CHEMICAL INTERACTIONS AMONG MILKWEED PLANTS
(ASCLEPIADACEAE) AND LEPIDOPTERAN HERBIVORES

By

JAMES A. COHEN

A DISSERTATION SUBMITTTED TO THE GRADUATE SCHOOL OF THE

UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
1983

ACKNOWLEDGMENTS

It is a pleasure to acknowledge the many friends and colleagues
whose aid and cooperation have been instrumental in carrying out this
work. My major professor, Lincoln P. Brower, has been a constant
source of stimulation, guidance, and encouragement. His contagious
enthusiasm for biological problems is truly inspirational. To my good
friend Lincoln I extend my sincerest thanks. The members of my
graduate and examining committees, Drs . H. Jane Brockmann, Richard A.
Kiltie, Frank Slansky Jr., and Norris H. Williams, have provided much
willing assistance during the research phase and many helpful
editorial suggestions, for which I am grateful. Drs. C. Covell, D.
Griffin, D. Habeck, J.N. Seiber, and S. Yu provided further advice and
consultation.

I am also fortunate to have had the cooperation and aid of Dr.
Norm Leppla and the staff of the USDA Insect Attractants, Behavior,
and Basic Biology Research Laboratory. Drs. K.S. Brown and D.J.
Futuyma kindly provided unpublished manuscripts. The hospitality and
assistance of Dr. R. Rutowski in collecting queen butterflies is also
gratefully acknowledged. Assistance in the laboratory was kindly
provided by J. Frey, T. van Hook, and M. Hoggard. Figures were
prepared by P. Ibarra and W. Adams.

I also wish to thank the Department of Zoology and the Graduate
School of the University of Florida for generous financial support
during my graduate program. Numerous other graduate students and
post-doctoral associates acted as sounding-boards for my ideas and

ii

provided an ideal atmosphere for pursuing these studies. Among these
I would especially like to thank J.B. Anderson, W.H. Calvert, S.B.
Malcolm, P.G. May, and N.E. Stamp. J.R. Lucas helped me greatly to
become computer-literate. He and S.A. Frank further instructed me in
the gentle art of statistics.

I reserve a separate paragraph to thank Craig S. Hieber (UVPD)
for engaging in countless late-night ravings with me and for an
infinite willingness to generate and explore all kinds of hypotheses.
I could not have asked for a finer office-mate, nor for a better
friend.

Finally, a word of thanks to my parents, Isaac and Marie Cohen,
for assistance in ways far too numerous to mention.

ill

TABLE OF CONTENTS

ACKNOWLEDGMENTS ii

LIST OF TABLES vi

LIST OF FIGURES vii

ABSTRACT i^

CHAPTER I INTRODUCTION 1

Plant/Herbivore Coevolution A

Specialization and Generalization in Herbivory 12

Chemical Defense of Milkweed Plants 16

The Metabolic Cost of Cardenolide Ingestion 22

CHAPTER II OVIPOSITION AND LARVAL SUCCESS OF WILD MONARCH

BUTTERFLIES (LEPIDOPTERA: DANAIDAE) IN RELATION

TO HOSTPLANT SIZE AND CARDENOLIDE CONCENTRATION 32

Introduction 32

Methods 34

Results 35

Discussion .38

CHAPTER III DIFFERENCES AND SIMILARITIES IN THE CARDENOLIDE
CONTENTS OF QUEEN AND MONARCH BUTTERFLIES IN
FLORIDA AND THEIR ECOLOGICAL AND EVOLUTIONARY
IMPLICATIONS ^'^

Introduction ^^

Methods ^7

Results A9

Discussion 59

CHAPTER IV CARDENOLIDE SEQUESTRATION BY THE DOGBANE TIGER MOTH

( CYCNIA TENERA; ARCTIIDAE) 74

Introduction 74

Methods 76

Results 79

Discussion 84

iv

CHAPTER V THE EFFECTS OF INGESTED CARDENOLIDE UPON
FOOD CONSUMPTION AND GROWTH OF SPECIALIST
AND GENERALIST LEPIDOPTERAN LARVAE 91

Introduction 91

Methods 93

Results 98

Discussion 103

CHAPTER VI GENERAL DISCUSSION AND CONCLUSION 112

General Discussion 112

Conclusion 130

LITERATURE CITED 134

BIOGRAPHICAL SKETCH 1'^''

LIST OF TABLES

Table 1. A survey of midstera leaf cardenolide concentrations
for six milkweed species in north-central Florida 21

Table 2. Eggs, larvae, larval success, and cardenolide
concentrations for 10 Asclepias humistrata plants 36

Table 3. Cardenolide concentrations for 7 matched sets of
partially eaten and uneaten Asclepias humistrata leaves 39

Table 4. Correlation coefficients for monarchs and plant
area, and for monarchs and cardenolide concentration 40

Table 5. Right wing lengths, weights, fat, and cardenolide
contents of wild-caught queen and monarch butterflies from
three sites in Florida 53

Table 6. Spearman correlation coefficients for cardenolide
concentration vs. body size, weight, and fat contents of
wild-caught queen and monarch butterflies from Florida 58

Table 7. Body sizes, weights, fat and cardenolide contents
of queens and monarchs (Dominican Republic stock) reared in
the laboratory on the milkweed, Asclepias humistrata... 60

Table 8. Spearman correlation coefficients for cardenolide
concentration vs. body size, weight, and fat content of
queen and monarch butterflies (Dominican Republic stock)
reared in the laboratory on Asclepias humistrata 61

Table 9. Means and standard deviations for the weight and
cardenolide contents of adult Cycnia tenera reared on the
milkweeds Asclepias humistrata and A^ tuberosa 80

Table 10. Relative growth rate, relative consumption rate,
and efficiency of conversion of ingested matter for fourth
instar monarch butterfly larvae reared on four Asclepias
tuberosa-based diets incorporating different cardenolide

. QQ

concentrations ^'

Table 11. Relative growth rate, relative consumption rate,
and efficiency of conversion of ingested matter for fifth
instar fall armyworm larvae reared on artificial diets
incorporating varying amounts of cardenolide 101

vi

Table 12. Additional data for fall armyworms reared on
artificial diets incorporating varying amounts of cardenolide. . . . 102

Table 13. Relative growth rate, relative consumption rate,
and efficiency of conversion of ingested matter for fifth
instar velvetbean caterpillars reared on artificial diets
incorporating varying amounts of cardenolide 104

Table 14. Additional data for velvetbean caterpillars
reared on artificial diets incorporating varying amounts
of cardenolide ^^^

vil

LIST OF FIGURES

Figure 1. The number of monarch eggs and larvae of each
instar on 10 Asclepias humlstrata plants, pooled over
11 observation days ^'

Figure 2. Frequency distributions of cardenolide
concentration for three populations of Florida queen
butterfly, and one population of monarchs ...51

Figure 3. Frequency distributions of total cardenolide
per individual butterfly 52

Figure 4. Thin layer chroraatogram of wild-caught monarchs
and queens from Miami area • ..55

Figure 5. Chromatograms of adult monarchs and queens
reared in the laboratory on Asclepias humistrata 63

Figure 6. An adult male Cycnia tenera and a late-instar larva.

Figure 7. TLC profiles of four adult Cycnia tenera reared
on either Asclepias humistrata or _A^ tuberosa

,77

.83

viii

Abstract of Dissertation Presented to the Graduate School

of the University of Florida in Partial Fulfillment

of the Requirements for the Degree of

Doctor of Philosophy

CHEMICAL INTERACTIONS AMONG MILKWEED PLANTS
(ASCLEPIADACEAE) AND LEPIDOPTERAN HERBIVORES

By
James A. Cohen
December, 1983

Chairman: Lincoln P. Brower
Major Department : Zoology

Milkweed plants are widely believed to be chemically defended
against insect herbivory by steroidal compounds known as cardenolides
(cardiac glycosides). Moreover, it has frequently been claimed that
milkweed-specializing insects have coevolved with their hostplants by
breaching a cardenolide-barrier over the course of evolutionary time.
However, there has been, until now, no evidence that ingested
cardenolides have any defensive properties against any invertebrate
herbivore. Thus, cardenolide-based models of coevolution among
milkweeds and insects have had little foundation. This study seeks to
provide such a foundation by comparing the ecological and
physiological effects of cardenolides upon lepidopteran species which
do or do not feed on milkweeds.

ix

In accordance with evolutionary predictions, the results provide
little evidence that milkweed-specializing insects pay metabolic costs
of handling cardenolides. These plant chemicals appear to influence
neither oviposition nor larval development in wild monarch butterflies
(Danaus plexippus; Danaidae) , nor to affect (even when in abnormally
high concentrations) consumption, growth, or food conversion
efficiency of fourth instar monarchs in the laboratory.

In contrast, the polyphagous fall arrayworms (Spodoptera
frugiperda; Noctuidae) exhibited reduced growth, food consumption, and
food conversion efficiency in the fifth instar, even when fed
moderate, natural cardenolide concentrations. Although no such
negative effects were found for fifth instar velvetbean caterpillars
(Anticarsia gemmatalis; Noctuidae), a species that specializes on
cardenolide-free plants (i.e., legumes), younger instars of this
species were severely affected by cardenolides. This serves as a
warning to other researchers who frequently test only older instars
and may thereby generate misleading results.

This study is the first to demonstrate that ingested cardenolides
may provide a chemical barrier to herbivory by certain insects.
However, a detailed review of the literature provides little support
for frequent contentions that milkweed-specialists were once deterred
by, and subsequently breached, such a barrier. An alternative
coevolutionary model is considered which predicts (inter alia) that

certain danaid and ithomiid butterflies should utilize cardenolides as
hostplant recognition cues. Although my data do not confirm this
prediction for monarch butterflies, this latter model nevertheless
holds considerable promise as a prime example of plant/herbivore
coevolution.

xi

CHAPTER I
INTRODUCTION

A major thrust of modern ecology Involves the study of
relationships between heterotrophic organisms and their food supplies.
Such interactions (e.g., predator vs. prey, host vs. parasite) occur
not only in ecological time but also in evolutionary time (e.g.,
Dawkins and Krebs, 1979; Thompson, 1982). Natural selection may be
presumed to act within populations to optimize simultaneously the
ability to acquire resources (e.g., Pyke at al., 1977; Krebs, 1978)
and the ability to avoid becoming the acquired resources of similarly
selected organisms of higher trophic levels (e.g., Edmunds, 1974;
Rhoades, 1979). Clearly, then, a major component of ecological
research must concern the study of the varied defenses utilized by
organisms to avoid being attacked by their enemies.

In this context, plants may be viewed as subject to the attacks
of both predators and parasites. Predation occurs when the plant is
killed outright by another organism(s) and parasitism occurs when, as
in most cases of herbivory, some of the plant's biomass is consumed,
lowering plant fitness, but the plant does not die in the process

1

(Gilbert, 1979; Price, 1980). The theory of plant/herbivore
coevolution (Ehrlich and Raven, 1964) has provided a theoretical
framework for the organization of a large and diverse body of
literature concerning the ability of plants to avoid enemy-attack, as
well as the counter-adaptations of attacking species. In essence,
plants have evolved a variety of defensive adaptations (e.g.,
morphological, phenological, and chemical traits) against predators
and parasites. In turn, such enemies have evolved means to circumvent
or counteract these defenses, leading to further elaboration of plant
defensive strategies, subsequent enemy offenses, and so on.

A key system, widely regarded as providing the classic, textbook
example of plant/herbivore coevolution, is that of milkweed plants
(Asclepiadaceae) and their specialized herbivores, most notoriously
the monarch butterfly ( Danaus plexippus; Danaidae) . Consider the
following excerpts from Harborne's (1983) text. Ecological
Biochemistry (emphases added) :

What is now the classic example of plant-animal
coevolution in which secondary compounds have a
key role is the interaction between milkweeds,
monarch butterflies, and blue jays. [p. 87]

The milkweed produces several cardiac glycosides
within its tissues _as a_ passive defense against
insect feeding, [p. 87]

The monarch butterfly caterpillar learns to adapt
to these toxins, [p. 88]

production of the toxins continues to provide the
plant with protection not only from most insects
but also from all grazing animals, [p. 89]

The present dissertation is concerned precisely with this interaction,
for several aspects of Harborne's summary have never been
substantiated by data, despite their general acceptance among
evolutionary biologists. In particular, I address the question of
whether the cardiac glycosides (cardenolides) present in certain
milkweed plants may, in fact, offer protection against generalist-
feeding insects, but not against purportedly coevolved specialists
such as monarchs. The crucial question is: Do generalists pay "costs"
of ingesting cardenolides that specialists do not pay? This is the
first, basic step that must be established before one can entertain
claims that milkweed-specialists have breached a cardenolide-barrier
during the course of coevolution.

In the remainder of this chapter, I shall briefly outline the
theory of plant/herbivore coevolution and the related question of
chemical defense in plants. As it is supposed that a breaching of
such defenses leads to the evolution of feeding specialization (e.g.,
Ehrlich and Raven, 1964; Feeny, 1975), I will then contrast the two
strategies of specialization and generalization, with emphasis on
biochemical (detoxification and tolerance) differences. By way of
introduction to the specific subject matter of this dissertation, I
shall then review the chemical defense of milkweed plants in
particular, focusing on cardenolides, and finally summarize what is
currently known about the metabolic costs, to specialists or
generalists, of ingesting these purportedly defensive chemicals.

Plant/Herbivore Coevolution

Ehrlich and Raven's (1964) formulation of plant/herbivore
coevolution was derived primarily from considerations of larval
feeding specialization in the Lepidoptera, the most thoroughly studied
order of insect herbivores. These authors summarized the large body
of observations showing that certain lepidopteran taxa (frequently
subfamilies or tribes) were restricted to feeding upon specific plant
taxa, and suggested that these patterns of relationships were most
easily interpreted in terms of the allelochemistry of the plants.
Allelochemics are plant compounds apparently not directly required for
the primary metabolic functions of the plant but having a variety of
inhibitory or (in some cases) stimulatory effects upon potential or
actual herbivores, pathogens, or other plants (Fraenkel, 1959, 1969;
Whittaker and Feeny, 1971; Levin, 1976). In many cases the compounds
have potentially toxic or digestibility-reducing effects and are
presumed to have evolved in response to herbivore pressure. Ehrlich
and Raven (1964) suggested that certain plants, through mutation and
recombination, have elaborated novel chemicals which reduce plant
palatability or suitability as food for herbivores. Thus freed from
previous herbivore pressure, these plants entered a new "adaptive
zone" wherein phylogenetic radiation was possible. In the course of
evolution, certain herbivore groups, also through a fortuitous genetic
event, breached the plant's chemical defense and, in so doing, were
also able to enter (and radiate within) a new adaptive zone. Ehrlich

and Raven (1964:602) further suggested that the ability of an
herbivore to tolerate one kind of defensive chemistry reduces its
ability to tolerate other kinds of chemical defense. Thus, the
breaching of a chemical barrier is presumably followed by the rapid
evolution of herbivore feeding specialization for plants having
similar defensive chemistry (commonly confamilials) . This hypothesis
is supported by the fact that when, in exceptional cases, oligophagous
lepidopterans feed on taxonomically disparate plant species, these are
frequently shown to share a defensive chemistry. In such cases the
herbivore may have evolved the ability to utilize the allelochemic for
hostplant recognition (Fraenkel, 1959; Smith, 1966; Rees, 1969; van
Eraden, 1972; Nielsen, 1978).

Extending this general coevolutionary scenario, Brower and Brower
(1964) demonstrated that many of the lepidopteran families feeding on
toxic plant families are themselves unpalatable and aposematically
colored, while those feeding on relatively innocuous plants were
typically cryptic and palatable. This suggested that, following the
breaching of a plant's chemical defense, certain insects might not
only be able to tolerate the allelochemics but might store them in
their tissues for their own defensive purposes. These compounds may
even be incorporated into the tissues of some invertebrate predators
feeding upon allelochemic-storing herbivores (e.g., Malcolm, 1981).
Thus, defensive chemicals may pass through a food web, having
ramifications at several trophic levels.

Berenbaum (1983) has provided an example in which each step of
the evolutionary sequence postulated by Ehrlich and Raven (1964) can
be supported by either direct or circumstantial evidence. She makes
the following argument. (1) The production of phototoxic
furanocoumarins (the toxicity of which is induced only in the presence
of ultraviolet light) in numerous plant families could plausibly have
evolved as a fortuitous elaboration of pre-existing biosynthetic
pathways (e.g., lignin synthesis). (2) These compounds are more toxic
to certain herbivorous insects than are their precursors. (3) Within
the Umbelliferae, the one subfamily containing coumarins is far more
diverse in number of species than are two subfamilies that lack
coumarins. This is circumstantial evidence for an evolutionary
radiation following the evolution of the chemical defense. (4) Various
biochemical and behavioral means of circumventing the plant's defense
have evolved among insects. Most striking among these is the habit of
leaf-rolling among the Oecophoridae, lepidopterans that feed
exclusively on Umbelliferae. While feeding within rolled leaves, the
larva is shielded from ultraviolet light and consequent phototoxic
effects. (5) Genera of oecophorids and papilionids which are
associated with furanocoumarin-containing plants have diversified (in
species numbers) more extensively than have those which feed on
furanocoumarin-free hostplants. This is circumstantial evidence for
an evolutionary radiation following the breaching of the plant's
chemical defense.

Berenbaum's (1983) work provides one of the best-developed
examples of what has been called "diffuse coevolution" (Gilbert, 1975;
Fox, 1981; Futuyma and Slatkin, 1983), i.e., that resulting from the
simultaneous interaction of several plant and animal species. Some
workers (e.g., Janzen, 1980), however, prefer to restrict the term
(and concept) of plant/herbivore coevolution to instances in which a
close evolutionary sequence of reciprocal counter-adaptation between
one specific plant population and one specific herbivore population
("paiirwise coevolution") can be demonstrated:

"Coevolution" may be usefully defined as an
evolutionary change in a trait of the individuals
in one population in response to a trait of the
individuals of a second population, followed by an
evolutionary response by the second population to
the change in the first. [Janzen, 1980:611]

By "evolutionary change" or "evolutionary response," Janzen refers to
genetic change. For example, specific alleles in wheat have been
identified which confer resistance to Hessian flies (Mayetiola
destructor) , and each of which corresponds to a specific fly allele
for counter-resistance (Hatchett and Gallun, 1970). While this has
been cited as one of the clearer examples of coevolution (Futuyma,
1979; Thompson, 1982), it still does not meet Janzen's (1980) strict
criteria, since the frequencies of alleles for resistance in wheat
populations have been determined by agricultural geneticists rather
than by Hessian fly herbivory. In another well-studied case, Williams
and Gilbert (1981) have described the production by certain Passiflora
plant species of structures which mimic the eggs of, and inhibit

oviposition by, their specialized herbivores, the Heliconius
butterflies. These structures exploit the behavioral tendency of
Heliconius females not to oviposit on plants which harbor previously
laid eggs, since early-hatching larvae frequently cannibalize
subsequently laid eggs. It seems plausible that the evolution of
Heliconius feeding specialization on Passiflora, with its increased
threat to the plant's fitness, has in turn selected for the counter-
evolution of these defensive pseudo-eggs. Note that even here the
strict criterion for coevolution will only be met if the butterflies
are shown to have a specific counter-adaptation to circumvent the
plant's deception, such as the ability to discriminate mimetic eggs
from real ones. Although such discrimination has not been directly
observed (Gilbert, 1983), it may be inferred from the close degree of
resemblance of the mimetic eggs to real Heliconius eggs. Surely the
first "mimetic eggs" on Passiflora were crude bits of tissue and their
present resemblance to real eggs must have evolved gradually as the
butterflies evolved an improved ability to detect the difference (even
if they no longer can do so) . This example of coevolution emphasizes
the importance of integrating direct observations in ecological time
with sound inferences about processes that may occur over evolutionary
t irae .

While coevolution has proven to be, both theoretically and
empirically, an elusive and controversial concept (e.g., Jermy, 1976),
it has fostered a great deal of work on the biochemical interactions
of plants and their enemies, especially herbivores. A major

organizing theory attempting to explain the kinds of plant chemical
defenses evolved has been provided simultaneously by Feeny (1976) and
Rhoades and Gates (1976). This theory, now generally known as
"apparency theory," assumes that ephemeral, early successional plant
species are more able than long-lived, prominent, late-successional
species to escape herbivory spatiotemporally. Such "unapparent"
species typically devote a large proportion of their energy and
nutrients to rapid growth, therefore presumably allocate less to
chemical defense and thus rely on what Feeny (1976) terms
"qualitative" chemical defenses. These are compounds which are
produced in low concentrations and have specific, highly toxic effects
against a broad range of herbivorous species. Examples of such
compounds include alkaloids, saponins, cyanogenic glycosides,
glucosinolates and non-protein amino acids (reviewed in Rosenthal and
Janzen, 1979). In contrast, long-lived, late-successional species are
less able to escape in ecological space and time, are "bound to be
found" (Feeny, 1976) by herbivores, and are therefore selected for a
greater investment in the amounts of chemicals allocated to defense.
Because some herbivores will eventually succeed (evolutionarily) in
breaching any qualitative defense, these "apparent" plants should be
selected to contain chemicals which are sufficiently general in mode
of action as to be effective, in a dose-dependent manner, against both
polyphagous and oligophagous species. The best-known examples of such
a "quantitative" chemical defense are the tannins, a group of
polyphenol compounds which, at least in vitro, precipitate proteins

10

(both dietary and enzymal) and thereby inhibit digestion (e.g., Feeny,
1970; but see Bernays, 1981, and Zucker, 1983, for a growing
controversy regarding their in vivo effects).

Several criticisms can be raised against the theory of plant
apparency. First, the prediction that early-successional species
should invest in qualitative rather than quantitative defenses rests
on the assumption that qualitative defenses are less costly to produce
than quantitative ones, simply because they are effective at, and
therefore produced in, lower concentrations. However, the cost of
chemical defense cannot be assessed simply by comparing tissue
concentrations when different kinds of compounds are involved. It is
not necessarily true that it costs a plant as much to synthesize a
milligram of tannin as to produce a milligram of, say, cyanogenic
glycoside. Furthermore, some toxins (e.g., alkaloids) may be broken
down and resynthesized many times in the same plant (requiring
repeated investments of energy), while digestibility-reducers such as
tannins may be synthesized only once and retained for life (Fox,
1981). It is not yet known which of these two strategies is the more
costly (and this too will surely vary with the chemical nature of the
particular compounds involved). There may also be costs associated
with the internal transport of the compounds or the production of
special sites for their storage (Chew and Rodman, 1979). Thus, while
it is reasonable to assume that there is some cost to the plant for
chemical defense, the a priori assumption that the cost to ephemeral
species is necessarily less than that paid by persistent species is
not justified (Fox, 1981).

11

Second, one should also be wary of the dichotomy between
qualitative and quantitative defenses (Feeny, 1976). Toxins may well
have dose-dependent effects (e.g., glucosinolates : Chew and Rodman,
1979; sesquiterpenes: Langenheim et al., 1980) and purported
digestibility-reducers such as tannins may have little or no effect
upon adapted specialists (Fox and Macauley, 1977; Bernays et al.,
1980).

Third, apparency itself can be quite an elusive concept. While
Rhoades and Gates (1976) base their definition on ecological
longevity, growth form, serai stage, etc., Feeny (1976) Includes the
host-finding ability of the herbivores as well. Rhoades (1979) has
argued that if host-finding adaptations are included as a component of
plant apparency, the entire concept of apparency is rendered circular.
This is because the intensity of selection for host-finding ability
should be greater for herbivores attacking rare or ephemeral plants
than for those attacking highly apparent plants. Once host-finding
ability is included, the apparencies (sensu Feeny) of the two plant
types converge. Thus, it is not clear that a short-lived herbaceous
plant is any less apparent _to its specialized herbivores (which may
have sophisticated host-finding adaptations) than is an ecologically
more persistent plant species. Yet, in order to make predictions
about the kinds of defense to expect among various plant groups, we
need to have some relative measure of the selection pressure exerted
by the plants' enemies. Clearly, this can only be appreciated by
taking into account enemy host-finding abilities.

12

Given these problems with apparency theory, it may be best to
restrict its application to discussions of the defenses of related
plants facing similar guilds of enemies. If, for example, most plants
within a family utilize similar allelochemics for defense (Ehrlich and
Raven, 1964), then it may be safe to assume that those species
producing greater quantities of the allelocheraic make greater
investments in chemical defense than do related species producing less
of the allelochemic. This is surely safer than assuming cost
differentials for qualitatively very different compounds in widely
divergent plant families (see above). Also, if the enemy guilds are
similar for the related plants, one might assume similar host-finding
abilities and then concentrate on making predictions about defensive
investments based on such considerations as growth habit (e.g., herbs
vs. shrubs), population density, and phenology. This approach will be
taken in a later section in discussing the chemical defense of
milkweed plants.

Specialization and Generalization in Herbivory

It has frequently been suggested that since generalist herbivores
use a broader range of resources than do specialists (by definition) ,
they are less susceptible to fluctuations in the availability of any
particular food species and are therefore "ecologically resilient"
(e.g., Levins, 1968; Schoener, 1970; MacArthur, 1972). They must,
however, cope with a broader variety of resource defenses and are
therefore thought to be less efficient at utilizing any one particular

13

resource type than are species which specialize on eating only one or
a few resource types (MacArthur, 1972; Feeny, 1975; Scriber and Feeny,
1979). One means by which generalist insects have adapted
biochemically to a broad range of dietary toxins is through the mixed-
function oxidase (MFO) system (Brattsten et al., 1977). This
microsomal system permits the rapid induction, upon contact with
specific toxins, of the specific oxidative enzymes required for their
detoxification. Krieger et al. (1971) demonstrated that the general
level of MFO activity was significantly greater in polyphagous
lepidopteran larvae than in oligophagous species.

Such biochemical adaptation is presumably effected at some
metabolic cost to the insect, i.e., requiring energy which could
instead be utilized for other fitness-promoting activities. Feeny
(1975) has conceptualized two kinds of biochemical metabolic costs
which may be incurred. The first is the cost of synthesizing
appropriate detoxification enzymes and any morphological structures
required for their storage. This is the "fixed cost" that is paid
regardless of the quantity of toxin encountered. The second kind of
cost is the "variable cost" of running the detoxification process
itself, the magnitude of which depends upon the quantity (and chemical
nature) of toxin to be handled. Feeny (1975) argues that while the
variable costs of detoxifying a particular compound are probably
similar for both specialists and generalists (since similar processes
would be required), the fixed costs should be lower for specialists
since they must construct and retain biochemical mechanisms for
dealing with a more limited range of toxic compounds. This leads to

14

the prediction that polyphagous species should utilize any particular
food type less efficiently than would oligophagous or monophagous
species.

This prediction was confirmed by Auerbach and Strong (1981) who
found that specialist insects that feed on Heliconia imbricata leaves
had higher food utilization efficiencies than did more polyphagous
species also fed H. imbricata. Scriber (1979) also provided data that
support the prediction. He showed that larvae of the Lauraceae-
specializing butterfly, Papilio troilus, had food utilization
efficiencies 2 to 3 times greater than did the more polyphagous P^
glaucus when also fed a lauraceous diet. Other studies have failed to
confirm the prediction, however. For example, Futuyma and Wasserman
(1981) fed leaves of Prunus serotina to the tent caterpillars,
Malacosoma americanum (a specialist on Prunus) and M^ disstria (a more
polyphagous species), finding no significant difference in their food
utilization efficiencies. [Fueling the debate further, studies by
Schroeder (1976, 1977) and Scriber and Feeny (1979) claim to show that
specialists are no more efficient food-converters than are
generalists. However, because these authors did not compare
efficiencies when the specialists and generalists were fed the same
plants, their observations are of limited value in the present
context. ]

One reason why conflicting evidence may be found in studying
differences between specialists and generalists has been suggested by
Fox and Morrow (1981). These authors show that many species commonly
regarded as "generalists" nevertheless exhibit a considerable degree

15

of specialization at the population level. Because the evolution of
feeding adaptations would normally occur at the population, rather
than entire species, level, it is possible that such populations are
as biochemically and digestively specialized as are populations of
species which feed on a single food type throughout their ranges.

A second possible explanation for the contradictory evidence is
suggested by the work of Smiley (1978) who found that larvae of the
butterfly Heliconius erato (which is essentially monophagous on
Passiflora biflora) grew faster on P^ biflora than did larvae of the
more oligophagous species H^ cydno (which feeds on at least five
Passiflora species). However, another monophagous species, H.
melpomene, grew no faster on its hostplant (JP^ oerstedii) than did H_^
cydno. Smiley (1978) suggests that H. melpomene has specialized on P.
oerstedii for ecological rather than biochemical or nutritional
reasons. These might include higher interspecific competition or
parasitism rates on other available host plant species. If such
"ecological monophagy" (Gilbert, 1979) is widespread (see also Singer,
1971), specialists may not necessarily exhibit greater food
utilization efficiencies than generalists.

The case of E^ erato described above exemplifies a second type of
monophagy, in which specific digestive/biochemical adaptations for
coping with the primary and secondary chemistry of the particular host
species have apparently evolved. Gilbert (1979) terms this "coevolved
monophagy," but "evolved monophagy" might be a preferable term since
such herbivore adaptation may exist even when "coevolution" per se can
not be demostrated.

16

While the basic distinction of evolved vs. ecological monophagy
is valid, it is probably best to regard these as opposite ends of an
evolutionary continuum. For example, if the selection pressures
encouraging ecological monophagy persist for sufficient time, then
there will presumably also be selection for an increase in the
efficiency with which the particular host type is utilized. Thus,
what begins as ecological monophagy may well result in evolved
monophagy. At any point in evolutionary time, we may expect to find
species (or populations) that are at different (and unknown) points
along this evolutionary path. This fact will certainly hamper our
ability to make predictions about the relative dietary efficiencies of
"specialists" and "generalists."

Chemical Defense of Milkweed Plants

The milkweed family, Asclepiadaceae (order Apocynales) , provides
a good example of an early-successional, herbaceous plant group which
is fed upon by several oligophagous insect species. It is comprised
of some 1700 species of perennial herbs, vines, and shrubs (Willis,
1931). While the family is predominantly pantropical, 108 species
within the genus Asclepias alone have been described from North
America (Woodson, 1954). Milkweeds are renowned for their toxic
properties, based upon a class of C-23 steroids called cardenolides
(Kingsbury, 1964). These typically occur in glycosidal form in the
plants (but are not present in all species; Roeske et al., 1976) and
are therefore often referred to as "cardiac glycosides." The

17

designation, "cardiac," refers to the well-known effect of these
compounds on vertebrate heart tissue, acting through an inhibition of
the Na-K-ATPase system (Hoffman and Bigger, 1980). In therapeutic
dosages, cardenolides cause the heart to beat more slowly and
regularly, but in excessive dosages, they are lethal (Hoffman and
Bigger, 1980).

Although the toxic effects of cardenolides to many species of
domestic mammals are well-established (e.g., Detweiler, 1967), their
toxicity to invertebrate herbivores is less clear. This is a serious
gap in our knowledge because there exist several groups of herbivorous
insects that specialize on asclepiads (Duffey and Scudder, 1972;
Scudder and Duffey, 1972; Rothschild and Reichstein, 1976) and it has
frequently been inferred that these have "coevolved" with milkweed
plants after breaching the presumed cardenolide defense (see, e.g.,
Brower and Brower, 1964; Dixon et al., 1978; Harborne, 1983). Thus,
it is obviously crucial to know whether cardenolides are, in fact,
toxic to herbivorous insects. The evidence for this will be reviewed
in detail in a later section.

While qualitative variation in cardenolide contents, within and
among milkweed populations, has been noted (e.g., Brower et al.,
1982), little effort has been made to relate such variation to
ecological and evolutionary forces. Roeske et al. (1976) summarize
work on the individual cardenolides isolated and identified from
various milkweeds and show that some populations of Asclepias
curassavica and A. syriaca contain specific cardenolides not reported
from other populations (see also Malcolm, 1981). Also, in A.

eriocarpa, the relative proportions of the various leaf cardenolides
appear to vary between populations. The authors do not speculate on
the ecological significance of such differences. However, because the
toxicity of most allelocheraics is positively related to their
lipophilicity (e.g., Harborne, 1983), this may suggest that those
populations containing more lipophilic cardenolides face more
resistant (more "highly coevolved"?) herbivores than do populations
having more hydrophilic (i.e., polar) compounds. This argument could
be extended to cover interspecific qualitative differences as well
(see below).

Quantitative variation in cardenolide concentration within
populations has also been described (e.g., Nelson et al., 1981; Brower
et al., 1982). One of the most variable species is Asclepias syriaca,
which may exhibit a 40-fold range of cardenolide concentration within
a single population (Roeske et al., 1976). Other populations of this
species are reportedly devoid of cardenolides altogether (e.g.,
Rothschild et al., 1975). The ecological significance of such
differences has never been studied and it would be instructive to
learn, for example, whether individuals or populations lacking or low
in cardenolide face a different set of herbivores than do those having
higher tissue concentrations. [It is possible that some plants
lacking cardenolides compensate for this by the storage of other
allelochemics. Although both saponins and alkaloids reportedly occur
in at least some milkweeds (see Brower and Brower, 1964), their
functions in these plants remain totally unstudied.]

19

Although nearly all milkweed species examined have leaf
cardenolide concentrations less than 1% of dry weight (Roeske et al.,
1976), interspecific variation should not be ignored. Several species
apparently lack cardenolides entirely, or contain undetectable amounts
(e.g., Gonolobus spp., Brower, 1969; A. viridiflora, Roeske et al.,
1976; Hoya, Stephanotis, Marsdenia, Tylophora , Cynanchum , Gymnema ,
Rothschild and Marsh, 1978). The most complete tabulation of milkweed
cardenolide concentrations is provided by Roeske et al. (1976: Table
2) for 24 species, and well illustrates the wide interspecific
variation. The highest recorded cardenolide concentration in leaves
is 14.7% of dry weight for A. masonii from California (Roeske et al.,
1976; but this is based upon a single determination and should be
replicated) .

How can these differences be explained? Using apparency theory,
one might predict that the most apparent species would, other factors
being equal, tend to have the highest cardenolide concentrations,
whereas the least apparent would have the lowest. Roeske et al.
(1976), using Woodson's (1954) data on species distributions and
density, have found that those species having wide geographic
distributions and growing densely tend to have low cardenolide
concentrations, while those of more restricted distributions,
occurring in low density, have higher cardenolide concentrations.
These trends are opposite to those expected from apparency
considerations, since widespread, densely growing plants are
presumably more apparent than restricted, sparsely growing plants.

20

However, the abundance and density of milkweed plants have probably
changed drastically with the development of North American agriculture
(e.g.. Fink and Brower, 1981), and so Woodson's (1954) biogeographic
(county record) information may no longer be valid for making
quantitative comparisons.

It is noteworthy that, of those milkweed species growing in
north-central Florida, only two of six studied produce leaf crops by
early spring (Cohen, unpubl. data), when large numbers of asclepiad-
specializing monarch butterflies re-enter eastern North Merica from
overwintering sites in Mexico (Urquhart and Urquhart, 1977). These
two species, Asclepias humistrata and A. viridis, tend to grow in
dense patches, and were located in each of the years 1981-1983 by the
migrants and utilized heavily as larval food sources (Cohen, pers.
observ.). Both species have relatively high leaf cardenolide
concentrations (Table 1). In contrast, the remaining four species (A.
tuberosa, _A^ amplexicaulis, A. tomentosa, and _A^ verticillata) grow
more sparsely, produce leaf crops too late for use by most migrant
monarchs, and have very low leaf cardenolide concentrations (Table 1).
While monarchs are surely not the only herbivores attacking milkweeds
in north-central Florida (see, e.g., Chapter IV), their massive
migrations into the area may provide a more acute risk than the more
regular, but lower level, herbivory by other species.

If the quantitative cardenolide differences within or among
milkweed species are indeed the result of differential risks to
herbivorous insects, it would suggest either that (1) some insects are
affected by cardenolides in a dose-dependent manner and, through

21

Table 1 : A survey of mldstem leaf cardenolide concentrations for six
milkweed species in north-central Florida. Data are expressed as
digitoxin equivalents (see Brower et al., 1972) as a percentage of dry
weight of leaf tissue.

Species X (%) Range

Asclepias humistrata

0.55

0.36-1.01

12

A. viridis^

0.38

0.19-0.54

10

A. tuberosa

0.02

0.00-0.05

4

A. amplexicaulis

0.03

0.01-0.04

2

A. tomentosa

0.06

0.03-0.09

3

A. verticillata

0.05

2

Route 24, ca. 8 km W of Gainesville (Alachua Co.) city limit;

collected 4 May 1981.
ca. 1 km from Alt. Route 27, ca. 3 km W of junction Route 41,

Williston (Levy Co.); 18 April 1981.
^ Route 24, at E. city limit Bronson (Levy Co.); 2 July 1981.

Devil's Millhopper State Geological Site, NW 53 Ave, ca. 1 km W.

Gainesville city limit; 2 June 1981.
^ NW 62 St., ca. 1 km W of Gainesville city limit; 7 June 1981.

Route 24, at E. city limit Bronson; 26 May 1981; leaves pooled from

two plants for a single cardenolide determination.

22

coevolution, have selected for higher defensive levels in their
hostplants, and/or (2) different insect species have different
threshold tolerances for cardenolides and those plant species having
greater concentrations may be adapted for defense against more
cardenolide-resistant herbivore species.

The Metabolic Cost of Cardenolide Ingestion

The two propositions above require that at least some herbivorous
insects be adversely affected by ingested cardenolides. Indeed, only
if it can be convincingly demonstrated that these compounds do present
a chemical barrier to herbivory by at least some species will it
become credible to think of milkweed-specialization by such
oligophagous groups as danaid butterflies (Brower, 1969; Brower et
al., 1975; Rothschild and Marsh, 1978) and lygaeid bugs (Scudder and
Duffey, 1972) as the possible result of a coevolutionary process
involving the breaching of such a barrier. Moreover, it should be
possible to demonstrate that milkweed-specialists have cardenolide-
tolerance capacities exceeding those of either polyphagous species or
species that specialize on cardenolide-f ree plant families.

Considerable attention has been paid to the question of whether
milkweed specialists suffer any negative physiological consequences
(which are here collectively called "metabolic costs") of ingesting,
metabolizing, detoxifying, or sequestering cardenolides. In contrast.

23

very little effort has been expended in testing the effects of these
compounds upon polyphagous species, despite the expectation that
generalists should be more likely than specialists to be affected by
toxins (see above; also Blau et al., 1978). Nevertheless, the first
suggestion of such a metabolic cost in an adapted specialist was made
by Brower et al. (1972), who found a progressive southward decrease in
the mean cardenolide concentration of migrating monarch butterflies
and suggested that those individuals having high cardenolide contents
may have had reduced viability or migratory ability relative to those
of lower cardenolide content. Rothschild et al. (1975) offered an
alternative hypothesis, suggesting that larvae developing in colder,
more northern locales would simply require a longer development time
than those in warmer, southern areas. As a result, they might ingest
more food and, consequently, more cardenolide. This suggestion was
later refuted by Dixon et al. (1978) who reared monarch larvae in the
laboratory at either 17° C or 28° C. Although those reared at 17°
took longer to complete development, they consumed less food, and
incorporated into their tissues both the nutrients and the dietary
cardenolide more efficiently, than did larvae reared at 28 . As a
consequence, rearing temperature had no significant effect upon final
cardenolide content of the insects.

A third possibility, not previously considered, is that the lower
cardenolide content of southern monarchs is due to a greater degree of
wing-scale loss during migration. It has been shown that the
cardenolide concentration in the wings is approximately twice that in

24

the remainder of the body (Brower and Glazier, 1975), and nearly 30
per cent of this wing cardenolide is localized in the wing-scales
(Nishio, 1980). It therefore seems possible that the further south a
monarch flies, the more scales it loses, and the lower its cardenolide
content becomes.

A second suggestion of metabolic costs of cardenolide ingestion
was provided by Brower and Moffitt (1974), who found a significant
negative correlation between the amounts of cardenolide stored by and
the sizes and dry weights of monarch females collected in
Massachusetts. The correlation approached significance for males
(which, on average, were 9 per cent lower than females in cardenolide
concentration) but disappeared entirely among both males and females
collected in California. On average, the California butterflies were
62 per cent lower in cardenolide concentration than were the
Massachusetts butterflies. It is thus possible that the purported
metabolic cost was seen only among the Massachusetts females because
only they contained sufficient concentrations to be affected.
Massachusetts males came next closest to exhibiting the effect,
perhaps because they contained the second highest cardenolide
concentrations, and California butterflies exhibited no apparent cost
because both sexes contained insufficient concentrations. Future
analyses of this problem should attempt to separate true geographic
and gender differences from cardenolide concentration differences with
which they may be correlated. It would also be valuable to learn
whether the apparent cardenolide/body size trade-off phenomenon
extends to other milkweed-feeding species, or is limited to monarchs.

25

Several recent studies have shown that monarchs (Erickson, 1973),
African queen butterflies (Danaus chrysippus; Smith, 1978) and
milkweed bugs (Oncopeltus fasciatus; Israan, 1977; Chaplin and Chaplin,
1981) tend to grow better when fed those plant species high in
cardenolide than when fed those low or lacking in cardenolide. These
authors (see also Blum, 1981) interpret such results as evidence
against a metabolic cost of cardenolide ingestion. The major fallacy
of such an interpretation is the confounding of cardenolide
differences among plant species with other nutritionally important
species differences. For example, other factors being equal, one
might expect those plant species high in nitrogen or water (two
important nutrients for insects; see Scriber and Slansky, 1981) to be
more desirable foodplants than those low in these nutrients. They
should therefore be under stronger selection for chemical (and other)
defense. Thus, we might expect a correlation between allelochemic
content and nutritional quality (e.g., furanocoumarins : Berenbaum,
1981), and the fact that some insects may grow better on high
cardenolide plants may be due to the correlated higher nutritional
quality of such plants, and may occur despite any metabolic costs that

may still be paid.

For this reason, a solution to the problem of metabolic costs
cannot be gained from studies in which several species of hostplant,
each having different cardenolide (and nutrient) contents, are fed to
insects. Rather, one must add purified cardenolide(s) , preferably in
varying dosages, to diets of standardized nutritional quality (e.g..

26

artificial diets). This has recently been done for monarchs fed
cardenolide-f ree milkweed leaves (Gonolobus rostratus) to which were
added increasing concentrations of the cardenolides calotropin,
uscharidin, uzarigenin, or digltoxigenin (Seiber et al., 1980). No
effects upon larval development time, food consumption, or body weight
were identified at any dose level for any of the four cardenolides.
The only suggestion of a metabolic cost was a dose-dependent melanism
occurring in larvae fed uzarigenin. In Lepidoptera, abnormal melanism
is a common indicator of stress such as crowding or starvation (Peters
and Barbosa, 1977). However, Seiber et al. (1980) housed their larvae
individually and showed that uzarigenin-f ed larvae consumed as much
food as controls, so that crowding and starvation cannot explain the
result. The melanistic response occurred only at dosages 4 to 61
times greater than that found in the hostplant (A. curassavica) from
which the uzarigenin was isolated. Thus, while there may be a dose at
which a cost is paid, it is rarely (if ever) encountered in nature and
it is unlikely that the present levels of uzarigenin in _A^ curassavica
are attributable to herbivore pressure from monarchs.

Seiber et al. (1980) also found a quantitative regulation by
monarch larvae of the amount of cardenolide stored in their tissues.
Thus, when fed low-cardenolide diets, proportionally more cardenolide
is sequestered than when high-cardenolide diets are fed (see also
Brower et al., 1982). As a result, the variation in the total amount
of cardenolide sequestered is damped. Although the mechanism
underlying this regulation has not been investigated, one may wonder
why the insects do not simply store as much cardenolide as they

27

ingest, or do not excrete (or metabolize to noncardenolide compounds)
a constant fraction of the ingested cardenolide. Again, there is a
suggestion that there may be an upper limit on monarch cardenolide
tolerance.

Using in vitro preparations, Vaughan and Jungreis (1977) found
that monarch neuronal tissues were some 300 times less sensitive to
cardenolide- (ouabain-) induced inhibition of the Na-K-ATPase system
than were tissues of two polyphagous lepidopteran species (Hyalophora
cecropia and Manduca sexta) . This study is important because it
represents the first serious attempt to ascertain whether a metabolic
cost may be paid by species not adapted as milkweed specialists. It
must be regarded with caution, however, because in vitro preparations
circumvent a good part of the potentially protective physiology of the
organisms. For example, it is possible that the digestive systems of
Hyalophora and Manduca prohibit assimilation of ingested cardenolides
into the hemolymph. If so, one would not expect the evolution of
protection at the neuronal level. Since injection bioassays are
equally unrealistic, this caveat also applies to the results of von
Euw et al. (1967) and Rafaeli-Bernstein and Mordue (1978) that
milkweed-feeding orthopteran species are less sensitive to injections
of cardenolide than are species that do not feed on milkweeds. What
remains to be done is a comparative study of the effects of ingested
cardenolide upon the growth and metabolism of both milkweed-adapted
and non-adapted herbivorous insects.

28

In this dissertation, I present the results of several studies
bearing on the question of the metabolic costs of cardenolide
ingestion in lepidopteran species that are either adapted or not
adapted to feeding on milkweed plants. The specialized milkweed-
feeders studied were the monarch butterfly ( Danaus plexippus) , the
queen (Danaus gilippus) , and the dogbane tiger moth ( Cycnia tenera;
Arctiidae). The fall armyworm (Spodoptera frugiperda; Noctuidae) was
studied as an example of a highly polyphagous herbivore. Finally, the
velvetbean caterpillar (Anticarsia gemmatalis; Noctuidae) was studied
as an example of a specialist species which feeds on plants lacking
cardenolide (in this case, only plants in the family Leguminosae are
eaten). The species selected represent a wide taxonomic range (i.e.,
both moths and butterflies).

In Chapter II, I discuss the hostplant selectivity and larval
success of monarch butterflies in relation to plant cardenolide
concentration. If there is, in this species, a metabolic cost of
feeding on high cardenolide diets, then ovipositing females might be
expected to have evolved a preference for lower cardenolide plants
over higher ones. Furthermore, if the costs accrue in the larval
stage, then the survival of larvae on high-cardenolide plants might be
reduced relative to that of larvae on lower-cardenolide plants.
Alternatively, if no significant cost exists, females might instead
behave in such a way as to maximize the opportunities of their
offspring to sequester defensive chemicals, i.e., higher cardenolide
plants should be preferred. Finally, if larvae on high-cardenolide
plants are better protected from parasites and predators than those on

29

low-cardenolide plants, a differential in larval survivorship should
be measurable.

Chapter III presents a comparative analysis of the cardenolide
contents of wild queen butterflies (D^ gilippus berenice) collected
from three locations in Florida, and of monarchs from one of those
sites. Since it is knovra for other lepidopteran species that body
size is positively correlated with fecundity (Hinton, 1981), the
hypothesis that these danaid species pay fitness costs of cardenolide
ingestion would be supported if negative correlations between
butterfly cardenolide concentration and body size or weight were
found. In a separate experiment, also reported in Chapter III, these
two species were simultaneously reared in the laboratory on the
milkweed, Asclepias humistrata, and their cardenolide sequestration
abilities compared. Again, correlations between body size, weight,
and cardenolide content were sought.

It should be noted that even if such indications of metabolic
cost are found, this need not imply that the net benefit to the insect
is necessarily negative. Sequestered allelochemics may provide a
chemical defense that outweighs any costs paid to acquire them. As a
result, there may be a net gain in fitness. Nevertheless, a reduced
body size may be considered an investment made in order to achieve
this level of defense. It is assumed that, as adaptation to
allelochemics evolves, the magnitude of the investment required would
be reduced.

In addition to their relevance to the issue of metabolic costs,
the data on wild queens provide the first practical application of the

30

chromatographic cardenolide "fingerprinting" technique devised by
Brower et al. (1982) to identify the hostplants of wild-caught danaid
butterflies. The results may also shed some light on a long-standing
problem in the study of mimicry, viz., why the viceroy butterfly
(Limenitis archippus) in Florida mimics the queen instead of its usual
model, the monarch (e.g., Brower, 1958b).

In Chapter IV, I investigate the issue of cardenolide
sequestration in an oligophagous arctiid moth, Cycnia tenera. This is
an aposematic, apocynad/asclepiad-specializing species reported by
Rothschild et al. (1970) not to sequester cardenolides from its
hostplant (Asclepias syrlaca in their study). This raises the
interesting question of whether, even among specialists, some species
do not sequester cardenolides because they are more sensitive to these
compounds than are sequestering species such as danaids.
Unfortunately, Rothschild et al. (1970) did not confirm that the A.
syriaca fed to the C. tenera larvae indeed contained cardenolides. It
is thus necessary first to either confirm or refute their findings,
using hostplants of known cardenolide contents. In this chapter, I
report on the growth, fat storage, and both qualitative and
quantitative aspects of the cardenolide contents of _C^ tenera reared
on the milkweeds, _A^ humistrata and _A^ tuberosa.

In Chapter V, I report the results of a series of gravimetric
experiments (Waldbauer, 1968) designed to test whether dietary
cardenolides per se affect consumption rates, growth rates, body size,
and other important fitness indices of larval Lepidoptera. I will
compare the metabolic responses to ingested cardenolide of a milkweed

31

specialist (monarchs), a polyphagous species (fall armyworms), and a
species specializing on cardenolide-negative plants (velvetbean
caterpillars) .

Chapter VI summarizes the major results presented in the
dissertation and discusses their relevance to the theories of
plant/herbivore coevolution and chemical defense.

CHAPTER II

OVIPOSITION AND LARVAL SUCCESS OF WILD MONARCH BUTTERFLIES
(LEPIDOPTERA: DANAIDAE) IN RELATION TO HOSTPLANT SIZE AND CARDENOLIDE

CONCENTRATION*

Introduction

Larvae of the monarch butterfly (Danaus plexippus' L.) feed upon
milkweed plants ( Asclepiadaceae) , from which they may ingest and
sequester plant defense chemicals known as cardiac glycosides or
cardenolides (Parsons, 1965; Reichstein et al., 1968; Brower, 1969;
Roeske et al., 1976). In turn, these chemicals are important in
defending the monarch against some vertebrate predators by making the
butterflies unpalatable (Brower and Brower, 1964; Brower, 1969).
Therefore, one might expect the presence, concentration, or particular
types of cardenolides present in individual milkweed plants to be
among the factors influencing a monarch female's decision to oviposit

*Published 1982 in Journal of the Kansas Entomological Society 55 :343-

348.

32

33

on a particular plant. Dixon et al. (1978) investigated this question
on an interspecific level by comparing the numbers of eggs laid on
greenhouse plants of three milkweed species differing in cardenolide
concentration. They reported an oviposition preference for the
species having the lowest cardenolide concentration (Asclepias
curassavica) , although they concluded that factors such as plant age
and the presence of previously laid eggs and larvae are more important
determinants of oviposition. However, their study did not test the
independent effect of cardenolide concentration upon oviposition
preference for the following reasons : 1) milkweed species differ in
many ways other than cardenolide concentration, such as water and
nitrogen content (Erickson, 1973) or plant morphology (Woodson, 1954)
so that ovipositional preferences for a particular species cannot be
attributed to any one of these plant differences unless more
controlled studies are done; 2) the data on cardenolide concentrations
were not derived from the same individual plants upon which the
butterflies oviposited, and significant intraspecif ic differences in
milkweed cardenolide concentrations occur (Brower et al., 1982; and
this study); and 3) the study was carried out in captivity where
unnatural butterfly behavior commonly occurs (e.g., Dixon et al.,
1978 :443, 448).

To avoid the above problems, I carried out field observations of
oviposition and larval success of wild monarchs on individual
Asclepias humistrata Walt, plants which were then analyzed for
variation in cardenolide concentration.

34

Methods

Ten plants of Ascleplas humlstrata (Asclepiadaceae) , growing
along a sparse grassy roadside (Route 346) near Cross Creek, Alachua
Co., Florida, were marked for study. The total leaf area of each
plant was estimated as follows. A separate collection of 25 A.
humistrata leaves was first used in determining the relationship
between leaf length and leaf area. Lengths were measured to the
nearest 0.1 cm with a ruler and areas determined electronically (to

the nearest cm^) with a Li-Cor Model 3100 Area Meter. The regression

2
relationship was: area = 6.34 [length] - 15.14, with an r = 0.92.

Because A. humistrata has opposite leaves of approximately equal size,

I then measured (_in situ) the length of the least eaten of each pair

of leaves on the 10 marked plants. Their leaf areas were then

estimated using the regression formula, summed for each plant, and

then doubled to provide an estimate of the total plant leaf area.

I counted the total numbers of monarch eggs and larvae on each

plant on each of the following dates in April 1981: 4, 5, 6, 8, 10,

12, 14, 16, 18, 20, and 23. These total counts overestimate the

actual egg and larval numbers since each individual may be counted

more than once (i.e., on different dates). However, this problem

applies equally to all 10 plants and thus does not affect the

statistical analyses or conclusions. I therefore define larval

success as the total number of fifth instars per egg counted.

35

On the day final counts were made, I collected for cardenolide
analysis four midstem leaves from each of the nine surviving plants,
avoiding any partially eaten leaves. To assess whether this
collection of only uneaten leaves would bias the results (i.e., if
their cardenolide concentrations were not representative of the
plants' foliage in toto) , two partially eaten and two uneaten midstem
leaves were also taken from each of seven adjacent plants. All leaves
were then sealed in plastic bags, placed on ice, and kept in a deep-
freeze until analysis in mid-July. Cardenolide concentrations
(expressed as micrograms of digitoxin equivalent per 0.1 g dry weight
of leaf) were determined by standard spectrophotometric methods (see
Brower et al., 1972, 1975).

Tests of association between variables were done with Spearman's
rank order correlation, followed by t-tests for significance.

Results

The numbers of eggs and larvae, larval success rates, plant
sizes, and cardenolide concentrations for each plant are shown in
Table 2. Egg densities were also calculated in order to assess the
influence of cardenolide concentration, independent of plant size,
upon oviposition. It is evident from these data that a large
percentage of eggs (x=96%) failed to reach the fifth instar. Figure 1
shows that a constant (43%) proportion of the larvae appear to be
lost, presumably to mortality, during each instar.

36

Table 2. Eggs, larvae, larval success, and cardenolide concentrations
for 10 Asclenias humistrata p].ants.

^"^

Garden-

Glide

tration

Egg

Percent larval

success

(mO.1

leaf

Total

density

Total n

umber larvae

(No- lar

■aeegg) < 100

Plant

stems

leaves

(cm^)

eggs

100 cm^)

1

II

III

IV

V

I

II

III

IV

V

leaft

1

1?

362

8

2.21

4

2

1

0

0

50

25

13

0

0

438

2

18

322

21

6.52

5

8

3

3

2

24

38

14

14

10

205

I

11

300

7

2.33

3

0

0

0

0

43

0

0

0

0

560

4

8

202

27

13.37

4

0

0

0

0

15

0

0

0

0

602

S

8

122

5

4.10

3

0

0

0

0

60

0

0

0

0

415

6

11

77

1976

92

4.66

53

35

17

10

7

58

38

18

11

8

279

7

■!2

699

83

11.74

21

10

5

2

3

25

12

6

2

4

374

8

26

655

35

5.34

15

7

1

0

0

43

20

3

0

0

496

9

42

1250

24

1.92

4

10

5

5

2

17

42

21

21

8

601

10

64

1510

25

1.66

21

20

17

11

3

84

80

68

44

12

•

.?

■io

740

33

5.39

13

9

5

3

2

42

26

14

9

4

441

SD

24

630

31

4,12

16

11

7

4

->

49

44

35

29

20

140

' Not determined because plant died before termination of study.

Source: Cohen and Brov/er. 1982

37

400

EGG

LARVAL STAGE

Fie,. 1. The number of monarch egp.s and larvae of each ins tar
on 10 Asclepias humistrata plants, pooled over 11 observation
days. l:he least-squares regression line suggests a constant
^3% iportality rate at each instar. (Source: Cohen and Brovjer,
1982)

38

Cardenolide concentrations within the plant population varied
approximately three-fold (coefficient of variation = 32%; Table 2).
Since no significant difference in the cardenolide concentrations of
uneaten and partially eaten leaves was found (Table 3), the analysis
of only uneaten leaves from the marked plants was not biased by this
factor.

Significantly more eggs were laid on larger plants, with the
consequence that egg densities did not vary with plant area (Table 4).
Fifth instar larval success was positively correlated with plant size,
However, the number of eggs laid per plant, egg densities, and larval
success rates all varied independently of the cardenolide
concentrations of the plants.

Discussion

I found a positive correlation between the number of eggs on a
plant and its total leaf area. This is expected on purely statistical
grounds since larger plants present larger targets for oviposition. A
consequence of this relationship is that egg density does not vary
regularly with plant size (Tables 2 and 4). It may be surprising,
therefore, that larval success does correlate with plant size, since
this is not likely to be due to overcrowding and competition among
larvae for food or space. I suggest that larger plants, having more
overlapping stems and leaves, may offer greater protection against

39

Table 3. Cardenolide concentrations (ug/O.lg dry weight) for 7
matched sets of partially eaten (PE) and uneaten (UE) Asclepias
humistrata leaves.

PLANT

SD

PE

UE

390 497 681 205 723 434 513 492 176
395 639 613 231 211 807 438 476 221

t=0.15; P>0.05; Student's t-test, matched pairs design. Variances
are not significantly different: F-max=1.26; P>0.05. (Source: Cohen
and Brower, 1982)

40

Table 4. Correlation coefficients for monarchs and plant area, and
for monarchs and cardenolide concentration. Egg numbers and larval
success vary positively with plant area, but no correlations with
cardenolide concentration occur.

CARDENOLIDE

PLANT AREA

CONCENTRATION

(N=10)

P

(N=9*)

P

0.66

0.04

-0.17

NS

-0.35

NS

-0.20

NS

0.69

0.03

-0.25

NS

TOTAL NO. EGGS

EGG DENSITY

V LARVAL SUCCESS

Spearman rank order correlations; alpha=0.05.

* No cardenolide data available for plant 10 which died.

(Source: Cohen and Brower, 1982)

41

both biotic (e.g., parasites and predators) and physical (e.g., direct
sunlight and dessication) mortality factors. The curve in Fig. 1
suggests a constant proportional mortality rate. However, this is
based upon a mixed cohort of eggs and larvae of varying initial ages,
as well as some recruitment, summed over the 19 day period. Also,
unlike monarchs in California (Brower et al., 1982), these fifth
instars usually left their hostplants when ready to pupate, and thus a
few may have been missed. The data may therefore slightly
underestimate fifth instar larval success. Yet, despite these error
factors, it is remarkable that the data fit so well the logarithmic
relationship characteristic of Type II survivorship curves (Wilson and
Bossert, 1971).

No relationship between egg numbers or egg densities and the
cardenolide concentrations of the plants was found. This is in
contrast to the apparent oviposition preference for low-cardenolide
plant species reported by Dixon et al. (1978). Moreover, the
relatively high intraspecif ic variance in plant cardenolide
concentrations (Table 2), together with similar variability found by
Brower et al. (1982) for A. eriocarpa, reaffirms the necessity of
measuring concentrations in the particular plants for which
oviposition data are gathered, rather than generalizing for entire
species or even populations. Intraplant variation is also possible
(e.g., Rhoades and Gates, 1976). I have investigated the specific
hypothesis that intraindividual cardenolide differences exist between
uneaten and partially eaten leaves, but no such differences were found
(Table 3).

42

I measured plant sizes on 4 April but collected leaves for
analysis on 23 April. Plant sizes change over time as a function of
both herbivory and growth. Similarly, cardenolide concentrations may
vary with time (e.g., Nelson et al., 1981, for A. eriocarpa) .
However, my conclusions are based on rank-order, rather than
parametric, correlations and thus require only the assumption that
size and cardenolide rankings of the plants did not change over the
course of the 19-day study. At least for plant size, this accords
with subjective observations of the plants.

Brower et al. (1982) demonstrated that monarchs reared on low-
cardenolide _A^ eriocarpa plants sequester proportionately more
cardenolide than do those reared on high-cardenolide plants. Thus,
the monarchs effectively regulate their own chemical concentrations,
with the resultant variation in butterfly concentrations considerably
less than that in the plants upon which they had fed. This regulatory
ability suggests that the actual cardenolide concentration of the
plant on which an egg is laid is, within certain limits, of little
significance in determining the ultimate amount of cardenolide in, and
hence probable degree of protection of, the butterflies. However,
this regulatory ability appears to break down when plant species of
extremely low cardenolide content are eaten (Brower, Seiber, and
Nelson, in prep.). Since A. humistrata is a relatively high-
cardenolide species (see Roeske et al., 1976), the possibility remains
that females would be more selective in choice of hostplant when
ovipositing on lower-cardenolide species.

43

The lack of correlation between plant cardenolide concentration
and monarch egg deposition patterns or larval success rates provides
evidence against the hypothesis that specialist herbivores pay
metabolic costs of feeding on cardenolide-containing plants. These
are, however, but two possible measures of such costs, utilizing only
one specialist insect species. In the next chapter, I will examine
(inter alia) the relationship between the amount of cardenolide
sequestered by two specialist species and their body sizes and
weights. Since the latter variables are correlated with fecundity in
most Lepidoptera (Hinton, 1981), this will constitute a further
(indirect) test of the metabolic cost hypothesis. Moreover, since it
is unlikely that all herbivores are equally well adapted to plant
allelochemics (perhaps reflecting different degrees of coevolution?) ,
the addition of a second species will help provide a comparative basis
for understanding the evolution of such adaptations.

CHAPTER III

DIFFERENCES AND SIMILARITIES IN THE CARDENOLIDE CONTENTS OF QUEEN AND
MONARCH BUTTERFLIES IN FLORIDA AND THEIR ECOLOGICAL AND EVOLUTIONARY

IMPLICATIONS

Introduction

The butterfly family Danaidae has figured prominently in the
development of the theory of plant/herbivore coevolution (Brower and
Brower, 1964; Ehrlich and Raven, 1964) and has provided one of the
best examples of the chemical defenses of insects against vertebrate
predators (Brower, 1969; Brower et al., 1982; Dixon et al., 1978;
Reichstein et al., 1968). However, much of our understanding of the
ecology and evolution of plant/danaid/predator interactions is based
upon only one species in this tropical family, the monarch butterfly
(Danaus plexippus L.; review in Brower, in press). Because this
species is known to be unique among danaids in certain other respects
(e.g., migration: Urquhart, 1960; lack of plant-derived sex
pheromones : Edgar et al., 1976; Boppre, 1978), one must question
whether monarchs are representative of the Danaidae with respect to
hostplant adaptation and chemical defense, or whether they represent
just one point (or range) within a broader spectrum of danaid
adaptations. If such a spectrum is found to exist, it may ultimately

44

45

become possible to reconstruct some of the evolutionary steps leading
to the close association between danaids and their highly toxic
milkweed hostplants ( Asclepiadaceae) .

Danaid species differ in the amounts of toxic cardenolides which
they sequester from their foodplants. For example, Brower et al.
(1975) showed that the cardenolide concentrations of laboratory-reared
African queen butterflies (Danaus chrysippus L.) were only about 30
per cent that of monarchs simultaneously reared on the same milkweed
hostplants (see also Brower et al., 1978; Rothschild et al., 1975).
The cardenolide concentrations of queen butterflies (D^ gilippus
berenice Cramer) from Florida were, on average, about 75 per cent that
of monarchs reared in the laboratory on the same hostplants. Other
genera of danaids (e.g., Anauris, Euploea) appear to prefer as larval
hostplants those milkweed species lacking cardenolides (Rothschild and
Marsh, 1978).

Does this diversity in the storage of cardenolides reflect
differences among danaid species in their degree of adaptation to
these defensive plant allelochemics? Do queens, for example, store
less cardenolide than monarchs because they are "less coevolved" and
can tolerate these compounds less readily? Do the cardenolide
contents of various danaid species differ only quantitatively, as
described above, or are there also qualitative differences in the
particular set of hostplant cardenolides sequestered?

Here, I address these questions through a comparison of the
cardenolide contents of wild queen butterflies {Bj_ gilippus berenice)

46

from three populations in Florida and of monarchs (Danaus plexlppus)
from one of these populations. Following Brower and Moffitt (1974), I
will assess the "cost" of storing cardenolides by searching for
correlations between body size or weight and cardenolide concentration
of the butterflies. Since body size and weight are typically
correlated with fecundity in Lepidoptera (see Hinton, 1981), a
negative correlation with cardenolide concentration would suggest that
one component of fitness (i.e., fecundity) has been traded for another
(e.g., higher survival due to chemical defense from cardenolides).
While such a trade may well be of positive net value to the insect, it
nevertheless would require an investment or cost which, it is assumed,
should be lessened as adaptation to allelochemics evolves.

The subspecies of queen studied here is of further interest
because it is the apparent model for mimicry by the southern
subspecies of the viceroy butterfly (Limenitis archippus floridensis
Strecker) , which throughout the remainder of North America mimics the
monarch (Brower, 1958a, b; Klots, 1951; Remington, 1968). Thus, a
comparison of the chemical basis for defense in the queen and monarch
should aid in understanding both the selective rationale for the
switch in viceroy mimicry and the broader issues of plant/herbivore
coevolution mentioned above.

47

Methods

Wild queen butterflies were collected from three populations in
Florida, listed from north to south as follows: Lake Istokpoga,
Highlands County, 7 September 1981; Corkscrew Swamp Sanctuary, Collier
County, 6 September 1981; Miami, Dade County, 4 December 1981. In
addition, monarchs were collected from the Miami site where they were
sympatric with queens, and where only the milkweed Asclepias
curassavica grew abundantly. (However, a few A. incarnata plants were
also located.) These collections therefore permit a study of
population variation in cardenolide content of queens, as well as a
comparison of the relative value, with respect to cardenolides , of
monarchs and queens as models for viceroy mimicry. All butterflies
were placed on ice immediately following capture, killed by freezing,
and later dried for 16 h at 60° C. Dry weights were determined using
a Mettler AK-160 electronic balance. The right wing was removed with
forceps and the distance from the apex to the anterior notal process
was measured to the nearest 0.5 mm with hand calipers. Fat was
removed from the butterflies by petroleum ether extraction of each
entire insect for 1 h (methods in Walford and Brower, in prep.). This
procedure removes only negligible amounts of cardenolide from the
insect (Nelson and Brower, unpublished data; see also Nishio, 1980).
Lean weights were calculated by subtracting the weight of extracted
fat from the total dry weight of each insect.

Cardenolide content was determined by standard spectrophotometric
methods (Brower et al., 1972, 1975), with one modification. Soon

48

after beginning the spectroassay of queens from Corkscrew Swamp, it
became evident that many of the butterflies contained very little
cardenolide. In such cases it is frequently difficult to achieve a
stable absorbance reading. Consequently 0.3 ml of a 12.5 X 10 M
ethanolic solution of digitoxin was added to each butterfly extract in
the cuvette (replacing 0.3 ml of 95% ethanol; see Brower et al., 1972)
in order to artificially "boost" absorbance readings to a more stable
midrange. A pilot test demonstrated that the absorbance of the
"boost" digitoxin alone was 0.400 + 0.009 (X + SD; N=9). This mean
value was therefore subtracted from the total absorbance read for a
sample, the remainder being the absorbance due to the butterfly
extract alone.

In order to compare qualitatively the cardenolides present in
sympatric raonarchs and queens from the Miami sample, those butterflies
containing a total of at least 30 ug equivalents of digitoxin (as
determined from the spectroassay) were subjected to a lead-acetate
clean-up procedure in preparation for thin-layer chromatography (TLC).
The procedure used was that described by Brower et al. (1982) with the
exception that the final solution was filtered through a Millipore
filter (Millipore Corp., Bedford, MA) rather than through a funnel of
glass wool and anhydrous sodium sulfate. This clean-up procedure
removes much of the interfering pigments and other noncardenolide
compounds from the butterfly samples. TLC was then performed and
plates developed four times in a chloroform : methanol : formamide
solvent system (90:6:1). Further details of the TLC procedure are
available in Brower et al. (1982).

49

In addition to the wild-caught butterflies, eggs and first instar
larvae of both monarchs and queens were collected from milkweed plants
growing in La Vega province, Dominican Republic, during July, 1981.
These were brought back to the laboratory and reared to maturity on an
exclusive diet of A. humistrata leaves, collected wild in the vicinity
of Gainesville, Florida. The leaves of this species contain
relatively high concentrations of cardenolide (see Chapter I). From
these rearings, 10 adult monarchs and 7 queens were compared for
quantitative (via spectrophotometry) and qualitative (via TLC)
differences in cardenolide storage. For comparison, two arctiid moth
species (Cycnia tenera, an apocynale specialist; and Estigmene acraea,
a highly polyphagous species; Tietz, 1972) were reared on A.
humistrata and chromatographed along with the danaids.

Results

Wild-Caught Butterflies from Florida

The frequency distributions of cardenolide concentration and
total cardenolide per insect for the wild-caught butterflies are shown
in Figs. 2 and 3, respectively. Each distribution departs
significantly from normality (Shapiro-Wilk (W) tests; p < 0.01; Helwig
and Council, 1979). Thus, nonparametric statistical analyses were
used.

50

Males and females did not differ significantly either in
cardenolide concentration or total content in any of the queen or
monarch populations studied (Table 5; Wilcoxon 2-sample tests, p>0.05
for all pairwise comparisons). Consequently, the data from both sexes
of each population were pooled for all subsequent analyses.

Cardenolide concentration varied significantly among the three
queen populations (Kruskal-Wallis H=31.60, df=2, p<0.0001), as did the
total cardenolide content per butterfly (H=30.03, df=2, p<0.0001).
Pairwise comparisons revealed that the queens from Lake Istokpoga had
significantly lower cardenolide concentrations than either those from
Corkscrew Swamp (Wilcoxon 2-sample test; Z=5.17, p<0.0001) or those
from Miami (Z=4.72, p<0.0001), but that the latter two populations did
not differ significantly from one another (p>0.05). Similar results
were found for total cardenolide content per butterfly.

Analysis of the Miami samples shows that monarchs had
significantly greater cardenolide concentrations (Wilcoxon 2-sample
test; Z=4.82, p<0.0001) and total contents (Z=6.12, p<0.0001) than the
sympatric queens. Chromatography reveals a single cardenolide profile
common to both species (Fig. 4). This consists of nine spots,
including a major one at the approximate Rf of digitoxin, and one of
higher Rf . A few individuals (of both species) exhibit all of these
spots plus an additional two faint spots of still-higher Rf (numbered
spots 10 and 11 in Fig. 4). This chroraatogram is virtually identical
to that of monarchs reared in the laboratory on Asclepias curassavica
(Fig. 4 inset), providing strong evidence that this was the hostplant
utilized by the Miami monarchs and queens. To date, no other milkweed

51

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Fig. 4. Thin-layer chromatogram of wild-caught monarchs (M) and
queens ( Q) from Miami area. Developed four times in chloroform:
methanol: formamide (90:6:1 v/v/v). The great concordance of all
profiles below spot #10 suggests that these animals had all fed
upon the same larval hostplant species. Note the agreement of
this profile with that of monarchs reared in the laboratory on
Asclepias curassavica (Inset), a milkweed growing abundantly in
the collection area. Since nearly all milkweed species studied
to date produce qualitatively different TLC profiles in danaids
(Brower et al., 1982, in press, and in preparation; see also Fig.
5), A. curassavica is the likely larval hostplant of the Miami
butt'erflies. (Tnset from Brower, in press). For reference, a
1:1 (v/v) mixture of digitoxin (dig) and digitoxigenin (dgn) was
spotted in three channels. Numbers to the right of each channel
indicate the micrograms of cardenolide spotted, calculated prior
to lead-acetate clean-up.

55

t

MONTVALE N J 07645

b-

:llllilllllllllllllllllll.iiHllil,lil:i,),'!ll!i:: li,li:i.!;iililllll.iuill

A ^B Dlgitoxln/

^^ Digitoxigenin 5

D. plexippus 26

D. plexippus 40

D.g. berenice 51

«»^ ft ' A^ A ^* pl^^ippus 75

^^a) • I * ^* plexippus 40

g^ ^ ^ D.g. berenice 10

^^ D. plexippus 26

^ ^^ Digitoxin/

^^ 5P Digitoxigenin 5

D. plexippus 75

D. plexippus 48

D.g. berenice 40

D. plexippus 75

INSET

D. plexippus

A. curassavica

Digitoxin/
Digitoxigenin

56

species is known to produce this particular chromatographic profile in
danaid butterflies (Brower et al., 1982, in press, and in
preparation) .

The wing lengths, dry and lean weights, and fat contents of the
butterflies are summarized in Table 5. The correlations between these
variables and cardenolide concentration are indicated in Table 6. For
both male and female monarchs there was a highly significant negative
correlation between wing length and cardenolide concentration (Table
6C). Moreover, males also showed negative correlations between
cardenolide concentration and both body weight and fat content. For
queens, none of the correlations was significant in either sex (all 3
populations pooled; Table 6A).

These statistical differences between monarchs and queens could
reflect true species differences. However, the monarchs were all
collected in the Miami region, while the queens for this analysis were
pooled from 3 areas. It is possible that an unknown geographic effect
is operating here, such that butterflies from the Miami region,
regardless of species, would show these negative correlations (e.g.,
due to a common hostplant). To test this, the queens from Miami were
also analyzed separately from the other 2 populations (Table 6B) .
However, as before, no significant correlations emerged (p > 0.10 for
all tests). Thus, the observed species difference is not attributable
to a geographic difference.

It is possible that monarchs show negative correlations between
body size and cardenolide concentration, while queens do not, simply

57

because they store greater concentrations of these chemicals (i.e.,
queens may not store sufficient cardenolide to be adversely affected
by it). If this is the case, then the negative correlations should
vanish for that subset of monarchs having concentrations similar to
those of queens. To test this, I analyzed only those monarchs (sexes
pooled) having cardenolide concentrations equal to, or less than that
of, the most highly-concentrated queen (i.e., 226 ug/O.lg). In this
case, significant negative correlations between cardenolide
concentration and wing length, dry weight and lean weight still
occurred (Table 6D) . Thus, it appears that the difference between
monarchs and queens is not due merely to geographic or cardenolide
concentration differences. It is also not due to differences in
larval hostplant species, since Fig. 4 demonstrates that both species
in Miami had most likely developed on A. curassavica. Rather, the
negative correlations appear to represent inherent species
differences.

Laboratory-Reared Butterflies (Dominican Republic Stock)

When monarchs were reared in the laboratory on Asclepias
humistrata, the females developed significantly higher cardenolide
concentrations than did males (Wilcoxon 2-sample test; Z=2.02, p<0.05;
Table 7). No such sex difference was evident for queens reared under
identical conditions (Z=0.53; p>0.50). As in the wild-caught samples,
only the monarchs showed significant (again, negative) correlations

58

Table 6. Spearman correlation coefficients for cardenolide
concentration vs. body size, weight, and fat content of wild-caught
queen and monarch butterflies from Florida. The data for queens are
first shown for all three populations pooled (A), and then for the
Miami sample separately (B). Monarchs were first analyzed using the
entire data set (C) and then by truncating the set such that only
individuals having cardenolide concentrations equal to, or less than,
that of the most concentrated queen (226 ug/O.lg) were included (D) .
See text for explanation.

SAMPLE

SEX

CARDENOLIDE CONCENTRATION VERSUS

WING DRY LEAN PERCENT
LENGTH WEIGHT WEIGHT FAT

QUEENS

A.

Both

119

-0.004

0.01

0.03

-0.14

(all popu-
lations)

M

61

-0.13

0.14

0.13

-0.02

F

58

0.03

-0.08

-0.08

-0.25

B.

Both

43

-0.17

0.20

0.20

0.14

(Miami only)

M

26

-0.25

0.26

0.25

0.32

F

17

-0.20

0.32

0.23

0.14

MONARCHS

C.
(Entire
Miami
Sample)

oth

48

-0.53***

-0.22

-0.21

-0.25

M

25

-0.55**

-0.44*

-0.39

-0.53**

F

23

-0.50*

-0.04

-0.10

-0.06

D.

( Concen-
trations M
less than
226 ug/O.lg) F

Both 35

17

-0.40* -0.33*
-0.36 -0.46*
-0.38 -0.20

-0.43** -0.18
-0.49* -0.36
-0.40 -0.06

* P < 0.05; ** P < 0.01; *** P < 0.0001.

59

between cardenolide concentration and other size and weight parameters
(Table 8).

Thin-layer chromatography (Fig. 5) demonstrates that the two
danaid species (as well as two arctiid moth species) sequestered
virtually identical sets of cardenolides from _A^ humistrata. This TLC
profile is clearly distinguishable from that of butterflies reared on
A. curassavica (cf. Fig. 4 inset).

Discussion
Body Size and Cardenolide Content

Brower and Moffitt (1974) reported a negative correlation between
the body weight and cardenolide concentration of female raonarchs from
Massachusetts, and suggested that these individuals may have suffered
a "metabolic cost," in terms of growth, of sequestering cardenolides
for defense (see also Brower et al., 1972). Such negative
correlations were not found for males from Massachusetts (which were 9
per cent lower than females in mean cardenolide concentration) , or in
either sex collected in California (which were 62 per cent lower in
mean concentration than the Massachusetts females). However, these
data confound sexual and geographic differences with correlated
cardenolide concentration differences (see Chapter I). Here, I have
shown that negative correlations between cardenolide concentration and
various size and weight parameters, which occur in monarchs, but not

60

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61

Table 8. Spearman correlation coefficients for cardenolide
concentration vs. body size, weight, and fat content of queen and
monarch butterflies (Dominican Republic stock; both sexes pooled)
reared in the laboratory on Asclepias humistrata.

CARDENOLIDE CONCENTRATION VERSUS

WING DRY LEAN PERCENT
SAMPLE N LENGTH WEIGHT WEIGHT FAT

QUEENS 7 0.33 0.18 0.25 -0.57
MONARCHS 10 -0.63** -0.56* 0.05 -0.58*

* 0.05 < P < 0.10; ** P < 0.05.

Fig. 5. Chromatograms of adult monarchs and queens reared In the
laboratory on Asclepias humistrata (developed as In Fig. 4).
Note that the profiles of the two species are virtually
identical, yet differ from those shown in Fig. 3. All
butterflies were collected as eggs or first instars in the
Dominican Republic, except for the monarch labelled "FL," which
was collected wild in the egg stage in Florida and shows an
identical "fingerprint" pattern to those of Dominican Republic
stock. For comparison, extracts of 6 dogbane tiger moths ( Cycnia
t enera ) and 1 saltmarsh moth ( Es t igmene acraea ) , reared
simultaneously on A. humistrata, were spotted on the same silica
plate. Also spotted are an ethanolic leaf extract of A.
humistrata and a 1:1 (v/v) mixture of digitoxin (dig) and
d ig i t oxigeni n (dgn). Numbers to the right of each channel
indicate the micrograms of cardenolide spotted, calculated prior
to lead acetate clean-up.

63

Digitoxln/

Digitoxigenin

10

C. tenera

25

C. tenera

38

C. tenera

34

D. gilippus

75

D. plexippus

75

D. plexippus

75

(FL)

A. humistrata

75

Digitoxln/

Digitoxigenin 10
D. plexippus 75

D. gilippus

E. acraea
C. tenera
C. tenera

C. tenera
Digitoxln/
Digitoxigenin 10

r

S»9Z0 T'N 'l1VAil>

64

in queens, are independent of geographic, sexual, foodplant, or
correlated concentration differences.

While these negative correlations might well represent "metabolic
costs" of cardenolide ingestion, as suggested by Brower and Glazier
(1975), there is no direct evidence of a causal connection between
cardenolide differences and body size differences. Seiber et al.
(1980) found no effect of ingested digitoxin (added to a controlled
diet) upon the development time, food consumption, or body weight of
fourth instar monarch larvae (see also Chapter V). However, if it is
true that the negative correlations do reflect metabolic costs, then
we must inquire why such costs should be paid by monarchs and not by
queens. One possible explanation can be found in the different
population structures of these two species. Monarchs are migratory,
with most individuals in eastern North America flying to a few
restricted areas in Mexico to overwinter (Urquhart and Urquhart,
1977). Mating occurs during the remigration back to North Anerica
each spring and most probably results in many matings between
individuals which developed as larvae on different species of
milkweed, some of which lack cardenolide (see Roeske et al., 1976).
Many of these matings would therefore involve butterflies that had not
been subjected to selection for allelochemic-tolerance. This would
tend to recombine any evolving gene complexes for cost-free adaptation
to allelochemics. In contrast, queens are fundamentally non-migratory
(Young, 1982), making only limited regional movements (Brower, 1961;
Burns, 1983). This greater degree of philopatry should more often

65

result in inbred matings among individuals that fed, as larvae, on the
same hostplant species. In areas where high-cardenolide plants
predominate, this should facilitate the evolution of a more rapid,
fine-tuned adaptation to hostplant allelochemics.

Sexual Differences in Cardenolide Concentration

Previous studies of monarch butterflies have found that females
have higher cardenolide concentrations than males (Brower and Glazier,
1975; Brower and Moffitt, 1974; Brower et al., 1972). This was again
demonstrated here for the laboratory-reared monarchs. Brower and
Glazier (1975) suggested that such a dimorphism might reflect
relatively stronger selection for chemical defense in females which
must spend considerable time exposed to potential enemies while
searching for oviposition plants. However, males also incur certain
risks in searching for females, and it is not clear how these risks to
males are to be compared with risks to females when predicting
differences in defensive strategies. In any event, such risks would
presumably apply to the congeneric queen butterfly as well. The lack
of sexual difference in cardenolide content of queens shown here
therefore suggests that the "differential risk" hypothesis is not
sufficient to explain the dimorphism previously observed in monarchs.

Another possible explanation is that female monarchs store more
cardenolide than males as a means of providing for the defense of
their eggs (Brower et al., 1982). Thomashaw (in Brower, in press)

66

reported that each egg of a female monarch reared on _A^ curassavica
contained, on average, 0.97 ug of cardenolide. Since a female may lay
from 100 (Erickson, 1973) to 400 (Urquhart, 1960) eggs, these could
collectively contain as much as 97 to 388 ug of cardenolide. Adult
raonarchs reared on A. curassavica contain an average of 670 ug of
cardenolide (Roeske et al., 1976). Thus, the amount placed by females
into eggs constitutes a substantial proportion (14% to 58%) of this
total. One might therefore expect cardenolides to be effective
predator- or parasite-deterrents in monarch eggs but this has not yet
been tested. The lack of sexual difference in cardenolides of queens
may suggest that females of this species are not strongly selected to
store these compounds for egg-defense, and leads to the prediction
that they should allocate proportionally less cardenolide to eggs than
do raonarchs. Further work should be directed at this issue.

Interestingly, while laboratory-reared monarch (but, again, not
queen) females did have significantly higher cardenolide
concentrations than males, this was not observed in field-collections
from the Miami area. This may suggest that some of the wild-caught
females in the Miami sample had already laid some of their
cardenolide-rich eggs, thereby reducing their (initially greater)
cardenolide loads to a level similar to that of males. (This
suggestion is consistent with Dixon et al.'s (1978) laboratory finding
that female monarchs that had laid all of their eggs were less emetic
when force-fed to pigeons than were f reshly-eclosed females.) Since
females would likely oviposit at varying rates, this should lead to a

67

greater variation in cardenolide concentration of females, relative to
males. Indeed, females do tend to have higher variances than males
for this trait (see Table 5; F-max = 2.12, 0.05 < p < 0.10; Sokal and
Rohlf, 1981).

Cardenolide Variability and its Implications

The great intra- and interspecific uniformity in qualitative
cardenolide profiles of the Miami butterflies (see Fig. 4), suggests
that a single hostplant species had been utilized by larvae of both
species, but that there is individual variation in storage of certain
compounds, especially those of highest Rf value. That these TLC
profiles are virtually indistinguishable from those of monarchs reared
in the laboratory on A. curassavica (Fig. 4 inset) strongly suggests
that this was the hostplant species utilized by both danaid species in
the Miami sample (cf. Fig. 5 for A. humistrata-reared butterflies).
This represents the first practical application of the cardenolide
"fingerprinting technique" (Brower et al., 1982) to identify the
hostplants utilized, as larvae, by wild-caught danaid butterflies. It
also demonstrates the potential of the technique as an aid in
understanding the natural history and migration patterns of danaids.
Since the monarchs studied were collected in Miami in December and
developed on _A^ curassavica (an introduced milkweed with a North
American distribution restricted to southern states; Woodson, 1954),
we may conclude that they were not merely migrant butterflies from

68

northern states that had "become trapped" in peninsular Florida en
route to Mexico. Rather, this is strong evidence that monarchs breed
in south Florida during the winter months.

A large percentage of each queen sample consisted of butterflies
containing no measurable cardenolide (57% in Lake Istokpoga, 17% in
Corkscrew Swamp, and 21% in Miami). Since there were no significant
sex differences, such intrapopulational variability may instead
reflect localized differences in hostplant species availability,
intraspecific variation in plant cardenolide content (see, e.g..
Nelson et al., 1981), or individual butterfly differences in
cardenolide sequestration. Whatever its origin, such variability
suggests the existence of a cardenollde-based palatability spectrum
for queens, similar to that previously described for monarchs (Brower,
1969; Brower et al., 1968; Brower and Moffitt, 1974).

This result has important implications for understanding the
southern viceroy's apparent switch from mimicking the monarch (as it
does elsewhere in its range), to mimicking the queen in Florida (see
Chapter I). If queens had been found to contain, on average, either
more cardenolide than monarchs, or a different and potentially more
potent (e.g., more emetic; Brower, 1969) set of cardenolides , then
such a mimetic switch might be easily understood. However, the Miami
queens clearly contained lower cardenolide concentrations and total
amounts than did the sympatric monarchs. Since the two species had
virtually identical cardenolide "fingerprints," it cannot be argued
that queens stored a more noxious array of cardenolides than did

69

monarchs and were therefore more emetic even at lower concentrations.
Thus, when fed on the same plants, monarchs and queens store the same
cardenolides but monarchs concentrate these to a greater extent than
do queens. This conclusion is further supported by laboratory
rearings of the two species on Asclepias humistrata (Table 7 and Fig.
5). Moreover, Brower et al. (1975) have shown that, in order for an
A. curassavica-reared monarch (sexes pooled) to be emetic to an 85 g
blue jay on 50% of test trials, it must contain at least 76 ug of
cardenolide. Of the Miami butterflies analyzed here, 85% of monarchs
met this criterion of unpalatability, while only 30% of queens did so.
It therefore seems that, at least with respect to cardenolides, queens
are poorer models for viceroy mimicry than are monarchs.

Why then should the viceroy have abandoned its usual model in
favor of the queen? Since monarchs are migratory, "pulsing" through
Florida in large numbers only in the spring and fall (Urquhart and
Urquhart, 1976; Brower, Malcolm and Cockrell, unpubl. data), while
queens are more sedentary, the latter species would be
spatiotemporally more "available" than monarchs to act as models in
Florida. A theoretical model developed by Pough et al. (1973) showed
that mildly noxious species could serve as suitable models for mimicry
if they occurred in sufficient abundance. This may provide the
explanation for the switch in viceroy mimicry. If resident, Florida
monarch populations have been stable and predictable in their current
locations for sufficient time, then reversals in the trend of mimicry
might be expected, such that viceroys in those areas should tend to be

70

more "monarch-like" than those elsewhere in the state. A detailed
geographic analysis of wing patterns is needed to test this
prediction.

Alternatively, queens might, in fact, be superior models to
raonarchs, not because of their cardenolide content but, rather, due to
sequestered pyrrolizidine alkaloids (PA's) that adults ingest from
certain withering plants (Edgar, 1975; Edgar et al., 1979). While
both sexes typically store these compounds as adults, male queens
employ them further as precursors of their sex pheromone (Meinwald et
al., 1969; Pliske and Eisner, 1969). Male and female raonarchs are
also somewhat attracted to PA sources and may store the alkaloids but
males apparently do not use them as pheromone precursors (Edgar et
al., 1976). While there is as yet no experimental verification of PA-
based defense in danaid butterflies, K.S. Brown (unpublished
manuscript) reports that certain neotropical spiders will release
ithomiid butterflies from their webs unharmed if they contain PA's.
The dependence of male queens (and not monarchs) on PA's for sexual
competence suggests that, on average, queens may contain more of these
compounds than monarchs, and therefore possibly serve as better models
for viceroy mimicry. A comparative study of the PA concentrations of
wild-caught raonarchs and queens would shed further light on this
intriguing problem.

71

Plant/Herbivore Coevolution

With respect to the issues of plant/herbivore coevolution
presented in the Introduction, this study leads to the following
provisional conclusions: (1) There is no evidence that the lower
cardenolide concentrations sequestered by queens relative to nionarchs
reflects a poorer underlying tolerance for these allelochemics. On
the contrary, since only monarchs demonstrate significant negative
correlations between body size and cardenolide concentration, it might
be argued that monarchs are less adapted than queens for handling
cardenolides. However, a causative connection between cardenolide
sequestration and body size has not been established (see also Chapter
V); (2) Despite quantitative differences between monarchs and queens
in cardenolide storage, both species appear to sequester the same
individual cardenolide compounds from their hostplants. Indeed, when
reared on the milkweed _A^ humistrata, even dogbane tiger moths ( Cycnia
tenera; Arctiidae) , also apocynad/asclepiad-specialists, produced the
same characteristic TLC profile (Fig. 5) as did the polyphagous
arctiid moth, Estigmene acraea (although only in trace amounts; Fig.
5). Moreover, Marty (1983) has shown that gut homogenates of both
monarch and E_^ acraea larvae are capable of effecting a similar
enzymatic transformation of one milkweed cardenolide, uscharidin, to
two more polar metabolites (calactin and calotropin) . Such similarity
among taxonomically disparate Lepidoptera, whether oligo- or
polyphagous, suggests that there may exist only a single qualitative

72

route of cardenolide processing in this insect order but that further
evolution may involve a quantitative increase in tissue cardenolide
concentration in accordance with the defensive requirements of each
species. However, the basic biochemical processes shared by all these
species may represent a common preadaptation to feeding on milkweeds
or other cardenolide-containing plants.

In summary, this chapter has demonstrated that although monarch
butterflies are the most thoroughly-studied danaids, they may not be
representative of their family in respect to adaptation to hostplant
allelochemics. A better understanding of danaid/milkweed
relationships will therefore depend upon obtaining a broader
comparative data base to document differential species responses to
ingested or sequestered cardenolide. Here it was shown that although
raonarchs and queens sequester the same qualitative set of cardenolides
from milkweed plants, monarchs concentrate these in their tissues to a
greater extent than do queens. Furthermore, adult monarchs exhibit
significant negative correlations between the concentration of
sequestered cardenolide and both body size and weight, whereas queens
show no such correlations. Although this may suggest a metabolic cost
paid by monarchs and not by queens (and perhaps attributable to
different life-history characteristics; see above), there is, as yet,
no direct evidence of a causal relationship between cardenolide
differences and body size or weight differences. This question will
be explored more directly in Chapter V, following a consideration in
Chapter IV of cardenolide sequestration in a third milkweed-
specializing insect, the dogbane tiger moth (Cycnia tenera) . This

73

species was previously reported not to sequester hostplant
cardenolides (Rothschild et al., 1970). This raises the interesting
question of whether, even among specialists, some species do not
sequester cardenolides because they are more sensitive to these
compounds than are sequestering species. If so, then species such as
C. tenera might represent an intermediate stage in the evolution of
allelochemic-adaptation.

CHAPTER IV

CARDENOLIDE SEQUESTRATION BY THE DOGBANE TIGER MOTH
(CYCNIA TENERA; ARCTIIDAE)*

Introduction

Immature stages of monarch butterflies (Danaus plexippus L.;
Parsons, 1965; Reichstein et al., 1968; Brower, 1969) and milkweed
bugs (Hemiptera: Lygaeidae; e.g. Scudder and Duffey, 1972; Isman et
al., 1977a, b) are well-known for their ability to sequester plant
defense chemicals known as cardenolides (cardiac glycosides) from
their milkweed hostplants ( Asclepiadaceae) . Moreover, laboratory
studies have shown that these stored chemicals protect the insects
from some vertebrate predators (Brower and Brower, 1964; Brower, 1969;
Rothschild and Kellett, 1972). Several other insect species feed at
least occasionally on milkweed plants (Wilbur, 1976; Price and
Willson, 1979), and some of these have been assayed for the presence
or absence of cardenolides (e.g., Duffey and Scudder, 1972; Rothschild
and Reichstein, 1976). However, for relatively few species has it

*Published 1983 in Journal of Chemical Ecology 9;521-532. Reprinted
with permission.

74

75

been experimentally demonstrated that the cardenolides found in wild
and/or lab-reared insects are indeed derived from the hostplants
(allochthonous) rather than synthesized by the animals de_ novo
(autochthonous), as occurs, for example, in some chrysomelid beetles
(Pasteels and Daloze, 1977; Daloze and Pasteels, 1979). One may
distinguish these two possibilities by simultaneous rearings of
insects on cardenolide-rich and cardenolide-poor diets (e.g., Brower
et al., 1967; Rothschild et al., 1978). Differences in the
cardenolide contents of insects reared on such foods may then be
attributed to these dietary differences. This approach was taken in
the present study of the dogbane tiger moth, Cycnia tenera Huebner
(Arctiidae), a species previously reported not to sequester hostplant
cardenolides (Rothschild et al., 1970). Since a related species (C.
inopinatus) , which is also a specialist feeder on asclepiadaceous and
apocynaceous plants (Tietz, 1972; Nishio, 1980), has since been found
to store these chemicals (Nishio, 1980), a reevaluation of C. tenera
seemed desirable. Among other arctiids, two species (Arctia caja and
Euchaetias antica) are known to sequester hostplant cardenolides (see
Rothschild and Reichstein, 1976). While three others (Euchaetias
egle, Digama aganais, D. sinuosa) were reportedly devoid of
cardenolides, it remains unclear whether this was due to storage
inability or perhaps to feeding on milkweeds low, or lacking in these
compounds. Sufficient intraspecif ic and interspecific diversity of
milkweed cardenolide contents exists (e.g., Roeske et al., 1976;
Nelson et al., 1981; Seiber et al . , in press), so that it is possible

76

for an herbivore to be a milkweed specialist without being a
cardenolide specialist (see also Rothschild et al., 1970). However,
if C. tenera are found to sequester cardenolides then the hypothesis
that they pay metabolic costs of doing so may be evaluated by
searching for correlations between the concentration of sequestered
cardenolide and body weight (see Chapter III for rationale).

Cycnia tenera females lay clutches of 50 to 100 eggs (personal
observations), and larvae feed gregariously on their hostplants (we
have frequently seen groups of 5-7 larvae on a single _A^ humistrata
leaf). If the female has mated only once prior to oviposition, these
larvae will be full-siblings whereas, if she has mated more than once,
they will be at least half-siblings. This high degree of relatedness
permits the evolution of unpalatability and aposematism via kin
selection (Hamilton, 1964; Harvey et al., 1982; Brower, in press).
Thus, if cardenolides are, in fact, sequestered by the larvae and
retained into adulthood (both stages being aposematic; see Fig. 6),
then they may contribute importantly to the chemical defense and
evolution of this species.

Methods

In north-central Florida, Cycnia tenera larvae are commonly found
on Asclepias hxmiistrata, a milkweed species having a relatively high
cardenolide concentration in Its leaves (Nishio, 1980; cf. Roeske et

77

Fig. 6. An adult male Cycnia tenera and a late-instar larva. The
taxonomy of this genus has been disputed. Forbes (1960) considered
C. tenera as distinct from C. inopinatus, while Kimball (1965) merged
both species under the latter name. More recent treatments (e.g.,
Hedges, in press) maintain separate status for the two species.
Voucher specimens of larvae and adults used in this study have been
deposited in the Florida State Collection of Arthropods. Doyle Conner
Building, Gainesville. FL. (Photos by L.P. Rrover)

(Source: Cohen and Brower, 1983)

78

al., 1976; see also Chapters I and II). A clutch of 54 C. tenera eggs
was collected on 12 May 1981 from an _A^ humistrata plant growing wild
approximately 3 Km W of Gainesville (Alachua County), FL. The eggs
were brought to the laboratory and, upon hatching, each larva was
placed in an individual 250 cc closed (vented) plastic container and
reared to eclosion at 23+l°C. Of these, 7 were reared through to
adulthood on a total diet of A. humistrata leaves, while 12 fed only
on A. tuberosa, a local species known to contain only very slight
amounts of cardenolide (Chapter I; also Roeske et al., 1976). All
adult moths were killed by freezing between 12 and 24 hours after
eclosion and remained frozen until chemical analysis in March, 1982.

Prior to analysis, the moths were dried at 60 C for 16h in a
forced-draft oven. Dry weights were determined to the nearest 0.1 mg
using a Mettler AK 160 electronic balance. Fat was then removed from
each insect by petroleum ether extraction of the dried material for 30
min in a 35°C shaker bath (methods in Walford and Brower, in
preparation). Fat extraction removes only negligible amounts of
milkweed cardenolide from the insect material (C. Nelson and L.P.
Brower, unpublished data; see also Nishlo, 1980).

Cardenolides were extracted from the fat-free material and
concentrations determined (as microgram equivalents of digitoxin)
using spectrophotometric procedures described in Brower et al. (1972,
1975). A lead-acetate clean-up procedure (Brower et al., 1982) was
then used to remove interfering pigments and other noncardenolide
compounds from the extract remaining after spectroassay. Thin-layer

79

chromatography (TLC), employing an ethyl acetate , "methanol solvent
system (97:3 by volume; see Brower et al., 1982), was used to
visualize the cardenolides present in each hostplant species and in
two randomly selected moths that had developed on each species.

Results

The mean dry and lean weights, cardenolide concentrations and
total cardenolide contents of C^ tenera adults reared on A^ humistrata
and A^ tuberosa are shown in Table 9 . Two-way analyses of variance
demonstrate that females reach heavier dry weights than do males,
regardless of foodplant eaten (F=32.5, df=l,15, p<0.001), but that
both sexes are heavier when reared on _A^ humistrata than on A.
tuberosa (F=40.9, df=l,15, p<0.001). The interaction of sex and
foodplant was not significant (p>0.50). The results for lean weights
are similar, with females heavier than males (F=16.2, df=l,15,
p<0.005) and both sexes tending to be heavier on A. humistrata than on
A. tuberosa (F=5.6, df=l,15, 0.1>p>.05). Females contain slightly
more fat than do males (F=4.6, df=l,15, 0.1>p>.05) and both sexes
store more fat when fed A^ humistrata than when fed _A^ tuberosa
(F=21.2, df=l,15, p<0.001). Overall, the fat content of the adult
moths constitutes from 45 to 60 per cent of the total dry weight.

80

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81

Clearly, both sexes sequestered higher concentrations and total
amounts of cardenolide from _A^ humistrata than from _A^ tuberosa
(F=76.4 for concentration; F=50.0 for total amount; df=l,15 and
p<0.0001 for both tests). However, the sexes did not differ
significantly either in concentration (F=1.2, df=l,15, p>0.2) or in
total cardenolide content (F=2.4, df=l,15, p>0.2). No significant
interaction effects were identified (p>0.2). IVhen both sexes are
considered together, moths reared on _A^ humistrata had, on average, 10
times higher concentrations, and contained 15 times more total
cardenolide, than did moths reared on _A^ tuberosa.

Spearman rank order correlations demonstrate no significant
relationship between the concentration of sequestered cardenolide and
either dry weight (r=-0.28; P>0.50) or lean weight (r=-0.43; P>0.30).
This analysis was limited to those moths reared on A. humistrata (n=7;
sexes pooled) in order not to confound cardenolide-dif f erences with
other plant species differences (e.g., in nutritional quality; see
Discussion) .

Thin-layer chromatography (Fig. 7) clearly confirms that
sequestration of hostplant cardenolides occurred, since individuals
reared on A. humistrata contain virtually every cardenolide visualized
in their hostplants. However, the chroraatogram of _A^ tuberosa, a very
low-cardenolide species, consists only of a faint band ranging from
approximately Rf = 0.1-0.5. Much of this band is pink (i.e.,
noncardenolide interference) rather than blue (cardenolide) and it is
possible that these interfering compounds may have contributed to the

Fig. 7. TLC profiles of four adult Cycnia tenera reared on
either Asclepias humistrata (2? ? ) or _A^ tuberosa (1 cT , 1 ? ) .
For reference, a mixture of commercially procured digitoxin (dig;
Rf = 0.31) and digitoxigenin (dgn; Rf = 0.60) was spotted in
three channels. The amounts of cardenolide (ug equivalent to
digitoxin) spotted in each channel are shovra at the bottom of the
plate. (These are calculated prior to lead-acetate clean-up, a
procedure which sometimes reduces the amount actually spotted.
Thus, the female in channel 3 has a lighter trace than that in
channel 2, presumably having lost disproportionately more
cardenolide during clean-up.) For A^ humistrata there is a very
close correspondence between the cardenolides present in the leaf
tissue and those in the moths reared thereupon. However, A.
tuberosa has a very low cardenolide concentration and only faint
spots are produced from the leaf aad moth extracts. In the
original color prints, all cardenolides appear as blue spots.
However, much of the _A^ tuberosa leaf and moth chromatographs
appears pink in the color original, except for faint blue
cardenolide regions indicated by broken lines. No such
interference occurs in the chromatographs of _A^ humistrata, the
moths reared thereupon, and the commercial standards. (Source:
Cohen and Brower, 1983)

83

A. humtotf.t. A, tubSfOM
Moth Plant Plant Moth

% %

^ ?

dgn

OKI

II II
H II

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jug of cardenolide spotted

84

absorbances recorded for the moths reared on this species, thereby
overestimating cardenolide concentrations. If this is so, then the
true difference in cardenolide contents between moths reared on the
two hostplants may be even greater than reported above.

Discussion

These data demonstrate the sequestration of hostplant
cardenolides by Cycnia tenera larvae and the subsequent retention of
these chemicals into adulthood. Individuals reared on Asclepias
htmistrata, a cardenolide-rich milkweed species, attained much higher
concentrations and total amounts of cardenolide than did those reared
on a cardenolide-poor diet of A. tuberosa. Moreover, TLC analysis
demonstrates a close correspondence between the individual
cardenolides of _Aj_ humistrata and those of the moths reared thereupon.
If all of the cardenolide present in _C^ tenera were of autochthonous
origin, moths reared on both milkweed species would be expected to
contain similar amounts and kinds of cardenolide. Clearly, they do
not. However, the possibility that the small amount of cardenolide
present in A. tuberosa-reared moths may, at least in part, be of
authochthonous origin cannot be excluded.

Both sexes reached higher fat and lean weights when reared on the
higher cardenolide species, A. humistrata. Similar results for Danaus
plexippus (Erickson, 1973), D. chrysippus (Smith, 1973), and

85

Oncopeltus fasciatus (Isman, 1977; Chaplin and Chaplin, 1981) have
been interpreted as evidence against a metabolic cost of handling
hostplant cardenolides (see also Blum, 1981). However, this inference
is problematical because milkweed species differ in many ways other
than in cardenolide content (e.g., nitrogen and water content,
Erickson, 1973; leaf shape, growth form, texture and pubescence,
Woodson, 1954). Such differences contribute to the overall
suitability and quality of these plants as insect food (Scriber and
Slansky, 1981). Asclepias tuberosa is a relatively poor food source
due to low nitrogen and water content (Erickson, 1973) and is
infrequently used by herbivores in north-central Florida (Cohen and
Brower, unpublished observations), or elsewhere (e.g., Wilbur, 1976;
Price and Willson, 1979). It is to be expected that such poor
foodplant species will be under weaker selection for antiherbivore
adaptations than will species of higher nutritive value, and might
therefore have relatively lower cardenolide contents. Hence, it is
possible that herbivores develop more successfully on relatively-high
cardenolide species, such as A. humistrata, not because there is no
cost of handling these chemicals, but because the higher food quality
is great enough to offset any such costs that might exist.

This complication explains why correlations between insect
cardenolide concentration and body weight were sought only for those
individuals reared on A. humistrata. Clearly, had the A. tuberosa-
reared moths been included in the analysis, a positive correlation
would result. However, this would most likely be due to the

86

nutritional differences between the two plant species, rather than
their allelochemic differences. That the A. humistrata-reared moths
exhibited no significant correlations between cardenolide
concentration and body weight provides further (indirect) evidence
that milkweed-specializing lepidopterans pay little, if any, cost of
feeding on cardenolides.

It is interesting that the extracted fat from C^ tenera adults
may constitute from 45 to 60% of the total dry weight (Table 9). This
is sharply higher than the 5 to 20% common for freshly eclosed danaid
butterflies (Beall, 1948) and is similar to the levels found in
migrating monarch butterflies just prior to overwintering in Mexico
(Walford and Brower, in prep.). Moths of a related arctiid genus,
Euchaetias, do not feed as adults (Forbes, 1960; Schroeder, 1977) and
lifetime energy stores must be accumulated by the larvae prior to
pupation. However, unlike Euchaetias, adult Cycnia have well-
developed probosces (Forbes, 1960), and presumably feed. Thus their
apparently high fat storage remains enigmatic. While this result is
not directly relevant to the issue of cardenolide-def ense, it does
raise an intriguing question and it should prove interesting to
compare larval fat storage between those arctiid species which have a
feeding adult stage and those which do not.

The results on cardenolide sequestration are in contrast to those
of Rothschild et al. (1970), in which no cardenolides were found in C.
tenera reared on Asclepias syriaca. That plant species, however, is
extremely variable in cardenolide content (see Roeske et al., 1976),

87

and certain strains are reportedly devoid of cardenolldes (Rothschild
et al., 1975). It is thus possible that the particular plants fed to
the larvae in the experiments of these authors were of insufficient
cardenolide content for larval sequestration to occur. Alternatively,
intraspecific geographic differences in sequestration abilities in the
moths may be indicated. Such differences have been hypothesized for
the various geographic races of the African queen butterfly, Danaus
chrysippus (Rothschild, et al., 1975) but have not yet been
definitively established for any species. A third possibility is that
C. tenera is not adapted to sequester the particular array of
cardenolldes found in A. syriaca. Indeed, of 6 milkweed species
studied by Price and Willson (1979) in central Illinois, A. syriaca
was one of only two never utilized by C. tenera as a larval foodplant.
However, there is no evidence that this apparent rejection was
determined in any way by the cardenolldes of _A^ syriaca.

Cycnia tenera adults are conspicuously colored, with off-white
wings and black-spotted, yellow abdomens (Fig. 6). The bitter-tasting
cardenolldes they contain probably impart to them a noxious quality,
as is true in the monarch butterfly (Brower, 1969; Brower and Moffitt,
1974). Moreover, they produce both audible and ultrasonic sounds
(Fullard, 1977) which may serve to warn bats and other potential
predators of the unpalatability of the moths, thus serving an
aposematic function (e.g.. Dunning and Roeder, 1965).

Larvae, too, are aposematic, with bright orange bodies and
contrastingly-dark tufts of setae along the dorsum (Fig. 6).

88

Interestingly, the setae of a closely related species, C. Inopinatus.
are virtually devoid of cardenolides , while the underlying larval
cuticle is rich in these compounds (Nishio, 1980). This suggests the
potential operation of three separate lines of defense in larval
Cycnia. The setae may have a sensory function, permitting the larvae
to recognize the approach of a predator or parasite. Larvae respond
to tactile stimulation by dropping to the ground and curling the body
(unpublished observations). This behavior has the effect of exposing
the dorsal setae maximally. Should the larva nevertheless be found by
a vertebrate predator, a second, mechanical line of defense may come
into play: naive predators may attempt to eat a larva, but release it
unharmed when the mouth is irritated by the hairs. The setae may then
become an aposeraatic signal, preventing further attacks by experienced
predators. The evolution of such sensory and mechanical defense is
not problematic since larvae having such setae would presumably be
more likely than those lacking them to survive an attack. If,
however, these defenses should fail, the cardenolides present in the
larval tissues would provide an unpalatable, and possibly emetic,
experience (see, e.g., Brower, 1969), leading to later avoidance of
further larvae encountered. Indeed, it is possible that the setae,
once ingested, may irritate the gastro-intestinal lining (Bisset et
al., 1960; Frazer, 1965) and thereby facilitate absorption of
cardenolides, i.e., an interaction of mechanical and chemical
defenses! The larva would die in the process, however, and thus its
genes for cardenolide sequestration would not be propagated in the

89

population. Kin-selection (see Hamilton, 1964) would be required for
the evolution of such chemical defense and seems plausible in this
species, since eggs are laid in clutches and the larvae feed
gregariously on their hostplants. Thus, kin-groups of at least half-
siblings, and possibly full-siblings, feed together on the same plant.
If a predator were to sample and kill one or a few of these aposematic
larvae before learning to avoid them, the remaining siblings would be
spared and shared genes both for unpalatability and aposematism would
continue to spread within the population.

My work on cardenolides in _C^ tenera suggests that these compounds
may provide at least a partial basis for an underlying unpalatability
of both larvae and adults. However, this does not preclude the
possibility of other chemical defenses. Parsons and Rothschild (in
Rothschild et al., 1970) have noted the presence of histamine-like
and/or acetylcholine-like substances in _C^ tenera, although the manner
in which these compounds function in nature remains largely
unexplored. Moreover, several species of arctiid moths (including
Cycnia mendica; Rothschild, 1973) are known to sequester pyrrolizidine
alkaloids (PA's) from larval hostplants (although these have not yet
been identified from Asclepiadaceae) or, for those species with
feeding adult stages, from decomposing leaves (Rothschild et al.,
1979). Finally, it is possible that other noxious chemicals in these
moths may be of autochthonous origin, rather than derived from plant
sources (Rothschild et al., 1970, 1979). The relative contributions
and possible interactions of cardenolides, biogenic amines, PA's,

90

and/or other noxious substances in the defensive strategies of insects
can only be determined through controlled-rearing schemes followed by
ecologically relevant predation studies.

In this chapter, I have confirmed that, contrary to previous
reports, dogbane tiger moths do sequester milkweed cardenolides. Like
the queen butterfly (Danaus gllippus) discussed in Chapter III, but
unlike monarchs, these moths, when reared on Asclepias humistrata,
exhibited no correlation between sequestered cardenolide concentration
and body weight. This argues against the hypothesis that a metabolic
cost is paid by specialists. Moreover, those individuals reared on a
high-cardenolide milkweed species (_A^ humistrata) achieved
significantly higher body weights than did those reared on a very low-
cardenolide species (A. tuberosa) . While several other workers have
interpreted such results in other insect species as evidence against
the metabolic cost hypothesis, such interpretations confound
cardenolide differences among milkweed species with other
nutritionally relevant differences. These confounding factors were
eliminated in the experiments described in the next chapter, in which
purified cardenolides were added to otherwise uniform diets.

CHAPTER V

THE EFFECTS OF INGESTED CARDENOLIDE UPON FOOD CONSUMPTION
AND GROWTH OF SPECIALIST AND GENERALIST LEPIDOPTERAN LARVAE

Introduction

The theory of plant/herbivore coevolution (Ehrllch and Raven,
1964) has played a major role in organizing a very diverse literature
on the defensive strategies of plants against their enemies, as well
as enemy counter-offenses (see, e.g., Feeny, 1976; Rosenthal and
Janzen, 1979; Futuyma and Slatkin, 1983). In essence, plants are
viewed as defended by chemical and other means (e.g., mechanical and
phenological) against a broad array of potential attackers. In the
course of evolution, a few mutant individual herbivores may find
themselves able to circumvent the plant's defensive barrier and enter
a new adaptive feeding zone, relatively free from interspecific
competition. This, in turn, selects for subsequent counter-defenses
by the plant population, counter-offenses by the herbivore population,
and so on.

It has been widely held (e.g., Brower and Huberth, 1977; Dixon et
al., 1978; Harborne, 1983) that the classic example of coevolution

91

92

between Insect herbivores and their larval foodplants is that of the
monarch butterfly (Danaus plexippus L; Danaidae) , which develops on
milkweed plants ( Asclepiadaceae) . Many milkweed species contain
cardenolides (cardiac glycosides), chemicals which are potent
vertebrate heart poisons. However, there is no evidence (nor, indeed,
has it ever been tested) that ingested cardenolides provide any kind
of barrier to herbivory by non-adapted insects. Clearly, such a
barrier must be shown to exist before it becomes plausible to think of
adapted species, such as monarchs, as having "breached" a chemical
defense during the course of coevolution.

Here I test the effects of ingested cardenolide upon the
consumption rates, growth rates, and food utilization efficiencies of
three lepidopteran herbivores: a) a milkweed "specialist," the monarch
butterfly, Danaus plexippus L. (Danaidae); b) a highly polyphagous
species or "generalist," the fall armyworra, Spodoptera frugiperda
(J.E. Smith) (Noctuidae), and c) a species specializing on a plant
family lacking cardenolides (i.e., a "negative-specialist"), the
velvetbean caterpillar, Anticarsia gemmatalis Hubner (Noctuidae). A
similar three-way comparison by Blau et al. (1978), in which sinigrin,
the defensive compound of cruciferous plants, was added to controlled
diets, demonstrated no significant negative effects upon a crucifer-
specialist, moderate effects upon a generalist, and severe effects
upon a negative-specialist. Similar results were predicted in the
present study because generalists, which consume plants from many
diverse families, should be selected for an optimal, broad-scale

93

detoxification ability, but not to handle any one class of compounds
as effectively as would a specialist on those compounds (Feeny, 1975;
Rhoades and Gates, 1976). Moreover, a negative-specialist might be
most severely affected since, although it has a fine-tuned
detoxification ability, this is directed at a wholly different class
of compounds.

Methods

Three separate experiments were conducted, each with one of three
lepidopteran species (details provided below) , but all utilizing the
same basic experimental approach. This involved the addition of
varying amounts of cardenolide (digitoxin, Sigma Chemical Co.) to
otherwise standardized diets. Gravimetric analyses (Waldbauer, 1968)
were performed for the entire duration of the penultimate instar in
each case and yielded the following performance indices (all weights
are dry) :

- Relative Growth Rate (RGR) : This expresses the amount of
weight gained per unit mean body weight, normalized to a one-day time
period. An RGR of 1.0 thus indicates that an animal adds 100% of its
(dry) body weight in new growth each day. A decrease in RGR with
increasing cardenolide in the diet could result either from a decrease
in consumption rate (RCR) or from a decrease in the efficiency with
which ingested food is converted into new biomass (ECI) , or both.

94

- Relative Consumption Rate (RCR) : This expresses the weight of
food eaten as a proportion of the mean body weight, normalized to a
one-day time period. Thus, an RCR of 1.0 indicates that an individual
consumes its own (dry) weight in food each day. A decrease in RCR
with increasing cardenolide in the diet would indicate a behavioral
feeding deterrent effect of these chemicals.

- Efficiency of Conversion of Ingested Matter (ECI) : This
expresses the weight gained as a percentage of the weight of food
consumed. Thus, an ECI of 50% indicates that half of the food eaten
is converted into new body growth. A decrease in ECI with increasing
cardenolide would suggest a "cost" of handling these allelochemics
because additional time and energy expenditures, in terras of feeding
behavior, would be required to support the same amount of growth.

Larvae were housed individually in 12 ml stoppered glass vials
(Seiber et al., 1980) and maintained in the laboratory between 23 and
25 C and ambient light conditions. Gravimetric testing began upon
molting into the penultimate instar and ended upon molting into the
final instar. All determinations of cardenolide concentrations were
made by standard spectrophotoraetric procedures (Brower et al., 1972,
1975). Details specific to each experiment are as follows:

95

Experiment J_ - The Specialist ; Monarch Butterfly

Larvae used were the progeny of a single female, collected as a
larva in Gainesville, Alachua Co., FL, reared to maturity and mated in
the laboratory to a similarly-reared male. All larvae were fed from
hatching on an exclusive diet of Asclepias tuberosa leaves (root-stock
obtained from Gurney Seed & Nursery Co., Yankton, S. Dakota). Leaves
of this plant species contain very low to negligible amounts of
cardenolide (see Chapter 1; also Roeske et al., 1976). Upon molting
into the penultimate (fourth) instar, 23 larvae were randomly assigned
to treatment groups and given a weighed surfeit of leaves of one of
four kinds :

a) Undipped Control ; These were leaves identical to those fed
to the earlier instars. Mean cardenolide concentration was 0.03% by
dry weight (S.D.=0.02%; N=3) .

b) Water-Dipped Control : Leaves briefly dipped into distilled
water and allowed to air-dry before feeding. Mean cardenolide
concentration 0.03% (S.D.=0.02%; 1^^3) .

c) High Cardenolide : Leaves briefly dipped into an
(distilled) aqueous suspension of digitoxin. Mean cardenolide
concentration 1.6% (S.D.=0.28%; N=3).

96

d) Very High Cardenollde ; Leaves briefly dipped, as above, to
produce a mean concentration of 2.6% (S.D.=0.55%; N=3) .

Naturally occurring cardenollde concentrations in milkweeds rarely
exceed 1.0% (Roeske et al., 1976). As both concentrations used in
this experiment were beyond this limit, they should be sufficient to
reveal any negative effects which might accrue to the larvae.

Experiment _2 - The Generalist ; Fall Armyworm

Populations of this species are known to feed on plants from many
diverse families (e.g., Tietz, 1972). Eggs were collected from stock
of the U.S.D.A. Insects Attractants, Behavior, and Basic Biology
Research Laboratory (Gainesville, FL) and larvae fed ad lib from
hatching on one of four diets :

a) Control : This consisted of cardenolide-f ree artificial
diet, as described by Leppla et al. (in press).

b) Low Cardenollde ; Artificial diet into which powdered
digitoxin was mixed to produce a mean allelochemic concentration of
0.17% by dry weight (S.D.=0.02%; N=3) .

c) Medium Cardenollde ; Artificial diet brought to a mean of
0.56% cardenollde (S.D.=0.09%; N=3) .

97

d) High Cardenollde ; Artificial diet brought to a mean of
1.20% cardenolide (S.D.=0.33%; ^3).

In total, 49 larvae were tested gravimetrically upon molting into
the penultimate (fifth) instar. Additional larvae were maintained on
the various diets for up to 21 days in order to study survival (N=167)
and development time (N=145).

Experiment 3 - The Negative Specialist ; Velvetbean Caterpillar

This species feeds only on plants in the family Leguminosae
(Tietz, 1972), none of which is known to contain cardenolides. Eggs
were collected from U.S.D.A. stock (as above for fall armyworras) and
larvae fed ad lib from hatching on one of five diets :

a) Control : Cardenolide-free artificial diet (Leppla et al.,
in press).

b) Aerosol Control : Artificial diet to which was added a
slight amount of the wetting agent. Aerosol OT (Fisher Scientific Co.)
to improve the solubility and dispersion of digitoxin in the diet
(see, e.g., Nielsen, 1978). The total concentration of the wetting
agent in the diet was 0.008% by volume.

98

c) Low Cardenollde ; Artificial diet, Aerosol (as above), and
sufficient digitoxin to produce a mean cardenolide concentration of
0.21% by dry weight (S.D.=0.03%; N=4) .

d) Medium Cardenolide ; Artificial diet and Aerosol, brought
to a mean of 0.53% cardenolide (S.D.=0.06%; N=4) .

e) High Cardenolide ; Artificial diet and Aerosol, brought to
a mean of 0.97% cardenolide (S.D.=0.10%; N=4) .

In total, 66 larvae were tested gravimetrically upon molting i'nto
the penultimate (fifth) instar. Additional larvae were maintained on
the various diets for 17 days in order to study survival (N=150) and
development time (N=113).

Due to heteroscedasticity and non-normality of data, RGR, RCR,
and ECI values for all three species were tested for significance
using Kruskal-Wallis nonparametric analyses of variance (approximate
chi-squared distribution), as provided by Helwig and Council (1979).

Results

Monarch Butterflies

Values of relative growth rate (RGR) , relative consumption rate
(RCR) , and efficiency of conversion of ingested matter (ECI) are shown

99

Table 10. Relative growth rate (RGR) , relative consumption rate
(RCR), and efficiency of conversion of ingested matter (ECI) for
fourth instar monarch butterfly larvae reared on four Asclepias
tuberosa-based diets incorporating different cardenolide
concentrations. Data are X ± SD with sample sizes in parentheses,

% CAR-
TREATMENT DENOLIDE

Undipped

Water

High Car-
denolide

Very High
Cardenolide

0.03

0.03

1.6

2.6

RGR RCR ECI

(mg/mg/d) (mg/mg/d) (%)

0.18 + 0.15 1.24 + 0.53 15.11 + 4.81

(7) (7) (6)

0.22 + 0.16 1.36 + 0.68 18.37 + 1.06

(5) (5) (3)

0.24 ± 0.15 1.70 + 0.64 15.52 ± 2.15

(5) (5) (4)
0.26 + 0.09 1.72 ± 0.45 15.20 + 4.44

(6) (6) (6)

Kruskal-Wallis x
df

P

1.85

3

0.60

3.54

3

0.32

1.53
3

0.68

100

in Table 10. Kruskal-Wallis analyses of variance demonstrate no
significant heterogeneity among treatment groups for any of these
variables, even at cardenolide concentrations well above those
encountered in nature. There is, however, a slight (nonsignificant)
trend toward higher consumption and growth rates as the cardenolide
concentration increases. No such trend is evident for the conversion
efficiency.

Fall Armyworms

RGR, RCR, and ECI values are shown in Table 11. RGR was
unaffected at low cardenolide concentrations but was significantly
depressed at medium and high dosages. This decline can be traced to a
significant trend toward lower RCR as cardenolide level increased :
when pooled, the control and low-cardenolide groups had significantly
greater RCR values than did the pooled medium- and high-cardenolide
groups (Wilcoxon 2-sample test, p<0.002) . Moreover, as allelocheraic
concentration increased, there was a significant general decline in
ECI, which also contributed to the lower RGR values.

Additional data on the length of time required to develop from
hatching to the start of the sixth instar are shown in Table 12A.
This period was significantly lengthened as cardenolide concentration
increased. Measurements of body weight at the start of the fifth
instar (Table 12B) show that this prolongation of the development time
enabled the larvae to maintain an approximate "target weight," despite

101

Table 11. Relative growth rate (RGR) , relative consumption rate
(RCR), and efficiency of conversion of ingested matter (ECI) for fifth
instar fall armyworm larvae reared on artificial diets incorporating
varying amounts of cardenolide. Data are X ± SD with sample sizes in
parentheses. Asterisks indicate values that are significantly
different from control at the (*) 0.05 or (**) 0.01 level by Kruskal-
Wallis chi-square approximation (Helwig and Council, 1979).

% CAR-

RGR

RCR

ECI

TREATMENT

DENOLIDE

(mg/mg/d)

(mg/mg/d)

(%)

Control

0

0.52 + 0.08

1.65 + 0.35

32

.29 ± 4.77

(11)

(11)

(11)

Low Car-

0.17

0.52 ± 0.03

1.75 + 0.15

30

.06 + 2.58*

denolide

(11)

(11)

(11)

Medium Car-

0.56

0.43 + 0.11*

1.43 + 0.41

30

.62 + 3.67*

denolide

(16)

(16)

(16)

High Car-

1.20

0.44 + 0.07**

1.53 + 0.22

28

.84 + 3.32*

denolide

(10)

(10)

(10)

Kruskal-Walli

2

3 X

19.39

14.06

9.62

df

3

3

3

P

0.0002

0.003

0.02

102

Table 12. Additional data for fall armyworras reared on artificial
diets incorporating varying amounts of cardenolide. (A) Duration of
the larval period from hatching until the start of the sixth instar.
(B) Estimated dry weights of newly-molted, fifth instars. The
estimation is arrived at by multiplying the fresh weight by the dry
weight :f resh weight ratio determined on a similarly reared sample of
larvae. (C) 2X4 contingency table showing the numbers of larvae dead
or alive after 21 days feeding.

TREATMENT

CARDENOLIDE

CONTROL LOW MEDIUM HIGH
(0.17%) (0.56%) (1.20%)

(A) Larval Period (Hatching-VI)
Median Days
Range
N

10 11 12 12
9-13 9-16 10-20 11-20

32

38

43

Kruskal-Wallis x^=42.92, df=3, P=0.0001

32

(B) Estimated Dry Weight (V)
X

S.D.
N
One-way ANOVA F^ -,g=1.70, P=0.17

4.43 4.18 4.19 3.69

0.74 1.00 1.26 1.21

23 21 20 18

(C) Survival After 21 Days
No. Alive
No. Dead
x^ = 4.5, df = 3, P > 0.10

27
8

37
5

39
7

32
12

103

a tendency to be lighter when fed higher cardenolide diets. There was
no significant dose-dependent mortality (Table 12C).

Velvetbean Caterpillars

RGR, RCR, and ECI values are shown in Table 13. Kruskal-Wallls
tests demonstrate no significant heterogeneity among treatment groups
for any of these variables. However, the length of time required to
reach the sixth instar increased markedly when cardenolide was added
to the diet (Table 14A) . Despite this increase in development time,
estimated dry weights at the start of the gravimetric experiment
declined significantly with cardenolide (Table 14B), reflecting the
cumulative effect of the allelocheraic upon growth of the first four
instars. Direct mortality also increased with cardenolide
concentration (Table 14C) . Nearly 82% (9/11) of these mortalities
occurred in the first two instars.

Discussion

Previous studies of the effects of ingested cardenolides on
herbivores have focused entirely on adapted, milkweed-specializing
species (Danaus plexippus ; Erickson, 1973; Dixon et al., 1978; Seiber
et al., 1980; Danaus chryslppus ; Smith, 1978; Oncopeltus fasciatus

104

Table 13. Relative growth rate (RGR) , relative consumption rate
(RCR), and efficiency of conversion of ingested matter (ECI) for fifth
instar velvetbean caterpillars reared on artificial diets
incorporating varying amounts of cardenolide. Data are X ± SD with
sample sizes in parentheses.

% CAR- RGR RCR ECI

TREATMENT DENOLIDE (mg/mg/d) (mg/mg/d) {%)

Control 0 0.39 + 0.04 1.20 ± 0.10 32.78 + 2.69

(13) (13) (13)

Aerosol

0

0.39 + 0.03

1.26 ± O.IZ

Ji

/» + i.»D

Control

(13)

(13)

(13)

Low Car-

0.21

0.38 + 0.04

1.17 + 0.13

32

74 + 2.26

denolide

(12)

(12)

(12)

Medium Car-

0.53

0.39 + 0.06

1.26 + 0.16

30

.98 + 5.95

denolide

(14)

(14)

(14)

High Car-

0.97

0.37 + 0.08

1.23 ± 0.15

30

.30 + 5.91

denolide

(14)

(14)

(14)

Kruskal-Wallis

2

X

0.47

3.87

6.63

df

4

4

4

P

0.98

0.42

0.16

105

Table 14. Mditional data for velvetbean caterpillars reared on
artificial diets incorporating varying amounts of cardenolide. (A)
Duration of the larval period from hatching until the start of the
sixth instar. These observations terminated after 17 days. All
larvae in the Control and Aerosol groups had reached the VI instar but
1,2, and 5 larvae in the Low-, Medium-, and High-Cardenolide groups,
respectively, had not. The medians given for these three groups omit
these individuals and are therefore underestimated. (B) Estimated dry
weights of newly-molted, fifth instars. The estimation is arrived at
by multiplying the fresh weight by the dry weight :f resh weight ratio
determined on a similarly reared sample of larvae. Means followed by
the same letter are not significantly different at the 0.05 level by
Duncan's multiple range test. (C) 2X5 contingency table showing the
numbers of larvae dead or alive after 17 days feeding. Nine of these
larvae died in the I or II instar, one in the III, and one in the V.

TREATMENT

CARDENOLIDE

CONTROL AEROSOL LOW MEDIUM HIGH
(0.21%) (0.53%) (0.97%)

(A) Larval Period (Hatching-VI)

Median Days 13 13 14 13 14

Range 13-16 13-16 13-17+ 12-17+ 13-17+

N 22 22 25 23 21

(B) Estimated

Dry Weight

(V)

X

4.97^

4.98^

4.08^^

3.71^

3.76*'

S.D.

1.39

1.47

1.08

1.18

1.55

N

13

13

12

14

14

One-way

ANOVA

h

,65=

=2.94,

P=

=0

.03

(C) Survival After 17 Days
No. Alive
No . Dead

29
1

30
0

29
1

26
4

25
5

X = 9.2, df = 4, P = 0.05

106

[Heraiptera: Lygaeidae] : Israan, 1977; Vaughan, 1979; Chaplin and
Chaplin, 1981). In all but two cases (Vaughan, 1979; Seiber et al.,
1980), the authors compared metabolic performance of insects when fed
different species of milkweeds, that differed in mean cardenolide
concentration. The problems associated with this approach have been
elaborated elsewhere (see Chapter IV) and relate to a confounding of
cardenolide and other nutritionally- important plant species
differences. Other workers have studied cardenolide-ef f ects upon
lepidopteran neural tissue in vitro (Vaughan and Jungreis, 1977) or
have utilized injected (rather than ingested) cardenolide (von Euw et
al., 1967; Rafaeli-Bernstein and Mordue, 1977). While all three of
these latter studies concluded that milkweed-feeding insects are less
sensitive to cardenolide than are species that do not normally feed on
milkweeds, their results must be regarded with caution because J^
vitro and injection assays circumvent much of the natural biology of
the study organisms (see Chapter I). It is only when ingested by an
intact animal that cardenolides can be expected to have their natural
effects upon herbivores.

The data presented here demonstrate, for the first time, that
ingested cardenolide can have negative effects upon non-adapted
herbivores. Thus, the potential exists for cardenolides to constitute
a chemical barrier against herbivory by at least some insect species.
As predicted, the highly polyphagous fall armyworras grew more slowly
as dietary cardenolide concentration increased (Table 11). This
effect is attributable to two causes: (1) the larvae consumed less of

107

the medium- and high-cardenolide diets than of the control and low-
cardenolide diets, and growth is obviously dependent, in part, upon
consumption. A similar behavioral feeding inhibition was reported
(albeit non-quantitatively) by Nielsen (1978) for chrysomelld beetles
fed cardenolide-treated leaf discs. However, a behavioral inhibition
against ingesting a particular diet (in the absence of alternative
food sources) would only be adaptive if the diet is indeed detrimental
(an issue not addressed by Nielsen, 1978). That this is the case in
fall arrayworms is reflected by the (2) decreased conversion efficiency
of larvae fed the cardenolide-treated diets. Such larvae must spend
additional time and energy in feeding behavior, relative to control
larvae, in order to support the same amount of weight gain.

A second indication of a metabolic cost to fall armyworms of
ingesting cardenolide is seen in the prolongation of the median larval
period by as much as 20% (Table 12A). Indeed, some cardenolide-fed
individuals required fully twice as long as typical control larvae to
complete this developmental period. A slower development not only
would expose the larvae for longer periods of time to predators,
parasites, and other mortality agents, but can be related directly to
a decrease in fitness through a reduction in the intrinsic rate of
natural increase: r=(loggRo)/T, in which R^ is the net reproductive
rate and T is the mean generation (or development) time. Thus,
individuals having longer developmental periods will have lowered mean
fitness unless this is accompanied by a very substantial increase in
net reproductive rate (Green and Painter, 1975; Lanciani and May,

108

1982). However, since body weight and fecundity are well correlated
in Lepidoptera (see Hinton, 1981), the tendency of arrayworras fed high-
cardenolide diets to weigh less than control animals suggests that
such a compensatory increase in reproductive rate is highly unlikely.
If it is agreed that the most drastic effect an allelochemic can
have on an herbivore is to cause direct mortality, then the legume-
specializing velvetbean caterpillars, as predicted, were more severely
affected than the polyphagous fall arrayworms (compare Tables 12C and
14C). Moreover, while armyworms fed 1.2% cardenolide diets were able,
by prolonging development, to maintain their body weights within 83%
of control levels (Table 12B), velvetbean caterpillars fed only 0.53%
cardenolide diets suffered a significant 25% mean decline in fifth
instar body weight (Table 14B) , despite a comparable increase in
development time (Table 14A).

With these indications of a severe allelochemic impact on the
negative-specialist, it is perhaps surprising that dietary cardenolide
had no apparent effect upon consumption rate, growth rate, or
conversion efficiency in the penultimate instar (Table 13). Clearly,
then, the greatest effect is on the younger instars in this species.
This difference in age-specific susceptibility may reflect the
ontogenetic development of allelochemic-detoxif ication abilities (see
Brattsten et al., 1977). While younger instars may succumb to
cardenolide effects (Table 14C), those which survive to the
penultimate stage (on which the gravimetric study was performed) may
have had sufficient time to induce the detoxification enzymes

109

necessary to process cardenolldes (see also Ahmad and Forgash, 1975).
The truly surprising result may then be not that velvetbean
caterpillars are unharmed by cardenolide in the penultimate instar
but, rather, that fall armyworms are harmed, since polyphagous species
appear to have more active detoxification systems than do oligophagous
species (Krieger et al., 1971).

The strong effects of cardenolldes upon younger velvetbean
caterpillar instars, and their apparent absence in the penultimate
stage serve as an important warning to other workers utilizing the
gravimetric protocol (Waldbauer, 1968). Most studies of this nature
investigate allelochemic-eff ects only upon the penultimate or ultimate
instars (e.g., Erickson, 1973; Blau et al., 1978; Scriber and Feeny,
1979; Seiber et al., 1980; Futujnna and Wasserman, 1981). My data show
that a failure to test for effects on younger instars can lead to very
misleading results.

As also predicted, the milkweed-specializing monarch butterfly
showed no apparent response to dietary cardenolide, even at
concentrations 2.5 times greater than encountered in nature (Table
10). This confirms the results of Seiber et al. (1980) for both
fourth and fifth instar raonarchs. It should be noted that in both of
these studies the larvae were reared on diets containing low to
negligible cardenolide until the penultimate instar, when they were
suddenly dosed with abnormally-high concentrations. This rearing
design does not permit the testing of cardenolide-ef fects upon younger
instars (see above). However, it minimizes the opportunity for the
induction of detoxification enzymes by younger instars and thereby

no

maximizes the potential effect of the allelocheraic during the
gravimetric trials. Thus, the lack of an effect on monarch
consumption rate, growth rate, or conversion efficiency reported here
is even more dramatic.

The trend in monarchs toward higher consumption and growth rates
with increasing dietary cardenolide concentration, while not
statistically significant, may suggest an intermediate stage of
adaptation to these allelochemics. Many oligophagous insects utilize
hostplant allelochemics as feeding stimulants (e.g., Fraenkel, 1959;
Rees, 1969; van Emden, 1972; Nielsen, 1978) and the possiblity that
monarchs are now in an early stage of evolving such a capacity
warrants further research.

Cardenolide researchers have long speculated and argued over
whether these allelochemics evolved to defend plants against potential
invertebrate or vertebrate herbivores. The data presented herein are
the first to suggest that cardenolides may have evolved, at least in
part, as a defense against certain insects. However, the existence of
such a chemical barrier neither proves that the allelochemic
originally evolved as a plant defense mechanism at all, nor that it is
currently maintained because of its anti-insect effects. As correctly
noted by Moran and Hamilton (1980) and Fox (1981), a defense (like
most traits) will evolve only if it enhances the fitness of its
possessor; it is not sufficient merely to diminish the fitness of
one's enemies. Further work should next be directed at testing the
effects of cardenolides upon plant fitness.

Ill

Finally, it must be noted that whereas the demonstration of a
chemical barrier is necessary to coevolutionary models that assume
their breaching (see Introduction), this is not evidence that such a
breaching has necessarily occurred in the evolutionary history of
specialist herbivores. An alternative is that extant milkweed
specialists (or their progenitors) were biochemically preadapted for
handling cardenolides and did not have to evolve this capacity during
the course of evolutionary interaction with milkweed plants. This
subject is discussed more fully in Chapter VI.

CHAPTER VI
GENERAL DISCUSSION AND CONCLQSION

General Discussion

The four chapters presented In this dissertation bear on the
related questions of plant/herblvore coevolutlon and -chemical defense
In a system widely touted as a model for both. However, as described
In earlier chapters, previous research has provided little direct
evidence either for the coevolutlon of milkweed plants with their
specialized herbivores, or for their chemical defense against
generallst herbivores. It has been repeatedly stressed that a
demonstration of a chemical barrier is a prerequisite for claims that
specialist herbivores have breached such a defense during the course
of coevolutlon with their hostplants. Clearly, this alone will not
suffice to demonstrate coevolutlon (see below) but its absence (as has
been the case with cardenolldes until now) is sufficient to reject
such a coevolutionary scenario.

I have used a variety of both observational and experimental

techniques to investigate the effects of cardenolide upon the behavior

and physiology of both milkweed-adapted and non-adapted lepldopteran

species. Behavioral traits considered Included female ovlposition and

112

113

larval feeding rates. Physiological variables included larval
survival, growth rate, food conversion efficiency, cardenolide
sequestration, and relationships between adult cardenolide content and
body size or weight. In Chapters II-IV, I focused upon adapted
species since even these would be expected to show a range of
tolerance for cardenolides reflecting different degrees of adaptation
to these allelocheraics.

One method for assessing the physiological cost of ingesting and
storing cardenolides is to search for correlations between the amount
of the allelochemic sequestered and the adult body size or weight,
since these latter variables appear to be correlated with fitness.
Such a cost need not imply that the net benefit to cardenolide-
ingesting insects is necessarily negative. Sequestered allelocheraics
may defend the insect from predation or parasitism (see Chapters III
and IV) and thereby increase fitness. However, a reduced body size
may be considered an investment that the insect must make in order to
achieve this level of defense. It is assumed that, as adaptation to
allelocheraics evolves, the magnitude of the investment required would
be reduced. In Chapter III, I reported strong negative correlations
between the amount of cardenolide sequestered and both body size and
weight of monarch butterflies, whether wild-caught or laboratory-
reared. Since no such correlations were found for the congeneric
queen butterfly, these data suggest that monarchs may pay
physiological costs of cardenolide incorporation which queens do not.
As discussed in Chapter III, this difference may be related to

114

differences in movement patterns and consequent degrees of inbreeding
in the two species. Monarchs are highly migratory and congregate by
the millions in Mexico prior to mating. They are therefore more
likely than are queens to mate with individuals that have developed as
larvae on different milkweed species. Since there is such great
diversity in the amounts and kinds of cardenolides found in different
milkweeds (many species lack these compounds entirely; see Chapter I),
such interbreeding would tend to retard the rate of adaptation to
cardenolides among monarchs. In contrast, queen butterflies make only
regional movements within more localized geographic areas. This would
likely result in a higher frequency of raatings among individuals that
had fed on the same larval foodplant species and would tend to promote
a more rapid evolution of cardenolide-tolerance than would be expected
among monarchs. This hypothesis receives further support from the
case of the dogbane tiger moth (Chapter IV), another nonmigratory,
milkweed-feeding species that showed no significant correlation
between cardenolide content and body weight.

A second technique used to study the costs of feeding on
cardenolide-rich plants involves the distribution of eggs in relation
to the allelocheraic content of the hostplants (Chapter II). This
study was stimulated by the results of Dixon et al. (1978) who
reported an apparent preference of monarch butterflies to oviposit on
a lower-cardenolide milkweed species relative to higher-cardenollde
species. Such a preference, if confirraed by more careful study, might
suggest an aversion to plants of high allelocheraic content, and lead

115

to subsidiary hypotheses to explain its evolution (e.g., a metabolic
cost to the larvae). However, Dixon et al.'s experimental design
confounded cardenolide differences with other milkweed species
differences and the ovipositional preference could therefore not be
attributed to the lower cardenolide content per se of the preferred
milkweed species. In Chapter II, I approached this problem by
studying egg distribution patterns in relation to intraspecif ic
milkweed cardenolide variation, thereby removing a major source of
confoundment (i.e., other plant species differences). This study
failed to demonstrate any significant effect of plant cardenolide
concentration upon egg deposition patterns. Moreover, estimates of
larval success on these plants (which, if lowered on high-allelochemic
plants, could reflect metabolic costs) were unrelated to the
cardenolide content of the plants. The results on oviposition are in
contrast to those of Dixon et al. (1978) and they offer no support for
the hypothesis that monarchs pay metabolic costs of feeding on
cardenolide-rich plants. However, the results are not necessarily
incompatible with the earlier finding of a negative correlation
between adult monarch cardenolide content and body size, since
different life history stages were studied in each case. It is
possible that cardenolides may not adversely affect larvae but may
exert their effects during metamorphosis to the adult stage, thereby
producing smaller adults. This possibility remains to be
investigated.

116

A more direct, experimental test of cardenolide-ef f ects upon
behavior and physiology was reported in Chapter V in which one
cardenolide (digitoxin) was added to the diets of three lepidopteran
species: monarch butterflies, fall armyworms , and velvetbean
caterpillars. It was predicted that fall armyworms , because they are
highly polyphagous insects, should pay larger metabolic costs of
cardenolide ingestion than should raonarchs which, as a species,
specialize on cardenolide-containing plants. Moreover, velvetbean
caterpillars, which specialize on the Leguminosae (a plant family
lacking cardenolides) , should be most severely affected by
cardenolides because, although they should have a finely-tuned
detoxification ability, this would be directed at handling the
allelochemics of legumes rather than those of milkweeds. These
predictions were generally supported by the results. Fourth instar
monarchs showed no cardenolide-induced alterations in growth rate,
feeding rate, or food conversion efficiency, even when fed diets
containing cardenolide concentrations 2.5 times greater than those
ever encountered in natural milkweeds. In contrast, fifth instar fall
armyworms demonstrated reductions in growth, food consumption,
conversion efficiency, and developmental rate, even at very moderate,
natural cardenolide concentrations. While fifth instar velvetbean
caterpillars were unaffected even by unusually high cardenolide
concentrations, additional data revealed severe allelocheraic effects
upon the younger instars, including dose-dependent mortality.

117

Several aspects of this last study deserve further mention.
Firstly, fourth instar monarchs did show a trend toward higher
consumption (and hence, growth) rates as dietary cardenolide
increased. While this trend was not significant, it could reflect an
intermediate stage in the evolution of monarch adaptation to
cardenolides. As noted in Chapter I, some oligophagous insects have
been shown to utilize plant allelocheraics as attractants for either
oviposition, larval feeding, or both. In the case of monarchs,
cardenolides cannot be the critical feeding or oviposition stimulant,
since these insects will recognize and use cardenolide-f ree milkweed
species as food. However, cardenolides could nevertheless alter the
insects' motivational level to feed or oviposit, once these behaviors
have been released by the key stimuli. While ray data provide no
evidence that cardenolides influence oviposition behavior (Chapter
II) , the trend toward increased larval feeding rates shown in Chapter
V is intriguing and merits further study.

Secondly, the effects of cardenolide on fall arrayworms and
velvetbean caterpillars were more pronounced at higher allelochemic
concentrations than at lower levels. This dose-dependence serves as a
warning regarding Feeny's (1975, 1976) dichotomy of plant chemical
defenses as either qualitative or quantitative. According to Feeny's
view, such compounds as tannins, which may occur in very high
concentrations in plants (see Chapter I), are "quantitative" defenses
in the sense that ever-higher concentrations may be selected for in
plants in order to maintain a defense against herbivores which are

118

evolving, in parallel, ever-greater tannin-tolerance. In contrast,
Feeny views such compounds as cardenolides as "qualitative" defenses.
These typically occur in relatively low concentrations (<2% by dry
weight) in plant tissues and may be toxic to a broad range of
potential attackers. [They may be relatively easy barriers to
overcome evolutionarily, however, as studies of pesticide-resistance
suggest (see Futuyma, 1983).] Nevertheless, this need not mean that
cardenolides are any less "quantitative" than tannins, and their now-
demonstrated dose-dependent effects suggest that, like tanniniferous
plants, milkweeds could be involved in quantitative evolutionary races
against their enemies.

Are these races necessarily "coevolutionary"? As discussed in
Chapter I, a strict definition of coevolution requires that at least
three reciprocal evolutionary steps be identified. For example, a
plant may evolve a chemical defense in response to herbivore pressure
from an insect population (step 1). The insect may evolve a means of
tolerating or circumventing the defense (step 2). The plant evolves
yet another defense mechanism, or greater levels of the first
mechanism (step 3). What is the evidence that the chemical defense of
milkweeds has resulted from such a coevolutionary process? This
question can be conveniently dissected into three subquestions , each
corresponding to one of the above coevolutionary steps.

1) Against which species have milkweeds evolved cardenolides?
This can only be inferred from identification of potential enemies

119

that are presently deterred or harmed by cardenolides. The most
likely candidates for this are pathogenic organisms (e.g., bacteria or
viruses), vertebrate herbivores, and invertebrate herbivores (notably
insects). To date there is no evidence that cardenolide affects
pathogenic organisms. I-^ereas there have been, to my knowledge, no
studies with viruses as target organisms, Frings et al. (1948)
reported that hemolymph of the milkweed bug (Oncopeltus fasciatus) ,
extracted in saline, inhibited the in vitro growth of the bacteria
Staphylococcus aureus and one strain of Bacillus subtilis. These
results were later misinterpreted by Rothschild (1972) and by Price
(1980) to be due to the presence of cardenolides in the hemolymph.
However, a careful reading of the original study will show that saline
extracts of the milkweed seeds (upon which the bugs were reared) had
no antibacterial properties. Since the seeds are the source of
cardenolides for milkweed bugs (e.g., Isman et al., 1977a), it is
unlikely that the cardenolides were the antibacterial agent.
Moreover, after solvent-partitioning of the hemolymph, Frings et al.
(1948) found antibacterial action only in the water-soluble fraction
and not in the lipophilic fraction. Since milkweeds commonly contain
lipophilic cardenolides (e.g., Seiber et al., in press), this sheds
further doubt that the hemolymph cardenolides were responsible for the
antibacterial effects. In the only direct test to date of the
possible antibacterial action of cardenolides, Malcolm (1981) grew the
bacteria Escherischia coli and Bacillus cereus var. mycoides on
culture media incorporating the cardenolides ouabain or digitoxin.

120

His results show no significant allelochemic effects upon colony
growth for either bacterial species. Obviously, other pathogens might
be affected by cardenolides but, if so, they have not yet been
identified.

In contrast, there is a large body of pharmacological evidence
that cardenolides are toxic to many vertebrate species (see Chapter
I). However, most of this evidence is based upon intravenous
injections in domesticated mammals (see Detweiler, 1967), rather than
studies of the effects of ingested cardenolide on wild herbivores.
Although there have been cases of poisoning of domestic livestock that
have eaten milkweed plants (e.g., Tunnicliff and Cory, 1930; Campbell,
1931), such data for wild herbivores are lacking. Thus, it is not yet
possible to speculate about which wild vertebrate species might have
promoted the evolution of cardenolides in milkweeds. They may even
have been representatives of now-extinct groups (see Janzen, 1980).

Finally, cardenolides might have evolved as a response to
pressure from invertebrate herbivores. For example, one might
hypothesize that interspecific differences in milkweed cardenolide
concentration (Chapter I) reflect differential risks to milkweed-
specializing insects. This would be suggested by the fact that the
two north-Florida milkweed species (Asclepias humistrata and A^
viridis) which have re-established widespread, above-ground
populations by early spring (when large numbers of monarchs enter
Florida from Mexico) have substantially higher cardenolide
concentrations than do several other species which either grow more

121

sparsely or emerge later (after most of the migrants have left the
area) . If monarch herbivory has been a major cause of the high
allelochemic levels in the two early species, we would expect monarchs
to be either deterred or directly harmed by the levels of cardenolide
now found in these plants. However, Chapter V shows that even
considerably higher concentrations may have no adverse effect on
monarchs. (Note that since only digitoxin-ef fects were tested, this
result bears repetition with other cardenolides) . Thus, at this
stage, it is not possible to view present milkweed species cardenolide
differences as resulting from differential risks of the plants to
monarchs.

In contrast to monarchs, the fall arrayworms and velvetbean
caterpillars studied in Chapter V began to show adverse effects of
cardenolides at levels between 0.17% and 0.56% (Chapter V).
Interestingly, this range is approximately that found in the two
early-spring milkweed species mentioned above (see Table 1). This
suggests that non-adapted species, rather than specialists, may
account for the present levels of cardenolide in milkweed plants.
However, there is no evidence that any particular non-adapted species
has interacted with any milkweed population on a persistent basis for
sufficient time to have taken the next step in the coevolutionary
sequence, i.e., the evolution of a specific counter-defense to
cardenolides.

122

2) Have any milkweed enemies evolved specific counter-defenses to
cardenolides? As implied above, this would be most likely to occur in
oligophagous species, i.e., those that do not feed on other plant
families and have no choice but to face the cardenolides. While ray
data suggest that specialists have greater cardenolide tolerance than
generalists, it is by no means evident that they have evolved such
tolerance during the course of their evolutionary interactions with
milkweeds. One alternative is that specialists were biochemically
preadapted for handling cardenolides. This hypothesis can be
investigated via the comparative method. For example, certain genera
of Danaidae (e.g., Mauris, Euploea) are reputed to feed primarily on
cardenolide-free milkweed species (Rothschild and Marsh, 1978). If
this is true, then such butterflies will not have recently undergone
strong selection for cardenolide tolerance. Thus, if they are found
to be harmed by cardenolides, it will imply that monarchs and other
cardenolide-ingesting danaids have evolved cardenolide-tolerance
during an evolutionary history of exposure to these allelochemics. In
contrast, if Anauris and Euploea, despite their feeding habits, are
nevertheless found to have a cardenolide-tolerance similar to that of
monarchs, it will imply either that (a) the entire family was
preadapted for dealing with cardenolides and did not have to evolve
this capacity, or (b) the tolerance evolved prior to the divergence of
the genus Danaus from Amauris and Euploea and has not been lost
despite relaxed selection for its maintenance. Possibility (b) (i.e.,
cardenolide-tolerance by descent from species that evolved such

123

tolerance) can be potentially eliminated by testing the tolerance of
butterfly taxa not closely related to danaids and having no known
history of interaction with cardenolide-containing plants (e.g.,
Pieridae, Papilionidae, Lycaenidae; Ehrlich and Raven, 1964). To the
extent that these are nevertheless tolerant of cardenolides , it would
suggest a general biochemical preadaptation among butterflies for
dealing with these allelochemics. My data (Chapter V) show that such
preadaptation, if it exists, is not so broad among Lepidoptera as to
include certain noctuid raoths. However, it might well extend to
pierid butterflies: in preliminary experiments, Feeny (pers. comm.)
has found a considerable tolerance of Pieris rapae larvae to
strophanthin cardenolides, thus supporting the preadaptation
hypothesis. Of course, cardenolide-ingesting species could still
evolve improvements upon their preadaptations. If future work shows
this to be the case, it will suggest that the second step in the
coevolutionary sequence has been taken.

Although sufficient work has not yet been done to test fully the
preadaptation hypothesis in Lepidoptera, a revealing parallel
situation exists at a higher trophic level. Deer mice (Peromyscus
maniculatus labecula) are known to feed upon cardenolide-containing
monarch butterflies overwintering in Mexico (Brower et al., in prep.).
As in the case of insects attacking milkweeds (or birds attacking
raonarchs; Fink and Brower, 1981), one might well ask whether these
rodents have evolved this ability or were preadapted for it. In
laboratory experiments, Marty (1983) has shown that a subspecies of

124

deer mouse found in California (P_^ m^ gambelii) has a high tolerance
for ingested cardenolide, which is poorly absorbed across the gut.
Since P. m. gambelii does not feed on cardenolide-containing insects,
these results suggest that Peromyscus maniculatus may be a species
generally preadapted for ingesting cardenolides safely. (Indeed, even
laboratory rats are strongly resistant to cardenolides. Detweiler
(1967) reported that the LD-50 for intravenous ouabain in this species
was 671 times greater than that for domestic cats.) Whether P. m.
labecula has evolved further refinements of this preadaptation is an
important area for future research.

3) Have milkweed plants evolved any specific counter-defenses
against their specialist enemies? Presumed examples of such counter-
defense in non-milkweed species (described more fully in Chapter I)
include mechanical structures (e.g., the egg-mimicking structures or
hooked trichomes of Passiflora plants; Gilbert, 1983), higher
concentrations of existing chemical defenses (e.g., tannins; Feeny,
1976), the addition of new kinds of chemical defenses to the arsenal
(e.g., the addition of angular furanocoumarins to linear
furanocoumarins in Umbellif erae; Berenbaum, 1983), or displacement in
space and time to avoid specialist herbivores altogether (Rhoades and
Cates, 1976). While milkweeds may show certain of these features,
there is no evidence that these evolved as counter-adaptations to
their specialized enemies. For example, Asclepias tuberosa in north-
central Florida sprouts late in the spring, after most monarchs have

125

left the area on their northward migration (see Chapter I). While
this certainly makes the plants unavailable to these herbivores, it is
not known whether herbivore pressure is in any way responsible for
this phenological pattern rather than, say, competition for
pollinators (e.g., Pleasants, 1980). Ever-higher concentrations of
cardenolides would seem to be effective against generalist insects but
not as a counter-strategy against specialists (Chapter V). The
addition of new cardenolides (e.g., of lower polarity; see Chapter I)
might present a greater challenge to specialists but, again, there is
no evidence that milkweed species or populations that are under
greater specialist pressure have a predictably different set of
cardenolides from those under less pressure. This is a major line of
research that should be conducted.

The above considerations provide scant support for the contention
that milkweeds have coevolved with their specialized herbivores in the
kind of three-step manner described earlier. However, Edgar et al.
(1974) have proposed an alternative coevolutionary scenario in which
the progenitors of extant milkweed plants may have interacted with
their specialist herbivores. They hypothesize that such plants
contained (presumably, but not necessarily, for defensive purposes)
both cardenolides and pyrrolizidine alkaloids (PA's) and that females
of species comprising the basal stock of Danaidae and Ithomiidae
utilized these chemicals as host recognition cues. Whereas the
cardenolides (and possibly the PA's as well) provided the insects with
a potent chemical defense, the PA's became further necessary as

126

precursors of male sex pheromones (see references in Chapter III),
possibly because this allowed males to induce females to land away
from their hostplants (and therefore to be mated) . Once the
butterflies had become dependent upon both PA's and cardenolides for
these purposes, those plants which lacked either one or both of these
chemical classes would be favored by natural selection because they
would attract fewer herbivores. As a result, this group of plants
gave rise phylogenetically to three branches, one containing
cardenolides only (Apocynales: Asclepiadaceae and Apocynaceae) , one
containing PA's only (Boraginaceae and Corapositae) , and one containing
neither (Solanaceae) . [All five of these plant families are placed in
a single subclass, Asteridae (Cronquist, 1968; Takhtajan, 1969),
suggesting a common origin.] The divergence of the plants could then
have been followed by a divergence of the butterfly stock into
apocynale-specialists (danaids) and Solanaceae-speclalists
(ithomiids), with adults of both families still visiting PA-containing
plants to acquire pheromonal precursors and/or defensive compounds.
This hypothesis is coevolutionary in that the following three
fundamental steps are included. (1) The primitive butterflies evolved
pheromonal systems based upon chemicals already present in their
larval foodplants. [It is not necessary that these chemicals evolved
as defenses against these particular butterflies.] (2) The herbivore
pressure led to a deletion of either PA's or cardenolides (or both)
from the plants. Since selection would presumably act against hybrid
plants containing both chemical classes, an evolutionary divergence

127

resulted. (3) The butterflies diverged in parallel to the plants,
giving rise to the danaids and ithomiids. These three steps, if
documented, would satisfy even Janzen's (1980) rigorous criteria for
coevolution (see Chapter I).

The strongest support for Edgar et al.'s (1974) ingeneous
hypothesis comes from the observation that certain primitive, danaid-
like, extant ithomiids have been discovered to feed as larvae on PA-
containing plants in the tribe Parsonieae of the Apocynaceae. Thus,
neotropical ithomiids in the genera Tithorea and Aeria feed upon
plants in the genera Fernaldia and Prestonia (Edgar, 1982). Both
plant genera include species containing PA's and some Fernaldia
species are known to contain cardenolides (Edgar et al., 1974). It
therefore seems plausible that at least certain plants fed upon by
primitive-stock butterflies contained cardenolides and PA's
simultaneously, as required by the hypothesis. Moreover, whereas in
most danaid and ithoraiid butterflies it is predominantly adult males
which are attracted to withering PA-sources, in Tithorea females are
equally attracted (Pliske, 1975). This suggests that while females of
other genera have lost their presumably primitive responsivity to PA's
(because these no longer occur in their solanaceous hostplants),
females of species still feeding as larvae on PA-containing plants
have not. However, it has not yet been established whether Tithorea
females, as predicted, utilize PA's as hostplant recognition cues.

A further observation adding credence to the coevolutionary
hypothesis is of another primitive danaid-like ithomiid, this one from

128

Australia and New Guinea. Larvae of this species, Tellervo zoilus
zoilus, feed upon the PA-containing apocynad, Parsonsia velutina (also
in the tribe Parsonieae) . The fact that the Tellervo-Parsonsia
association is found in Australia, which has been separated from the
neotropics since the breakup of Gondwanaland in the Eocene, strongly
suggests that these associations between primitive ithoniiines and PA-
containing plants in the Parsonieae, both in the new and old world,
are representative of an ancestral condition. Whereas Edgar et al.
(1974) and Edgar (1982) do not present evidence that the tribe
Parsonieae, like its ithomiine herbivores, is primitive, the placement
of all five of the plant families concerned in the hypothesis into the
single subclass, Asteridae, implies the kind of phylogenetic radiation
hypothesized by these authors. Moreover, certain PA's found in
Parsonsia are typical of the Boraginaceae and of the genus Eupatorium
of the Corapositae (Edgar and Culvenor, 1975). These facts lend yet
further support to Edgar et al.'s (1974) hypothesis.

The above hypothesis has been challenged by Boppre (1978), who
offered a non-coevolutionary alternative interpretation of the data.
He suggests that primitive-stock danaid/ithomiid adults (of both
sexes) fed occasionally on PA-containing nectar. Those individuals
with a tendency to accumulate PA's in external tissues (which would
permit them to be tasted by predators and released unharmed; see
Brower, in press) would be favored by selection. Eventually, wilting
leaves of certain plants were discovered by the butterflies to be more
reliable PA-sources. Boppre (1978) further suggests that females may

129

have evolved an attraction to males disseminating "PA-odors" as a
means of avoiding courtship and possible hybridization with males of
mimicking species. This could have led to the incorporation of PA's
into the pheromonal system. Finally, he proposes that the use by
danaids of cardenolide-contalning plants was an independent
evolutionary development.

As evidence for his hypothesis, Boppre (1978) points out that
PA's have been found in the nectar of certain Senecio plants
(Compositae) (Deinzer et al., 1977) fed upon by adult danaids. He
further notes that the (occasional) use by danaids of Crotalaria
(Leguminosae) and some Moraceae as adult PA-sources and larval
foodplants, respectively, does not fit with Edgar et al.'s (1974)
hypothesis and suggests that the use of PA's in the pheromonal systems
of these butterflies evolved independently of the phylogeny of larval
foodplants.

It is important to note that Edgar et al.'s (1974) hypothesis
does not assume that cardenolides (or PA's) evolved in the plants as
deterrents to lepidopteran (or other) herbivores but, rather, that
they were deleted from some plants because they made the plants more
conspicuous to females searching out larval hostplants. Such a
deletion would presumably have resulted from selection favoring those
individual plants in a population that had somewhat lower cardenolide
concentrations than others (assuming heritability of plant cardenolide
content). If female butterflies utilized cardenolides for host-
finding, then such lower-cardenolide plants would presumably have been
located and oviposited upon less frequently than higher-cardenolide

130

plants. This situation was effectively modelled in Chapter II in
which it was found that oviposition rates of monarch butterflies are
independent of plant cardenolide concentration. These results
therefore do not support Edgar et al.'s (1974) hypothesis.

Boppre's (1978) alternative, however, cannot adequately explain
the occurrence, in both Australia and the neotropics, of primitive
ithoraiines that utilize PA- (and probably also cardenolide-)
containing larval hostplants. Edgar et al.'s (1974) hypothesis,
having the benefit of phylogenetic and biogeographic data, is more
persuasive in this respect and provides a reasonable, testable
scenario in which the primitive hostplants of danaid and ithomiid
butterflies may have coevolved (sensu stricto) with their specialist
herbivores.

Conclusion

In conclusion, there is little evidence that the cardenolides of
milkweed plants have evolved as deterrents to specialist herbivores,
whether through a stepwise coevolutionary process or otherwise.
However, ray data show that these allelochemics are deterrent and toxic
to at least one generalist lepidopteran species and to another
lepidopteran specialized for feeding on a plant family lacking
cardenolides (Chapter V). Furthermore, the observed concentrations of
cardenolides in the leaves of many milkweed species (Chapter I) are
sufficient to cause negative metabolic effects upon such non-adapted

131

species but are apparently insufficient to harm adapted specialists.
In the case of one non-adapted species (velvetbean caterpillar)
cardenolide-effects were seen only in the younger instars but not in
the penultimate stage. This serves as an important warning to other
researchers utilizing the gravimetric protocol (Waldbauer, 1968) since
most studies of this nature investigate allelocheraic-ef fects only in
the penultimate or ultimate instars (e.g., Erickson, 1973; Blau et
al., 1978; Scriber and Feeny, 1979; Seiber et al., 1980; Futuyraa and
Wassennan, 1981). My data show that failure to test for effects on
younger instars can lead to very misleading results. Furthermore, the
dose-dependence of the responses to cardenolide in the two non-adapted
species caution against a strict dichotomy of qualitative vs.
quantitative allelochemlcs (Feeny, 1975, 1976).

It is important in coevolutionary studies to specify the
particular coevolutionary model being tested. In this dissertation I
have considered two such models that have been proposed to account for
the relationships between milkweeds and their specialist herbivores.
The first of these might be called the "barrier-breach" model, i.e.,
the plants evolved cardenolides as a chemical barrier to insect
herbivory. It is proposed that this barrier was later breached by the
progenitors of extant milkweed specialists, which then led to the
evolution of further plant defenses, etc. I have presented the first
evidence that such a cardenolide barrier exists and may deter
herbivory by certain non-adapted insects. It is, however, not known
whether the cardenolide-tolerance exhibited by specialists evolved

132

during the course of evolutionary interaction with their hostplants
(suggesting "barrier-breach") or reflects, instead, a biochemical
preadaptation for coping with cardenolides. Recent evidence from
other workers lends credence to the preadaptation hypothesis. Thus,
claims of "barrier-breach" coevolution among milkweeds and their
specialized herbivores are not supported by the available evidence.

A second model of coevolution among these organisms (Edgar et
al., 1974) carries the weight of both phylogenetic and biogeographic
evidence. This is a "co-speciation" model which does not require that
cardenolides evolved in plants as insect-deterrents. Rather, it is
hypothesized that herbivores utilized cardenolides and PA's for
hostplant recognition and that this led to a divergence of the basal
plant stock into three branches (details above). This, in turn, was
followed by a divergence of the herbivore stock to specialize on the
new plant diversity. One prediction of this "cospeciation model" is
that cardenolides are at least partly responsible for hostplant
attractiveness to specialist herbivores. Whereas ray data on monarch
oviposition rates in relation to plant cardenolides (Chapter II) argue
against this, similar tests should be undertaken with other danaid
(and ithomiid) species. Of particular interest are the primitive
species that utilize the apocynaceous tribe Parsonieae as larval
f oodplants .

Finally, although milkweed plants and their herbivores (notably
monarch butterflies) comprise one of the most thoroughly studied
plant/herbivore associations, providing many insights into chemical

133

ecology, we are only now beginning to explore the details of these
interactions. Because the chemistry of cardenolides is so well known
(thanks to their medicinal properties), and techniques for their study
are straightforward, one can expect that further work on the milkweed
system will indeed be valuable in providing a clearer understanding of
the evolution of plant/herbivore interactions.

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BIOGRAPHICAL SKETCH

James A. Cohen was born in New York in 1952 and grew up on Long
Island. In 1974 he received his B.A. in psychology from Washington
University (St. Louis, Mo.), where he graduated summa cum laude. His
M.S. in Zoology was awarded three years later by the State University
of New York, College of Environmental Science and Forestry (Syracuse,
N.Y.). After a year's appointment as Scientific Consultant to the
World Federation for the Protection of Animals (Zurich, Switzerland),
he returned to do further graduate work in ecology and animal behavior
at The Johns Hopkins University and the University of Florida. In
addition to his work on the chemical interactions of plants and
animals, Cohen's research has included studies of animal
communication, motivational control, sexual selection, and behavioral
ecology in a wide diversity of both invertebrate and vertebrate
species, spanning three continents. Much of his spare time is devoted
to worship of the pedal steel guitar.

147

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate in scope and quality, as a dissertation for the degree
of Doctor of Philosophy. ,.■

Lincoln P. Brower, Chairman
Professor of Zoology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.

X-- >^^^-^'^'",:

.^:^-^.

'C-'

H . Jane Brockmann

Associate Professor of Zoology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate in scope and quality, as a dissertation for the degree
of Doctor of Philosophy. / \

FranV; Slansky Jr. |
Assistant Professor iojj
and Nematology

il

omology

This dissertation was submitted to the Graduate Faculty of the
Department of Zoology in the College of Liberal Arts and Sciences and
to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.

December, 1983

Dean for Graduate Studies
and Research

UNIVERSITY OF FLORIDA

3 1262 08553 5309