation could influence diapause induction in a herbivore. If feeding behavior of X* luteola were 106 negatively influenced by senescing or low nutrient content foliage, it could result in the beetles experiencing a period of starvation during the period in which they are sensitive to diapause induction.
Results of starvation experiments using U. pumila foliage or standard 30% protein diet as the eventual food for starved adult X* luteola indicated that starvation did not induce diapause in the host condition and quality experiments. None of the starvation treatments resulted in significant numbers of beetles initiating diapause (Tables
12 & 13). Even beetles that nearly starved to death did not enter diapause. This suggests that without input from the host plant, the beetle must rely on the environmental input that it does receive (i.e., photoperiod). Thus, host plant influences on diapause induction in X* luteola are most likely factors of nutritional quality rather than presence or absence and/or quantity consumed by the end of the sensitive period. Perhaps the beetle is physiologically influenced by an imbalance in certain essential amino acids or a failure to acquire enough of a certain nutrient to manufacture an important molecule(s) required for reproductive activity by a given point in development triggering diapause induction.
Influence of synthetic diets. Percent diapause in X* luteola fed the different diets or foliage is listed in
Table 14 and shown in Figure 14. No adults entered 107 diapause when fed actively-growing U. pumila foliage in the long day chamber. Nearly 100% of adults fed foliage or different synthetic diets entered diapause in the short day chamber. Various amounts of diapause were induced in the adults fed the different diets, but no statistically significant differences were found among the different groups of adults (Fprotein (p) = 1*44/ df = 2«4# p = 0.26;
Fflavonol (f) = O'2*' df = 2 ,4 , P = 0.76; Fprotein x flavonol (p x f) = °*89' df = 4'18' p = °*49>
P = 0.95; Fp x f = 1.35, df = 4,18, P = 0.29) (Fig. 15).
Significant differences were found to be due only to the effect of protein content of the diets. Females fed the
10% protein diets had a significantly higher mean percent diapause induction than females fed the 20% and 30% protein diets. Mean percent diapause induction in females fed the
20% and 30% protein diets were not' significantly different.
No statistically significant differences were found among male X. luteola fed on the same diets (Fp = 0.01, df = 2,4,
P = 0.99; Ff = 0.02, df = 2,4, P = 0.98; Fp x f = 0.34, df
= 4,18, P = 0.85) (Fig. 16). Thus, protein content of diet consumed by adult beetles contributes to diapause induction in female X» luteola. The flavonols in the diets did not appear to influence diapause induction. These flavonols were used, because:
(1) several flayonoid compound have been shown to be present in elm foliage including quercetin and rutin
(Santamour 1972), (2) these compounds are classified as secondary plant compounds which may be involved in the defense of the plant, (3) these compounds could potentially be an indicator of season that £. luteola could monitor, and (4) they were commercially available. The lack of a detectable response to the flavonols added to the diets may have been the result of one of the following: (1) the compounds may have no effect on X. luteola or (2) the quantities used in the diets were too low to produce detectable responses.
The differential sensitivity between female and male
X. luteola to condition of the host plant and quality of the diet consumed may in part be explained by differences in nutritional needs of females and males. Oogenesis is more metabolically demanding than spermatogenesis.
Therefore, reproductive success of females would be more highly influenced by host plant conditions than it would be in males. Thus, there may be a greater advantage to females to monitor and respond to host plant condition
(quality) than to males. The relative insensitivity of male X. luteola may also be indicative of a limited need for large numbers of males to inseminate females of a 109 population (i.e., a few males can mate successfully with many females). Furthermore, because male and female beetles aggregate in overwintering sites, they are already associated with one another as they leave the overwintering site, increasing the probability of finding a mate even if one sex is represented less than the other.
Mid-season reductions in population sizes of X» luteola feeding on heavily infested trees were noted by
Eikenbary and Raney (1968) and Wene (1968). Wene (1968) also noted that population sizes continued to rise on less heavily infested trees. Causes of decline in X. luteola numbers were attributed to depletions of suitable food supplies. Eikenbary and Raney (1968) also mentioned that a disease killed many X* luteola larvae and pupae of late season populations. Thus, it can be inferred from these observations that the declines in population sizes were due to adult migrations and/or larval-pupal mortality. It is now hypothesized that a third possible contributing factor to mid-season population decline could be mid-season adults entering an early diapause in response to a declining condition of host plants. Further research into the relationship between X» luteola diapause induction and host plant condition, both in the laboratory and the field, could be very fruitful. In addition to continuing to examine the adults response to the host plant, examination of the response of the larvae to the host plant should also be done. Recent studies on £. decemlineata have found that environmental factors to which the larvae are exposed can influence diapause induction that occurs in the adult (Hare 1983, de
Kort and Khan 1984). This could happen in X. luteola as well. Because the adult stage is the last stage in development, physiological decisions made in this stage may be the product of the cumulative environmental experience of all life stages. Influences of the larval host plant on ovariole number is one example that can be cited for X. luteola as evidence that events that occur in larval stages have influences in the adult stage. Therefore, future research on X* luteola diapause should at some time include an overall life experience factor as well as field confirmation studies to produce a more ecologically realistic understanding of X. luteola diapause and the role it plays in seasonal cycling. LIST OF REFERENCES
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Denno, R. F., and M. S. McClure. 1983. Variable plants and herbivores in natural and managed systems. Academic Press, New York.
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Feeny, P. 1970. Seasonal changes in oak leaf tannins and nutrients as a cause of spring feeding by winter moth caterpillars. Ecology 51: 565-581.
Hall, R. W. 1986. Preference for and suitability of elms for adult elm leaf beetle (Xanthoaaleruca luteola) (Coleoptera: Chrysomelidae). Environ. Entomol. 15:143-146.
Hall, R. W., and L. E. Ehler. 1980. Population ecology of Aphis nerii on Oleander. Environ. Entomol. 9: 338-344.
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Hare, J. D. 1983. Seasonal variation in plant-insect associations: utilization of Solanum dulcamara by Leotinotarsa decemllneata. Ecology 64: 345-361.
Ikeda, T., F. Matsumura and D. M. Benjamin. 1977. Chemical basis for feeding adaptations of pine sawflies, Neodiprion ruaifrons and Neodiprion swainei. Science 197: 497-498.
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Luck, R. F., and G. T. Scriven. 1979. The elm leaf beetle, Pyrrhalta luteola. in southern California: its host preference and host impact. Environ. Entomol. '8: 307-313.
McClure, M. S. 1980. Foliar nitrogen: a basis for host suitability for elongate hemlock scale, Florinia externa (Homoptera: Diaspididae). Ecology 61: 72-79.
Maier, C. T. 1983. Influence of host plants on the reproductive success of the parthenogenetic two-banded Japanese weevil, Callirhopalus bifasciatus (Roelofs) (Coleoptera: Curculionidae). Environ. Entomol. 12: 1197- 1203.
Mason, R. R., and J. W. Baxter. 1970. Food preference in a natural population of the douglas-fir tussock moth. J. Econ. Entomol. 63: 1257-1259.
Mattson, W. J., Jr. 1980. Herbivory in relation to plant nitrogen content. Annu. Rev. Ecol. Syst. 11: 119-161.
Mattson, W. J., and N. D. Addy. 1975. Phytophagous insects as regulators of forest primary production. Science 190: 515-522.
Raupp, M. J., and R. F. Denno. 1983. Leaf age as a predictor of herbivore distribution and abundance, pp. 91- 123. £ q R. F. Denno and M. S. McClure [eds.], Variable plants and herbivores in natural and managed systems. Academic Press, New York.
Raupp, M. J., and R. F. Denno. 1984. The suitability of damaged willow leaves as food for the leaf beetle, Plaaiodera verslcolora. Ecol. Entomol. 9: 443-448.
Rhoades, D. F., and R. Cates. 1976. Toward a general theory of plant antiherbivore chemistry. Recent Adv. Phytochem. 10: 168-213. 113
Santamour, F. S., Jr. 1972. Flavonoid distribution in Ulmus. Bull. Torrey Bot. Club 99: 127-131.
Scheltes, P. 1978. The condition of the host plant during aestivation-diapause of the stalk borers Chilo oartellus and Chilo orlghalPQcj U s H a (Lepidoptera, Pyralidae) in Kenya. Ent. exp. & Appl. 24: 679-688.
Schultz, J. C., and I. T. Baldwin. 1982. Oak leaf quality declines in response to defoliation by gypsy moth larvae. Science 217: 149-151.
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Sokal, R. R. , and F. J. Rohlf. 1981. Biometry: the principles and practice of statistics in biological research, 2nd ed. W. H. Freeman and Co., San Francisco.
Tauber, M. J., C. A. Tauber, and S. Masaki. 1986. Seasonal adaptations of insects. Oxford University Press, New York.
Usua, E. J. 1973. Induction of diapause in the maize stemborer, Busseola fusca. Ent. exp. & Appl. 16: 322-328.
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Weber, R. G. 1976. Sexing the elm leaf beetle, Pyrrhalta luteola (Coleoptera: Chrysomelidae). Ann. Entomol. Soc. Am. 69: 217-218.
Wene, G. P. 1968. Biology of the elm leaf beetle in southern Arizona. J. Econ. Entomol. 61: 1178-1180.
White, T. C. R. 1984. The abundance of invertebrate herbivores in relation to the availability of nitrogen in stressed food plants. Oecologia 63: 90-105.
Williams, K. S., and J. H. Myers. 1984. Previous herbivore attack of red alder may improve food quality for fall webworm larvae. Oecologia 63: 166-170.
Young, C. E., and R. W. Hall. 1986. Factors influencing suitability of elms for elm leaf beetle, Xanthoqaleruca luteola (Coleoptera: Chrysomelidae). Environ. Entomol. 15: 843-849. 114
Table 6.
Adult X* luteola standard synthetic diet ingredients.
Ingredient Weight (g)
Fraction A:
Elm Leaf Powder3 10 Sucrose 9 Corn Starch 3 Egg White (dried) 6.6 Casein 6.6 Celufil 4.0 Brewer's Yeast 0.5 Wesson's Salt Mix 0.5 Bacto-Agar 0.5 Flavonols 0 Deionized Water (65 ml) 65
Fraction B:
Vanderzant's Vitamin Mix 1 Ascorbic Acid 0.5 Cholesterol 0.01 Sitosterol 0.01 Ergosterol 0.01 Olive Oil (2 ml) 1.8 Methyl Paraben 0.15 Formalin (2 ml of 10%) 2
Total Weight of Diet 111.18
aUninfested, field-collected Ulmus pumila foliage, air- dried, ground and sifted through 50 mesh screen. 115
Table 7.
Number of diapausing and non-diapausing adult X. luteola fed lush-green actively-growing ("high" quality) or tough- bronzed senescing ("low" quality) 12. pumila foliage for 14 days after eclosion under 15:9 L:D at 25°C.
U. pumila Host Eemates Males______Quality Diap Non-d1ap Oiap Non-diap
High 1 40 1 44
Low 8 40 4 45 116
Table 8.
Number of diapausing and non-diapausing adult X. luteola fed U. pumlla foliage that was actively-growing, senescing from the field or senescing from the greenhouse for 14 days after eclosion under 15:9 L:D at 25°C.
U- pumlla Host Females Males Condition Diap Non-diap Diap Non-diap
Actively 0 71 0 67 Growing
Senescing 3 40 0 29 (Field)
Senescing 20 47 10 32 (Greenhouse) 117
Table 9.
Percent leaf water content, leaf protein content and soluble leaf carbohydrate content of U. pumila foliage of different conditions.
u. pumila Water Protein Carbohydrate Host Content Content Content Condition (%) (mg/g DW) (mg/g DW)
Actively 72.4 25.3 6.92 Growing
Senescing 62.1 28.4 10.61 (Field)
Senescing 62.5 6.8 10.76 (Greenhouse) 118
Table 10.
Percent leaf water content and leaf protein content of 'Urban' elms under different fertilization treatments.
Water Protein Treatment Content Content (%) (mg/g DW)
Fertilized 66.5 40.0
Non-fertilized 63.6 19.2 119
Table 11.
Number of diapausing and non-diapausing adult X. luteola reared under 15:9 L:D at 25°C and fed for 14 days after eclosion on foliage from 'Urban' elms conditioned with different fertilization treatments.
Females Males Treatment Diap Non-diap D1ap Non-d1ap
Fertilized 0 25 1 15
Non-fertllized 4 23 4 18 120
Table 12.
Percent diapause induction in male and female £. luteola reared under 15:9 L:D at 25°C and starved for 0, l, 2, 3, 4 or 5 days after eclosion then fed H. pumila foliage.
Consecutive ______Diapause Incidence______Days of Females Males Females & Males Starvation H % H % ti %
0 19 0 8 0 27 0
1 17 0 17 0 34 0
2 20 0 18 0 38 0
3 16 0 16 6.2 32 3.1
4 19 5.3 16 0 35 2.9
5 10 0 3 0 13 0 121
Table 13.
Percent diapause induction in male and female X. luteola reared under 15:9 L:D at 25°C and starved for 1, 2, 3, 4, 5 or 6 days after eclosion then fed 30% protein diet or were alternatively starved and fed 30% protein diet over a 2 week period.
Starvation Diapause Incidence Treatment
Consecutive Females Males Females & Males Days of N % tl % Starvation
1 38 0 37 0 75 0
2 39 0 28 0 67 0
3 38 0 35 0 73 0
4 24 0 19 0 43 0
5 19 0 13 0 32 0
6 3 0 0 0 3 0
Non-consecutlve c+yS ° L Females Males Females & Males Starvation ^ ^ U % U % Days Off:Days On
1:1 35 2.9 28 0 63 1.6
1:2 36 0 33 0 69 0
2:1 30 0 29 0 59 0
2:2 35 0 32 0 67 0
Foliage Females Males Females & Males Controls N % N % %
15:9 37 0 31 0 68 0
12:12 75 89.3 70 98.6 145 93.8 122
Table 14.
Mean percent diapause induction in female and male X. luteola fed foliage or synthetic diets with different levels of protein supplements (%) and flavonols (g) and exposed to 15:9 L:D or 12:12 L:D at 25°C.
Females and Females Males Males Diet N % Diapi N % Diap N % Diap
ULl2 L lQ
Foliage 39 0 43 0 82 0
30%, Og 38 36.2 45 35.6 83 35.9
30%, 0.5g 30 54.1 37 28.6 67 41.3
30%, l.Og 33 19.7 45 17.8 78 18.8
20%, Og 52 40.5 36 21.1* 88 30.8
20%, 0.5g 43 25.5 44 17.9 87 21.7
20%, l.Og 42 51.1 43 38.1 85 44.6
10%, Og 45 60.0 45 17.8 90 38.9
10%, 0.5g 42 61.9 42 29.7 84 45.8
10%, l.Og 40 76.8 40 27.8 80 52.3
12;12 Lifi
Foliage 44 97.8 45 100 89 98.9
30%, Og 44 100 43 100 87 100
30%, l.Og 44 100 42 100 86 100
10%, Og 44 100 41 100 85 100
10%, l.Og 43 100 44 100 87 100 Figure 14.
Percent diapause induction in female and male X. luteola fed synthetic diets with three levels of protein supplements and added flavonols or foliage for 14 days after adult eclosion under 15:9 L:D at 25°C and 2 levels of protein supplement and flavonols or foliage under 12:12 L:D at 25°C.
123 I
100 % VS1 Og F lar. D I I H U 0.5ff Flar. A 80- ESJ Iff F lar. P A U 60- S I E
I 40- N D U
C 2 0 - T I O I N r 10 30 Foliage 30 Foliage 12:12 L3> 15:9 L:D
Figure 14. 124 Figure 15.
Percent diapause induction in female X. luteola fed synthetic diets with three levels of protein supplements and added flavonols or foliage for 14 days after adult eclosion under 15:9 L:D at 25°C and 2 levels of protein supplement and flavonols or foliage under 12:12 L:D at 25 C.
125 i
% 100 E22 Og F lar. D H I 0.6a F lar. I 8 0 - A ES3 lg F la r. P A U 6 0 - S E
I 4 0 - N D U
C 2 0 - T I O
N “ I r 10 30 Foliage 20 30 Foliage 12:12 L:D 15:9 L:D
Rgure 15. 126 Figure 16.
Percent diapause induction in male X. luteola fed synthetic diets with three levels of protein supplements and added flavonols or foliage for 14 days after adult eclosion under 15:9 L:D at 25°C and 2 levels of protein supplement and flavonols or foliage under 12:12 L:D at 25 C.
127 100 % 0 E 3 Og F la r. D H 0.5g Flar. I 8 0 - A K 3 lg Flar. P A U 6 0 - S E
I 4 0 - N D U
C 2 0 - T I O
N I 10 SO Foliage 20 SO Foliage 12:12 L:D 15:9 L:D
Figure 1 6. to CO CONCLUSION
The success of an insect species is dependent upon the ability of the species to survive through all the seasonal environmental changes of its habitat and to synchronize its active feeding stages with the availability of food. The state of diapause is one of the most important adaptations that contributes to the survival of insect species. In X. luteola. diapause: (1) provides a means to survive through prolonged adverse environmental conditions; (2) developmentally synchronizes the members of a population; and (3) synchronizes a population/s activity with environmental conditions (e.g., temperature and food resources) suitable for growth and development.
X. luteola overwinters as an adult in a state of diapause. Thus, the seasonal cycling of X. luteola begins with a synchronous adult emergence in the spring at about the time elm foliage begins to expand and daily temperatures are usually increasing. The adults leave their overwintering sites, return to the host plants and begin feeding on newly-flushed foliage. In Columbus, Ohio, this occurs around mid-April. When the adults first arrive at the host plants, their reproductive organs are not
129 matured. Beetles roust eat before they can complete reproductive organ maturation. After maturing the
reproductive organs, the beetles mate and begin oviposition
in early May. Eggs begin to hatch about 7 to 10 days after deposition. In early to mid-June, mature third-instar
larvae abandon the foliage and descend from the tree to pupate, usually on the soil surface at the base of the tree. Survivorship and developmental rates of the eggs and
larvae are dependent on temperature (King et al. 1985) and suitability (quality) of foliage consumed (Wene 1968, Luck and Scriven 1979, Hall and Young 1986, Young and Hall
1986). In about a week, adult eclosion occurs. Newly- eclosed adults spend a short period of time in the pupation site, then crawl or fly back into the tree and begin
feeding in mid- to late June.
At this time, 2£. luteola adults reach a decision fork
in their development where one of two developmental options
is taken: (1) remain reproductively active and complete an additional generation to the next decision fork or (2) halt reproductive activity and enter development towards diapause, postponing reproduction until the following year.
The developmental decision is influenced by environmental factors to which the newly-eclosed adult is exposed. These environmental factors are reliable cues (token stimuli) of future environmental conditions. Like most insect species, the primary environmental cue used by X. luteola is 131 photoperiod. The newly-eclosed X* luteola adult is sensitive to photoperiod during the first week of adulthood. The critical photoperiod for diapause induction in X* luteola was determined to fall between 14.5:9.5 L:D and 14.25:9.75 L:D. In Columbus, this photoperiod range would be encountered sometime in early August. The adults that eclosed in June are exposed to photoperiods with daylengths longer than those of critical photoperiod and would not be expected to enter diapause. Thus, X- luteola would be expected to remain reproductively active and produce a second generation.
The second generation beetles' pass through the same progression of development, starting as eggs in late June and reaching the adult stage and the decision fork in early
August. Thus, these adults are likely to encounter daylengths shorter than the critical photoperiod for diapause induction. Most second generation adults do enter diapause and overwinter.
The host plant also influences aspects of X* luteola7s life cycle. Larval host suitability (quality) has been shown to influence larval survivorship and developmental rates (e.g., Wene 1968, Luck and Scriven 1979, Young and
Hall 1986). Adult host suitability has been shown to influence adult longevity, developmental rates and fecundity (e.g., Hall 1986, Young and Hall 1986). Larval host suitability apparently also carries over into the 132 adult stage as was shown by differences in ovariole numbers in females beetles reared as larvae on host plants of different host suitabilities. Host suitability also appears to influence diapause induction in X- luteola.
However, unlike photoperiod which has its main influence in the newly-eclosed adult, host suitability may elicit its response through a cumulative experience starting with the newly-hatched larvae and ending in the newly eclosed adult.
This has been shown to occur in £. decemlineata (Hare
1983). In this dissertation, only the adult host influence on diapause induction was investigated. Results of some experiments suggest that environmental factors (i.e., host quality) experienced by larvae may also influence decisions made in the adult stage of £. luteola. Continued research into the influences of host plant quality on diapause induction in X* luteola needs to be done with greater emphasis on the whole life experience.
With further laboratory and field validation research into )£. luteola responses to host plant quality, my laboratory studies on X. luteola diapause, adult host influences on diapause induction and larval host influences on ovarian development, may ultimately translate into differences in reproductive fitness and population dynamics of £. luteola under seasonal, field conditions. They might also, in part, explain differences in seasonal activity of
X. luteola in different geographical locations. This research may eventually be important in the successful development of biological control of X* luteola and/or the development of novel pest control methods of X. luteola.
In hopes of developing the most ecologically-minded methods of controlling insect pests of any kind, agricultural, medical, household or landscape ornamental, as was so aptly stated by de Wilde (1962) and quoted by Chippendale (1982):
"We may only hope to reach this goal on the basis of profound knowledge of the interactions between insect and environment. Diapause research contributes to this understanding." APPENDIX
DATA RELATIVE TO CHAPTER III
134 135
Table 15.
Percent mortality in X* luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) U. pumlla and exposed to different photoperiods at 25°C, July 1986.
% Mortality Host ------Quality Photoperiod Female Male Female And Male
H 15:9 .67.1 54.3 60.7
I 14.25:9.75 34.3 34.3 34.3
G 13.75:10.25 20.2 17.0 18.6
H 12:12 15.7 22.9 19.3
15:9 57.1 45.7 51.4 L 14.25:9.75 37.1 25.7 31.4 0 13.75:10.25 19.4 22.0 20.7 W 12:12 28.6 24.2 26.4 136
Table 16.
Percent diapause induction in X. luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) U. pumila and exposed to different photoperiods at 25°C, July 1986.
% Diapause Host Quality Photoperiod Female Male Female And Male
H 15:9 27.6 27.8 27.7
I 14.25:9.75 80.0 81.6 80.8
G 13.75:10.25 93.2 90.9 92.1
H 12:12 100 96.5 98. 3
15:9 33.3 34.9 34.2 L 14.25:9.75 96.0 91.4 93.7 0 13.75:10.25 94.8 100 97.4 W 12:12 100 100 100 Figure 17.
Percent mortality and percent diapause induction in £. luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) U. pumila and exposed to photoperiods of (1) 15:9 L:D, (2ji 14.25:9.75 L:D, (3) 13.75:10.25 L:D or (4) 12:12 L:D at 25° July 1986.
137 ■ ■ % M ortality P7J E23 % D iap aas* y/
FERTILIZED NON-FERTILIZED
Figure 17. 138 139
Table 17.
Percent mortality in X* luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) /Urban/ elms and exposed to different photoperiods at 25°C, August 1986.
% Mortality Host ------Quality Photoperiod Female Male Female And Male
H 15:9 34.4 31.1 32.7
I 14.75:9.25 21.1 25.6 23.3
G 14.5:9.5 21.4 15.4 18. 3
H 14.25:9.25 20 8.9 14.4
15:9 20 22.2 21.1 L 14.75:9.25 16.7 17.8 17.2 0 14.5:9.5 20 24.4 22.2 W 14.25:9.75 11.1 21.1 16.1 140
Table 18.
Percent diapause induction in 2C. luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) 'Urban' elms and exposed to different photoperiods at 25°C, August 1986.
% Diapause Host ------Quality Photoperiod Female Male Female And Male
H 15:9 42.4 30.6 36.4
I 14.75:9.25 54.9 47.8 51.4
G 14.5:9.5 64.3 50.6 57.1
H 14.25:9.75 84.7 80.5 82.5
15:9 56.9 51.4 54.2 L 14.75:9.25 57.3 43.2 50.3 0 14.5:9.5 65.3 54.4 60 W 14.25:9.75 83.8 87. 3 85.4 Figure 18.
Percent mortality and percent diapause induction in }£. luteola adults fed foliage from fertilized (high protein content) or non-fertilized (low protein content) 'Urban' elms and exposed to photoperiods of (1) 15:9 L:D, (2) 14.75:9.25 L:D, (3) 14.5:9.5 L:D or (4) 14.25:9.75 L:D at 25°C, August 1986.
141 100
H % M ortality -9 0 * ZZ3 % D iapaaa* D I A P A U S E I N D U C T I O N
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 FERTILIZED NON-FERTILIZED
Figure 18. 142 LIST OF REFERENCES
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