Ecological Entomology (2009), 34, 551–561
DOI: 10.1111/j.1365-2311.2009.01107.x
Crowding and disease: effects of host density on
response to infection in a butterfly–parasite interaction
E L I Z A B E T H L I N D S E Y 1 , 2 , M U D R E S H M E H TA 2 , VA R U N
D H U L I PA L A 2 , K A R E N O B E R H A U S E R 3 and S O N I A A LT I Z E R 4
1
Graduate
Program in Population Biology, Ecology, and Evolution, Division of Biological and Biomedical Sciences, Emory University, Atlanta,
Georgia, U.S.A., 2 Department of Environmental Studies, Emory University, Atlanta, Georgia, U.S.A., 3 Department of Fisheries,
Wildlife and Conservation Biology, University of Minnesota, St. Paul, Minnesota, U.S.A. and 4 Odum School of Ecology, University
of Georgia, Athens, Georgia, U.S.A.
Abstract. 1. Hosts experiencing frequent variation in density are thought to benefit
from allocating more resources to parasite defence when density is high (‘densitydependent prophylaxis’). However, high density conditions can increase intra-specific
competition and induce physiological stress, hence increasing host susceptibility to
infection (‘crowding-stress hypothesis’).
2. We studied monarch butterflies (Danaus plexippus) and quantified the effects
of larval rearing density on susceptibility to the protozoan parasite Ophryocystis
elektroscirrha. Larvae were inoculated with parasite spores and reared at three density
treatments: low, moderate, and high. We examined the effects of larval density on
parasite loads, host survival, development rates, body size, and wing melanism.
3. Results showed an increase in infection probability with greater larval density.
Monarchs in the moderate and high density treatments also suffered the greatest negative
effects of parasite infection on body size, development rate, and adult longevity.
4. We observed greater body sizes and shorter development times for monarchs reared
at moderate densities, and this was true for both unparasitised and parasite-treated
monarchs. We hypothesise that this effect could result from greater larval feeding
rates at moderate densities, combined with greater physiological stress at the highest
densities.
5. Although monarch larvae are assumed to occur at very low densities in the wild, an
analysis of continent-wide monarch larval abundance data showed that larval densities
can reach high levels in year-round resident populations and during the late phase of the
breeding season. Treatment levels used in our experiment captured ecologically-relevant
variation in larval density observed in the wild.
Key Words. Danaus plexippus, density-dependent prophylaxis, host–parasite interaction,
melanism, monarch butterfly, neogregarine protozoan, Ophryocystis elektroscirrha.
Introduction
It is often assumed that animals living in larger groups or at
higher population densities should experience a greater risk of
acquiring infectious diseases (Alexander, 1974; Freeland, 1976;
Correspondence: Sonia Altizer, Odum School of Ecology, University
of Georgia, Athens, GA 30602, U.S.A. E-mail: saltizer@uga.edu
2009 The Authors
Journal compilation 2009 The Royal Entomological Society
Møller et al., 1993; Krause & Ruxton, 2002; Moore, 2002;
Altizer et al., 2003). This is mainly because host contact rates
and the transmission of parasites spread by close proximity
among individuals are predicted to increase with host population density (Anderson & May, 1979, 1981; McCallum et al.,
2001; Lloyd-Smith et al., 2005). Several field and experimental studies support this assumption; mammals (Freeland,
1979; Hoogland, 1979, 1995; Wilkinson, 1985), birds (Brown &
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E. Lindsey et al.
Brown, 1986; Shields & Crook, 1987), and insects (Dwyer &
Elkinton, 1993; Knell et al., 1996; Ryder et al., 2005) exhibit
positive relationships between measures of parasite prevalence
or intensity, and host population density or group size. For example, in the African army worm (Spodoptera exempta), high
host density results in increased host activity (Reeson et al.,
2000), which directly affects contact rates between susceptible
hosts and pathogens.
As a result of increased parasite risk, animals living at
higher population densities are predicted to invest more resources in resistance to infection, including behavioural and
immune defences (Møller & Erritzoe, 1996; Møller et al.,
2001). Within a species, increased host resistance in response
to crowding or higher host density has been termed ‘densitydependent prophylaxis’ (Reeson et al., 1998; Wilson &
Reeson, 1998). Under this scenario, greater investment in parasite resistance is presumed to counter the risk of increased
transmission (Barnes & Siva-Jothy, 2000; Wilson et al., 2001).
There is substantial evidence that animals experiencing higher
densities do indeed show greater resistance to infectious
diseases (Kunimi & Yamada, 1990; Goulson & Cory, 1995;
Reeson et al., 1998; Wilson et al., 2002). For example, one
study showed that in a phase-polymorphic moth species, darkcoloured larvae (the high density phenotype) exhibited greater
immune defences than pale-coloured larvae (based on higher
haemolymph and cuticular phenoloxidase activity and a
stronger encapsulation response; Cotter et al., 2004). Other
studies have shown that larvae reared under higher densities tend to develop darker cuticular melanism (Simmonds &
Blaney, 1986; Hagen et al., 2003; Lee & Wilson, 2006). This
is important because for some species, an increase in external
melanism correlates with an increase in immune effector traits
(Reeson et al., 1998; Barnes & Siva-Jothy, 2000; Wilson et al.,
2001; Cotter et al., 2004), although this trend does not hold for
all insect species (Robb et al., 2003; Pie et al., 2005; Hagen
et al., 2006). It is also important to note that some studies have
found no effect of host density on measures of immunity, including field crickets (Adamo, 2006) and termites (Pie et al.,
2005).
Animals living in high density populations might also experience more intense competition for resources. Thus, a second
key hypothesis is that high density leads to physiological or nutritional stress, and that animals in crowded conditions will be
more susceptible to infectious diseases relative to less crowded
hosts. In insects, this hypothesis was initially examined by
Steinhaus (1958) in studies of caterpillars and their natural
pathogens. More recent experimental studies on lepidopteran
hosts have shown that animals reared at higher densities experience reduced disease resistance and/or decreased time to death
(Goulson & Cory, 1995; Reilly & Hajek, 2008). However,
Brown et al. (2003) found no effect of host-resource stress on
infection or immunity in bumblebees.
Here we ask how host rearing density affects the outcome
of infection by a common protozoan parasite, Ophryocystis
elektroscirrha, in monarch butterflies, Danaus plexippus.
Parasitism by O. elektroscirrha occurs in all monarch populations examined to date, but prevalence varies widely both
within and among populations (Leong et al., 1997; Altizer
et al., 2000). Monarch densities in the wild vary over space and
time (Ackery & Vane-Wright, 1984; Prysby & Oberhauser,
2004). Hence, it is possible that effects of host density on susceptibility to infection will affect patterns of infection in the
wild. We reared monarch larval stages under low (single larva),
moderate, and high densities and compared infection probability, development, and survival among treatments. We expected
that monarchs reared under the highest density treatment would
develop the fastest (based on time to pupation and adulthood),
and show smaller body sizes than monarchs reared at lower
densities. Monarchs reared under the highest densities could
show greater susceptibility to infection, in support of the stressdisease hypothesis, and would thus experience greater lethal
and sub-lethal effects of parasitism on host fitness. Alternatively,
monarchs reared under high densities could invest more in disease resistance, in support of the density-dependent prophylaxis hypothesis. This could be manifested by darker wing
coloration, which may correlate with resistance to infection
(Barnes & Siva-Jothy, 2000).
Materials and methods
Host–parasite system
Monarch butterflies inhabit islands and continents worldwide (Ackery & Vane-Wright, 1984), migrate annually in temperate North America and Australia (Urquhart & Urquhart,
1978; James, 1993; Brower, 1995), and form resident populations that breed year-round in tropical locations such as South
Florida and Hawaii (Stimson & Berman, 1990; Knight, 1998).
Although monarchs generally lay eggs singly on host plants
(Zalucki & Kitching, 1982; Farrey & Davis, 2004; Prysby &
Oberhauser, 2004), multiple larvae can occupy the same plant,
especially in areas where host plants are patchily distributed or
rare. In support of this, observations of monarchs breeding
year-round in South Florida indicate that it is not unusual to
find plants with several larvae feeding on them (e.g. Brower,
1964; Farrey & Davis, 2004). By comparison, across the large
breeding range of monarchs in North America, host plants (including common milkweed, Asclepias syriaca) are common
and widespread, and larval densities per plant can be exceedingly low, with a single larva occurring on roughly one out of
every 30–50 host plants examined (Prysby & Oberhauser,
2004). Other studies have shown that per-plant larval densities
can vary over time and space in response to weather-related
abiotic factors (Zalucki & Rochester, 1999; Fig. 1), and as
monarch numbers increase over the course of a breeding season (Prysby & Oberhauser, 2004).
The neogregarine protozoan parasite Ophryocystis elektroscirrha occurs naturally in wild monarch populations (Leong
et al., 1997; Altizer et al., 2000) and is transmitted when
adult butterflies scatter parasite spores on eggs and milkweed
leaves. After spores are ingested by larvae, emerging sporozoites penetrate the gut wall, migrate to the larval hypoderm,
and undergo vegetative schizogony (McLaughlin & Myers,
1970). During the host pupal stage, the parasite undergoes
sexual reproduction and haploid spores are formed 2–3 days
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Density and infection in monarch butterflies
553
sites. In support of a role for monarch density in affecting
parasite prevalence, prevalence of O. elektroscirrha in eastern
N. America increases from early spring to late summer, as
might occur with increases in adult and larval abundance during the summer months (S. Altizer, unpubl. data, http://www.
monarchparasites.org).
Monarch sources and mating design
Fig. 1. Fifth instar monarch larvae feeding on Asclepias syriaca
(common milkweed) in a field near Forestport, New York, U.S.A., during
summer 2007. Wild monarch larvae at this location are typically scattered at low density (a single larva per plant; Maureen Clark, MLMP
pers. Obs.). However, in 2007, up to 8 eggs were laid on single plants,
and multiple late instar larvae were seen feeding on some milkweeds,
probably owing to low rainfall and scarcity of milkweeds during this
year. (Photograph: Maureen Clark).
before adult butterflies eclose from their pupal cases. Infected
butterflies emerge covered with dormant parasite spores on the
outside of their bodies, concentrated primarily on the abdomen
(McLaughlin & Myers, 1970; Leong et al., 1992). Negative effects of O. elektroscirrha depend on the initial dose and the
stage at which hosts are infected (De Roode et al., 2007). They
include pre-adult mortality, shorter adult longevity, smaller
adult body sizes, smaller forewings, and lower flight ability
(Altizer & Oberhauser, 1999; Bradley & Altizer, 2005;
De Roode et al., 2007; Lindsey & Altizer, 2008).
All monarch populations examined to date have been parasitised by O. elektroscirrha, and prevalence is highly variable
across different regions (Leong et al., 1997; Altizer et al., 2000).
Monarchs in resident populations that breed year round (i.e. in
southern Florida and Hawaii) bear the highest parasite loads
(over 70% heavily infected). Approximately 30% of monarchs
from a migratory population in western North America are
heavily infected (Leong et al., 1992; Altizer et al., 2000). Less
than 8% of monarchs from the eastern migratory population
(longest-distance migrants) are heavily infected (Altizer et al.,
2000). These differences among populations have persisted for
many years and could be caused by differences in monarch migratory behaviour (Altizer et al., 2000; Bradley & Altizer, 2005),
local population densities, or environmental variation among
Monarchs used in this experiment were the great-grand progeny of monarchs collected as larvae and adult butterflies from
three sites in eastern N. America during August to October
2004: Virginia (Giles County), Georgia (Dekalb County), and
New York (Tompkins County). All monarchs were examined for
the presence of O. elektroscirrha according to Altizer et al.
(2000) and only uninfected individuals were used to obtain
progeny (Ninitial = 33 adults). Captive monarchs were reared
from egg to adult using a breeding design that eliminated the
possibility of full-sib mating and maximised the contribution of
initial founders to each generation (with N per generation
>200). Eggs for this experiment were obtained from 15 females
that oviposited onto potted greenhouse-reared Asclepias incarnata. Plants were transferred to a laboratory and maintained at
24 °C, and larvae remained on their natal plants until they
reached the second instar.
Inoculation and host rearing
We used a fully factorial design where infection treatment
(parasitised and control) and larval rearing density (low, moderate, and high) were experimental factors (Table 1). Parasite
inoculum was derived from the abdomen of a monarch captured in Atlanta, GA, U.S.A. Following Altizer and Oberhauser
(1999), we vortexed the abdomen for 5 min in 10 ml of distilled water and calibrated inoculum to a dose of 300 spores
per larva using a haemocytometer. Control inoculum was prepared by vortexing the abdomen of an uninfected eastern
adult monarch. We inoculated second instar larvae individually by pipetting 10-l drops of inoculum onto 1-cm2 milkweed pieces placed on dampened filter paper inside sterile
8.5-cm Petri dishes. Larvae were maintained singly in the Petri
dish until they consumed all of the plant material, which occurred within 48 h.
After inoculation, larvae were transferred to plastic 3.8-l containers with mesh-screen lids and reared to adulthood in a laboratory exposed to ambient light (∼LD 14:10 h) and maintained
at 26 °C. A total of 420 larvae were randomly assigned to density
treatment groups as follows: one larva/container (low density),
five larvae/container (moderate density), and 10 larvae/container
(high density; Table 1). We checked containers twice daily, and
at least once per day added fresh cuttings of greenhouse-raised
milkweed (A. incarnata) to each container, removed frass, and
maintained a clean, moist paper lining. We adjusted the total
food supply so that the number of leaves per larva remained
relatively constant by provisioning approximately five leaves
per larva to each container per feeding. Milkweed cuttings were
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554 E. Lindsey et al.
Table 1. Number of monarchs used to initiate the experiment, and per cent surviving to adult eclosion, shown separately for each parasite treatment
and larval rearing density.
Density treatment
Parasite treatment
Control
Initial number
Third Instar
Adult
Egg to adult survival*
Third instar to adult survival*
Parasitised
Parasitised
Initial number
Third instar
Adult
Egg to adult survival*
Third instar to adult survival*
Parasitised
Low (1 larva per container)
Moderate (5 larvae per container)
40
40
22
55%
55%
0
60
55
28
47%
51%
0
40
39
18
45%
46%
14 (78%)
60
56
40
67%
71%
34 (85%)
High (10 larvae per container)
110
88
47
43%
53%
0
110
105
33
30%
31%
29 (88%)
Total
210
183
97
46%
53%
0
210
200
91
43%
46%
77 (85%)
*Survival estimates used in the statistical analysis were based on the number of adult monarchs divided by the number of third instar larvae, because
deaths of larvae from earlier instars were difficult to observe (larvae were frequently missing but no carcass was found). Overall survival (egg to
adult) is shown for comparison.
held in florist tubes and sterilised by soaking in a 20% bleach
solution for 20 min, and rinsing thoroughly in tap water prior
to use.
After all monarchs in a container had pupated, containers were
transferred to an adjacent laboratory maintained at 26 °C to avoid
contaminating the larval rearing area with parasite spores. Pupal
mass was measured on an analytic balance to the nearest 0.0001 g.
Pupae were transferred to single 0.5-l plastic containers to avoid
transfer of parasite spores among individual butterflies. We recorded the development time of monarchs based on the number
of days from oviposition to pupation and eclosion. After adults
emerged, we recorded the sex of each butterfly and placed
adults individually into glassine envelopes 6–12 h post-eclosion.
Monarchs were held at 24 °C without feeding, and mortality
counts were taken daily to record adult longevity (in days). We
used latex gloves to handle milkweed, monarchs, and inoculum;
gloves were frequently changed and laboratory surfaces and utensils were sterilised with 20% bleach solution to prevent unintentional transmission of parasite spores.
Quantifying infection and monarch wing parameters
We assessed the infection status of each adult by estimating
the total number of spores on the insects’ abdomens. Upon
death, the abdomen of each monarch was removed and placed
into a vial containing 5 ml of deionised water. After vortexing at
high speed for 15 min, a haemocytometer counting chamber
was used to estimate the number of spores per butterfly based on
replicate counts for each sample.
We used digital image analysis to quantify adult monarch
wing size and the degree of melanism (dark coloration). We
removed left and right forewings from preserved adults and
scanned them using a flatbed HP scanner set to 300 dpi using
the same exposure settings for each scan. Measurements were
made using Adobe Photoshop software with the Image Processing
Tool Kit plugin (Reindeer Graphics, Inc., Asheville, NC, U.S.A.).
Total forewing area (mm2) and two measures of wing melanism
were obtained for both forewings of each adult butterfly,
according to Davis et al. (2005). First, we quantified the proportion of forewing area encompassed by black pigmentation.
Second, we estimated the density of black pigmentation, an
indicator of the intensity or level of opacity of black. The scoring measurement of density is on a 0–255 scale, with 0 being
completely black (greatest colour density). Average measures
per individual were based on results for L and R forewings.
Regional and temporal variation in monarch density
We used data from the Monarch Larva Monitoring Project
(MLMP; Prysby & Oberhauser, 2004; Oberhauser & Prysby,
2008), a citizen science programme, to further indicate the
degree to which monarch butterfly larval densities vary over
time and space in eastern N. America. Volunteer observers for
the MLMP began collecting weekly abundance data during the
monarch’s breeding season in 1997, with per plant densities of
monarch egg and larval (reported to individual stadia) stages on
milkweed plants available for 32 states and provinces across
North America. Data included the total number of larvae and
milkweed plants observed at a specific time and location. As a
high proportion of monarchs die as eggs and early instar larvae,
we calculated average larval density per site based on count data
for the final three instars (3, 4, and 5) only. All sites used in the
analysis had been monitored for more than 1 year with a minimum of 4-weekly observations per year.
We divided observations from 1997 through to 2006 in the
eastern U.S. and Canada into three geographic regions: Midwest
2009 The Authors
Journal compilation 2009 The Royal Entomological Society, Ecological Entomology, 34, 551–561
Density and infection in monarch butterflies
(MN, WI, MI, IA, IL, IN, MO, OH, and NE), Northeast (VT,
MA, NY, NJ, PA, MD, ON, and DC), and South (TX, GA, NC,
VA, and TN), and three temporal breeding phases: early (before
June first), middle (June first–July 31st), and late (after July 31st)
to examine changes in larval abundance. Geographical regions
were selected based on previously described patterns of monarch
spring re-colonisation, whereby adults returning from Mexico
lay eggs in the southern-most states during April–May (here represented by the region denoted ‘South’), and a second generation
continues the journey north followed by a brief time lag (Malcolm
et al., 1993; Howard & Davis, 2004; Davis & Howard, 2005). In
addition, northeastern and mid-western states were examined
separately, because these areas are associated with two major fall
migratory flyways at the end of the breeding season (Howard &
Davis, 2009). We analysed the average larval density per site
based on the total number of larvae divided by the number of
milkweed plants examined each week, and averaged the weekly
density values for each site within a given phase. We then excluded zero density reports (where sites were monitored but no
larvae were reported for a given phase), and log-transformed the
remaining density estimates prior to analysis.
555
was used to examine treatment effects on development time and
adult longevity, and multinomial logistic regression was used to
examine treatment effects on the proportion of monarchs that
survived to adulthood, the proportion of adults infected with
O. elektroscirrha, and the proportion of adults with deformed
wings. For analyses of adult measures, monarch sex (M/F) was
included as a fixed factor, and the final density of larvae (based on
the actual number of monarchs that survived to pupation per container) was included as a continuous covariate (full model: dependent variable = parasite treatment + density treatment + final
desity + sex + parasite*density + infection*sex + density*sex).
Analyses were performed in SPSS (ver. 15.0; SPSS, Inc.,
Chicago, IL, U.S.A.) and we used comparisons of Akaike’s
Information Criterion (AIC) and Hurvich and Tsai’s Criterion
(AICC) for model simplification according to Crawley (2002).
In Table 2, we report significance values only for variables
included in the final minimum adequate model. Bonferroni’s
pairwise comparisons of means were used to further examine
differences between the three density treatments in cases where
rearing density was significant, and results are reported in the
figure legends. We examined the distribution of residuals for
each minimum adequate model, and in most cases, found that
these approximated a normal distribution.
Statistical analysis
Analyses to examine the effects of parasite treatment and
rearing density were conducted using average values for all
monarchs reared in a container as the unit of observation.
Dependent variables included pupal mass, adult forewing area,
development time from inoculation to adulthood, adult spore
load, adult longevity, and two measures of wing melanism (proportion of black and density of black on forewings). Count data
were log-transformed prior to calculating container means. We
tested for equal variances between density treatment groups using Fisher’s F-test, for normally distributed data, and Levene’s
Test, for non-normally distributed data, at significance levels of
␣ = 0.05. Variances between density treatments were equal for
spore load data and all continuous response variables.
Analysis of variance was used to examine effects of design
variables on one count variable, final spore load, and all continuous variables: pupal mass, adult forewing area, and two
measures of wing melanism (proportion of black and density of
black on forewings). The non-parametric Kaplan–Meier analysis
Results
Regional and temporal variation in monarch density
Analysis of MLMP data indicated that the average number of
larvae per plant differed among breeding phases (early, middle,
and late) and between regions in eastern N. America (Midwest,
Northeast, and South). The final data set included a total of 641
density estimates by sampling location and breeding phase, as
recorded by 78 observers over all 10 years. The number of density values (calculated for individual sites within a given breeding phase) used for each region by phase combination ranged
from 14 (Northeast, early phase) to 307 (Midwest, middle phase),
after reports of zero density were removed from the data set.
Average densities per location were highest in the South during
the early phase of the breeding season (Fig. 2). During the middle of the breeding season, monarch density was very low in the
South, and increased again late in the breeding season. In the
Table 2. Analysis of pupal mass and adult forewing characteristics as a function of experimental design variables (density and infection treatment) and
sex (full model: Response variable = rearing density + parasite treatment + sex + rearing density*treatment + final larval density + rearing
density*sex + treatment*sex + error term). Model simplification was performed as described in Methods text. F- and P-values are shown only for
those explanatory variables that remained in the final model; adjusted R2 is shown for the final reduced models. All analyses use container means as the
unit of observation.
Independent variable
Pupal mass
Wing area
Rearing density
Parasite treatment
Sex
Final density
Rearing density*parasite treatment
Adjusted R-square
F2,104 = 10.38 P = 0.000
F1,104 = 4.80 P = 0.032
F1,104 = 39.07 P = 0.000
F2,102 = 12.71 P = 0.000
F1,102 = 2.13 P = 0.148
F1,102 = 4.97 P = 0.028
F1,102 = 3.88 P = 0.052
F2,102 = 3.94 P = 0.022
0.254
F2,104 = 4.14 P = 0.019
0.389
Wing proportion black
Wing black density
F1,107 = 3.81 P = 0.053
F1,107 = 388.99 P = 0.000
F1,108 = 209.94 P = 0.000
0.788
0.657
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556 E. Lindsey et al.
Fig. 2. Average densities of monarch butterfly larvae reported by
MLMP (measured as the total number of larvae divided by the number
of milkweed stalks examined per site) for three regions in eastern North
America: Northeast (open bars), Midwest (gray bars), and South (black
bars). Phases are early (before Jun 1st), middle (Jun 1st - July 31st), and
late (after July 31st). Error bars represent standard errors.
d.f. = 6, P = 0.320) or across density treatments (2 = 17.43,
d.f. = 12, P = 0.134), and the interaction between rearing density
and parasite treatment was also not significant (2 = 35.24,
d.f. = 12 P = 0.998). Based on variation in survival within the
parasite-treated monarchs (Table 1), we ran a separate analysis
focused on survival within this treatment group, which showed
that the effect of rearing density was only nearly significant
(2 = 19.83, d.f. = 12, P = 0.070).
All surviving control monarchs were parasite-free (N = 97),
whereas 85% of the parasite-treated monarchs (N = 91) were
infected with O. elektroscirrha. The proportion of infected
monarchs within the parasite-treated class increased with larval
rearing density (Table 1). This effect of density on infection
status was significant (2 = 23.32, d.f. = 10, P = 0.010). The
average parasite load per infected monarch was 3.08 × 105
spores (range: 5.56 × 103–1.16 × 106), and we noted a trend
of increasing spore load with increased rearing density (average spore load by rearing density: Low 2.46 × 105; Moderate
2.91 × 105; High 3.39 × 105). However, analysis within the
subset of parasitised monarchs showed that average spore load
was not significantly affected by either rearing density
(F2,47 = 0.73, P = 0.490) or sex (F1,47 = 0.32, P = 0.573).
Pupal mass and development time
Northeast and Midwest, average larval density increased from
early to late in the breeding season (Fig. 2). Effects of phase
of breeding season (F2,627 = 3.71, P = 0.025) and the two-way
interaction between breeding phase and region (F4,627 = 2.82,
P = 0.024) were highly significant, but the main effect of region was not significant (F2,627 = 0.56, P = 0.574).
It is important to note that average larval densities reported in
Fig. 2 underestimate the actual numbers of larvae per host plant.
This is because plants with larvae and those without were
included equally in the count of plants examined. Since the
number of larvae on a single plant for sites with monarchs present
must be at least 1.0, averages shown in Fig. 2 could be higher if
only those plants with larvae had been counted. Moreover, within
each region, the maximum larval density for any given site was
greater than 1.0 in several cases, leaving no doubt that some
plants carried >1 larva (e.g. Fig. 1). Finally, an observer in
Delray Beach, FL (excluded from the analysis here because of its
close proximity to the S. Florida resident monarch population)
reported average numbers of 7.0 and 6.5 larvae per plant examined during the middle phase of the breeding season in both 2005
and 2006, respectively. This observation indicates that the numbers of larvae per plant could reach higher levels in year-round
resident populations as compared with the relatively low larval
densities experienced by the eastern migratory population.
Survival, infection status, and parasite load
Monarchs in the density-infection experiment experienced
high larval and pupal mortality across all treatments; only 49% of
all monarchs survived from third instar to the adult stage (Table 1).
On average, the probability of survival to adulthood did not differ
between control and parasite-treated monarchs (2 = 7.01,
Pupal mass was greatest in the moderate density treatment
(Fig. 3a) and this effect was highly significant (Table 2). Average
pupal mass was lower among parasite-treated monarchs in both
Fig. 3. Effects of larval rearing density and parasite treatment on
(a) pupal mass and (b) development time. Data are shown for unparasitized (open bars) and parasitized (gray bars) treatment groups within
each larval rearing density (low: 1 larva per container; moderate: 5 larvae per container; high: 10 larvae per container). Error bars represent
standard errors. Bonferroni pairwise comparisons showed that mean pupal mass for monarchs reared under moderate densities were significantly greater than means for high and high densities (p < 0.05). whereas
means for high and low density treatments were statistically similar
(p > 0.50). Bonferonni comparisons also showed that mean development
time for monarchs reared at moderate densities were significantly shorter
than means for monarchs reared at high densities (p < 0.03), whereas
means for all other combinations were statistically similar (p > 0.30).
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Density and infection in monarch butterflies
the moderate and high density treatments, but parasite-treated
and control monarchs had similar pupal mass in the low density
treatment (Fig. 3a). Statistical analysis showed a significant
main effect of parasite treatment and a significant two-way
interaction between parasite treatment and density (Table 2).
Pupal mass was also greater in males than females across all
treatment categories, and this effect was highly significant
(Table 2).
Parasite-treated monarchs developed more slowly (based on
time from hatching to adult eclosion) than monarchs in the control treatment (Log Rank: 2 = 6.89, d.f. = 1, P = 0.009), and
this effect was strongest in the low and high density treatments
(Fig. 3b). Moreover, larvae in the moderate density treatment
developed faster than those in the low and high density treatments, and this effect of rearing density on development time
was highly significant (Log Rank: 2 = 8.77, d.f. = 2, P = 0.013).
There was also a significant interaction between parasite treatment and density (Log Rank: 2 = 15.11, d.f. = 5, P = 0.013).
Specifically, development time differed across rearing densities
only within parasite-treated monarchs, as demonstrated by further analysis within control (Log Rank: 2 = 1.01, d.f. = 2,
P = 0.605) and parasite-treated monarchs (Log Rank: 2 = 9.70,
d.f. = 2, P = 0.008). In addition, female monarchs developed
more quickly than males across all parasite and density treatments (Log Rank: 2 = 8.447, d.f. = 1, P = 0.004).
Adult longevity and wing traits
Parasite-treated monarchs experienced a 23% reduction in
adult longevity as compared with control monarchs, and this effect of parasite treatment was highly significant (Fig. 4; Log Rank:
Fig. 4. Effects of larval rearing density and parasite treatment on adult
longevity (in days). Data are shown for unparasitized (open bars) and
parasitized (gray bars) treatment groups within each larval rearing density (low: 1 larva per container; moderate: 5 larvae per container; high:
10 larvae per container). Error bars represent standard errors.
557
Fig. 5. Effects of larval rearing density and parasite treatment on
(a) adult forewing area and (b) proportion of back pigmentation on adult
forewings. Data are shown for unparasitized (open bars) and parasitized
(gray bars) treatment groups within each larval rearing density. Error
bars represent standard errors. Bonferroni pairwise comparisons showed
that mean forewing area for monarchs reared under moderate density
was significantly greater than means for monarchs reared at low and
hogh densities (p < 0.002), whereas means for high and low density
treatments were statistically similar (p = 0.098). Rearing density was
not significantly associated with the proportion of back pigmentation on
monarch forewings.
2 = 48.06, d.f. = 1, P < 0.001). Adult longevity was not affected
by larval density alone (Log Rank: 2 = 3.95, d.f. = 2, P = 0.139).
However, there was a significant interaction between parasite
treatment and density (Log Rank: 2 = 58.31, d.f. = 5, P = 0.000;
Fig. 4), such that adult longevity decreased with increasing larval
density within parasite-treated, but not control monarchs.
Only two unparasitised monarchs (n = 105) emerged with
wing deformities, whereas five parasitised adults (n = 77)
had deformed wings. Four out of the five parasitised monarchs
with wing deformities were in the high density treatment.
However, neither parasite treatment nor rearing density was significantly associated with wing deformities (parasite treatment:
2 = 5.03, d.f. = 3, P = 0.170; rearing density: 2 = 9.93,
d.f. = 6, P = 0.127).
Adult forewing area was significantly influenced by rearing
density, parasite treatment, and sex (Table 2). Males were larger
than females, and monarchs in the moderate density treatment
had the largest forewings, whereas those in the low density
treatment had the smallest forewings (Fig. 5a). Forewing area
was also affected by the density of parasite treatment interaction
(Table 2). Specifically, control monarchs had larger forewings
than parasite-treated monarchs in both the moderate and high
density treatments, whereas parasite-treated monarchs had
larger forewings in the low density treatment (Fig. 5a).
The proportion of black coloration on monarch forewings
was greater for control monarchs than for parasite-treated
butterflies (63% vs 61%), but this trend was marginally nonsignificant (P = 0.053). Females were darker than males across
all treatment groups (Fig. 5b, Table 2). The density of wing pigmentation, a measure of the opacity or intensity of black, was
greater for female than for male monarchs, but this variable was
not significantly affected by parasite treatment (Table 2). Finally,
neither measure of wing coloration was significantly affected by
rearing density based on main or interaction effects (Table 2).
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558 E. Lindsey et al.
Discussion
Both larval rearing density and infection by the protozoan
O. elektroscirrha affected measures of size and development
in monarch butterflies. First, we observed significant negative
effects of parasite treatment on monarch fitness, with infection
resulting in decreased monarch pupal mass, slower development rate, reduced wing area, and shorter adult longevity. This
is consistent with previous studies demonstrating that high
replication of O. elektroscirrha within monarch hosts results
in substantial negative consequences for adult lifespan, body
size, wing size, mating success, and flight ability (Altizer &
Oberhauser, 1999; Bradley & Altizer, 2005; De Roode et al.,
2007; Lindsey & Altizer, 2008).
While monarch infection probability increased significantly
with increasing larval densities, average spore loads after exposure to O. elektroscirrha increased only slightly with larval
densities. The effect of density treatment on host infection status, weakly suggests increased susceptibility to infection with
increasing larval density. Thus, in the case of rearing density,
results here do not support the ‘density-dependent prophylaxis’
hypothesis, which predicts that increased resistance to parasitism can result from increased rearing density (Reeson et al.,
1998; Barnes & Siva-Jothy, 2000; Wilson et al., 2003). On the
one hand, as monarchs appear to experience ecologically relevant variation in density in the wild, they should, in theory,
benefit from tailoring levels of immunity or resistance to
variation in host density. This is because infection patterns by
O. elektroscirrha in wild populations show that prevalence
increases from early to late summer within migratory populations (S. Altizer, unpubl. data) and is higher in year-round
breeding populations as compared with migratory populations
(Leong et al., 1997; Altizer et al., 2000, 2004). Since both of
these situations also correlate with higher numbers of larvae on
plants (e.g. Fig. 2), it seems likely that monarchs in high-density
populations could also experience higher risks of infection.
On the other hand, the two species for which the ‘densitydependent prophylaxis’ hypothesis has found the most support
(armyworm and desert locust; Reeson et al., 1998; Wilson
et al., 2002; Wilson et al., 2003) are both polyphenic outbreak
species, with specific high and low density morphs. These insects experience much greater fluctuations in population density on a regular basis than monarchs, thus the selective benefit
of greater immune defences under high density conditions is
likely to be higher in these species than in monarchs.
Additionally, monarch immune defences can be costly (Lindsey &
Altizer, 2008). Hence, although greater measures of immunity
correlate with higher host survival after infection with O. elektroscirrha, these defences might not be readily mobilised under
high-risk conditions.
An alternative hypothesis to density-dependent prophylaxis
is the ‘crowding and stress’ hypothesis, which predicts that individuals reared at lower population densities will be in better
physical condition, and hence will be better able to resist the
negative effects of parasitism (Goulson & Cory, 1995; Adamo,
2006; Reilly & Hajek, 2008). This idea is based on the assumption that increased stress and intra-specific competition will
make hosts more susceptible to infection, or less able to tolerate
the negative consequences of parasitism. In support of this
hypothesis, monarchs in the moderate and high density treatments generally experienced slightly higher infection rates and
suffered the greatest negative effects of infection (based on differences between the parasite-treated and untreated groups).
Moreover, monarchs in the moderate and high density rearing
treatments also suffered the greatest negative effects of infection
on development and body size (based on relative differences between the parasite-treated and untreated class). By comparison,
parasitised monarchs in the low density treatment had mean values of pupal mass, development rate, and adult wing size that
were similar to or slightly greater than uninfected monarchs.
Collectively, these effects of rearing density on host infection
and fitness measures suggest that monarchs under high-density
conditions are more susceptible to parasite infection and its
costly effects.
Contrary to our initial expectations, we observed a non-linear
relationship between larval density and measures of development and body size for both parasite-treated and untreated
larvae. Specifically, each of these variables was greatest for
monarchs reared under moderate density, and averages were
10–20% lower for monarchs reared singly and at the highest
density. Previous studies of Lepidoptera and other insect species
have shown that immature stages reared under high density conditions experience decreased survivorship, slower development
rates, and/or achieve smaller adult body sizes (Mercer, 1999;
Tammaru et al., 2000; Gibbs et al., 2004). On the other hand,
cabbage moth Mamestra brassicae larvae reared both singly
and at the highest densities weighed less than larvae reared at
intermediate densities (Goulson & Cory, 1995). Our findings
are consistent with those of Goulson and Cory (1995), and suggest that for both healthy and infected monarchs, the ideal rearing conditions are to be neither solitary nor in a large group, but
to occur at moderately low densities, provided that food resources are not limiting.
Observations of greater body size and faster development
rate for intermediate density conditions, irrespective of host infection status, might be best explained by two different mechanisms. Given that high densities could cause greater intra-specific
competition under natural conditions, larvae might feed more
rapidly to attain a large body size before food supplies are depleted. As food supplies, ultimately, were not limited in this
experiment, this could have resulted in both more rapid development and greater size at pupation when compared with solitary larvae. In our experiment, monarch larvae reared under the
highest density conditions (10/container) could have suffered
from physical or developmental stress associated with overcrowding or interference competition. For example, Gibbs et al.
(2004) observed aggressive encounters, including head and tail
biting and head butting, among speckled wood butterfly larvae
(Pararge aegeria) reared at high densities. Although we did not
quantify feeding behaviours in this study, monarchs engage in
similar aggressive interactions in captivity, and larvae can also
bear integument scars and missing tubercules from previous injuries (S. Altizer, pers. obs.). Finally, we note that in experiments described here, a greater number of host plant leaves
provisioned to containers with more larvae could have altered
the micro-environmental conditions in ways that enhanced
2009 The Authors
Journal compilation 2009 The Royal Entomological Society, Ecological Entomology, 34, 551–561
Density and infection in monarch butterflies
monarch survival and development in the moderate density
treatment.
External melanism (i.e. dark body coloration) is often thought
to correlate positively with resistance to infection (Barnes &
Siva-Jothy, 2000), but not in all insect species (Robb et al.,
2003). Moreover, previous work has shown that body or wing
melanism can increase under crowded host conditions (Wilson
et al., 2001). For example, polyphenism in Lepidoptera can occur such that the high density phenotype is darker than the low
density phenotype (e.g. as demonstrated for Spodoptera littoralis; Cotter et al., 2004). Within monarchs, our results did not
support an effect of rearing density (or a density by parasite
treatment interaction) on measures of dark coloration in adult
butterflies. In addition, although we found that unparasitised
adults had darker forewings (based on the proportion of black
pigmentation) than parasite-treated butterflies, this trend was
not statistically significant.
Field data from the MLMP do not allow us to determine per
plant larval densities for occupied plants alone, and thus it is
impossible to determine how often the experimental larval densities examined here occur in the wild. However, MLMP data do
clearly demonstrate that per-plant monarch densities vary up to
five-fold across space and time in North America, and we have
anecdotal reports of densities of late-instar larvae similar to
those used in our study (Fig. 1). It is therefore possible that the
faster development observed here for monarchs in the moderate
density treatment is an evolved response to crowding that occurs
in nature.
Ultimately, hosts that live in variable environments can experience changes in population structure and extrinsic forces that
influence variation in disease risk. These risks are particularly
relevant to monarch butterflies that have been threatened at their
overwintering sites and breeding habitats in recent years (Brower &
Malcom, 1991; Zalucki & Rochester, 1999; Brower et al., 2002;
Oberhauser & Peterson, 2003). Specifically, destruction of overwintering sites, climate warming, and planting of tropical milkweed host plant species in non-native regions can alter the
ecological dynamics of migratory populations, potentially resulting in the replacement of the large migratory populations
with smaller remnant populations that breed year-round. These
non-migratory populations could experience higher local population densities, and are also likely to become heavily parasitised (e.g. Altizer et al., 2000, 2004).
As indicated by this study, both parasite infection and larval
density can affect monarch butterfly fitness, although results
reported here were not always consistent with predictions from
previous work. Specifically, we found that contact with other
larvae at moderate (but not high) local densities could increase
pre-adult survival, stimulate larval growth, and increase pupal
and adult body size, irrespective of host infection status.
However, it is important to note that this finding probably relies on non-limiting food resources given modest increases
in density. Moreover, our study indicates that highly crowded
conditions can increase the potential for monarchs to suffer
from the negative consequences of disease, probably owing to
increased stress and intra-specific competition. In summary,
being in a group may have beneficial impacts on a number of
fitness traits, even for species such as monarchs that do not
559
have gregarious larval stages. However, high density conditions appear to have negative effects on monarch resistance and
tolerance to infection, and results such as those reported here
can help reveal the mechanisms that underlie the positive
and negative consequences of host density for individual
performance.
Acknowledgements
We thank Nick Vitone, Mindy Edelson, Andrew Davis, Laura
Gold, and Jaap de Roode for help with the experiments, Les
Real and the Emory University Biology Department for providing greenhouse space for rearing milkweed host plants, and
two anonymous reviewers for comments on the manuscript.
The National Science Foundation (ISE-0104600), the Xerces
Society, the University of Minnesota Extension Service and
Monarchs in the Classroom have provided financial support
for the MLMP. Financial support to S.A. was provided by a
National Science Foundation grant (DEB-0643831), support
to E.L. was provided by the Graduate Division of Biological
and Biomedical Sciences at Emory University and a PRISM
Fellowship (NSF DGE-0231900), and for M.M. and V.D. by
the Summer Undergraduate Research Program (SURE) and
the Scholarly Inquiry Research Experience (SIRE) at Emory
University.
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Accepted 29 February 2009
First published online 8 June 2009
2009 The Authors
Journal compilation 2009 The Royal Entomological Society, Ecological Entomology, 34, 551–561