Detrimental effects of latex and cardiac glycosides on ...biology/Classes/269/monarchs.pdfDetrimental effects of latex and cardiac glycosides on survival and growth of ﬁrst-instar

Detrimental effects of latex and cardiac glycosides onsurvival and growth of ®rst-instar monarch butter¯ylarvae Danaus plexippus feeding on the sandhillmilkweed Asclepias humistrata

M Y R O N P . Z A L U C K I , 1 L I N C O L N P . B R O W E R 2 and

A L F O N S O A L O N S O - M 3 1Department of Zoology and Entomology, The University of Queensland,

Australia, 2Department of Biology, Sweet Briar College, Virginia, U.S.A. and 3Smithsonian Institution, Washington, DC, U.S.A.

Abstract. 1. A novel experimental method was developed to study negative

physical and chemical effects of latex and cardiac glycosides on ®rst-instar

monarch butter¯y larvae in their natural environment in north central Florida.

Forceps were used to nibble through the petioles of leaves of the sandhill milkweed

Asclepias humistrata, mimicking the behaviour of mature monarch larvae. This

notching cut off the supply of latex to the leaves without signi®cantly reducing

either their cardiac glycoside concentration or water content.

2. The mean cardiac glycoside concentration in larvae that fed on intact leaves

was nearly two and a half times greater than in larvae that fed on notched leaves.

This was probably because more latex is present in the gut of the larvae that fed on

the intact leaves. Supporting this is the fact that the mean concentration of cardiac

glycosides in the latex was 34±47 times that in the leaves.

3. Wet weights, dry weights, and growth rates of ®rst-instar larvae that fed on

intact leaves over a 72-h period were less than half those of larvae that fed on

notched leaves.

4. Mortality due to miring in the latex was 27% on the intact leaves compared

with 2% on the notched leaves.

5. Latex, cardiac glycosides, and other as yet undetermined plant factors all have

a negative effect on ®rst-instar larval survival.

6. Video-analyses indicated that ingestion of latex caused the larvae to become

cataleptic and increased their chances of being mired on the leaf by the setting

latex glue. Dysfunction resulting from latex ingestion may lead to the larvae falling

off the plant and being killed by invertebrate predators.

7. The dif®culty of neonate monarch larvae surviving on A. humistrata ± one of

the principal milkweed species fed on each spring as monarchs remigrate from

Mexico into the southern U.S.A. ± is evidence that a co-evolutionary arms race is

operating in this plant±herbivore system.

Key words. Catalepsis, co-evolutionary arms race, experimental study, feeding

behaviour, miring, mortality, novel technique, petiole notching, plant defences,

toxicosis.

Introduction

The monarch butter¯y Danaus plexippus (L.) (Nymphalidae) is

a specialised larval herbivore on plants of the family

Asclepiadaceae, commonly known as milkweeds. Larvae have

Correspondence: Professor L. P. Brower, Department of Biology,

Sweet Briar College, Sweet Briar, VA 24595, U.S.A. E-mail:

[email protected]

212 # 2001 Blackwell Science Ltd

Ecological Entomology (2001) 26, 212±224Ecological Entomology (2001) 26, 212±224

been reported feeding on at least 27 of the 108 known North

American species in the genus Asclepias (Woodson, 1954;

Ackery & Vane-Wright, 1984; Malcolm & Brower, 1986).

These plants contain differing arrays of cardiac glycosides that

vary in amount and type within and between species, within

various plant parts, and through time (Nelson et al., 1981;

Brower et al., 1982; Malcolm, 1991, 1995). Asclepias species

are noted for milky latex that is contained under pressure in a

non-articulated, sealed system of vessels known as laticifers

(Lucansky & Clough, 1986). When any part of the plant is

punctured, latex ¯ows rapidly out of the laticifers and

coagulates on contact with air. Milkweed latex may contain

high concentrations of cardiac glycosides (Seiber et al., 1982),

amyrin (a precursor of rubber), and other noxious chemicals

(Van Emon & Seiber, 1985; Farrell et al., 1991). The system of

pressurised canals bearing a mixture of toxic chemicals in a

quick-setting glue has been interpreted as a plant defence,

particularly against generalist herbivores that lack latex canal

sabotaging behaviour (Dussourd & Eisner, 1987; Dussourd,

1993; Dussourd & Denno, 1994).

Monarch caterpillars have evolved the ability both to

circumvent the latex defence of milkweeds (Dussourd &

Eisner, 1987) and to appropriate the cardiac glycoside

chemical defences of the plant (Brower, 1984). All larval

instars show sabotaging behaviours that effectively disable the

latex ¯ow in milkweeds: early instars trench and cut small

moats through the leaves (Dussourd, 1990; Dussourd & Denno,

1991; Zalucki & Brower, 1992), while later instars sever the

petioles or midribs of the leaves before consuming them

(Brewer & Winter, 1977; Zalucki & Brower, 1992). Monarch

larvae feeding on milkweeds generally concentrate cardiac

glycosides above the level found in the plant leaves (Malcolm

& Brower, 1989; Nelson, 1993) and conserve the compounds

through to the adult stage, possibly as a form of storage

excretion (Brower et al., 1988). This aspect of monarch

biology has attracted considerable attention, particularly with

respect to adult cardiac glycoside content and its deterrence of

predation by vertebrates (Brower, 1969, 1984; Brower & Fink,

1985; Glendinning & Brower, 1990).

In general, females lay most of their eggs on plants with

intermediate cardiac glycoside levels (Zalucki et al., 1989,

1990; Oyeyele & Zalucki, 1990; Van Hook & Zalucki, 1991)

by displaying post-alighting discrimination against plants with

low or high cardiac glycoside concentrations.

Survival of ®rst instars on various milkweeds in the ®eld is

poor, ranging from 3 to 40%, with most studies ®nding low

survivorship (Zalucki & Kitching, 1982; Zalucki & Brower,

1992). Using direct ®eld experiments, Zalucki and Brower

(1992) found that only 3±11% of newly hatched larvae

survived through the ®rst instar on Asclepias humistrata, a

major southern U.S.A. host of monarchs remigrating from

Mexico in late March and early April (Malcolm et al., 1987;

Knight et al., 1999). Larval survival was correlated negatively

with the concentration of cardiac glycosides in the leaves (see

also Zalucki et al., 1990) but was not affected measurably by

ground-dwelling predators. A major source of mortality was

that about 30% of the larvae that died were glued by the latex

to the leaf surface. This occurred even though all the neonate

larvae engaged in trenching behaviour to sabotage the latex

out¯ow. The ®rst bite into leaves of this milkweed by neonate

larvae is dangerous.

This sabotaging behaviour of the ®rst-instar larvae involves

mandibular chewing and slashing through the leaf surface. The

larva breaks the latex vessels, encounters latex out¯ow that

adheres to the mouth parts and head, then attempts to clean

itself vigorously. The larva frequently imbibes the latex and

becomes cataleptic (Zalucki & Brower, 1992). The 34±47-fold

higher concentration of cardiac glycosides in the latex than in

the leaves (see below) may be responsible for the catalepsis,

although it is not clear whether cardiac glycosides per se or

some other chemicals in the latex cause catalepsis and

contribute to the subsequent high mortality.

Previous research on the latex defences of plants has focused

largely on the ability of specialist herbivores, including

Danaus plexippus and Danaus gilippus berenice (Cramer), to

defeat the system (Dussourd & Eisner, 1987; Dussourd &

Denno, 1991, 1994; Dussourd, 1993). At the time their

experiments were conducted, no studies had measured the

effect of the latex system on the survivorship and growth rates

of dietary specialists on latex-containing plants (but see

Dussourd, 1995). While this manuscript was in preparation,

Zalucki and Malcolm (1999) utilised the experimental protocol

developed in this paper to investigate the effects of latex on

®rst-instar larvae feeding on three additional milkweed species

(see discussion). Here this issue is addressed using ®rst-instar

monarch butter¯y larvae and A. humistrata, the sandhill

milkweed, growing naturally in a pasture in north central

Florida.

A novel experimental method that mimics ®fth-instar larval

behaviour by disrupting the ¯ow of latex into the leaves

allowed the partitioning of three effects of the plant's defences

on ®rst-instar larval survival and growth: the lethal effects of

miring and gluing by the latex, the toxic effects of cardiac

glycosides in the latex, and the toxic effects of cardiac

glycosides in the leaves.

Materials and methods

The research site and egg sources

All ®eld experiments were conducted near Cross Creek,

Alachua County in north central Florida (29°31¢44²N,

82°12¢00²W) during April 1993. The site is a 5.3 ha sandhill

habitat used for cattle grazing, with a population of at least 500

healthy sandhill milkweeds, and has been used for research on

monarch population biology dating back to 1981 (Cohen &

Brower, 1982; Malcolm et al., 1987; Zalucki et al., 1990;

Zalucki & Brower, 1992; Knight et al., 1999).

For all experiments, eggs were obtained from remigrating

monarch females netted at Cross Creek. Three to 15 females

were maintained on sucrose solution and con®ned inside silk

organza bags over the stems and leaves of potted Asclepias

curassavica (L.) plants in an insectary. Leaf discs (200±300),

each with one egg, were cut individually from leaves daily

using a metal leather hole punch (5 mm diameter), placed onto

# 2001 Blackwell Science Ltd, Ecological Entomology, 26, 212±224

Milkweed latex vs. ®rst-instar monarch larvae 213Milkweed latex vs. ®rst-instar monarch larvae 213

moistened ®lter paper and either kept at 25±28 °C or cooled

(4±15 °C) to slow development until required.

Experimental leaf notching to reduce latex ¯ow

Depressurisation of the latex system was achieved by

mimicking the petiole notching behaviour of ®fth-instar larvae

by nibbling with blunt-nosed metal forceps about two-thirds of

the way ventrally through the petiole at the base of the leaf

(Fig. 1, inset, A). Out¯owing latex was removed continuously

during this process with a cotton-wool ball to prevent the

accumulation of the exudate and its contained chemicals on the

leaf surface. This operation achieved a marked reduction of

latex ¯ow (see below) without leaf wilting. Henceforth these

petiole-nibbled leaves are referred to as notched leaves (= on

the experimental stems), in contrast to intact leaves (= on the

control stems), which were not notched.

Preliminary experiment

From 8 to 11 April, a preliminary experiment was conducted

to investigate: ®rst-instar larval behaviour on notched vs. intact

leaves, cardiac glycoside concentration of notched vs. intact

leaves, and the effect of notching on latex out¯ow distal to the

notch. On two leaf pairs of each stem of three separate plants,

either all four leaf petioles were notched, or left intact, or two

were notched and two left intact. A small drop of latex

obtained from an adjacent plant (or non-experimental stem)

was used to glue leaf discs bearing one monarch egg onto the

surface of each experimental or control leaf (see Fig. 1; Zalucki

& Brower, 1992). All eggs selected were on the verge of

hatching, i.e. with the black head capsule of the neonate larva

clearly visible through the egg shell. All discs were glued in

place between 10.30 and 13.00 hours on 8 April. On 9±11

April, all stems were checked to determine the number and

location of larvae and feeding damage on each plant part

(leaves, stems, buds, and/or in¯orescences). All notched and

intact leaves were tested with a light pinprick for the presence

or absence of latex out¯ow on 9 April. The numbers of

stems per plant and the number of treatment leaves were

as follows: plant 1 had six stems with 12 intact and 12

notched leaves, plant 2 had three stems with six intact and

six notched leaves, plant 3 had ®ve stems with 12 intact and

eight notched leaves.

Main experiment

The preliminary experiment established that several ®rst-

instar larvae wandered from where they hatched on their initial

Fig. 1. Diagrammatic representation of two stems of an Asclepias humistrata plant showing the experimental and control stem treatments. Each

stem is shown with ®ve leaf pairs and an in¯orescence. The number 4 denotes the stem number, N denotes that the leaf petioles are notched,

while U denotes that the petioles are unnotched, i.e. left intact. Leaf pairs 2±4 of each experimental stem were notched (enlarged inset A), and

three eggs on individual leaf discs (enlarged inset B) were glued to the undersides of leaf pairs 2 and 3, for a total of 12 eggs per treated stem.

The fourth leaf pair 4 was also notched but received no eggs and served to expose any upward-wandering larvae to the same experimental

treatment. The left stem (4U) shows the control stem (no leaves notched) with 12 eggs on the same two leaf pairs. One leaf of leaf pair 1 on

both the notched and unnotched stems was punctured to obtain the initial latex volume samples. At the end of the experiment, two additional

sets of samples were taken from each plant: latex from the other leaf of leaf pair 1 for cardiac glycoside concentration and moisture analyses;

and a single unnotched or notched leaf was removed from each treatment or control stem (from leaf pair 2±4) for cardiac glycoside concentration

and moisture analyses. As indicated in Table 6, the actual numbers of leaf pairs ranged from 6 to 10.


214 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M214 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M

leaves onto other leaves, buds, and/or ¯owers. Consequently it

was impossible to compare notched and intact treatments on

any one stem. For the main experiment, an altered procedure

based on 31 matched stem pairs from 29 plants (two large

multi-stemmed plants with two pairs of matched stems) was

used. Stems were matched for length, the number of leaf pairs,

and ¯owering status. In a non-systematic fashion, each stem

was designated as either experimental, on which three leaf

pairs were notched, or control, on which all the leaf petioles

were left intact (compare stems labelled 4U and 4N in Fig. 1).

The experimental leaves were assigned as follows: the two

leaves of the ®rst major leaf pair counting from the base of the

stem were not notched; one leaf was used to assess latex

volume at the beginning of the experiment by a single puncture

of the midrib and by collecting the total latex out¯ow in a

capillary tube. The opposite leaf was pricked one or more

times at the end of the experiment and latex was collected to

assess latex cardiac glycoside concentration and per cent

moisture. The next ascending leaf pairs (Fig. 1, numbers 2±4)

were notched. Three egg-bearing leaf discs were glued onto

each of leaf pairs 2 and 3, for a total of 12 eggs per

experimental stem (Fig. 1). The fourth notched leaf pair was

left without eggs to assure that upwardly wandering larvae

would encounter other notched leaves. Leaves on the control

stems were not notched, and the placement of 12 eggs was the

same as on the experimental stems.

The numbers, locations, and fates of all larvae (live and

feeding, dead because of becoming mired in the latex, or

missing and presumed dead) were assessed for each plant/stem

twice: once » 24 h after all the eggs had hatched, and again

when all larvae were harvested, » 72 h after they hatched.

Larvae were harvested from the plants in approximately the

order in which they were placed out.

Larval weights, growth rates, and cardiac glycoside

concentrations

Harvested larvae from the experimental and control plants in

the ®eld were placed individually into 64 dram plastic cups,

labelled by plant number, treatment, and plant part from which

they were collected. Each cup was put into a cold ice chest and

each larva was weighed (wet weight, mg) in the laboratory

later the same evening on a Mettler AE240 balance (Mettler

Corporation, Jacksonville, Florida). Each larva was then frozen

to death in its cup in a laboratory freezer, dried for 16 h at

60 °C in a forced draught oven, and re-weighed (dry weight,

mg). Based on the approximate times the larvae had hatched

and were harvested, individual growth rates per hour spent on

the plant were calculated. Initial wet and dry weights of a

separate set of 25 newly hatched larvae were determined

individually within 1±2 h of hatching. Individual larval growth

rates of the ®eld larvae were calculated by subtracting the

mean wet weight of the 25 newly hatched larvae from the ®nal

wet weight of each individual harvested ®eld larva. This

weight gain for each ®eld larva was converted to a dry weight

basis, using the measured per cent moisture content of each

respective larva. These wet and dry weight values were divided

by the time the larvae had spent on the plant to give individual

wet and dry growth rates (mg h±1).

The larvae collected from the notched leaves on the

experimental stems and from the intact leaves on the control

stems were pooled separately to determine their two mean

cardiac glycoside concentrations as digitoxin equivalents per

0.1 g dry weight, using a standard spectrophotometric assay

(Brower et al., 1975).

Plant characteristics, leaf and latex samples

All experimental plant stems were measured (length, cm),

the number of leaf pairs counted, and the ¯owering status

recorded (early bud to freshly opened ¯owers). Before glueing

the egg-bearing leaf discs onto the leaves, the initial volume of

latex produced was measured by piercing the midrib vein of

one leaf of leaf pair 1 (see Fig. 1) using a number 5 insect pin,

and collecting the exuding latex in 100-ml capillary tubes.

Immediately after the larvae were harvested (72 h), three

additional samples were taken. Latex was again collected from

leaf pair 1, using the leaf opposite that from which the ®rst

latex sample had been collected. A pin was used to puncture

both the petiole and the midrib vein and the latex was taken up

using a Pasteur pipette. Each sample was deposited into pre-

weighed 10-ml ¯asks and kept on ice in a cooler. Back in the

laboratory, the ¯asks were weighed again to obtain the wet

weight of the latex. The latex was dried in the ¯asks for 16 h at

60 °C, and the ¯ask re-weighed a third time to obtain the dry

weight of the latex. Each dry latex sample was then extracted

in the ¯ask with 95% ethanol for cardiac glycoside analysis.

The second sample consisted of one notched leaf collected

from each experimental stem (usually from leaf pair 4, Fig. 1),

and the third sample consisted of one unnotched leaf, similarly

harvested from each control stem. Each leaf was placed into a

plastic bag, labelled, and stored on ice for transport back to the

laboratory. Here the leaves were weighed wet, dried for 16 h at

60 °C, and re-weighed, ground to a ®ne powder, weighed

again, then extracted for analysis of cardiac glycoside

concentration.

Natural cohort survival and behavioural observations

The fates of naturally laid eggs and larvae on 45 plants

located randomly on a north±south and east±west transect that

traversed the ®eld were recorded. Each plant was censused

every 3±4 days, from 7 to 25 April. Newly laid eggs found on

leaves were circled using a pen and their fates and the fates of

larvae were determined at each subsequent census.

To study general behavioural responses of larvae to the

latex, newly hatched larvae were placed onto intact and

notched leaves of plants not used in the above experiments.

Behaviours were recorded using a Canon L-1 Hi-8 video-

camera (Cannon Corp., B&H Photo-Video-Pro Audio Co.,

New York) with a CL 8±120 mm zoom lens attached to a CL

23 macro extender. From the video recordings, the time



individual larvae spent feeding, their reactions to the latex, and

other aspects of their behaviour were measured.

Larval feeding experiments

To assess the effect of latex on early stage larval feeding

preference, four 15-mm diameter discs from A. humistrata

leaves were placed equidistant from the centre and from

each other into each of 18 clear plastic dishes (11.4 cm

diameter 3 3.8 cm in depth) lined with moist tissue paper. A

drop of latex from A. humistrata was placed and smeared over

the dorsal surface of each alternate leaf disc. Four newly

hatched larvae were placed in the centre of each dish at

16.00 hours on 26 April. Dishes were kept at » 25 °C in the

dark. The location of larvae and the percentage damage to each

disc was assessed 41 h later.

Results

Survival rates from eggs to second instar on non-

experimental plants

Of 141 eggs laid naturally on the A. humistrata plants, 25

second-instar larvae (18%) were recovered. On 34 plants on

which there was at least one egg, the average survival per plant

from egg to second instar was 22%. On 14 plants, no larvae

survived beyond the ®rst instar.

Preliminary experimental trials

The distal portion of the leaves of A. humistrata whose

midribs had been notched with forceps, produced no, or

unmeasurably small amounts of, latex out¯ow when pricked

with a pin. Intact adjacent and lower leaves produced the usual

rapid out¯ow that characterises this milkweed (Zalucki &

Brower, 1992). Notched and intact leaves that were opposite

each other did not differ in cardiac glycoside concentration,

and while both appeared to have lower concentrations than the

intact leaf below, the differences were not signi®cant (Table 1).

Thus while leaves from different positions on a stem may

differ in cardiac glycoside concentration, the leaf notching

methodology cut off the latex ¯ow successfully without

changing the cardiac glycoside level in the leaf signi®cantly.

Note that the mean cardiac glycoside concentration in the latex

was 76±92 times higher than in the leaf samples (Table 1).

Forty-four per cent of the 112 larvae set out wandered off

the leaf on which they had been placed, and were mostly found

on the young leaves and/or ¯ower buds at the top of the stem

on which they had been set out. Larvae placed on intact leaves

to which the latex ¯ow had not been cut off were much more

likely to wander: only 23% of the initial 60 larvae were

recovered on the intact leaves. In contrast, nearly twice as

many placed on the notched leaves were recovered on notched

leaves (44% of the initial 52; test of proportions, z = 2.35,

P < 0.05).

The main experiment: effects of notched and intact leaves on

larvae

Survival of larvae. Twenty-eight per cent of the 372 larvae

on the intact leaves survived, compared with 59% of the same

number set on the notched leaves (c2 = 124, 1 d.f., P < 0.001;

Table 2). Larvae on the intact leaves were 14 times more likely

to be mired than were those on the notched leaves (c2 = 192,

d.f. = 1, P < 0.001). The proportion of missing larvae was also

slightly higher on the intact leaves (45 vs. 40%) but the

difference was not signi®cant (c2 = 2.7, d.f. = 1). Clearly,

survival of ®rst-instar larvae is much higher on stems with

Table 1. The preliminary experiment: cardiac glycoside concentrations (mg g±1 dry weight) in the notched leaves, the intact leaves opposite those

notched, the intact leaves immediately below the notched leaves, and in the latex collected from the notched leaves.

Sample Sample Mean

category size concentration SD Minimum Maximum

Notched 9 161 89 55 285

Opposite (intact) 9 168 56 109 275

Below (intact) 9 195 61 80 288

Latex (notched) 9 14 777 5506 5912 26 367

Paired t-test comparisons: notched vs. opposite: t = 0.323, P = NS; notched vs. below: t = 1.099, P = NS; opposite vs. below: t = 2.295, P = 0.05.

Table 2. The main experiment: numbers and fates of 744 ®rst-instar larvae originally placed on notched or on intact leaves.

Experimental leaf Larvae set Live larvae Larvae Larvae

category out recovered mired in latex missing

Notched 372 218 (59%) 7 (2%) 147 (40%)

Intact 372 103 (28%) 101 (27%) 168 (45%)



notched leaves, and a major cause of mortality on the intact

leaves is becoming mired in latex. This was supported further

by the fact that seven larvae that had originally been put on

notched leaves were found mired after having wandered onto

intact leaves or ¯ower buds.

Wandering behaviour of the larvae. The presence of normal

latex ¯ow into the leaves also affected the behaviour of the

larvae: they were more than twice as likely to wander off intact

leaves than off notched leaves. Thus, of the 86 larvae

recovered from those initially set out on intact leaves, 45%

had moved off the original leaf. In contrast, of those set out on

notched leaves, only 21% of 207 that were recovered had

moved (2 3 2 contingency table, c2 = 17.0, 1 d.f., P < 0.001;

Table 3).

Temporal change in survival rates. The pattern of survival

differed between the two treatments. On intact stems, 44% of

the 372 larvae had died (or disappeared) on the ®rst day and

51% of the 208 remaining had died (or disappeared) after 72 h.

The corresponding values for stems with notched leaves were

much lower: 28 and 18% respectively. This pattern justi®ed

ending the experiment after 72 h; if the larvae had been left on

the plants for a further 24 h, the numbers remaining on the

control stems would have been too small for comparing weight

changes.

Larval weights and growth rates. The surviving 293 ®rst-

instar larvae recovered after 72 h from the 744 eggs initially set

out were weighed individually. Because 82 of these larvae had

wandered off their original leaves (28 + 34 + 11 + 9; Table 3),

the data were broken down into the three categories

summarised in Table 4: larvae that stayed on the original leaf

pairs 2±4, larvae that wandered from their original placement

upwards onto the in¯orescences, and larvae that wandered

from their original placement onto the other intact leaves (see

Fig. 1).

The differences in wet and dry weights (Fig. 2) and growth

rates between larvae originally set out on notched vs. intact

Table 3. The main experiment. Wandering behaviour of ®rst-instar larvae recovered alive on various plant parts on the experimental and control

stems². As shown in Fig. 1, leaf pairs 2±4 were notched on the experimental stems and left intact on the control stems.

Plant parts on which the larvae were recovered²

Larvae initially Remained on Moved to Moved to other Total larvae

set on: leaves 2±4 ¯owers intact leaves³ recovered

Intact leaves 47 (55%) 28 (33%) 11 (13%) 86

Notched leaves 164 (79%) 34 (16%) 9 (4%) 207

²The lower number of larvae recovered here compared with Table 2 is because all recovered larvae could not be assigned to a particular plant

part.

³Most larvae that wandered off their original leaves moved upwards.

Table 4. The main experiment. Effect of notched vs. intact leaf treatments on mean ®rst-instar larval wet weights (mg), dry weights (mg), and

growth rates (mg h±1) for the total of 293 live larvae recovered from notched leaves on the experimental stems (164 larvae), and from intact

leaves on the control stems (47 larvae), from in¯orescences on the experimental stems (34 larvae) or control stems (28 larvae), and from other

intact leaves on the experimental (nine larvae) or control stems (11 larvae). Figure 2 is based only on the numbers of larvae recovered from

notched leaves on the experimental stems and intact leaves on the control stems.

Leaf treatment: originally on leaves that were:

Notched Intact

Notched/intact

n Mean SD n Mean SD ratio

Weights and growth rates for larvae recovered on notched and intact leaves

Wet weight 164 174 75 47 79 30 2.2

Dry weight 164 26 12.8 47 10 4.5 2.6

Growth 164 1.65 0.91 44 0.51 0.37 3.2

Weights and growth rates for larvae that moved onto in¯orescences

Wet weight 34 144 70 28 110 43 1.3

Dry weight 34 21 11 28 17 8 1.2

Growth 34 1.26 0.81 28 0.83 0.56 1.5

Weights and growth rates for larvae that moved onto other intact leaves

Wet weight 9 104 54 11 80 25 1.3

Dry weight 9 16 10 11 10 5 1.6

Growth 9 0.74 0.62 10 0.53 0.27 1.4



leaves in all three groups were highly signi®cant (two-way

ANOVA F1,287 = 19.47, P < 0.001). All three data sets indicate

that leaf notching enhanced larval weights and growth rates

signi®cantly. When the larvae stayed on their original notched

leaves (Table 4), the wet and dry weights and the growth rates

were 2.2±3.2 times greater than those that remained on intact

leaves.

The experiment indicated a highly signi®cant interaction

between treatment (notched or intact) and plant part where the

larvae were ultimately found (F2,287 = 6.89, P = 0.001;

Table 4), even though the effect of plant part per se was not

signi®cant (F2,287 = 2.17, P = NS; Table 4). Thus larvae origin-

ally set out on intact leaves that moved onto other intact leaves

grew at virtually the same slow rate as larvae that stayed on

their original intact leaves (Fig. 3, right, square vs. triangle).

Larvae originally on intact leaves that moved up onto

in¯orescences grew slightly faster than those that stayed on

the intact leaves (Fig. 3, right, square vs. circle). In contrast,

larvae originally on notched leaves that moved up onto

in¯orescences grew more slowly than those that stayed on their

original notched leaves (Fig. 3, left, square vs. circle), and

larvae that moved from notched onto other intact leaves grew

even more slowly (Fig. 3, left, circle vs. triangle).

The growth rates of larvae that had been placed on notched

leaves and later found on any plant parts were always faster

than those that remained on intact leaves, presumably

re¯ecting both the higher initial growth on the notched leaves

and the variable times spent wandering on the intact leaves.

The decline in growth rates of larvae originally on notched

leaves but recovered on other plant parts appears to be

correlated positively with the cardiac glycoside concentration

in the latex (see below).

Plant variables. Even though latex ¯ow was effectively

stopped by notching the leaf petioles, the intact and notched

leaves sampled at the end of experiment did not differ

signi®cantly either in cardiac glycoside concentration (Table 5,

Fig. 4a) or in per cent water (Table 5, Fig. 4b). This is

extremely important because it indicates that the principal

effect of the experimental notching was to cut off the latex

¯ow to the leaves without changing their water content or

cardiac glycoside concentrations signi®cantly.

The experimental and control stems also did not differ in the

initial number of leaf pairs per stem, stem length, or in the

volume of latex collected from leaf pair 1 at the beginning of

the experiment (Table 6). At the completion of the experiment,

both the per cent water and the concentration of cardiac

glycosides in the latex were higher in leaf pair 1 from stems

Fig. 2. (a) Frequency distributions of the wet weights of 211

surviving late ®rst-instar larvae. The histograms compare the 47

survivors of the 372 that had been set out as eggs on intact leaves

(grey bars) with the 164 survivors of the 372 that had been set out

on leaves with notched petioles (cross-hatched bars) in order to cut

off the latex ¯ow. The data are from Table 4. The mean difference

in wet weight is highly signi®cant: larvae are much heavier when

they feed on leaves to which the latex ¯ow has been stopped.

(b) Frequency distributions of dry weights comparing the same

surviving larvae shown in (a). The data are from Table 4. The mean

difference in dry weight is highly signi®cant: larvae are much

heavier when they feed on leaves to which the latex ¯ow has been

stopped.

Fig. 3. Growth rates of ®rst-instar larvae that stayed put or

wandered onto three different plant parts after they were initially set

out either on notched leaves (left side of diagram) or on intact

leaves (right side of diagram). The larvae are grouped by the plant

parts on which they were recovered: the original treatment leaves

(j), in¯orescences (d), or other intact leaves (m). The error bars

are 6 1 SD. The data are from Table 4. The growth rates are based

on the 72 h duration of the experiment. (See text for explanation.)



with notched leaves (Table 6, P < 0.01), suggesting a possible

response to the damage caused by notching.

Effects of latex on ®rst-instar larval feeding behaviour.

During 279 min of video recording of three larvae on notched

leaves, feeding bouts lasted on average 6.9 min (range

2±14 min). In contrast, the average feeding bout for two larvae

recorded for 244 min on intact leaves was 0.8 min (range

0.05±5 min), a greater than eight-fold difference. Larvae fed on

intact leaves until they encountered latex then ceased feeding,

displayed various avoidance behaviours (see Zalucki &

Brower, 1992), or wandered and found another feeding site.

Their inter-feeding times averaged 11.2 min (range 1±40 min).

On notched leaves, the inter-feeding periods were longer

(mean = 23.4 min, range 8±46 min), probably re¯ecting the

ingestion of more leaf material. Rather than tending to wander,

these larvae generally remained quiescent beside the small

hole they had chewed, defecated, then resumed feeding. This

resulted rapidly in a large hole or trench in the leaf and was in

marked contrast to the behaviour on intact leaves on which

they rarely managed to produce a major incision.

The separate leaf disc experiment provided additional

insight into the latex avoidance behaviour. The experimental

leaf discs smeared dorsally with latex were barely eaten,

suggesting that the larvae can avoid A. humistrata latex on the

basis of taste. The few discs eaten (nine of 36) had been

attacked mostly from the ventral side (six of nine), i.e. the side

lacking smeared latex. The proportion of leaf material eaten

per disc also differed markedly between the latex treated

(n = 18, mean = 0.04, SD = 0.067) and the untreated control

(n = 18, mean = 0.16, SD = 0.097) discs (t-test on arcsin-

transformed data, t = ±3.528, P < 0.01). Overall, only 10 of 72

larvae were found on eight of 36 latex-smeared discs,

compared with 31 larvae on 22 of 36 untreated discs (c2

corrected = 9.7, d.f. = 1, P < 0.01, for larvae on discs with vs.

without latex).

Relationships among variables

To investigate the relationship between larval growth rates

and plant variables, mean larval growth rates on the plants for

stems with notched vs. intact leaves were calculated. For the

intact group, this was frequently based on only one larva per

treated stem. There was signi®cant variation in larval growth

rates among plants (F30,43 = 2.19, P < 0.01) and, as expected,

signi®cant treatment effects on growth rate (F1,43 = 44.2,

P < 0.01), but the treatment 3 plant interaction was not

signi®cant (F28,43 = 1.13, P = NS), suggesting that the treatment

effects are consistent across plants but that plants differed in

the degree of the effect.

For 24 plants, there were data for larvae that survived on

both notched and intact leaves. These grew at different rates,

Table 5. The main experiment: cardiac glycoside concentration

(mg g±1 dry weight) and per cent water in 31 intact leaves from the

control stems and 31 notched leaves from the experimental stems.

The leaves were harvested at the end of the experiment from leaf

pairs 2±4 (see Fig. 1).

n Mean SD

Cardiac glycoside concentration

In intact leaves 31 350.5 108

In notched leaves 31 323.1 106

Paired t-test: d.f. = 30, t = ±1.14, P = NS

Per cent water

In intact leaves 31 86.9 1.4

In notched leaves 31 86.4 1.3

Paired t-test, arcsin transformation, d.f. = 30, t = ±1.92, P = NS

Fig. 4. (a) Frequency distributions comparing cardiac glycoside

concentrations in two groups of 31 leaf samples taken at the end of

the experiment. The cross-hatched bars are concentrations in the

notched leaves from the experimental stems. The solid bars are

concentrations in the intact leaves from the control stems. The

leaves were from leaf pairs 2±4 as shown in Fig. 1. The data are

from Table 5. The difference is not statistically signi®cant.

(b) Frequency distributions comparing per cent water in two groups

of 31 leaf samples taken at the end of the experiment as in (a). The

cross-hatched bars are the per cent water in the notched leaves from

the experimental stems. The solid bars are the per cent water in the

unnotched leaves from the control stems. The leaves were from leaf

pairs 2±4 as shown in Fig. 1. The data are from Table 5. The

difference is not statistically signi®cant.



but the effect was not uniform in magnitude on all plants. To

explore why the notching of some plants had a greater effect

than others, the growth rate differences (growth on notched

minus growth on intact for each plant) were regressed against

four measures of plant quality: per cent leaf moisture, per cent

latex moisture, latex cardiac glycoside concentration, and leaf

cardiac glycoside concentration.

There was no effect of per cent moisture in either the latex

or leaves on the growth rate differences, but the cardiac

glycoside concentration in both the latex (Fig. 5) and leaves

(Fig. 6) had signi®cant negative effects. Thus, the higher the

concentration of cardiac glycosides in either the latex or leaf

tissue, the greater the difference between growth rates on the

intact vs. notched leaves.

These data provide the ®rst de®nitive evidence that

monarchs incur a physiological cost due speci®cally to

ingesting cardiac glycosides from milkweeds; however, the

percentage of the variance accounted for by the regressions in

Table 6. The main experiment. Three plant measures taken on 31 control stems (with leaves intact) and on 31 treated stems (with leaves

notched) at the beginning of the experiment, and the cardiac glycoside concentration and per cent water in the latex of leaf pair 1 for the same

62 stems measured at the end of the experiment. All latex samples were taken from leaf pair 1.

Stem treatment

Leaves intact Leaves notched

Parameter Mean Range SD Mean Range SD Signi®cance

Measures taken at beginning of experiment

Leaf pairs per stem 7 6±9 0.9 7 6±10 1.0 NS1

Stem length (cm) 12.8 8±17 2 12.7 9±17 2.2 NS1

Latex volume (ml) 309 66±654 144 312 48±691 157 NS1

Measures taken at end of experiment2

Latex cardiac glycoside3

concentration 11 808 a 5089 15 191 b 4847 P < 0.014

Latex percentage water5 89 66±95 7 94 93±96 1 P < 0.016

1Not signi®cant by inspection.2All samples are from leaf pair 1 (see Fig. 2).3As mg per 0.1 g dry weight.4Paired t-test; t = 3.56, d.f. = 30, P < 0.01.5Water as per cent of wet weight of latex.6Paired t-test on arcsin-transformed data: t = 4.39, d.f. = 30, P < 0.01.aRange = 2889±23087.bRange = 7898±25069.

Fig. 5. The negative effect of latex cardiac glycoside concentration

on the difference in the growth rates of larvae collected from stems

with intact leaves and stems with notched leaves (see text for

explanation).

Fig. 6. The negative effect of leaf cardiac glycoside concentration

on the difference in the growth rates of larvae collected from stems

with intact leaves and stems with notched leaves (see text for

explanation).



each case was low (r2 = 0.18 for latex, r2 = 0.37 for leaf cardiac

glycoside concentration). The large residual variation suggests

that other plant factors also in¯uenced larval growth rates.

These could be plant nutrient levels or other secondary plant

chemicals in the leaves and/or latex.

Discussion

Latex reduces ®rst-instar larval growth rates

There was a severe detrimental effect of the latex of

A. humistrata on the growth rate of ®rst-instar monarch larvae.

Larvae feeding on leaves whose petioles had been notched

experimentally to cut off the latex out¯ow grew at more than

three times the rate of larvae on intact control leaves. This

resulted in the wet and dry weights of these larvae being more

than twice those for larvae feeding on intact leaves during the

72-h period of the experiment. Whether this growth advantage

continues beyond the ®rst instar needs investigating.

Latex increases ®rst-instar larval mortality

Only 28% of the ®rst-instar larvae survived up to 72 h when

originally placed as eggs on intact leaves of A. humistrata,

compared with 59% survival when originally placed on

notched leaves. Moreover, 27% of the mortality on the intact

plants was due to the larvae becoming mired in the latex,

compared with only 2% miring on the notched stems. Thus

feeding on the intact leaves was nearly 15 times more likely to

kill the larvae than when they fed on leaves to which the ¯ow

of latex had been cut off by notching.

Growth reduction factors include the time involved by the

larvae in latex avoidance behaviour, in moving to other plant

parts, and in becoming inactive (cataleptic) after ingesting

latex. Because mortality of early instars due to ants,

Neuroptera, and other entomophages must be time dependent,

any factors that slow growth rates will probably also reduce

survival by exposing larvae to various predators for longer.

Had the experiment continued to the end of the ®rst instar,

survival would have been lower, comparable with that

recorded earlier on this host, i.e. about 10% (Zalucki &

Brower, 1992). Life is dif®cult for neonate monarch larvae

feeding on A. humistrata.

Death due to miring was more likely on plants with lower

latex cardiac glycoside concentrations and may have resulted

from larvae imbibing more latex when feeding on lower

concentration plants and becoming cataleptic and/or dis-

oriented, and thus being more likely to be glued to the leaf

as the latex dried.

Many studies of Lepidoptera have found that loss in early

instars accounts for most mortality per generation (Cohen &

Brower, 1982; Zalucki & Kitching, 1982; Dempster, 1983;

Kyi et al., 1991). As with growth rates, factors associated with

the food plant, the physical environment, predators, and

competitors all interact in affecting survival.

Latex avoidance behaviours by ®rst-instar larvae

While ®rst-instar larvae attempted to circumvent the

A. humistrata latex by vein snipping, trenching, and by

moving to feed away from the latex vessels (cf. Dussourd,

1993), whenever they encountered latex their feeding was

disrupted and none of these ploys seemed very effective. Their

behaviour in this study stands in sharp contrast with that

reported by Rothschild (1977) and Dixon et al. (1978), who

stated that ®fth-instar larvae actively seek out and drink milky

latex oozing from injured leaves and stems.

In the above choice experiment, ®rst instars avoided latex-

treated leaf discs (probably on the basis of taste), and on wild

intact stems they often moved to ¯owering structures away

from the latex-producing leaves. Growth rates were higher for

these larvae, which may indicate that ¯owers and buds have

lower latex out¯ow, are nutritionally richer (e.g. Chew &

Robbins, 1984), or provide better micro-conditions (e.g.

Willmer, 1986). Feeding on ¯ower petals did not initiate

copious latex out¯ow, although feeding on the ¯ower petioles

did.

Detrimental effects of various plant constituents on the

larvae

Apart from the physical miring caused by the latex, the

cardiac glycoside concentration in the latex and leaves is partly

responsible for lowering ®rst-instar larval growth rates. Larvae

that had moved onto ¯owers above the notched leaves grew

more slowly than those on notched leaves, whereas those on

¯owers above the intact leaves grew faster than those on the

intact leaves. These differences in growth rate were correlated

with higher cardiac glycoside levels in the latex of the

experimentally damaged plants. This may indicate a plant

response to damage and constitute an induced defence

(Malcolm & Zalucki, 1996).

Growth rates of Lepidoptera on their host plants are

in¯uenced by many factors including moisture, nutritional

contents, and multiple effects of secondary plant compounds

(Scriber & Slansky, 1981; Herms & Mattson, 1992; Slansky,

1992). The last will vary among plants, plant parts, and

developmental stages of the plant (Nelson et al., 1981; Brower

et al., 1982). The experimental design enabled some plant

effects to be controlled. Growth on treated stems estimated the

maximum possible rate in the absence of latex on plants under

®eld conditions. The difference between this rate and that

achieved on intact stems indicated the effect of plant variables

on growth rates. The difference was greater on plants with

higher concentrations of latex and leaf cardiac glycoside

concentrations (Fig. 6). There were other effects on growth

rates, as indicated by the scatter of points and by the low

variance explained by cardiac glycoside measures alone. These

may include latex constituents (e.g. rubber compounds) and/or

leaf chemistry, moisture, physical attributes, and nutrient

levels.

Most studies showing negative correlations between

secondary plant compounds and larval performances or growth



rates were carried out under arti®cial laboratory conditions,

used late-instar larvae, or incorporated compounds into

arti®cial diets. The experiments reported here demonstrated

negative effects of both latex and cardiac glycosides directly

under natural ®eld conditions and used ®rst-instar larvae that

are far more ecologically relevant (cf. Chapman et al., 1983;

Eigenbrode et al., 1991).

The neonate larvae did not perform well either on the intact

leaves or on plants with higher cardiac glycoside concentra-

tions. Moreover, the mean cardiac glycoside concentration in

the 86 larvae recovered from intact leaves was more than twice

that of the 206 larvae recovered from the notched leaves (1350

vs. 550 mg/0.1 g dw). Much of this difference may be due to the

presence of more latex in the gut of the larvae recovered from

intact leaves. Although survival was lower on intact than on

notched leaves, the question of whether larvae on intact leaves

may be better protected from predators due to this higher

cardiac glycoside level was not addressed. The possible trade-

off of lower predation rate with higher mortality caused by the

latex and cardiac glycosides requires further research. Another

untested effect of latex ingestion is that catalepsis and

disorientation may cause the far more ecologically relevant

®rst-instar larvae to fall off the plants and increase the

probability that they will be killed by predators.

Caveat on utilising late-instar larvae in bioassays

The use of late-instar larvae to determine effects of physical

and chemical plant characteristics on growth and food

consumption may be misleading (e.g. Schroeder, 1976). Thus

later instars may be effectively unaffected by plant variables

because of body size, their ability to select plant parts that are

less toxic, and by being less subject to toxicosis and physical

effects, including miring in the latex. A case in point is a study

by Erickson (1973) that utilised fourth-instar monarch larvae

reared on four species of milkweeds Asclepias curassavica,

A. syriaca, A. incarnata L., and A. tuberosa L. While these

plants vary substantially in latex production, cardiac glycoside

concentration, and constituent cardiac glycosides, Erickson

found no correlations of these parameters with larval growth or

survival. Larger larvae are easier to handle but using them can

miss crucially important effects on neonates and therefore lead

to ecologically irrelevant conclusions.

Comparison of Asclepias humistrata with three other North

American milkweeds

Zalucki and Malcolm (1999) undertook a similar leaf

petiole notching experiment on three different milkweed

species in Michigan. They found that ®rst-instar survivorship

and growth were dependent both on the species and the high or

low volumes of latex produced. While survival rates were

similar, growth was more rapid on notched than on intact

leaves of the high-latex volume and low-cardenolide milkweed

Asclepias syriaca. On the low-latex volume and low-

cardenolide milkweed A. tuberosa, growth and survival were

affected marginally, while neither growth nor survival was

affected on the low-latex volume and low-cardenolide milk-

weed A. incarnata.

These results contrast sharply with the ®ndings on

A. humistrata, which has both high latex and high cardenolide.

When the authors compared larval growth rates on a common

degree-day time scale among all four milkweed species, they

found that growth rates were identical on leaves with notched

petioles, supporting the contention that the latex and the

included cardenolides are both important in affecting ®rst-

instar monarch larval growth rate and survivorship negatively.

Is a co-evolutionary arms race still occurring?

The monarch is considered a milkweed specialist (Ackery &

Vane-Wright, 1984; Malcolm & Brower, 1986; Borkin, 1993)

with various adaptations for feeding on and overcoming the

latex-based defences of these plants (Dussourd, 1990, 1993).

The results presented here indicate, however, that ®rst-instar

monarchs have dif®culty surviving on A. humistrata. The latex

has a strong effect on larval feeding ef®ciency and behaviour,

and acts both as a poison and a glue. The small size of ®rst-

instar larvae seems to impose a physical constraint on their

ability to handle A. humistrata and probably, to some degree,

all latex-bearing milkweeds. Once the hurdle of the early

instars is passed, monarch larvae appear better able to deal

with the latex-based defences of the milkweed, i.e. vein

snipping and avoiding ingestion of the latex is physically

easier and more effective. The phenology of larvae becoming

less constrained by latex needs further research.

The migratory biology of monarchs (Brower, 1996) may be

such as to preclude the evolution of optimal larval perfor-

mances on all milkweed species. In North America, the eastern

population of monarchs migrates from overwintering sites in

Central Mexico to the south-eastern U.S.A. in early spring.

Here females oviposit on several milkweed species, including

A. viridis Walt, Fl. Carol. and A. humistrata (Malcolm et al.,

1993), that differ in latex and cardiac glycoside levels and

other characteristics (Zalucki & Malcolm, 1999).

Monarchs normally spend only one generation on the

southern milkweeds before high temperatures force their

continued northward movement (Cockrell et al., 1993;

Knight et al., 1999). In the northern range, the predominant

host is A. syriaca (Malcolm et al., 1989, 1993; Wassenaar &

Hobson, 1999), which produces large amounts of latex but has

low cardiac glycoside concentrations (Zalucki & Malcolm,

1999). Thus selection due to high cardiac glycoside concen-

trations in the southern milkweeds may be relaxed as the

butter¯ies move northwards, i.e. the monarchs' annual

migration cycle (Brower, 1996) probably precludes close

adaptation to any one host. The prediction from this scenario is

that in those populations where migration does not occur, and

where the species is largely restricted to a single host as in

southern Florida (Knight et al., 1999), and in island popula-

tions, larval performance on the restricted hosts should

improve.



The work reported here has shown that there is a major cost

to monarch butter¯ies that oviposit on A. humistrata.

Nevertheless, nearly 19 years of study at Cross Creek have

shown that monarchs breed here regularly each spring (Knight

et al., 1999; L. P. Brower, in prep.) and frequently defoliate

entire milkweed plants. This milkweed is in the line of ®re of

numerous specialist milkweed herbivores during the spring in

the southern U.S.A. and may have evolved such effective

chemical defences that it is a suboptimal milkweed host for

monarchs. Many of the insects associated with milkweeds

sabotage the latex system in various ways (e.g. Tetropis and

Rhysomatus beetles). This suggests that the presence of two or

more specialist herbivores on plants at one time may improve

each other's abilities in dealing with the latex. Taken together,

the evidence suggests that co-evolutionary interactions of the

monarch butter¯y with milkweeds are occurring (cf. Dussourd,

1990).

Acknowledgements

We thank Mr and Mrs Zane Hogan and Zac for access to their

land; Tonya Van Hook for helping with the ®eld research,

Laurie Walz for Fig. 1, Anthony Clarke, Steve Malcolm and

Linda Fink for comments on the manuscript; and Robert

Lederhouse for discussion during the design of the experiment.

Videotaping was made possible by the generosity of Mr Glenn

L. Allen Jr of Walnut Creek, California. This research was

supported by grants to M.P.Z. from UQ and to L.P.B., principal

investigator of NSF GB1624545-12 to the University of

Florida.

References

Ackery, P.R. & Vane-Wright, R.I. (1984) Milkweed Butter¯ies: their

Cladistics and Biology. Cornell University Press, Ithaca, New York.

Borkin, S.S. (1993) Rejection of Apocynum androsaemifolium and

A. sibiricum (Apocynaceae) as food plants of larvae of Danaus

plexippus: refutation of early accounts. Biology and Conservation of

the Monarch Butter¯y (ed. by S. B. Malcolm and M. P. Zalucki),

pp. 125±128. Natural History Museum of Los Angeles County

Science Series no. 38, Los Angeles, California.

Brewer, J. & Winter, D. (1977) Short-lived phenomena. News of the

Lepidopterists' Society, 1977, 7.

Brower, L.P. (1969) Ecological chemistry. Scienti®c American, 220,

22±29.

Brower, L.P. (1984) Chemical defence in butter¯ies. The Biology of

Butter¯ies (ed. by R. I. Vane-Wright and P. R. Ackery),

pp. 109±134. Academic Press, London.

Brower, L.P. (1996) Monarch butter¯y orientation: missing pieces of a

magni®cent puzzle. Journal of Experimental Biology, 199, 93±103.

Brower, L.P., Edmunds, M. & Mof®tt, C.M. (1975) Cardenolide

contents and palatability of a population of Danaus chrysippus

butter¯ies from west Africa. Journal of Entomology, 49, 183±196.

Brower, L.P. & Fink, L.S. (1985) A natural toxic defense system:

cardenolides in butter¯ies vs. birds. Experimental Assessments and

Clinical Applications of Conditioned Food Aversions (ed. by

N. S. Braveman and P. Bronstein), pp. 171±188. New York

Academy of Sciences, New York.

Brower, L.P., Nelson, C.J., Seiber, J.N., Fink, L.S. & Bond, C. (1988)

Exaptation as an alternative to co-evolution in the cardenolide-based

chemical defence of monarch butter¯ies (Danaus plexippus L.)

against avian predators. Chemical Mediation of Coevolution (ed. by

K. C. Spencer), pp. 447±475. Academic Press, New York.

Brower, L.P., Seiber, J.N., Nelson, C.J., Tuskes, P. & Lynch, S.P.

(1982) Plant-determined variation in the cardenolide content, thin

layer chromatography pro®les, and emetic potency of monarch

butter¯ies, Danaus plexippus reared on the milkweed, Asclepias

eriocarpa in California. Journal of Chemical Ecology, 8,

579±633.

Chapman, R.F., Woodhead, S. & Bernays, E.A. (1983) Survival and

dispersal of Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) in

two cultivars of sorghum. Bulletin of Entomological Research, 73,

65±74.

Chew, F.S. & Robbins, R.K. (1984) Egg-laying in butter¯ies. The

Biology of Butter¯ies (ed. by R. I. Vane-Wright and P. R. Ackery),

pp. 65±79. Academic Press, London.

Cockrell, B.J., Malcolm, S.B. & Brower, L.P. (1993) Time,

temperature, and latitudinal constraints on the annual recolonization

of eastern North America by the monarch butter¯y. Biology and

Conservation of the Monarch Butter¯y (ed. by S. B. Malcolm and

M. P. Zalucki), pp. 233±251. Publications of the Los Angeles

County Museum of Natural History, Los Angeles, California.

Cohen, J.A. & Brower, L.P. (1982) Oviposition and larval success of

wild monarch butter¯ies (Lepidoptera: Danaidae) in relation to host

plant size and cardenolide concentration. Journal of the Kansas

Entomological Society, 55, 343±348.

Dempster, J.P. (1983) The natural control of populations of butter¯ies

and moths. Biological Reviews, 58, 461±481.

Dixon, C.A., Erickson, J.M., Kellett, D.N. & Rothschild, M. (1978)

Some adaptations between Danaus plexippus and its food plant,

with notes on Danaus chrysippus and Euploea core (Insecta:

Lepidoptera). Journal of Zoology London, 185, 437±467.

Dussourd, D.E. (1990) The vein drain; or how insects outsmart plants.

Natural History, 90, 44±49.

Dussourd, D.E. (1993) Foraging with ®nesse: caterpillar adaptations

for circumventing plant defenses. Caterpillars: Ecological and

Evolutionary Constraints on Foraging (ed. by N. E. Stamp and

T. M. Casey), pp. 92±131. Chapman & Hall, New York.

Dussourd, D.E. (1995) Entrapment of aphids and white¯ies in lettuce

latex. Annals of the Entomological Society of America, 88, 163±172.

Dussourd, D.E. & Denno, R.F. (1991) Deactivation of plant defense:

correspondence between insect behavior and secretory canal

architecture. Ecology, 72, 1383±1396.

Dussourd, D.E. & Denno, R.F. (1994) Host range of generalist

caterpillars: trenching permits feeding on plants with secretory

canals. Ecology, 75, 69±78.

Dussourd, D. & Eisner, T. (1987) Vein-cutting behavior: insect

counterploy to the latex defense of plants. Science, 237, 898±901.

Eigenbrode, S.D., Espilie, K.E. & Shelton, A.M. (1991) Behaviour of

neonate diamondback moth larvae (Plutella xylostella (L.)) on

leaves and on extracted leaf waxes of resistant and susceptible

cabbages. Journal of Chemical Ecology, 17, 1691±1704.

Erickson, J.M. (1973) The utilization of various Asclepias species by

larvae of the monarch butter¯y, Danaus plexippus. Psyche, 80,

230±244.

Farrell, B., Dussourd, D. & Mitter, C. (1991) Escalation of plant

defense: do latex/resin canals spur plant diversi®cation? American

Naturalist, 138, 891±900.

Glendinning, J.I. & Brower, L.P. (1990) Feeding and breeding

responses of ®ve mice species to overwintering aggregations of

the monarch butter¯y. Journal of Animal Ecology, 59, 1091±1112.



Herms, D.A. & Mattson, W.J. (1992) The dilemma of plants: to grow

or to defend. Quarterly Review of Biology, 67, 283±335.

Knight, A.L., Brower, L.P. & Williams, E.H. (1999) A population

study of spring remigrating monarch butter¯ies (Danaus plexippus

L., Lepidoptera: Nymphalidae) in North Central Florida. Biological

Journal of the Linnean Society, 68, 531±556.

Kyi, A., Zalucki, M.P. & Titmarsh, I.J. (1991) An experimental study

of early stage survival of Helicoverpa armigera (Lepidoptera:

Noctuidae) on cotton. Bulletin of Entomological Research, 81,

263±271.

Lucansky, T.W. & Clough, K.T. (1986) Comparative anatomy and

morphology of Asclepias perennis and Asclepias tuberosa sub-

species rolfsii. Botanical Gazette, 147, 290±301.

Malcolm, S.B. (1991) Cardenolide-mediated interactions between

plants and herbivores. Herbivores: their Interactions with

Secondary Plant Metabolites, 2E Vol. 1: the Chemical

Participants (ed. by G. A. Rosenthal and M. R. Berenbaum),

pp. 251±296. Academic Press, New York.

Malcolm, S.B. (1995) Milkweed, monarch butter¯ies and the

ecological signi®cance of cardenolides. Chemoecology, 5±6,

101±117.

Malcolm, S.B. & Brower, L.P. (1986) Selective oviposition by

monarch butter¯ies (Danaus plexippus L.) in a mixed stand of

Asclepias curassavica L. and A. incarnata L. in south Florida.

Journal of the Lepidopterists' Society, 40, 255±263.

Malcolm, S.B. & Brower, L.P. (1989) Evolutionary and ecological

implications of cardenolide sequestration in the monarch butter¯y.

Experientia, 45, 284±295.

Malcolm, S.B., Cockrell, B.J. & Brower, L.P. (1987) Monarch

butter¯y voltinism: effects of temperature constraints at different

latitudes. Oikos, 49, 77±82.

Malcolm, S.B., Cockrell, B.J. & Brower, L.P. (1989) Cardenolide

®ngerprint of monarch butter¯ies reared on common milkweed,

Asclepias syriaca L. Journal of Chemical Ecology, 15, 819±853.

Malcolm, S.B., Cockrell, B.J. & Brower, L.P. (1993) Spring

recolonization of eastern North America by the monarch butter¯y:

successive brood or single sweep migration? Biology and


M. P. Zalucki), pp. 253±267. Natural History Museum of Los

Angeles County, Los Angeles, California.

Malcolm, S.B. & Zalucki, M.P. (1996) Milkweed latex and cardenolide

induction may resolve the lethal defence paradox. Entomologia

experimentalis et applicata, 80, 193±196.

Nelson, C.J. (1993) Sequestration and storage of cardenolides and

cardenolide glycosides by Danaus plexippus plexippus and

D. chrysippus petilia when reared on Asclepias fruticosa: with a

review of some factors that in¯uence sequestration. Biology and


M. P. Zalucki), pp. 91±105. Natural History Museum of Los

Angeles County, Los Angeles, California.

Nelson, C.J., Seiber, J.N. & Brower, L.P. (1981) Seasonal and

intraplant variation of cardenolide content in the California

milkweed, Asclepias eriocarpa, and implications for plant defense.

Journal of Chemical Ecology, 7, 981±1010.

Oyeyele, S.O. & Zalucki, M.P. (1990) Cardiac glycosides and

oviposition by Danaus plexippus on Asclepias fruticosa in south-

east Queensland (Australia) with notes on the effect of plant

nitrogen content. Ecological Entomology, 15, 177±185.

Rothschild, M. (1977) The cat-like caterpillar. News of the

Lepidopterists' Society, 1977, 9.

Schroeder, L.A. (1976) Energy, matter and nitrogen utilization by the

larvae of the monarch butter¯y Danaus plexippus. Oikos, 27,

259±264.

Scriber, J.M. & Slansky, F.J. (1981) The nutritional ecology of

immature insects. Annual Review of Entomology, 26, 183±211.

Seiber, J.N., Nelson, C.J. & Lee, S.M. (1982) Cardenolides in the latex

and leaves of seven Asclepias species and Calotropis procera.

Phytochemistry, 21, 2343±2348.

Slansky, F., Jr (1992) Allelochemical±nutrient interactions in herbivore

nutritional ecology. Herbivores: their Interactions with Secondary

Plant Metabolites, 2E, Vol. 1: the Chemical Participants (ed. by

G. A. Rosenthal and M. R. Berenbaum), pp. 135±174. Academic

Press, New York.

Van Emon, J.V. & Seiber, J.N. (1985) Chemical constituents and

energy content of two milkweeds, Asclepias speciosa and A.

curassavica. Economic Botany, 39, 47±55.

Van Hook, T. & Zalucki, M.P. (1991) Oviposition by Danaus

plexippus (Nymphalidae; Danainae) on Asclepias viridis in northern

Florida. Journal of the Lepidopterists' Society, 45, 215±221.

Wassenaar, L.I. & Hobson, K.A. (1999) Natal origins of migratory

monarch butter¯ies at wintering colonies in Mexico: new isotopic

evidence. Proceedings of the National Academy of Sciences USA,

95, 15436±15439.

Willmer, P. (1986) Microclimatic effects on insects at the plant

surface. Insects and the Plant Surface (ed. by B. Juniper and

T. R. E. Southwood), pp. 65±80. Edward Arnold, London.

Woodson, R.E., Jr (1954) The North American species of Asclepias L.

Annals of the Missouri Botanical Garden, 41, 1±211.

Zalucki, M.P. & Brower, L.P. (1992) Survival of ®rst instar larvae of

Danaus plexippus (Lepidoptera: Danainae) in relation to cardiac

glycoside and latex content of Asclepias humistrata

(Asclepiadaceae). Chemoecology, 3, 81±93.

Zalucki, M.P., Brower, L.P. & Malcolm, S.B. (1990) Oviposition by

Danaus plexippus in relation to cardenolide content of three

Asclepias species in the southeastern U.S.A. Ecological

Entomology, 15, 231±240.

Zalucki, M.P. & Kitching, R.L. (1982) Temporal and spatial variation

of mortality in ®eld populations of Danaus plexippus L. and

D. chrysippus L. larvae (Lepidoptera: Nymphalidae). Oecologia, 53,

201±207.

Zalucki, M.P. & Malcolm, S.B. (1999) Plant latex and early stage

monarch larval growth and survival on three North American

milkweed species. Journal of Chemical Ecology, 25, 1827±1842.

Zalucki, M.P., Oyeyele, S. & Vowles, P. (1989) Selective oviposition

by Danaus plexippus L. (Lepidoptera: Nymphalidae) in a mixed

stand of Asclepias fruticosa and A. curassavica in southeast

Queensland. Journal of the Australian Entomological Society, 28,

141±146.

Accepted 5 August 2000



Documents

Detrimental effects of latex and cardiac glycosides on ...biology/Classes/269/monarchs.pdfDetrimental effects of latex and cardiac glycosides on survival and growth of ﬁrst-instar