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:
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.
# 2001 Blackwell Science Ltd, Ecological Entomology, 26, 212±224
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
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Milkweed latex vs. ®rst-instar monarch larvae 215Milkweed latex vs. ®rst-instar monarch larvae 215
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%)
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216 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M216 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M
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
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Milkweed latex vs. ®rst-instar monarch larvae 217Milkweed latex vs. ®rst-instar monarch larvae 217
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.)
# 2001 Blackwell Science Ltd, Ecological Entomology, 26, 212±224
218 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M218 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M
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.
# 2001 Blackwell Science Ltd, Ecological Entomology, 26, 212±224
Milkweed latex vs. ®rst-instar monarch larvae 219Milkweed latex vs. ®rst-instar monarch larvae 219
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).
# 2001 Blackwell Science Ltd, Ecological Entomology, 26, 212±224
220 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M220 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M
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
# 2001 Blackwell Science Ltd, Ecological Entomology, 26, 212±224
Milkweed latex vs. ®rst-instar monarch larvae 221Milkweed latex vs. ®rst-instar monarch larvae 221
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.
# 2001 Blackwell Science Ltd, Ecological Entomology, 26, 212±224
222 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M222 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M
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.
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Accepted 5 August 2000
# 2001 Blackwell Science Ltd, Ecological Entomology, 26, 212±224
224 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M224 Myron P. Zalucki, Lincoln P. Brower and Alfonso Alonso-M