Retrospective Theses and Dissertations
1997
Relationship between poplar leaf chemicals and
cottonwood leaf beetle adult feeding preferences
Sisi Lin
Iowa State University
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Lin, Sisi, "Relationship between poplar leaf chemicals and cottonwood leaf beetle adult feeding preferences " (1997). Retrospective
Theses and Dissertations. Paper 11483.
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Relationship between poplar leaf chemicals
and Cottonwood leaf beetle adult feeding preferences
by
Sisi Lin
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Entomology
Major Professors: Elwood R. Hart and Bradley F. Binder
Iowa State University
Ames, Iowa
1997
UMI Number: 9725434
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Graduate College
Iowa State University
This is to certify that the Doctoral dissertation of
SisI Lin
has met the dissertation requirements of Iowa State University
Signature was redacted for privacy.
Professor
Signature was redacted for privacy.
Co-Major Professor
Signature was redacted for privacy.
For the Major Program
Signature was redacted for privacy.
For th
uate College
iii
TABLE OF CONTENTS
ABSTRACT
iv
CHAPTER 1. GENERAL INTRODUCTION
Introduction
Dissertation Organization
Literature Review
1
1
2
3
CHAPTER 2. INSECT FEEDING STIMULANTS FROM THE LEAF
SURFACE OF POPULUS
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgments
References
26
26
27
28
31
34
37
37
CHAPTER 3. CHEMICAL ECOLOGY OF COTTONWOOD LEAF
BEETLE FEEDING PREFERENCES ON POPULUS
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgments
References
44
44
45
47
53
55
58
58
CHAPTER 4. GENERAL CONCLUSIONS
71
APPENDIX A. STRUCTURES OF BEETLE FEEDING STIMULANTS
73
APPENDIX B. STRUCTURES OF PHENOLIC GLUCOSIDES
74
APPENDIX C. PLANT MATERIALS USED IN THE RESEARCH
75
REFERENCES
76
ACKNOWLEDGMENTS
91
iv
ABSTRACT
The Cottonwood leaf beetle, Chrysomela scripta F. (Coleoptera:
Chrysomelidae), is considered to be one of the most serious defoliators of Populus
plantations in North America. This research was conducted to understand the
relationship between poplar leaf secondary chemicals and adult beetle feeding
preferences.
Leaf surface chemicals from a cottonwood leaf beetle-preferred poplar
clone, 'Eugenei' fPopulus deltoides x Populus nigra), was found to induce feeding
in the adult cottonwood leaf beetle. The feeding stimulants were isolated and
identified as n-beheryl alcohol (C22), n-lignoceryl alcohol (C24), n-hexacosanol
(Cjg). n-octacosanol (Cgg), n-triacontanol (C30), and a-tocopherylquinone (a-TQ), (2(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-3,5,6-trimethyl-<1,4>ben2oquinone).
it is the first time that long chain fatty alcohols and a-TQ have been identified as
cottonwood leaf beetle adult feeding stimulants. And it is the first time that a-TQ
has been reported as a feeding stimulant for an Insect. Fatty alcohols or a-TQ
alone do not induce beetle feeding significantly, but a mixture of alcohols and a-TQ
synergistically stimulates beetle feeding.
Field-planted University of Washington poplar pedigree materials (parent
clones ILL-129, Populus deltoides. and 93-968, Populus trichocarpa. F, clones,
53-242 and 53-246, and 87 Fg selections) were used for leaf disc feeding tests.
Field cage feeding tests were performed with parent and F, clones. Leaf surface
V
chemicals, long chain fatty alcohols and a-TQ, and the phenolic glycosides
tremulacin and salicortin, were analyzed to correlate chemical abundance with
Cottonwood leaf beetle adult feeding preference. The beetles showed varied
feeding preferences among parent clones, F, clones, and Fg clones. Contents of
the alcohols or the phenolic glucosides did not explain adult beetle feeding
preference. Content of a-TQ on the leaf surface, in the presence of the alcohols,
could explain the adult beetle feeding preference. The beetle prefered to feed on
clones with a-TQ rather than on clones without a-TQ. As the amount of a-TQ
increased, the feeding preference increased, and then decreased as the amount of
a-TQ increased further.
1
CHAPTER 1.
GENERAL INTRODUCTION
Introduction
In North America, Populus species and hybrids are leading selections for
use in short-rotation woody crop systems (Dickmann and Stuart, 1983). These
plantations attract many defoliators, borers, sucking insects, and gall insects.
Among defoliators, the cottonwood leaf beetle, Chrysomela scripta Fabr.
(Coleoptera: Chrysomelidae), is one of the most serious pests in nurseries and
young plantations (1-3 years) in North America (Buri<ot and Benjamin, 1979;
Wilson, 1979; Harrell et al., 1981; Solomon, 1988). Plantations of Populus are
especially susceptible to cottonwood leaf beetle damage during the establishment
period because a large proportion of the leaf and stem tissue is succulent and
provides sufficient nutritional material for the cottonwood leaf beetle (Bingaman
and Hart, 1992).
In the development of integrated pest management strategies for this beetle
in poplar short rotation woody crop systems, host plant resistance is one of the
possible components. Increased endogenous pest resistance can reduce pest
damage and the costs of pest management, and increase the feasibility of
silvicultural prescriptions for maximizing biomass productivity. A wide range of
clonal resistance to the cottonwood leaf beetle has been reported for selected
clones (Oliveria and Copper, 1977; Caldbeck et al., 1978; Harrell et al., 1981;
Bingaman and Hart, 1992). Adults of the cottonwood leaf beetle show varied
2
feeding and oviposition preference among tested poplar clones and among leaves
of different developmental stages (Caldbeck et al., 1978; Harrell et al., 1981;
BIngaman and Hart, 1992). The mechanisms that underlie beetle preferences,
however, have not been understood. It has been shown that there is no correlation
between beetle preference and poplar leaf moisture, nitrogen, carbohydrate levels,
leaf thickness, toughness, or surface physical characteristics (Harell et al., 1981).
The purpose of this study was to determine any existence of and to identify
any feeding stimulants in the leaf surface chemicals from cottonwood leaf beetle
adult-preferred poplar clones, to understand the correlation between host-plant
selection and any feeding stimulants, and to elucidate further the effect of phenolic
glycosides, Salicaceae family specific secondary compounds, on host-plant
selection.
Dissertation Organization
The altemative dissertation fomiat consisting of completed papers was
chosen to facilitate the publication of results from this research. Chapter 2 and
chapter 3 of the dissertation each represents material that was included in
manuscripts. A general conclusion follows the second paper. References cited in
the general introduction are listed following appendix c.
The research included in this dissertation was conducted with the guidance
and assistance of Dr. B. F. Binder and Dr. E. R. Hart, Department of Entomology.
They will be listed as second and third authors on the manuscripts.
3
Literature Review
To meet the growing demand for fuel, fiber, and shelter by an expanding
human population living on a shrinking land base, and to reduce adverse effects of
intensive energy consumption and production, interest in bioenergy and other
forms of renewable energy have been increasing (Burwell, 1978; Ceulemans,
1990). The growing of both annual and perennial crops to produce biomass for
energy and industry under short rotation intensive culture (SRIC) systems has been
the subject of much research since the 1970's (Ceulemans, 1990). In the United
States, SRIC forestry as a new silvicultural system was first studied intensively in
the mld-1960's. In 1978, a comprehensive national program was initiated by the
United States Department of Energy. Use of wood as an energy resource has
grown in importance in the United States since the 1960's. In 1981, wood supplied
about 6 percent of the industrial and 10 percent of the residential heating
requirements nationwide (Geyer et al., 1985). As much as 25% of the harvested
roundwood in the United States was used for fuel in the 1980's (Koning and Skog,
1987). At present, however, most SRIC plantings supply raw materials for the pulp
industry and for the wood-based panel board industry, as well as a wide range of
local craft industries (Ceulemans, 1990).
SRIC refers to wood or biomass production in carefully managed
plantations, using fast growing hardwoods of good coppicing ability for rotations of
3 to 10 years (North America) or 3 to 15 years (Europe) (Ceulemans, 1990). SRIC
forestry is a silvicultural system using agronomic techniques such as site
preparation, fertilization, irrigation, and weed competition control to maximize
4
biomass production over a very short rotation length, typically 4-8 years (Lortz et
al., 1994), It is similar to mechanized farming and is characterized by high energy
inputs, high biomass yields, and complete mechanization. Management objectives
center on maximum annual woody biomass yield per hectare. Trees are harvested
at relatively short intervals and under specific regimes. In some systems, the next
crop is allowed to grow from the cut stump as coppice shoots (Ceulemans, 1990).
Rapid initial growth, consistent coppicing, and resistance to environmental stresses
are essential for practical commercial use. There are several fast-growing
deciduous tree genera or species, such as silver maple, Siberian elm, honeylocust,
and poplars, with potential uses for commercial short rotation plantations (Geyer,
1989). Among them, Populus species have proved to be extremely well-suited for
SRIC biomass production (Ceulemans, 1990; Lortz et al., 1994). Populus sop, also
have received much more study than any other fast-growing tree species (Wright et
al., 1992).
The poplars, genus Populus. together with the willows, genus Salix.
constitute the family Salicaceae. The Salicaceae are widely distributed in the
northem hemisphere, from the polar circle to latitude 30°; occasional species
range further to the south and even occur in the southern hemisphere (FAO, 1980).
The genus Populus is comprised of five sections with wide distribution in the
northem hemisphere: Abosa, Turanga, Leucoides, Populus (aspens and white
poplars), Tacamahaca (the balsam poplars), and Aigeiros (the cottonwood and
black poplars) (Dickmann and Stuart, 1983; Eckenwalder, 1996). The members of
Populus, Tacamahaca, and Aigeiros selections are of economic importance.
5
Poplar has a suite of distinctive biological characteristics (Ceulemans,
1990): 1. rapid juvenile growth and biomass production. On the best sites in the
Mississippi River Delta, the height growth of eastern cottonwood (Populus
deltoides) can average over 4 m per year for the first few years, and 145
of
pulpwood may be produced during a 12-year rotation (McKnight, 1970). In the
U.S. Pacific Northwest, hybrids of the native black cottonwood fPopulus
trichocarpa) with eastem cottonwood attained 27.6 dry Mg/ha/yr on im'gated plots
with excellent soil conditions (Heilman and Stettler, 1985). Average productivity of
the hybrids and the native black cottonwood are 20.3 and 12.5 dry Mg/ha/yr,
respectively. In Michigan, trees in a plantation with four hybrid poplar clones
reached 3.2 m height and 3.1 cm stem diameter, and biomass yields of 7.8 to 10.7
metric tons/ha after 2 years (Dickmann et al., 1979). Four-year height growth for
hybrids and selections grown in the state of Washington ranged from 4.66 m to
11.27 m and diameters ranged from 2.95 cm to 11.57 cm (Stettler et al., 1988).
Trees exhibited growth rates only slightly reduced from these levels over a growth
period of 25-30 yr. For the first 20 yr, annual growth rates of 2.5 cm in diameter and
1.5 m in height are possible in black cottonwood plantations (Dickmann and Stuart,
1983). For two P. trichocarpa X P. deltoides hybrids grown in France, after the first
growing season total tree height was 3.2 and 3.5 m. Above-ground biomass per
area was 601 and 637 g/m (FAO, 1980; Barigah et al., 1994). 2. ease of vegetative
propagation and convenient clonaiity, ready resprouting after harvest, and
therefore good coppicing ability. Populus has a rather amazing ability to reproduce
6
readily by asexual or vegetative means. All poplars sprout from stump or root
collars. Aspens also reproduce through shoots fomied from adventitious root buds.
Aigeiros and Tacamahaca poplars can be reproduced through hardwood cuttings
from dormant stems (Dickmann and Stuart, 1983). Softwood or green wood
cuttings from actively growing shoot tips'also root easily (Dickmann and Stuart,
1983; Faltonson et al., 1983; Sellmer et al., 1989). Some species can be
propagated through root cuttings (Hall et al., 1990). 3. amenability for tissue and
cell culture. Populus selections have demonstrated an ability for somatic
embryogenesis (Michler and Bauer, 1987; Cheema, 1989). They can be
reproduced easily through tissue culture. 4. ease of breeding. Populus are
dioecious, with the male and female flowers on separate individuals. They
produce good seed crops yearly when mature. Populus species hybridize both
spontaneously and artificially. Hybridization is frequent between trees of different
species. Nearly all crosses between species in the same section are possible.
Crosses between Aigeiros x Tacamahaca are easy to achieve and progeny are
highly fertile. Although crosses between Populus and Aigeiros or Populus and
Tacamahaca poplars are typically difficult and often produce low-vigor seedlings,
some vigorous progeny have been achieved in these crosses (Zsuffa, 1973; FAO,
1980; Dickmann and Stuart, 1983). 5. large species diversity. The poplars
constitute a large and diverse group of trees that consist of nearly 30 species with
wide natural distribution in the northem hemisphere (Dickmann and Stuart, 1983).
Species are native to Europe, Asia, and North America with an even much larger
number of varieties within and hybrids between them. Many species show a wide
7
degree of genetic variation (Ceulemans, 1990; FAO, 1980), 6. availability of
unmanaged. natural populations. 7. excellent growth on a wide range of sites.
These biological characteristics make Populus nearly an ideal model of a
tree genus for SRIC (Dickmann, 1985). The wood of poplars, because it is light in
weight, light colored, and uniform in texture, is very suitable for the pulp-and-paper
industry (Dickmann and Stuart, 1983). Poplar wood has been used as lumber for
construction and fumiture production, for packing and box manufacturing, as
veneer for plywood, matches, match-boxes, chip baskets, fiber and particleboard,
and as raw material for chemical, mechanical, and chemi-mechanical pulp for
printing and tissue paper. Poplars also are planted for erosion control, as
windbreaks, as ornamental trees, and more recently as an excellent converter of
wastewater and sludge (Ceulemans, 1990). Populus are also well-suited for
genetic and physiological research in forest trees (Stettler and Ceulemans, 1993)
and for tree improvement breeding programs (Ceulemans, 1990).
Short rotation woody crop systems are designed to obtain high productivity,
and are managed much like conventional agricultural systems. Fast growing,
genetically similar poplar clones of uniform age are planted at uniform spacing
over extensive areas. The trees are often located on sites selected more for ease
of access and cultural operations than for suitability to the biological requirements
of the specific trees. Cultivation, fertilizer, irrigation, and chemical or mechanical
control are often used to enhance growth and control competing vegetation. These
homogeneous monoculture systems, with reduced complexity compared with
natural stands, are vulnerabile to a large number of pests. Loss to insect pests can
8
be one of the factors that limit productivity (Shea, 1971; Wilson, 1976). Although
Populus species are well suited to SRIC systems, the genus has species of trees
that rank among the most preferred hosts for insects (Wilson, 1979). There are 24
insect species that are considered to be injurious at present, and as many as 150
species that are considered to be potentially injurious (Dickmann and Stuart,
1983). They include defoliators, borers, sucking insects, and gall insects.
Among defoliators, the cottonwood leaf beetle, Chrysomela scripta F.. is one
of the most serious pests in nurseries and young plantations (1-3 y) (Solomon,
1988). The cottonwood leaf beetle belongs to the order Coleoptera, family
Chrysomelidae, section Trichostoma, and subfamily Chrysomelinae (Jolivet, 1988).
The distribution range of the cottonwood leaf beetle extends over the entire
continental United States and into Mexico and southwestem Canada (Brown,
1956). The reported hosts of the cottonwood leaf beetle are principally species
and hybrids of Populus (section Aigeiros and Tacamahaca) and Salix (Lowe, 1898;
Brown, 1956), although they also include species of Acer (Lowe, 1898) and AInus
(Anon., 1985). The beetle is a multivoltine species, having four to six generations
each growing season in the north central states and up to seven in the southem
states (Buri<ot and Benjamin, 1979; Caldbeck et al., 1978). The final generation
oven/vinters as sexually-immature adults in leaf litter and debris. In eariy spring, in
approximate synchrony with the bud break of their host plants, adults emerge and
begin feeding, mating, and ovipositing. Adults lay egg masses, about 50-70 eggs
per egg mass, mostly on the abaxial surface. The average female fecundity under
laboratory conditions was 510 eggs (Burkot and Benjamin, 1979). Larvae have
9
three instars, each instar possessing two rows of dorsolateral reversible glands that
secrete defensive chemicals. One major component, salicylaldehyde, repels
generalist predators and benefits larvae and pupae of the beetle by offering some
chemical protection (Wallace and Blum, 1969). Both larvae and adults feed on
young, immature foliage of the same poplar plants. The first instars feed
gregariously, and feed by windowpaning the lower surface of the leaves, while the
latter two instars skeletonize or consume the entire leaves. Adults feed in a shothole pattern. Both larvae and adults also feed on buds and tender bark at the tip of
the shoots when suitable leaf tissue is in short supply. Repeated defoliation and
meristem damage by the beetle can decrease vitality, reduce growth and biomass
(Reichenbacker et al., 1996), induce defomiity, and in extreme cases, cause
mortality in poplar trees.
Large numbers of generations per year, high female fecundity, chemical
defense against general predators, indeterminate growth property of poplar that
provides abundant nutritional food material, and the properties of SRIC systems all
contribute to rapid outbreak conditions for the cottonwood leaf beetle. Plantations
of Populus are especially susceptible to cottonwood leaf beetle damage during the
establishment period because a large proportion of the leaf and stem tissue is
succulent and provides sufficient nutritional material for the cottonwood leaf beetle
(Bingaman and Hart, 1992). Although small, heavily infested areas may be
controlled with an insecticide such as carbofuran, disulfoton, or phorate (Bingaman
and Hart, 1992), direct control in large areas often Is not feasible (Boysen and
10
StrobI). Chemical control may cause problems such as environmental pollution,
insect resistance, natural enemy depression, and secondary pest outbreaks.
In the development of integrated pest management strategies for short
rotation woody crop systems, insect resistance is one of the possible components.
Mastering resistance mechanisms could make it possible to reduce or avoid insect
damage. Increased endogenous pest resistance can reduce pest damage and the
costs of pest control, and increase the feasibility of silvicultural prescription for
maximizing biomass productivity. Clonal forestry provides significant opportunities
to achieve these goals. The selection of productive clones with different modes
and mechanisms of pest resistance will facilitate the design and deployment of
more stable and sustainable plantations (Hart et al., 1992; Robison and Raffa,
1994).
In Populus. selection and breeding techniques and genetic engineering
have been used to increase pest resistance (Thomburg, 1990; Hart et al., 1992;
Nef and Duhoux, 1992; Klopfenstein et al., 1993). A wide range of clonal
resistance to the cottonwood leaf beetle has been reported for selected clones
(Oliveria and Copper, 1977; Caldbeck et al., 1978; Harrell et al., 1981; Bingaman
and Hart, 1992). The adults of the cottonwood leaf beetle showed varied
preference among tested poplar clones and among leaves of different
developmental stages (Caldbeck et al., 1978; Harrell et al., 1981; Bingaman and
Hart, 1992). The adults showed significant non-preference for aspen foliage when
compared to the Aigeiros and Tacamahaca foliage; on the same clones, the beetle
showed preference for immature leaves for feeding and oviposition.
11
Understanding the mechanisms that underlie the host plant preference variation of
the Cottonwood leaf beetle will help us incorporate resistant characters into our
pest resistance breeding programs more efficiently.
Present complex interactions between phytophagous insects and their host
plants are the result of long evolutionary processes of each species for their own
best survival strategies. Host recognition or host plant selection is a key factor in
the relationship between phytophagous insects and their host plants. Host plant
selection is a process by which an insect distinguishes between host and non(unsuitable) host plants and chooses some plants and not others within its host
range (Panda and Khush, 1995). Host selection includes a sequence of
behavioral events in the plant stimuli-insect response paradigm. The process
involves dispersal, host finding, host recognition, host acceptance or consumption,
and host suitability for food or oviposition. The process is the result of an
integration of numerous plant factors and intemai factors. In each step, the central
nervous system, various peripheral receptors, intemai physiological conditions,
and chemical and physical attributes of the plant are involved in the initiation and
determination of the actions (Miller and Strickier, 1984).
In the course of evolution, most phytophagous insects exhibit specialized
feeding habits, and they become adapted to specific plant taxa or to a group of
related families to the exclusion of others. As each insect species shows a series
of adaptations to its host plants, the diversity of host plant selection is
overwhelming (Panda and Khush, 1995). A multitude of chemical and physical
plant characteristics may interact to affect host acceptance. Although responses to
12
individual compounds may exert a dominant influence for some herbivores, it now
seems that plant perception is based normally on an integration of complex
stimulus patterns (Harrison, 1987). Although it has become clear that host
selection is influenced by many plant stimuli, such as color, leaf shape, and other
physical factors, chemical cues are of paramount importance (Stadler, 1986;
Visser, 1986). Plant chemicals involved in host plant selection by phytophagous
insects are of two broad categories: (1) those influencing the behavioral proceses;
and (2) those influencing the physiological processes. The chemicals that affect
insect behavior in host plant selection are categorized according to the sensory
response that they elicit, for example, attractants, arrestants or stimulants, and
repellents or deterrents. Plant chemicals affecting physiological processes may be
classfied as antifeedants, physiological inhibitors, or toxicants (Panda and Khush,
1995).
The fact that the plant surface is an important interface between
phytophagous insects and the host plant is gaining more and more attention in the
research of plant-insect relationships (Stadler and Roessingh, 1991). The surface
of all higher plants is covered by a protective cuticle composed of a lipid polymer
(cutin) and a mixture of extractable lipids (epicuticular waxes) synthesized by the
epidennal cells and deposited on their outer walls (Kolattukudy and Walton, 1972;
Walton, 1990 ). The primary role of epicuticular lipids on the aerial plant surface is
prevention of water loss. They also serve other ecological effects, one of them
being the interaction with insect herbivores. The physical stmcture of plant surface
lipids may affect insect attachment, locomotion, and visual input by different light
13
reflection spectra (Jeffree, 1986). Plant surface chemicals have been proved to be
involved in enhancing or deterring oviposition, movement, and feeding, and
affecting herbivores indirectly by influencing predatory and parasitic insects
(Chapman and Bemays, 1989; Espelie et al., 1991; Eigenbrode and Espelie,
1995).
A number of different insect species show that, before feeding on a plant,
they explore its surface. It is possible that all phytophagous insects make some
sensory exploration of the leaf surface as a prelude to biting (Chapman and
Bemays, 1989; Stadler and Roessingh, 1991). Once the insect has made physical
contact with a plant, contact chemoreceptors located on the antennae, mouthparts,
tarsi, or ovipositor perceive stimuli from the plant surface. Surface testing by
touching, biting, probing, or piercing with sensory organs can assess the host's
degree of fitness as a substrate for feeding or oviposition. The behavior of many
herbivorous insects immediately after arriving on a plant indicates that they are
evaluating the plant as a potential food source or as an oviposition site (Panda and
Khush, 1995). This behavior includes the fluttering or fanning of butterflies with
their wings before and after landing on the host plant, palpating with labial and
maxillary palpi by lan/al and adult beetles and moths, and in some cases also
palpating with the antennae. Following palpation, grasshoppers sometimes make
a test bite without removing tissues, whereas beetles compress the leaf with their
mandibles, tap with the proboscis, drum, or do exploratory running and ovipositor
contact (Chapman and Bemays, 1989; Stadler and Roessingh, 1991). There are
obvious advantages to the plant in protecting itself from potential predation by
14
depositing cliemicals that discourage biting or oviposition before any damage
occurs (Chapman, 1977). It also benefits insect herbivores because they can save
time and energy by rapid assessment of host quality on the basis of surface cues
(Panda and Khush 1995).
Some research has demonstrated that plant secondary compounds or
primary nutrients occumng on the leaf surface may act as deterrents or
phagostimulants (Woodhead and Chapman, 1986). There have been 20 free
amino acids and soluble carbohydrates found on the com plant leaf surface. Their
profiles on the leaf surface were very plant species-specific and some of them were
characteristic of the plant species studied. They also vary with developmental
stage and growth condition. These primary metabolites show that there is great
diversity of infonnation available on the leaf surface for the insects (Derridj et al.,
1996). Fructose and other water extractable nutrients on the leaf surface are
probably the major stimulatory compounds on com plants that stimulate European
com borer, Ostrinia nubilalis. feeding and oviposition (Derridj et al., 1987; Derridj et
al., 1990). Secondary chemicals, produced by leaf trichomes and then deposited
on the leaf surface, deter Colorado potato beetle feeding (Yencho et al., 1994).
Secondary plant components, present in the carrot, Dacus carota. leaf surface wax,
extracted by methylene chloride, synergistically stimulate carrot fly, Psila rosae.
oviposition (Stadler and Buser, 1984).
In most cases, however, the active fractions or compounds are non-polar
and the chemicals involved are of widespread occurrence, and insects do have the
capacity to perceive non-polar substances on the dry leaf surface (Chapman and
15
Bemays, 1989). Some chrysomelid beetles were stimulated to feed by dominant
wax components of their corresponding host plants. An alkane fraction from
soybean stimulated Atrachya menetriesi F. feeding. An alcohol fraction from
mugwart stimulated the feeding of Chrvsolina aurichalcea. Galerucella vittaticollls
responded to both alkane and carboxylic acid fraction from strawberry. The
feeding of Pvrrhalta humeralis was stimulated by an alkane fractions from viburnum
(Adati and Matsuda, 1993). n-hexacosanol and n-octacosanol from mulberry,
Morus alba, leaves have a stimulating effect on the feeding of the silkworm,
Bombyx mori. larvae (Mori, 1982). Lipid-soluble material from the leaf surface of
Poa annua induces nymphs of the migratory locust, Locusta migratoria. to bite
(Bemays et al., 1976). Hamilton et al. (1979) observed that hydroxy p-diketone, the
wax component from the shoot surface of oats, acts as an attractant for the frit fly,
Oscinella frit, synergistically with hexane-insoluble materials in the plant.
In some cases, extracts of the surface lipids deter feeding. Leaf surface
chemicals, p-hydroxybenzaldehyde, short chain n-alkane, and ester fractions from
seedlings of Sorghum biocolour cultivar 65D deter the third instars of Locusta
migratoria feeding (Woodhead, 1983). Leaf epicuticular waxes affect leaf-cutting
ant substrate selection, perhaps because leaf epicuticular waxes from some plant
species have a deterrent effect on the growth of fungi (Sugayama and Salatino,
1995). Locusta migratoria can associate the presence of the internal deterrent
plant constituents with the chemical characters of its surface wax (Blaney and
Winstanley,1982).
16
Leaf surface chemicals also affect female oviposition. The Hessian fly,
Mayetiola destructor, oviposits preferentially on the paper strips coated with
epicuticular lipid extracts of wheat (Foster and Harris, 1992). The spruce budworm,
Choristoneura fumiferana. also was stimulated to oviposit on filter paper treated
with a host plant epicuticular lipid extract (Grant and Langevin, 1994). Paraffin and
alkanes that are common components of leaf surface wax (Kolattukudy and Walton,
1972; Walton, 1990) strongly influence diamondback moth, Plutella xylostella.
oviposition in response to host-specific chemical cues. Both paraffin and alkanes
cause a marked increase in the relative acceptability of aqueous host plant
homogenate and sinigrin, a glucosinate that is an important primary feeding and
oviposition stimulant (Spencer and Joseph, 1996).
Insect cuticular lipids also frequently play an important role in predator-prey
relationships. The various surface chemistry of herbivorous insects is partly
detennined by the diet, or the surface lipids of heriDivorous insects are similar to
those of their hosts. Insect predators and parasitoids are able to recognize these
lipophilic components on the cuticular surface of their preys (Eigenbrode and
Espelie, 1995; Espelie et al., 1991).
The composition of plant epicuticular lipids, both in quality and in quantity,
change with the developmental stage, as well as in response to different types of
environmental stress such as water, cold, and salt stress (Atkin and Hamiton,
1982). In some plants, lipids found in the cuticle of the abaxial surface are
dramatically different from those found in the adaxial surface (Holloway et al.,
1977). Insect behavior certainly might be affected by these differences.
17
The behavior and food acceptance of phytophagous insects with different
food specialties are affected by the leaf surface chemicals. They have finely-tuned
sensilla to detect polar and nonpolar chemicals deposited on the plant surface.
Chapman and Bemays (1989) pointed out that host-plant recognition by some
oligophagous insects does not depend on the presence of secondary chemical
characteristics of host taxa, and in these cases chemicals on the leaf surface are
important phagostimulants. It also is possible for the insects to associate the
chemicals on the plant surface with nutrition and secondary chemicals within the
plants. The leaf surface chemicals also play roles in tritrophic interaction
(Eigenbrode and Espelie, 1995; Espelie et al., 1991). Understanding the effect of
the leaf surface on herbivory would help understand the interaction between plants
and herbivores.
In Salicaceae, Salix spp. and Populus sop., phenolic glycosides such as
salicin, and its derivatives salicortin and tremulacin (Lindroth et al., 1987a), are
family-specific secondary chemicals. In fact, among the plant chemicals that have
been examined, phenolic glycosides are the only group of secondary compounds
present in significant amounts in the leaves of Salicaceae (Palo, 1984). Both the
quality and quantity of the phenolic glycosides in plants may change according to
the season, age, sex, growing conditions, among the leaves on an individual plant,
among individuals of the same species, and among different species. Change in
phenolic biosynthesis, storage, or metabolism may influence the susceptibility of
poplar or willow trees to insects and mammals (Palo, 1984; Larsson et al., 1986;
Lindroth et al., 1987b; Orians et al., 1993; Orians and Fritz, 1995). Species in Salix
18
with phenolic glycosides tend to be the food of specialized herbivores and to be
avoided by generalists. Conversely, species in Salix without phenolic glycosides
tend to be eaten by more generalist insects and avoided by the more specialized.
The faunas of the different Salix species with phenolic glycosides in their leaves
are more similar to each other than to the faunas of the Salix species having no
phenolic glycosides in their leaves (Rowell-Rahier, 1984).
Phenolics can be toxic to animals and microbes or harmful to the digestion
of herbivores. Although the presence of phenolic glucosides had a positive effect
on the growth rates of some specialists and no effect on some other specialists,
presence of phenolic glucosides had, in general, negative effects on growth rates
of non-specialists (Matsuki and Maclean, 1994). The phenolic glycosides salicin
and salicortin were found to influence Phratora vulqatissima larval growth and
adult feeding preference. Salicin and salicortin were found to depress adult beetle
feeding. Survival was unaffected by salicin treatment, but developmental rate was
significantly retarded by salicin. Salicortin was more toxic to larvae than was
salicin. Pupation rate and prepupal mass were reduced by salicortin (Kelly and
Curry, 1991). Salicortin and tremuloidin also are toxic to the southem armywonn
and the tiger swallowtail subspecies Paoilio alaucus olaucus L which normally
does not feed on aspen (Lindroth, 1991). Even insects adapted to aspen also may
experience reduced performance when fed phenolic glycosides. The phenolic
glycosides, salicortin and tremulacin, that exist in aspen dramatically reduced
larval growth and negatively affected performance of the forest tent caterpillar and
the gypsy moth (Lindroth and Weisbrod, 1991; Lindroth, 1991; Lindroth and
19
Hemming, 1990). The mode of acton of these compounds, although not fully
described, seems to involve the fomnation of lesions in the midgut epithelium in
Lepidoptera (Lindroth, 1991). In Lepidoptera, esterase activity in the midgut is
responsible for phenolic glucoside detoxification. Variation in insect susceptibility
to phenolic glycosides can be explained largely on the basis of differences in
metabolic detoxification capacity (Lindroth and Weisbrod, 1991; Lindroth, 1991;
Lindroth and Bloomer, 1991).
The phenolic glucosides seem to have a protective function for plants.
Some phenolic glucosides can be effective feeding deterrents to non-adapted
insects. More specialized herbivore species may overcome the harmful effects of
plant secondary substances and may become adapted to use them as positive
cues in orientation and feeding, and even to exploit them as precursors for their
own defensive compounds.
Females of specialized sawflies are highly selective in their oviposition. The
females of Euura amerinae. a monophagous shoot galler, use phenolic glucosides
as a cue to recognize and choose their host willow. Phenolic glucosides,
especially 2'-0-acetyl-salicortin, stimulated ovpositional behavior in E. amerinae
when applied to a neutral substrate (Kolehmainen et al., 1994).
The Chrysomelidae as a group synthesize their own chemical defenses
(e.g., iridoids, cardenolides, isoxazolinones), or sequester their defenses from a
food plant (e.g. salicylaldehyde, juglone, cucurbitacins) (Pasteels and RowellRahier, 1990). They have nine pairs of dorsal exocrine glands located on the meso
and metathorax segments and on abdominal segments 1-7 (Pasteels et al., 1989).
20
Insect species with larvae producing chemical defenses de novo usually feed on a
large range of plants in which no obvious direct precursors are known (Pasteels et
al., 1983). On the other hand, a few chrysomeline species use direct host plant
precursors for production of the main components of their lan/al secretions
(Pasteels et al., 1989; Pasteels et al., 1990). Several Chrysomela species and
Phratora vitellinae. which feed on Salicaceae, use salicin and its derivatives from
their host plants to make their own chemical defenses. The concentration of
salicylaldehyde in the secretion is positively correlated with the amount of salicin in
the larval food (Pasteels et al., 1983). The secretions may be strikingly different
when the lan/ae feed on different food plants (Hilker and Schuiz, 1994). The
transformation of salicin into salicylaldehyde occurs within the defensive glands.
This phenolic glucoside is hydrolyzed by p-glucosidase. Most of the glucose from
the hydrolysis of salicin is recovered for nutrition. The aglycone is oxidized by a
specific enzyme to salicylaldehyde, which is then discharged by the exocrine larval
glands (Pasteels et al., 1990). The defensive chemicals are deterrents to
generalist predators, such as ants, ladybird beetles, and spiders (Pasteels et al.,
1990; Pasteels et al., 1989; Rank, 1994), but do not affect specialists (Rank and
Smiley, 1994).
Lan/al and adult feeding preferences and larval performances of
Salicaceae-feeding leaf beetles should depend on the host chemicals and whether
the larvae secrete the defensive compounds or not. Inter and intraspecific variation
in the salicylate content of leaves should have a clear impact on the distribution of
21
the leaf beetles. Some investigations on the relationship between the food habits of
leaf beetles and the phenolic glucoside contents of different willow species indicate
that phenolic glucosides may remarkably affect the feeding behavior of these
Insects.
The food selection pattems of the tested leaf beetles followed closely the
phenolic glycoside spectra of the willow species. The concentration and the
composition of the different phenolic glucoside blends are species specific in
willows. Both the total amount and the composition of phenolic glycosides affected
beetle feeding. Phenolic glycosides seemed to have both stimulatory and
inhibitory influences on beetle feeding, depending on the degree of adaptation of a
particular insect (Tahvanainen et al., 1985).
Larvae of Plaqiodera versicolora. a chrysomelid Salix feeder, did not use
salicylate from the food plant for defense, but secreted iridoid monoterpenes
instead of salicyaldehyde (Jones et al., 1977). Salicin and salicylalcohol were
secreted with the feces (Pasteels et al., 1990). The avoidance of some Salix and
PoDulus spp. by P. versicolora is mainly caused by their pilosity. It seems that
trichomes play a more important role in food preference of P. versicolora than do
the contents or absence of tannins and salicin, although chemical factors may also
play a role in more subtle preferences (Bogatko, 1990).
In Phratora vitellinae. a specialist chrysomelid, the larvae secret
salicyaldehyde. It feeds mainly on Salix and occasionally on Populus spp. Adults
are found mainly feeding and ovipositing on salicylate-containing tree species,
although salicin is not obligatory for food acceptance (Rowell-Rahier and Pasteels,
22
1982; Denno et ai., 1990). Adults of Ph. vitellinae were attracted to feeding by a
chemical fraction from the host willow species and by two related salicylate
glucosides, tremulacin and salicortin. Phenolic glucoside extracts stimulated more
feeding than did individual pure glucosides (Kolehmainen et al., 1995). But,
female oviposition preferences did not only depend on salicylate content. The
females avoided species with high concentrations of simple phenolic compounds,
although these plants had high concentrations of salicylate (Denno et al., 1990).
Rowell-Rahier et al. (1987) found that Ph. vitellinae larvae that fed on host material
with phenolic glucosides weighed more than larvae fed on host material without
phenolic glucosides. Denno et al. (1990) reported that there was no consistent
relationship between the host plant and larval growth In the laboratory in Ph.
vitellinae. Adult oviposition preferences did not correspond well to pattems of
larval performance. It seems that females may choose their host not to insure larval
performance, but for secretion against predators.
Kolehmainen et al. (1995) also found that two willow-feeding generalists,
Galerucella lineola and Lochmaea capreae. were also stimulated strongly by total
glucoside fractions from their host plant, especially by its major glucoside,
salidroside. It seems that willow leaf beetles select their food based on the
phenolic glucosides of their host plants, and different compounds have synergistic
effects in the feeding behavior of the leaf beetles.
Chrysomela aeneicollis is also a specialist and uses host phenolic
glycosides for the production of salicylaldehyde. In feeding-choice tests, both
larvae and adults preferred salicylate-rich species over salicylate-poor species.
23
regardless of their previous feeding experience (Rank, 1992). Adults were
stimulated to feed by salicin itself (Rank, 1992). In the field, the relative abundance
of Q. aeneicollis adult and egg clutches among these species corresponded to the
adult feeding preferences in the laboratory (Rank, 1992). Adult abundance and
damage caused by herbivory was related to salicin content and plant size. A
preference for salicylate-rich willows is partly responsible for the increased level of
attack on these selections (Smiley et al., 1985; Rank, 1992). Although in the field
the beetle also showed ovipositlon specificity, in oviposition-preference tests gravid
females showed no preference between salicylate-rich willow species and
salicylate-poor willow species. This may indicate that the apparent specificity in
oviposition actually reflects a feeding preference. The willow leaf beetle seems to
locate favorable hosts through adult feeding behavior (Rank, 1992). In the field, the
larvae develop equally well on salicylate-poor or rich Salix species. Larval growth
is not affected by the salicylates, and more likely is related to the water contents in
the plants (Rank, 1994). The importance of beetle defense to larval survival rate
depends not only on phenolic glycosides in the food plants, but also on the natural
enemies, if general predators (Smiley et al., 1985) or specialists (Rank, 1994) are
the more important factors.
Although the phenolic glycosides are important secondary chemicals
involved in the relationship between the Salicaceae and herbivory, other
chemicals also are involved in the mediation of Salicaceae-feeding beetle
behavior. Chlorogenic acid (Matsuda and Senbo, 1986) in the leaf exudates from
Salix integara. S.. chaneomeloides. and
koriyanagi leaf surfaces deterred the
24
feeding of Lochmaea caoreae cribrata. and stimulated the feeding of Plaoiodera
versicolora distincta. which feeds on these salicaceous plants. Three Salicaceaefeeding beetles, P. y. distincta. Chrysomela viaintipunctata costella. and L c.
cribrata responded to a flavonoid, luteolin-7-glucoside (Matsuda and Matsuo,
1985).
The Cottonwood leaf beetle, Chrysomela scripta. feeds on Pooulus spp. and
Salix spp. and the larvae secrete the defensive chemical, salicylaldehyde (Wallace
and Blum, 1969). Generalist predators are important mortality factors of the
Cottonwood leaf beetle. Burkot and Benjamin (1979) found the most important
predator on the cottonwood leaf beetle in Wisconsin was a generalist ladybird
beetle, Coleomegilla maculata. The cottonwood leaf beetle showed different
feeding preference among different willow clones. The amount of defoliation
varied significantly by species and clones. The overall average defoliation was
63%, and the clone means ranged from 5 to 95%. Male clones were damaged
significantly more than female clones (Randall, 1971). The adults showed varied
preferences among tested poplar clones and among leaves of different
developmental stages. The beetle usually rejects species or clones from the Leuce
section for feeding, and shows preference for clones containing more Aigeiros and
Tacamahaca parentage (Caldbeck et al., 1978; Harell et al., 1981; Bingaman and
Hart, 1992), although the reports on beetle feeding preference on Aigeiros and
Tacamahaca parentage differ (Harell et al„ 1981; Bingaman and Hart, 1992). The
beetle also discriminates among leaf age classes, preferring young leaves over
mature leaves for feeding (Harell et al., 1981; Bingaman and Hart, 1992). The
25
oviposition preference patterns correspond to feeding preference patterns, both
among clones and leaf ages (BIngaman and Hart, 1992). As with some willow
feeding beetles, such as Galerucella lineola. Phratora sp. (Denno et al., 1990), and
Chn/somela aeneicollis (Rank. 1992), no correlation between feeding preference
and leaf moisture, nitrogen, carbohydrate levels, leaf thickness, toughness, or
surface physical characteristics were found in the relationship between the
Cottonwood leaf beetle and the poplars (Harell et al., 1981). It was suggested that
a high concentration of foliage salicortin and tremulacin causes beetles to reject
foliage in the Leuce section, whereas leaves from the sections Tacamahaca and
Aigeiros, having lower levels of salicortin and no or lower tremulacin, are attractive
to beetles (Lindroth et al., 1987b). It was found that salicin and salicortin content
did not negatively influence host selection, but that tremulacin may negatively
influence such behavior (BIngaman and Hart, 1993), but no clear and direct
correlation between phenolic glycosides and feeding or oviposition preferences
has been established for the cottonwood leaf beetle.
26
CHAPTER 2. INSECT FEEDING STIMULANTS FROM THE
LEAF SURFACE OF POPULUS
A paper submitted to the Journal of Chemical Ecology
S. Lin, B.F. Binder, and E.R. Hart
Abstract
Leaf surface chemicals from a cottonwood leaf beetle-preferred poplar
clone, 'Eugenei' (Populus deltoides x Populus nigra), induce feeding in the adult
cottonwood leaf beetle, Chrysomela scripta Fabr. (Coleoptera: Chrysomelidae).
The feeding stimulants were isolated and identified as n-beheryl alcohol (Cgg), nlignoceryl alcohol (C24), n-hexacosanol (Cge), n-octacosanol (Cgg), n-triacontanol
(C30), and a-tocopherylquinone (2-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)3,5,6-trimethyl-<1 ,4>benzoquinone, a-TQ). It is the first time that a-TQ has been
reported as a feeding stimulant for an insect. Fatty alcohols or a-TQ alone do not
induce beetle feeding significantly, but a mixture of alcohols and a-TQ
synergistically stimulates beetle feeding. The role of these feeding stimulants in
insect feeding behaviors and possible use in a pest management program is
discussed.
Key words Populus: cottonwood leaf beetle; Chrvsomela scripta F. insect feeding
stimulant; leaf surface chemicals; n-primary alcohols; a-tocopherylquinone.
27
Introduction
It is generally accepted that host-plant selection behaviors or feeding
preferences of phytophagous insects are largely mediated by the presence and
distribution of secondary chemicals in plants. Plant leaf surface chemicals have an
important role as mediators of insect-plant interactions because, after being
attracted by visual and olfactory cues, insects first contact those compounds
present on the plant surface. Plant epicuticular waxes have been reported to affect
feeding behavior differently for various insects (Stadler, 1986; Chapman and
Bemays, 1989; Espelie et al., 1991; Eigenbrode and Espelie, 1995).
Phytophagous insects usually make a sensory exploration of the leaf surface as a
prelude to biting. Insects have a finely tuned sensory capacity to perceive nonpolar substances on the leaf surface (Chapman and Bemays, 1989). Epicuticular
lipids have been reported to stimulate insect feeding (Stadler, 1986). For example,
the surface lipids of the host plant Poa annua induce nymphs of Locusta migratoria
to bite, whereas hydrocarbons stimulate the pea aphid, Acyrthosiphon pisum. to
feed on an artificial membrane (Eigenbrode and Espelie, 1995). n-Hexacosanol
and octacosanol in the leaves of the mulberry, Morus alba, stimulate feeding in
larvae of the silkworm, Bombyx mori (Mori, 1982). To some extent, plant surface
chemicals may help insects to determine if the plant is suitable as a host.
Several species of chrysomelid beetles also are stimulated to feed by nalkanes and fatty alcohols isolated from the leaf surface of their host plants (Adati
and Matsuda, 1993). Similarly, we find that the leaf surface chemicals from
PoDulus clones preferred by the cottonwood leaf beetle induce adult feeding.
28
Adults of the cottonwood leaf beetle, Chrvsomela scripta F. (Coleoptera:
Chrysomelidae), show varied feeding preference among poplar clones, Populus
sg. (Salicaceae), and among leaves of different development stages (Bingaman
and Hart, 1992; Han'ell et al., 1981; Caldbeck et al., 1978; Bingaman and Hart,
1993). We posit that leaf surface chemicals play an important role in host plant
selection by the cottonwood leaf beetle. In this report, we characterize the beetle
feeding stimulants from the leaf surface of Populus.
Materials and Methods
Plant material. A beetle-preferred poplar clone, 'Eugenei' (£. deltoides x P.
nigra), was planted at the Hind's Farm, Ames, Iowa, in summer 1994. During the
growing season in both1994 and 1995, immature leaves were harvested for
biological evaluations and characterization of leaf surface feeding stimulants.
Biological evaluations. Egg masses or larvae of the cottonwood leaf beetle
were collected from a plantation near Ames, Iowa, and reared in a growth chamber
under a photoperiod of 16 : 8 (L : D) and a temperature regime of 24 : 16°C (L : D)
on greenhouse-grown P. deltoides leaves. Adults, age 36-48 h post eclosion, with
no prior feeding experience, were used to evaluate plant residues and
chromatographic fractions or authentic standards as described.
Three pairs of adult males and females were exposed to artificial leaf discs;
each disc was made from half pieces of Whatman No. 1 filter paper, 42.5-mm
diameter. The discs were fixed to a wax-coated glass petri dish (100 x 15 mm) with
insect pins. Two layers of towel paper and one layer of Whatman No. 1 filter paper
29
(7-cm diameter), completely soaked with deionized water, were used to keep the
humidity high in each petri dish. For each dish, two of the discs were treated with
plant residue (21.1 ^ig/cm^) or chromatographic fraction (11.2 or 8.5 |i.g/cm^) and
positioned altemately with two discs treated with solvent control (chloroform) for the
biological activity screening tests. Assays were conducted at 24 :16°C (16 : 8 h) in
the dark for 24 h. Dark condition was used to avoid any possible visual cues.
Three replications were run for each test. Bite marks were counted for each test.
A final test with authentic standards was made as just described with four
randomly arranged artificial leaf discs: 50 (xg n-beheryl alcohol (C22) (Sigma), 5 ^ig
synthetic a-TQ, a mixture of 50 |j.g n-beheryl alcohol and 5 |ig a-TQ, and the
solvent control (chloroform). The assay was replicated 70 times under the same
conditions as those for the screening tests. The bite marks were counted and
evaluated by analysis of variance (ANOVA) and t-tests. Percentages of responders
were tested with
(SAS, 1985).
Reference compounds. Synthetic a-TQ and n-beheryl alcohol (C22), nlignoceryl alcohol (C24), n-hexacosanol (Cjs), n-octacosanol (Cgg), and ntriacontanol (Cgo) (Sigma) were used as chromatographic standards and in
biological evaluations. a-TQ was prepared from d-a-tocopherol (Issidorides, 1951;
Weng and Gordon, 1993), and the acetates of authentic alcohols and alcohols from
the plant were prepared by using acetate anhydride.
30
Instrumentation and general methods. Intact fresh leaves were dipped into
hexane for 30 s at room temperature and filtered. The filtrate was concentrated
under reduced pressure at 35°C. Open-column chromatography: 5.1 g extract was
applied on 200 g Florisil (60-100 mesh, Fisher) deactivated with 7% water by
weight and eluted with hexane, benzene, chloroform, and methanol (Figure 1).
The fraction that eluted with benzene retained the biological activity. Then, 1.98 g
of the concentrated material was applied on 200 g Florisil and eluted with an
ascending series, 20%, 25%, 33%, and 50% chloroform in hexane. The fraction
that eluted with 50% chloroform in hexane held the activity. This fraction was
purified further by applying 40 mg on 10 g Florisil and eluting with chloroform.
Preparative thin-layer chromatography (TLC): 14.5 mg of the active fraction from
open column chromatography was applied on silica gel plates (Merck 60 F254,
250 |im thick, 20 x 20 cm), and eluted with solvent system I (hexane : diethylether:
formic acid, 50 : 50 : 0.5) followed with solvent system II (chlorofomri). Fractions
were scraped from the plate and desorbed with chlorofonn. The procedure was
repeated until 1.2 mg of a-TQ was obtained.
Gas chromatography/mass spectrometry (GC/MS): A sample of TLC
fractions possessing biological activity was injected on a 30-m x 0.25 mm- ID, 0.25^.m film thickness, DB-5, 5% phenyl methyl silicone column (J & W Associates) on a
HP 5890 series II gas chromatograph. Gas flow rate for He, 1.3 ml/min;
temperature program: 100°C for 1 min, then to 295°C at 10°C/min and maintained
31
at 295°C for 10 min; flame ionization detector; detector temperature, SOO'C, injector
temperature 250°C: split ratio 100 : 1 .
GC-MS data were recorded with a HP 5890 series II gas chromatograph
coupled with a HP 5972 mass spectrometer by using 70 ev. Chromatography was
perfonned on a DB-1 column, temperature: lOO'C (0 min), lO'C/min, 325°C.
Helium was the carrier gas at 1 ml/min. MS data also were recorded on Magnum
GC/MS with a DB-5, 0.25 mm -ID, 0.25 jxm film thickness column; He as carrier gas,
70 ev. GC program: 100°C, 1 min, 15°C /min, and 298°C 15 min.
Fourier Transfonned-lnfrared Spectrometry ( FT-IR ): An FT-IR spectrum of
the active fractions was obtained on a Buker IR 98 spectrophotometer, samples
were on a NaCI crystal, and the evaluation was between 4000 cm'^ and 500 cm \
UV spectra were detennined in absolute ethanol with a UV 160u Shimadza UV/Vis
spectrophotometer, scan range 200 nm to 400 nm. A 300-MHz 'H NMR was
recorded in deuterated chloroform (CDCI3) with tetramethylsilane (TMS) as an
internal standard with a Varian Vax 300 MHz NMR.
Results
The yield of leaf surface wax varied through the growing season and ranged
from 10 to 20 |xg/cm^ leaf area. The extract applied at 21.2 |ig/cm^ on the filter
paper induced adult beetle feeding, whereas the solvent chloroform did not. The
protocol outlined in Fig. 1 was used to isolate and identify the components with
feeding stimulant activity. After open-column chromatography and qualitative
32
analysis by TLC, the active fraction contained a UV (254 nm) quenching band and
a band with no UV absorbtion, suggesting the presence of at least two classes of
compounds.
Characterization of the active components. The compound with no UV
absorbtion had a TLC Rf = 0.58 in solvent system I and Rf = 0.32 in system II. The
purified compounds were colorless crystals in chloroform. IR absorbtion at 3300,
2920, 2850, 1465, and 720 (cm'^) indicated that the compounds were alcohols.
Analysis by GC showed that five compounds were present and they coeluted with
C22, C24, C26, C28, C30 -primary alcohols (Sigma). The acetates of the alcohols from
plants also had five peaks that coeluted with the acetates of C22, C24, Cgg, C28, C30primary alcohols. Final confirmation of the five primary long-chain alcohols was by
GC/MS. The mass fragmentation patterns of the plant alcohols and their
corresponding acetates were identical with those of authentic long-chain alcohols
and their acetates.
The band that quenched UV (254 nm) on the TLC had a Rf = 0.58 in solvent
system I and Rf = 0.18 in system II. The pure compound was a lemon-yellow oil.
The compound was identified as a-TQ by its UV, IR, GC-MS, and
NMR spectra
and confinned by comparison with the synthetic a-TQ.
Ultra-violet (nm): 203, 262, 268. Infra-red (cm '): 3522.7, 2952.8, 2925.2,
2851.5, 1682.7, 1643.8, 1463.4, 1375.8, 1307.2, 714.8. GC-MS (m/z): 447 (M+1,
1.3), 446 (M, 4.3), 431 (2.1), 430 (3.2), 428 (5.1), 221 (100), 203 (11.7), 180 (25.3),
33
179 (21.1), 178 (73.6), 165 (22.4), 150 (27.2), 135(9.6) .
NMR (5 ppm): all
proton signals were between 0.80 and 2.60.
Biological activities. a-TQ (56.3 ng/cm^) fronn the plant together with (8.45
|ig/ cm^) alcohols from the plant at a ratio, 1:150, showed high feeding-stimulant
activity; the mean number of bite marks was 74 ± 17. The activity decreased with
higher or lower amounts of a-TQ with a fixed amount of alcohols. At the ratio, 1 :
60, 1 :100 and 1 : 200, the mean numbers of bite marks were 10 ± 5, 11 ±6 and 1
± 1, respectively.
Commercially available Cja, C24, Cgg, Cgg, C30 -primary alcohols, each when
mixed with synthetic a-TQ, stimulated beetle feeding. Bioassays with C^z and
synthetic a-TQ showed that the beetles had varied responses to the chemicals, but
a significantly higher percentage of beetles responded to a mixture of
alcohol
and a-TQ than to either compound alone (Figure 2). C22 alcohol or a-TQ alone
showed no significant difference in the number of bite marks from that of the
control, but a mixture of both compounds had a significantly higher number of bite
marks than the control or either of the compounds alone (Figure 3). These
observations confirm the synergistic feeding-stimulant activity of the plant natural
products.
34
Discussion
Long-chain fatty alcohols are found commonly as components in plant
epicuticular lipids (Walton, 1990). The fatty alcohols stimulate feeding in larvae of
the silkworm Bombyx mori (Mori, 1982), in the chrysomelid beetle, Chrvsolina
anrichalcea (Adati and Matsuda, 1993), and the chrysomelid beetle, Chrvsomela
scripta. in our study. a-TQ, which has not been identified previously from the
poplar leaf surface, acts as a feeding synergist for C. scripta when combined with
the fatty alcohols from the epicuticular lipid of Populus. Seemingly, C. scripta
recognizes at least two classes of poplar natural products and uses a blend of
these compounds to evaluate the host plant suitability during gustation. To our
knowledge, it is the first time that a-TQ has been reported as an insect feeding
stimulant.
The main composition of the fatty alcohols from 'Eugenei' are Cgg,
Cgg,
Cgg, C30 -primary alcohols. The beetles respond to each of them when mixed with
a-TQ. Data from the bioassays indicate the possibility that the beetles can
differentiate among the aliphatic alcohols and that not all concentrations and ratios
of alcohols and a-TQ induce strong feeding activity. More tests are in progress to
evaluate the optimum ratio or the optimum concentration of these compounds. We
speculate that poplar clones with a high concentration of fatty alcohols and a-TQ,
and at an appropriate ratio for optimum gustatory stimulation, are susceptible to
heavy attack by the cottonwood leaf beetle.
35
Thirty percent of the beetles did not respond to the mixture of C22 alcohol and
a-TQ. This may indicate that the beetles have genetic differences in gustatory
responses or that physiological regulation made the beetles unresponsive to the
chemicals. Also, it is likely that there still may be other unidentified feeding
stimulants in PODUIUS leaf surface extracts. In bioassays in which synthetic a-TQ
was used either with authentic primary alcohols or with plant-extracted alcohols to
induce beetle feeding, a higher concentration of synthetic a-TQ was always
needed. The GC profile of the plant a-TQ fraction from the TLC purification step
showed that, besides a-TQ, there were several compounds in much smaller
concentrations that had GC retention times close to a-TQ. Some of these minor
compounds may be analogs of a-TQ and also have some stimulating effect on
beetle feeding. Lack of these compounds in the synthetic material, or the
difference between stereoisomer components of synthetic a-TQ and naturally
occuring a-TQ stereoisomers, may account for the observed lower feeding
responses.
The yield of the wax from poplar varied during the growing season, although
we do not know how the composition of the leaf surface feeding stimulants
changes. Plant leaf surface chemicals can vary in quality and quantity among
different plant tissues, during maturation, and because of fluctuating environmental
conditions (Stadler, 1986; Walton, 1990). The cottonwood leaf beetle adults feed
on the adaxial surfaces of leaves and prefer immature leaves (Bingaman and Hart,
36
1992); therefore, these leaves probably have a high concentration of alcohols and
a-TQ. Whether the chemicals have a stimulating effect on larval feeding has not
yet been tested. Evaluating how leaf surface chemicals are distributed in poplar
plants and their tumover dynamics will help us better understand the relationship
between the poplar leaf surface chemicals and the beetle feeding behavior.
a-TQ occurs widely in photosynthetic organisms, in animal tissues, and in
bacteria, and it has been ascribed to metabolic functions, including participation in
photosynthetic electron transport and antioxidative pathways. Many investigators
have suggested that, in plants, a-TQ is limited to the chloroplasts and that the
content of a-TQ is dependent on light intensity, tissue age, and other factors (Kruk
and Strzalka, 1995). The extraction method of the leaf surface chemicals in this
study makes us confident that the a-TQ was from the leaf surface, showing that the
distribution of a-TQ in plant tissues varies and may depend on genetics and
environmental conditions. Because a-TQ is widely distributed in plants, it is
possible that it may act as a feeding stimulant or synergist for other insects as well.
Populus selections, because of their distinguished biological characteristics
(Dickmann and Stuart, 1983; Ceulemans, 1990) - rapid juvenile growth and
biomass production, ease of vegetative propagation, tissue and cell culture
propagation, ease of breeding, large species diversity, availability of unmanaged
natural populations, and excellent growth on a wide range of sites - have been
developed as the preferred choice for short-rotation woody crop plantings
37
throughout much of the world. The cottonwood leaf beetle Is recognized as one of
the potential limiting factors for poplar plantations in North America (Wilson, 1976;
Brown, 1956; Burkot and Benjamin, 1979). The phagostimulants existing on the
leaf surface of the host poplar leaves and their variation may regulate beetle
feeding behavior and can be used as an important tool in the management of C.
scripta. Through breeding, selection or genetic transformation, we speculate that
we may be able to reduce or remove the feeding stimulants from the leaf surfaces
and thus improve poplar resistance to the cottonwood leaf beetle.
Acknowledgements
We thank A. Cosse, R. Cao and K. A. Harrata for technical assistance.
Research was supported in part by the Oak Ridge National Laboratory operated by
Martin Marietta Energy Systems, Inc. for the U. S. Department of Energy, contract
no. DE-AC05-840R21400. Journal Paper No. J-17207 of the Iowa Agriculture and
Home Economic Experiment Station, Ames, Iowa. Project No. 2976, 3443 and
supported by McEntire-Stennis and State of Iowa funds.
References
ADATI, T., and MATSUDA, K. 1993. Feeding stimulants for various leaf beetles
(Coleoptera: Chrysomelidae) in the leaf surface wax of their host plants.
Appl. Entomol. Zool. 28:319-324.
BINGAMAN, B.R., and HART, E.R. 1992. Feeding and oviposition preferences of
adult Cottonwood leaf beetles (Coieoptera: Chrysomelidae) among Populus
clones and leaf age classes. Environ. Entomoi 21:508-517.
BINGAMAN, B. R., and HART, E.R. 1993. Clonal and leaf age variation in
Populus phenolic glycosides: implications for host selection by
Chrysomela scripta (Coieoptera: Chrysomelidae). Environ. Entomoi.
22:397-403.
BROWN, W. J. 1956. The new world species of Chrysomela L. (Coieoptera:
Chrysomelidae). Can. Entomoi. 88:Supplement 3; 54p.
BURKOT, T.R., and BENJAMIN, D.M. 1979. The biology and ecology of the
Cottonwood leaf beetle, Chrysomela scripta (Coieoptera: Chrysomelidae),
on tissue cultured hybrid Aigeiros {PopulusXEuramericana) subclones
tissue cultured hybrid Aigeiros {PopulusXEuramericana) subclones in
Wisconsin. Can. Entomoi. 111:551-556.
CALDBECK, E.S., McNabb, H.S.Jr., and HART, E.R. 1978. Poplar clonal
preferences of the cottonwood leaf beetle. J. Econ. Entomoi. 71:518-520.
CEULEMANS, R. 1990. Genetic Variation in Functional and Structural
Productivity Determinants in Poplar. University of Antwerp, Amsterdam.
CHAPMAN, R.F., and BERNAYS, E.A. 1989. Insect behavior at the leaf surface
and leaming as aspects of host plant selection. Experientia 45:215-222.
DICKMANN, D.I., and STUART, K.W. 1983. The Culture of Poplars in Eastern
North America. Michigan State University, East Lansing, Ml.
EIGENBRODE, S.D., and ESPELIE, K.E. 1995. Effects of plant epicuticular lipids
on insect herbivores. Annu. Rev. Entomol. 40:171-194.
ESPELIE, K.E., BERNAYS, E.A., and BROWN, J.J. 1991. Plant and insect
cuticular lipids serve as behavioral cues for insects. Arch. Insect Biochem.
Physiol. 17:223-233.
HARRELL, M.O., BENJAMIN, D.M., BERBEE, J.G., and BURKOT, T.R. 1981.
Evaluation of adult cottonwood leaf beetle, Chrysomela scripta
(Coleoptera: Chrysomelidae), feeding prafsrence for hybrid poplars.
Great Lakes Entomol. 14:181-184.
ISSIDORIDES, A. 1951. The antioxygenic synergism of various acids with atocopherol. J. Am. Chem. Soc. 73: 5146-5148.
KRUK, J., and STRZALKA, K.J. 1995. Occurrence and function of atocopherol quinone in plants. Plant Physiol. 145:405-409.
MORI, M. 1982. n-Hexacosanol and n-octacosanol: feeding stimulants for larvae
of the silkworm, Bombyx mori. J. Insect Physiol. 11:969-973.
SAS 1985. SAS Institute Inc. Gary, NC. USA.
STADLER, E. 1986. Oviposition and feeding stimuli in leaf surface waxes, pp.
105-121, in B. Juniper and S.R. Southwood (eds.). Insects and the Plant
Surface. Edward Amold, Maryland, USA.
WALTON, T.J. 1990. Waxes, cutin and suberin, pp. 106-158, in J. L. Hanwood and
J.R. Bowyer (eds.). Methods in Plant Biochemistry, Vol. 4. Academic Press
Inc. San Diego.
40
WENG, X.C., and GORDON, M.H, 1993. Antioxidant synergy between
phospatidyl ethanolamine and a-tocopheryl quinone. Food Chem. 48:165168.
WILSON, L.F. 1976. Entomological problems of forest crops grown under
intensive culture. Iowa State J. Res. 50: 277-286.
41
cmde extract in hexane
open column fractionation
hexane
benzene
chloroform
methanol
open column fractionation
hexane: chloroform
4:1
3:1
2:1
1:1
open column
fractionation
chlorofomri
10ml fractions
Preparative thin layer chromatography
n-primary alcohols
a-tocopherylquinone
Fig. 1. The separation and purification scheme for the beetle feeding stimulants.
42
70
^60
"O
o 50
a
(0
£ 40
0
0) 30
O)
(0
c 20
0)
u
o 10
Q.
0
control
quinone
alcohol
mixture
Treatment
Fig. 2. Percentage of beetles responding to four treatments (control, Cjg alcohol, aTQ, C22 alcohol and a-TQ) in 24 h. Beetles were categorized as responders when
bite marks >10 and as non-responders when the bite marks were <10 in one
replication. Values with different letters indicate means (n = 70) that are
significantly different, (a = 0.01).
43
45
40
0)
X
1
35
(Q
E 30
0)
•••
£1
40
25
20
4)
£ 15
E
3
z 10
5
0
control
quinone
alcohol
mixture
Treatment
Fig. 3. Mean number of bite marks on disks (n = 70) with four treatments (control,
C22 alcohol, a-TQ, C22 alcohol and a-TQ) in 24 h. The mean number of bite marks
on disks is significantly different (F = 38.43, P = 0.0001). Error bars are SE. Values
with different letters indicate means that are significantly different (a = 0.01).
44
CHAPTER 3. CHEMICAL ECOLOGY OF COTTONWOOD LEAF
BEETLE ADULT FEEDING PREFERENCES ON POPULUS
A paper submitted to the Joumal of Chemical Ecology
S. Lin, B.F. Binder, and E.R. Hart
Abstract
Field planted University of Washington poplar pedigree materials, parent
clones ILL-129, fPopulus deltoides). and 93-968, (Populus trichocanja^.
clones,
(53-242 and 53-246), and 87 Fg selections were used. Both field cage feeding
tests with parent and F^ clones, and leaf disc feeding tests with all 91 clones, were
perfomned. Feeding stimulants on the leaf surface, long chain fatty alcohols and atocopherylquinone (a-TQ), and phenolic glycosides, tremulacin, and sallcortin,
were analyzed to correlate chemical abundance with cottonwood leaf beetle,
Chrysomelascripta Fabr. (Coleoptera: Chrysomelidae), adult feeding preference.
The beetles showed varied feeding preferences among parent clones, F, clones,
and Fg clones. In the presence of alcohols, contents of alcohols, tremulacin, and
salicortin did not explain adult beetle feeding preference. Content of a-TQ on the
leaf surface could explain the adult beetle feeding preference. The beetle preferred
to feed on the clones with a-TQ rather than the clones without a-TQ. As the amount
of a-TQ increased, the feeding preference increased, and then decreased as the
amount of a-TQ increased further.
45
Key words Populus: cottonwood leaf beetle; Chrysomela scripta F.: insect feeing
stimulants; leaf surface chemicals; long chain fatty alcohols; a-tocopherylquinone;
phenolic glycosides; tremulacin; salicortin.
Introduction
The cottonwood leaf beetle, Chrysomela scripta Fabr. (Coleoptera:
Chrysomelidae), is a major defoliating pest of Populus in North America (Burkot
and Benjamin, 1979; Wilson, 1979; Harrell et al., 1981). Both adults and lan^ae
feed on young leaves. The adult stage is highly mobile and well-adapted for the
wide dispersal of the species and for host-plant selection. The beetle usually
rejects species or clones from the Leuce section, and shows a preference for
clones containing Aigeiros and Tacamahaca parentage (Caldbeck et al., 1978;
Harrell et al., 1981; Bingaman and Hart, 1992). The beetle also discriminates
among leaf age classes, preferring to feed on young leaves (LP! 3-5) over mature
leaves (Harrell et al., 1981; Bingaman and Hart, 1992). No correlation between
beetle preference and poplar leaf moisture, nitrogen, carbohydrate levels, leaf
thickness, toughness, or surface physical characteristics have been found (Harrell
etal., 1981).
In Salicaceae, Salixspp. and Populus spp.. phenolic glycosides are familyspecific secondary compounds (Palo, 1984). The compounds are well known to
influence the susceptibility of plants to both insect and mammalian herbivory
(Rowell-Rahier and Pasteels, 1982; Smiley etal., 1985; Rowell-Rahier et al., 1987;
46
Denno et al., 1990; Lindroth, 1991; Kelly and Curry, 1991; Rank, 1992 and 1994;
Matsuki and Maclean, 1994; Kolehmainen et al., 1994 and 1995).
It was found
that salicin and salicortin content did not negatively influence cottonwood leaf
beetle host selection, but tremulacin may negatively influence such behavior
(Bingaman and Hart, 1993). Although these studies focused on salicin, salicortin,
and tremulacin leaf content, no clear and direct correlation between phenolic
glycosides and feeding or oviposition preference has been established.
After observing cottonwood leaf beetle feeding behaviors, we sumnised that
evaluation of the plant surface is an important step in host plant selection by this
phytophagous pest. Numerous studies have shown that plant surface chemicals
are involved in enhancing or detering movement, oviposition, and feeding, and
also affect herbivores indirectly by influencing predatory and parasitic insects. In
most cases, the behaviorally active components are common lipophilic compounds
(Stadler, 1986; Woodhead and Chapman, 1986; Chapman and Bemays, 1989;
Espelie et al., 1991; Stadler and Roessingh, 1991; Eigenbrode and Espelie, 1995).
In some chrysomelid beetles, dominant wax components of their corresponding
host plants stimulate them to feed (Adati and Matsuda, 1993). We found, however,
that long chain fatty alcohols and alpha-tocopherylquinone (a-TQ) in the leaf
surface of a preferred Populus clone 'Eugenei' (Populus deltoides x Populus nigra)
stimulated cottonwood leaf beetle adult feeding (unpublished data).
In this study, we investigated the variation of feeding stimulants in the leaf
surface to detemnine if changes in levels of long chain fatty alcohols and a-TQ
47
among poplar plants could explain the beetle feeding preference on trees.
Moreover, we elucidated further the role of phenolic glycosides on host-plant
selection. By using University of Washington poplar pedigree material, we
evaluated the effects of phenolic glucosides, fatty alcohols, and a-TQ on the
Cottonwood leaf beetle adult feeding preference.
Materials and Methods
Plants. Poplar pedigree materials generated by the University of
Washington were used for the research: two parent clones, 93-968 (Populus
trichocarpa^ and ILL-129 (Populus deltoidesh two F, generation clones, 53-246
and 53-242; and 87 Fj generation selections.
All trees were grown from softwood cuttings taken from greenhouse stock
plants. The cuttings were dipped into 500 ppm indolebutyric acid for 5 s and
planted in saturated peat pellets under mist until roots formed. The plants were
transplanted into pots containing vermiculite, periite, and milled peat ( 1 : 1 : 1 )
(Faltonson et al., 1983). In May, 1994, two parent clones and two F, clones were
planted at the Iowa State University (ISU) Hind's Research Farm, in Ames, Iowa, for
field cage feeding tests. Four plants were planted as a group, with one of each of
the four clones planted randomly within the group. There were 2 m between each
group and 1 m between each plant within a group. In April, 1995, all 91 clones
were planted in the field at the ISU Institute for Physical Research and Technology,
in Ames, Iowa for leaf disc feeding tests. The trees were planted in 8 blocks; within
48
each block, one of each of the 91 clones was planted randomly with interplant
spaces of 1 m within the row and 3 m between the rows.
Insects. For each experiment, lan/ae of the cottonwood leaf beetle were
collected from a plantation near Ames, Iowa, and reared in a growth chamber
under a photoperiod of 16 : 8 (L : D) and a temperature regime of 24:16 °C (L : D)
on greenhouse-grown P. deltoides leaves. In our preliminary tests, we found that
the larval feeding experience on different acceptable poplar clones did not affect
the adult feeding preference (unpublished data). After emergence, age 36-48 h
post eclosion adults with no prior feeding experience were used in the bioassays.
Bioassays. For field cage feeding preference tests, parent and F^ clones
planted at the ISU Hind's Research Farm were used. In September, 1994, the four
trees in each group were covered with a 2 m by 2 m cage. Five pairs of male and
female beetles were released into the center of the cage. After 4 d, the leaves were
collected and the amount of leaf area consumed from each plant was measured
with an area meter in the laboratory. The test was replicated three times and in two
years,1994 and 1995.
For leaf disc feeding tests among the 91 clones, plants from three field
blocks were used. Each field block was considered a replication. For each block,
branches from each tree were collected, placed on ice, and transferred to the
laboratory. Leaf discs, 224 mm^, were punched from leaves (LPI 3-5) with a #12
cori< borer. An incomplete Latin square design was used. One plastic petri dish
(100 x 15 mm) with two layers of paper towel moistened with distilled water, was
49
used as a block, resulting In a total of 91 blocks. In each block, 10 leaf discs, each
from a different clone, were placed in a circle around the perimeter of the petri dish.
Leaf discs from each clone, therefore, appeared in 10 out of the total of 91 blocks or
petri dishes (Cochran and Cox, 1992). Five pairs of beetles were released in the
center of each petri dish. The dishes were placed in the growth chamber under the
same conditions as described. After 24 h, the leaf area consumed from each disk
was measured with an area meter. For all three field blocks, the bioassays were
finished within 8 d, allowing 2 d for each field block.
Chemical analysis. Leaves for bioassay and chemical analysis were
collected at the same time. Leaves were chemically analyzed immediately
following collection. For the parent and F, clones used in the field cage feeding
tests, leaves were collected from four trees of the same clone and mixed together,
then two leaf samples were used for chemical analysis. For the 91 clones used in
the leaf disc feeding tests, leaves from each individual tree were used for chemical
analysis.
The fatty alcohols and a-TQ were analyzed by GC after solid phase
extraction. Erucyl alcohol (cis-13-docosen-1-ol) (Sigma), ca. 99% by capillary GC,
was used as an intemal standard (IS). Intact fresh leaves, 300-600 cm^, as
measured with an area meter, were dipped into 250 ml hexane at room
temperature for 30 s. Each sample was filtered with Whatman No.1 filter paper,
and then dried under a stream of nitrogen gas. The plant extract (ca.10 mg), along
with 5 |xg of erucyl alcohol, was dissolved in 220 p.1 5% ethyl ether in hexane. The
50
sample and IS were washed onto ca. 335 mg Supelclean* LC-Florisil (Supeico)
solid phase in a 15 cm long disposable/flint Pasteur glass pipet (Fisher). The
feeding stimulants and minor impurities were eluted from the solid phase with 4 ml
30% ethyl ether in hexane (Figure 1). The eluant was dried under a stream of
nitrogen gas. The residue was dissolved in 10 ^,1 chloroform. A 1 ^il aliquot was
injected on a 30-m x 0.25 mm- ID, 0.25-nm film thickness, DB-5, 5% phenyl methyl
silicone column (J & W Associates) on a HP 5890 series II gas chromatograph.
Gas flow rate for He, 1.3 ml/min; temperature program: 100°C for 1 min, then to
295°C at 10°C for 15 min; flame ionization detector; detector temperature, 300°C,
injector temperature 250°C; split ratio 100 : 1. Standard curves were determined
for erucyl alcohol, C22',
24* C,„,
26' C,„,
28 C30-alcohols (Sigma), and synthetic a-TQ
(unpublished data). The GC retention times relative to erucyl alcohol were used to
identify the feeding stimulants. The integrated area of each feeding stimulant was
compared to its standard cun/e to determine the amount in the sample injection.
The amount of the stimulant was adjusted to match the recovery of erucyl alcohol
from the solid phase for each injection. The amount of each stimulant was then
calculated for a 10 mg leaf-wax sample. Finally, the concentration (ng per leaf area
cm^) was obtained according to the leaf area used to get the 10 mg leaf-wax
sample.
The phenolic glycosides were analyzed by following their UV absorption at
220 nm. Fresh leaves, ready for extraction after they were cut at the petiole, were
placed on ice, transported to the laboratory and vacuum-dried (Orians, 1995). The
51
dry leaves were pulverized in a plastic bag and the leaf powder (25 mg) was
sonicated (Sonicor ultrosonic tank, Whatman) in 1 ml ice cold MeOH for 15 min.
The sample was centrifuged (5000 g, 6 min) and filtered through cotton to remove
particles, and 15 |il of the extract was applied to analytical grade silica gel plates
(Merck 60 F254, 250 nm thick, 10 cm x 10 cm). Two-dimensional TLC (solvent I;
CH^CI^: MeOH, 80 : 20, and solvent II;
: MeOH : THF, 60 :10 : 10) was used
to separate the phenolic glycosides. The chemicals were then desoriaed from the
salica in 1 ml ethanol, filtered, and measured at 220 nm. Standard curves were
detemnined for tremulacin and salicorin. To detennine the recoveries for tremulacin
and salicortin from TLC plates, 15 ul of 0.5 mg/ml tremulacin and salicortin
standards in MeOH were applied to TLC plates. After two-dimensional
development, the chemicals were desorbed in 1 ml ethanol, filtered, and measured
at 220 nm (n = 3). The concentrations were determined using the standard curve,
and recoveries were calculated: for tremulacin, 0.73; for salicortin 0.61. The
amount of tremulacin and salicortin in the plant samples were calculated according
to the standard curves, modified by the overall recovery, and expressed as ug per
mg leaf dry weight.
Isolation of phenolic glycoside standards. To obtain tremulacin and
salicortin, tips of twigs with immature leaves attached were collected from a wild
PoDulusalba hybrid, put on ice, transported to the laboratory, and vacuum-dried.
The dried leaves and twigs (490 g) were powdered and extracted in cold MeOH
with sonication for 20 min. After filtration, the extract was concentrated at 35°C. A
52
hexane wash was used to eliminate lipid materials from the residue. The method
of Picard et al. (1994) was used to recover the crude phenolic glycoside extract.
The residue (45.5 g) was dissolved in 900 ml HgO and 800 ml 40% (NH4)2S04.
Undissolved material was filtered away with Whatman No. 4 filter paper. The
solution was partitioned with three volumns of hexane, and the water phase was
extracted with four volumes of ethyl acetate. The ethyl acetate extract was dried
with MgS04, and concentrated. Nine grams of phenolic glycoside crude extract
was recovered. Vacuum liquid column chromatography on silica gel (10-40 nm,
Sigma) was used to separate the phenolic glycosides. An ascending series of
MeOH in CHCI3 (0, 10%, 20%, and 30%) was used to elute the material.
Preparative TLC (Merck 60 F254, 250 ^.m thick) was used to purify tremulacin and
salicortin. The solvent system, CHCI3: MeOH (80 : 20), was used for tremulacin
purification. The solvent system, CHCI3: MegCO : MeOH (70 : 15 : 15), was used
for salicortin. Chemicals from TLC were desorbed in acetone. ^H NMR (CDCI3,
400 MHz) of both tremulacin and salicortin was identical to those reported by
Lindroth et al. (1987).
Statistical Analysis. The percentage of leaf area consumed among parent
and F, clones in field cage feeding tests was evaluated by ANOVA and t-tests. Leaf
area consumed in leaf disk feeding tests for 91 clones was evaluated by GLM.
Beetle feeding activity and chemical variations among individuals of the same
clones planted in the three field blocks were evaluated by correlation analysis
(SAS, 1985). The relationships between the feeding preference and chemical
53
contents were analyzed by using data from each of the three blocks instead of
using the average data of the three blocks.
Results
Parent and F, clones. The field cage feeding test results showed that the
beetles easily distinguished among the four clones. The order of preference for
consumption was 53-242, 53-246, ILL-129, and 93-968. The percentage of leaf
area consumed was 59.0 ± 1.9, 31.2 ± 1.4, 8.2 ± 1,1, and 1.6 ± 1.6, respectively (F
= 286.2, p = 0.0001) in 1994, and 62.1 ± 10.9, 30.5 ± 11.1, 5.5 ± 1.4 and 2.7 ± 2.0,
respectively in 1995.
The leaf content of alcohol and a-TQ varied among the four clones (Table 1).
Parent clone ILL-129 had the highest content of alcohol on the leaf surface. 53242 and 53-246, the two F, clones, had alcohol contents between the parents.
Clone ILL-129 had a high content of a-TQ on the leaf surface, while a-TQ could not
be detected in the leaf surface of clone 93-968. The two F^ clones had a-TQ
content between the two parents.
The content of tremulacin and salicortin also varied among clones (Table 1).
The content of tremulacin and salicortin in F, clones and parents was variable. The
chemical variation pattem, however, did not correspond to the feeding preference
pattern.
54
Parents, F1, and F2 clones. The beetle showed significantly different
feeding responses to the 91 clones, p = 0.0001, for all three blocks. Average leaf
area consumed ranged from 18.4 ± 8.8 to 210.8 ± 16.3 mm^ in block one, 0.3 ± 0.9
to 208.6 ± 10.1 mm^ in block two, and 6.6 ± 7.5 to 193.3 ± 29.4 mm^ in block three.
The correlation coefficients were low for beetle feeding activity measured by
the average (n = 10) leaf area consumed, for the content of a-TQ on the leaf
surface, and for the tremulacin content per leaf dry weight, although the coefficients
were relatively higher for the amount of alcohol on the leaf surface and salicortin
per leaf dry weight (Table 2). Beetles showed varied feeding responses to
individuals of each clone, and the chemicals, especially a-TQ and tremulacin, were
also highly variable among individuals within each clone.
Leaf surface alcohol content does not show obvious correlation with beetle
feeding (Figure 2). The relationship of beetle feeding activity and leaf surface a-TQ
content show that the beetles had predictable responses to the clones with a-TQ
different from those clones without a-TQ (Figure 3). The beetle preferred to feed on
the clones with a-TQ over the clones without a-TQ. Moreover, as the amount of aTQ increased, the beetles consumed more leaf area. When the amount of a-TQ
increased further, however, the amount of leaf area consumed did not increase
further, and instead, decreased.
Leaf content of tremulacin and salicortin (Figures 4 and 5) did not correlate
with the amount of leaf area consumed by the beetles. Although contents of
55
tremulacin and saiicortin varied among clones and among individuals within
clones, especially for tremulacin, the variation did not explain beetle feeding
preference differences.
Discussion
A combination of leaf surface long chain fatty alcohols and a-TQ stimulated
adult beetle feeding (unpublished data). Field cage feeding tests and leaf disc
feeding tests also showed that the beetles preferred to feed on clones with a-TQ
present and only within a range of concentration of 1.00- 5.00 ng/cm^. In the field
cage feeding test and in leaf disc feeding tests, the beetles preferred to feed on
poplar plants with a-TQ on the leaf surface together with long chain fatty alcohols
present, and they could discriminate among clones with different ratios of a-TQ and
alcohols. The beetles preferred to feed on poplar plants with a high content of aTQ, but when the amount of a-TQ increased further, feeding decreased. The
variation of a-TQ content on the leaf surface explained cottonwood leaf beetle adult
feeding preference variation among poplar plants.
In the cottonwood leaf beetle, the oviposition preference patterns
correspond to feeding preference patterns both among clones and leaf ages
(Bingaman and Hart, 1992). In our feeding bioassays for gravid females, the beetle
showed feeding preference between artificial discs with and without feeding
stimulants, but they laid eggs randomly on all the disks and on the petri dish. It may
56
be true that, as with the willow-feeding leaf beetle, Chrvsomela aeneicollis (Rank,
1992), the cottonwood leaf beetle also locates favorable hosts through acceptance
or rejection by feeding adults. Adult feeding activity may be important in
detemnining the beetle abundance in the field.
Current practices for population suppression of cottonwood leaf beetle rely
heavily on the use of insecticides. With the knowledge that the beetles prefer to
feed on poplar plants with a-TQ and long chain fatty alcohols together on the leaf
surface, we may be able to find altemative ways to manage the beetle. In both the
field and the laboratory, we found that the adult beetle exhibited aggregation
behavior (unpublished data). Beetle feeding stimulants, a-TQ and long chain
alcohols, together with knowledge of the beetle aggregation behavior, may allow
us to develop an effective bait for the beetle, as has been done for diabroticite
beetles (Metcalf and Metcalf, 1992). The poplar trees with optimum a-TQ and long
chain alcohol content could also be used as a trap crop for the beetle. Selecting
and breeding poplar clones without a-TQ on the leaf surface, or using molecular
biology tools to block secretion of a-TQ onto the leaf surface could increase poplar
natural resistance to the cottonwood leaf beetle.
Larvae of the cottonwood leaf beetle use salicin and its derivatives from their
host plants to make their own defensive chemicals against generalist predators
(Wallace and Blum, 1969; Pasteels et al., 1989). In some willow-feeding salicinusing leaf beetles, the phenolic glucosides influence beetle feeding behavior
(Kolehmainen et al., 1995; Rank, 1992). In our observation and in the study of
Bingaman and Hart (1993) it seems that for the cottonwood leaf beetle, phenolic
glycosides do not influence adult feeding behavior. Tney may have some subtle
effects on oviposition behavior, and most probably much more significant effects on
tritrophic relationships, such as offering chemical protection from predators and
pathogens (Wallace and Blum, 1969; Pasteels et al., 1989).
Fatty alcohols are very common leaf surface wax components (Walton,
1990). They are abundant in the poplar leaf surface. The alcohol content is more
stable than a-TQ among individuals of the same clones. a-TQ also is a commonly
occuring phytochemical (Kruk and Strzalka, 1995). In some poplar clones, it is
somehow secreted onto the leaf surace wax matrix. It may be possible that this
secretion is very susceptible to environmental effects. In the summer of 1996,
when our poplar plantation was affected by flooding, together with some other
possible microclimatic and biological factors, different clones and individuals of the
same clones might have undergone varied microenvironmental conditions. These
may have significantly affected a-TQ secretion onto the leaf surface, and change
susceptibility to the beetles. Understanding how a-TQ, together with long chain
fatty alcohols, change during the season, with different leaf ages, and how the
abiotic and biotic environmental factors affect its expression on the leaf surface will
increase our knowledge of beetle feeding behavior and strengthen our pest
resistance breeding program.
58
Acknowledgements
We thank Paul N. Hinz and David F. Cox for their assistance with statistical
analysis, Michael J. Anderson, Elizabeth E. Butin, and Chris Feeley for technical
assistance. The salicortin standard was generously provided by Professor Colin M.
Orians, Tufts University. Research was supported in part by the Oak Ridge
National Laboratory operated by Martin Marietta Energy Systems, Inc. for the U.S.
Departnnent of Energy, contract no. DE-AC05-840R21400. Journal Paper No. J17353 of the Iowa Agriculture and Home Economics Experiment Station, Ames,
Iowa, Project No. 2976, 3443, and supported by McEntire-Stennis and State of
Iowa funds.
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Appl. Entomol. Zool. 28:319-324.
BINGAMAN, B.R., and HART, E.R. 1992. Feeding and oviposition preferences of
adult Cottonwood leaf beetles (Coleoptera: Chrysomelidae) among Populus
clones and leaf age classes. Environ. Entomol. 21:508-517.
BINGAMAN, B.R., and HART, E.R. 1993. Clonal and leaf age variation in Populus
phenolic glycosides: implications for host selection by Chrysomela scripta
(Coleoptera: Chrysomelidae). Environ. Entomol. 22:397-403.
BURKOT, T.R., and BENJAMIN, D.M. 1979. The biology and ecology of the
Cottonwood leaf beetle, Chrysomela scripta (Coleoptera: Chrysomelidae),
on tissue cultured hybrid Aigeiros {Populus x Euramericana) subclones in
Wisconsin. Can. Entomol. 111:551-556.
CALDBECK, E.S., MCNABB, H.S.JR., and HART, E.R. 1978. Poplar clonal
preferences of the cottonwood leaf beetle. J. Econ. Entomol. 71:518-520.
CHAPMAN, R.F., and BERNAYS, E.A. 1989. Insect behavior at the leaf surface
and learning as aspects of host plant selection. Experientia. 45:215-222.
COCHRAN, W.G., and COX, G.M. 1992. Experimental designs 2nd ed. p. 535.
DENNO, R.F., LARSSON, S., and OLMSTEAD, K.L 1990. Role of enenny-free
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71(1):124-137.
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17:223-233.
FALTONSON, R.R., THOMPSON, D., and GORDON, J.G. 1983. Propagation of
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HARRELL, M.O.. BENJAMIN, D.M., BERBEE, J.G., and BURKOT, T.R. 1981.
Evaluation of adult cottonwood leaf beetle, Chrysomela scripta (Coleoptera:
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KELLY, M.T., and CURRY, J.P. 1991. The influence of phenolic compounds on the
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63
WILSON, L.F. 1979. Insect pests of Populus in the Lake States, pp. 75-81, in
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Insects and the Plant Surface. Edward Amold, Maryland, USA.
64
Table 1. Content of secondary plant compounds in parent and F, clones (1994)
Feeding stimulants (ng/cm^)
Phenolic glycosides (|ig/mg)
Clone
Alcohol
a-TQ
53-242
1370.7
5.4
17.07
42.31
53-246
1429.8
2.5
12.87
34.90
ILL-129
1772.7
17.2
12.79
21.23
93-968
1187.9
14.19
40.53
not detected
Tremulacin
Salicortin
65
Table 2. Pearson correlation coefficient between blocks
Block one
vs. Block two
Block one
Block two
vs. Block three
vs. Block three
Feeding activity
0.10
0.19
0.43
Alcohol
0.66
0.54
0.66
a-TQ
0.35
0.25
0.45
Tremulacin
0.40
0.42
0.53
Salicortin
0.53
0.62
0.65
66
2
Fig. 1. GC profile for leaf surface feeding stimulants. IS, internal standard. 1, CggOH. 2, C24-OH. 3, Cgg-OH, 4, Cgg-OH. 5, a-TQ. 6, C30-OH. Between the second
slashes indicates where the attenuation was changed from 2" = 1 to 2" = -3.
67
250.0 T
Block one
ZOO.O
:
1SO.O
A
100.0
SO.O
N= 60
O.O
O.OO
2SO.O
1000.00
2000.00
3000.00
4000.00
5000.00
Block two
200.0
^
150.0
100.0
SO.O
N = 61
0.0
0.00
250.0
1000.00
2000.00
3000.00
4000.00
5000.00
Block three
200.0
150.0
100.0 i
5
^
N = 60
SO.O
0.0
Amount of alcohol
per leaf area (ng/cm^)
Fig. 2. Relationship of amount of leaf area consumed (n = 10) and content of long
chain fatty alcohol for data from block one (n = 60), block two (n = 61), and block
three (n = 60).
68
2SO.O -
Block one
N = 60
200.0 -
•«so.o <
•
100.0 i ^
I*
ft
SO.O t
E
"O
(D
E
ZJ
^
o.o
O.OO
250.0 -
5.00
200.0 -
20.00
2S.OO
N = 61
•»
1SO.O
CO
100.0 - •
*
CO
50.0 » A
CD
1S.OO
Block two
8
_
CO
10.00
»
^
•
'
*
ft A
ft
o.o
O.OO
5.00
10.00
15.00
20.00
25.00
C
Z3
O
C
250.0 -
<
Block three
200.0
-
N = 60
150.0 ^
*
100.0 AT*"
%
^ ^
*
*
*
50.0 -
o.o
O.OO
S.OO
10.00
15.00
20.00
25.00
Amount of alpha-TQ
per leaf area (ng/cm2)
Fig. 3. Relationship of amount of leaf area consumed (n = 10) and content of a-TQ
for data from block one (n = 60), block two (n = 61), and block three (n = 60).
69
2SO.O -
Block one, N = 52
200.0 -
^
A A •
A
^ f^ ^ »
150.0 •
100.0 -
^ t
SO.O
CM
E
O.O
O.OO
10.00
S.OO
15.00
20.00
25.00
30.00
35.00
25.00
30.00
35.00
"O
I
w
c
o
o
2SO.O -
Block two, N = 56
-
^°° ° '
/
. .
1S0.0 *
CO
03
100.0 -
* •
CO
SO.O -
<D
0.0
0.00
o
E
<
5.00
10.00
15.00
20.00
' Block three, N = 58
aoo.o
*
150.0 -
-w
100.0 -
•a
^
SO.O -
0.0
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
Amount of tremulacin
per leaf dry weight (ug/mg)
Fig. 4. Relationship of amount of leaf area consumed (n = 10) and content of
tremulacin for data from block one (n = 52), block two (n = 56), and block three (n =
58).
70
°'
Block one. N = 52
200.0 «
150.0 -
A ^
*
CM
C
^
O.O
O.OO
U
250.0 -
-*
200.0 -
®
CO
150.0 -
o
m
5;
O
is
CO
^
100.0 -
S=
80.00
100.00
120.00
Block two, N = 56
-
»
•
* . - • • •
- *
* »
*
•
50.0 -
A
0.0
0.00
*^ - *
*
20.00
40.00
60.00
ao.oo
100.00
120.00
2SO.O -
o
£
60.00
-40.00
A
o
CD
20.00
*
Block three, N = 58
200.0 -
<C
«
1S0.0 -
0.0
0.00
•
20.00
40.00
60.00
»
ao.oo
100.00
120.00
Amount of saiicortin
per leaf dry weight (ug/mg)
Fig. 5. Relationship of amount of leaf area consumed (n = 10) and content of
saiicortin for data from block one (n = 52), block two (n = 56), and block three (n =
58).
71
CHAPTER 4. GENERAL CONCLUSIONS
The cottonwood leaf beetle, Chrysomela scripta Fabr., is considered to be
one of the most serious defoliators of poplar nurseries and young plantations (1-3
years) in North America (Burkot and Benjamin, 1979; Wilson, 1979; Harrell et al.,
1981; Solomon, 1988). The nature of a short rotation woody crop system may
predispose the plantation to such pest problems (Dickmann and Stuart, 1983).
The use of Populus that exhibit a degree of resistance to the cottonwood leaf
beetle is a main component in the development of integrated pest management
strategies for this beetle in poplar short rotation woody crop systems. Increased
endogenous pest resistance can reduce pest damage and the costs of pest control,
and increase the feasibility of silvicultural prescription for maximizing biomass
productivity. A wide range of clonal resistance to the cottonwood leaf beetle has
been reported for selected clones (Oliveria and Copper, 1977; Caldbeck et a!.,
1978; Harrell et al., 1981; Bingaman and Hart, 1992). The adults of the cottonwood
leaf beetle showed varied feeding and oviposition preference among tested poplar
clones and among leaves of different developmental stages (Caldbeck et al., 1978;
Harrell et al., 1981; Bingaman and Hart, 1992). No correlation between beetle
preference and poplar leaf moisture, nitrogen, carbohydrate levels, leaf thickness,
toughness, or surface physical characteristics was found (Harrell et al., 1981). The
mechanisms that underiie these preferences have not been understood, so
screening for nonpreferred poplar clones is difficult.
72
My study was designed to understand the mechanisms for beetle feeding
preference and eludicate the relationship between poplar leaf secondary
chemicals and cottonwood leaf beetle adult feeding preferences.
Using the cottonwood leaf beetle preferred poplar clone 'Eugenei', the adult
beetle feeding stimulants, long chain fatty alcohols and a-TQ, were identified from
the poplar leaf surface. Fatty alcohols or a-TQ alone do not induce beetle feeding
significantly, but a mixture of both long chain fatty alcohols and a-TQ synergistically
stimulates beetle feeding.
University of Washington poplar pedigree materials, parent, F^, and 87 Fg,
were available for cage feeding tests, disc feeding tests, and chemical analysis.
The beetles showed varied feeding preference among the 91 different clones. The
beetle preferred to feed on clones with a-TQ present and within a detennined
range of concentration on the the leaf surface together with long chain fatty
alcohols present, and they also could discriminate among clones with different
contents of a-TQ. The variation of a-TQ content on the leaf surface could explain,
at least in great part, cottonwood leaf beetle adult feeding preference variation
among poplar plants.
In some willow-feeding, salicin-using leaf beetles, the phenolic glucosides
influence beetle feeding behavior (Kolehmainen et al., 1995; Rank, 1992). Unlike
these leaf beetles, in the cottonwood leaf beetle the phenolic glucosides tremulacin
and salicortin do not explain the adult feeding behavior very well.
73
APPENDIX A. STRUCTURES OF BEETLE FEEDING
STIMULANTS
alcohols
/VVS/VVWWV^ OH
/WWVWWW^OH
/VVVS/WWWW^oH
/VSA/WWWWW^ OH
/WWVWWWSA/V^OH
alpha-tocopherylquinone (alpha-TQ)
0
74
APPENDIX B.
STRUCTURES OF PHENOLIC GLUCOSIDES
CKpH
CF®H
Salicortin
Tremulacin
75
APPENDIX C. PLANT MATERIALS USED IN THE RESEARCH
The University of Washington poplar pedigree materials used in the research:
1057
1058
1061
1060
1062
1063
1064
1065
1067
1068
1069
1071
1072
1073
1076
1077
1078
1079
1080
1081
1082
1083
1084
1086
1087
1089
1090
1095
1096
1101
1103
1104
1106
1108
1111
1112
1114
1115
1118
1120
1121
1122
1125
1126
1127
1128
1130
1131
1133
1135
1136
1140
1149
1151
1156
1158
1162
1163
1165
1169
1173
1174
1179
1180
1181
1182
1088
1186
1579
1580
1581
1582
1583
1584
1586
1587
1590
1591
1592
1593
1594
1059
1600
1601
1603
1605
1820
93-968
ILL-129
53-246
53-242
76
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91
ACKNOWLEDGMENTS
My major professors, Dr. Elwood R. Hart and Dr. Bradley F. Binder gave me
advice, encouragement, and support throughout the whole program.
Without the assistance of many individuals, this research could not have
been finished. Numerous people have contributed their knowledge, ideas, time,
and equipment toward this project and should be recognized.
The members of my graduate committee. Dr. Richard B. Hall, Dr. Richard C.
Schultz, Dr. Jon J. Tollefson, Dr. Walter S. Trahanovsky deserve my gratitude for
their advice and their time. I extend a special thanks to Rich Faltonson, Department
of Forestry, for his instruction on greenhouse culture and for his help with field
planting; to Roger D. Hanna, Department of Forestry, for his help with field planting;
to Dr. Richard L. Wilson, Department of Entomology, and Plant Introduction Station,
for allowing me to use his equipment and laboratory facilities; to Dr. David F. Cox
and Dr. Paul N. Hinz, Department of Statistics for their assistance with statistical
analysis: to the faculty in the USDA-ARS, Com Insect Research Unit for allowing
me to use their facilities and their friendship: I especially wish to thank James C.
Robbins for his help, and kindness; also special thanks are given to Dr. Allard
Cosse, Dr. Rong Cao, and Dr. Kamel A. Harrata for their advice and techanical
assistance.
I aslo would like to thank the faculty and graduate students of the
Department of Entomology who provided their support, ideas, and frienddship. In
92
particular, I want to thank the Forest Entomology research group, Jennifer A,
Jarrard and Ying Fang, for their help and support. Numerous undergraduate
employees Including Chris Feeley, Elizabeth E. Butin, Christine Hall, and Michael
J. Anderson, have contributed greatly to my research; their assistance was greatly
appreciated.