Current Zoology
59 (3): 393–402, 2013
Lipid reserves and immune defense in healthy and diseased
migrating monarchs Danaus plexippus
Dara A. SATTERFIELD, Amy E. WRIGHT, Sonia ALTIZER*
Odum School of Ecology, University of Georgia, Athens, GA 30602, USA
Abstract Recent studies suggest that the energetic demands of long-distance migration might lower the pool of resources
available for costly immune defenses. Moreover, migration could amplify the costs of parasitism if animals suffering from parasite-induced damage or depleted energy reserves are less able to migrate long distances. We investigated relationships between
long-distance migration, infection, and immunity in wild fall-migrating monarch butterflies Danaus plexippus. Monarchs migrate
annually from eastern North America to central Mexico, accumulating lipids essential for migration and winter survival as they
travel southward. Monarchs are commonly infected by the debilitating protozoan parasite Ophryocystis elektroscirrha (OE). We
collected data on lipid reserves, parasite loads, and two immune measures (hemocyte concentration and phenoloxidase activity)
from wild monarchs migrating through north GA (USA) to ask whether (1) parasite infection negatively affects lipid reserves, and
(2) greater investment in lipid reserves is associated with lower immune measures. Results showed that monarchs sampled later in
the fall migration had lower but not significantly different immune measures and significantly higher lipid reserves than those
sampled earlier. Lipid measures correlated negatively but only nearly significantly with one measure of immune defense
(phenoloxidase activity) in both healthy and infected monarchs, but did not depend on monarch infection status or parasite load.
These results provide weak support for a trade-off between energy reserves and immune defense in migrants, and suggest that
previously-demonstrated costs of OE infection for monarch migration are not caused by depleted lipid reserves [Current Zoology
59 (3): 393–402, 2013].
Keywords
Butterfly, Hemocytes, Migration, Neogregarine, Infection, Energy reserves
Each year, thousands of species migrate long distances to track seasonal changes in resources and climate (Dingle, 1996; Bowlin et al., 2010). Migration can
be energetically costly. For example, flying insects have
a metabolic rate 20-100 times greater than insects at rest
(Rankin and Burchsted, 1992), and migratory birds expend thousands of kilojoules of energy during their
journeys (Wikelski et al., 2003; Schmaljohann et al.,
2012). To prepare for demanding migrations, some animals invest up to 50% of their lean body mass in lipid
reserves (Dingle, 1996). Long-distance journeys can
also be risky, with many animals facing increased mortality (e.g., Sillett and Holmes, 2002). Mortality risk
could be especially high for migrants infected with
parasites (reviewed in Altizer et al., 2011), in part because diseased animals could suffer from physical
weakness or the loss of energy reserves due to parasite
consumption of tissues and nutrients. As such, infected
migrants might travel more slowly, stop more frequently,
or die during the journey (Bradley and Altizer, 2005;
Altizer et al., 2011). In support of this idea, Bewick’s
Received Feb. 23, 2013; accepted June 2, 2013.
∗ Corresponding author. E-mail: [email protected]
© 2013 Current Zoology
swans Cygnus columbianus bewickii infected by
low-pathogenic avian influenza viruses postponed the
onset of migration and traveled shorter distances compared with uninfected individuals (van Gils et al., 2007).
Similarly, migrating mallard ducks Anas platyrhynchos
infected with low-pathogenic avian influenza at a stopover location in Europe had lower body mass than uninfected birds (Latorre-Margalef et al., 2009).
Because infection might lower survival during migration, migrating animals could experience pressure to
ward off infection by maintain high immune defenses
(Buehler et al., 2010). Yet, two decades of research
show that immune defenses are themselves energetically
expensive and trade off against other functions, especially reproduction (Demas et al., 2011). On the one
hand, immune defenses may not change during migration if robust migrants or those with access to resources
can invest both in immunity and in energetic reserves to
fuel migration. For example, captive red knots Calidris
canutus provided with high-quality food showed no
change in costly immune defenses during the
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Current Zoology
pre-migratory period (Buehler et al., 2008). On the other
hand, the energetic demands of migration might require
many animals to down-regulate immune responses or
experience reduced efficacy of immune pathways (reviewed in Altizer et al., 2011). For example, in a study
of three thrush species, actively migrating birds had
lower measures of innate immunity compared to individuals tested during a non-migratory period (Owen and
Moore, 2006). Likewise, captive Swainson’s thrushes
Catharus ustulatus showed diminished cell-mediated
immunity during the period of migratory restlessness
that immediately preceded migration (Owen and Moore,
2008). Thus, understanding how migratory animals
balance the energetic costs of both migration and immunity remains an open question. Amid concerns about
the movement of pathogens by migratory species
(Rappole et al., 2000; Koehler et al., 2008) and worldwide declines in animal migrations (Wilcove, 2008),
investigating the interactions between migration, immune defenses, and infection could facilitate advances
in human health and wildlife conservation (Altizer et al.,
2011; Klaassen et al., 2012).
While most work on immunity and infection in migrating animals has focused on a vertebrates, especially
birds, many insect migrations outweigh vertebrate migrations in abundance and biomass (Holland et al., 2006).
Insects, like vertebrates, can deploy multiple lines of
immune defenses (Schmid-Hempel, 2005). Two commonly studied measures of insect innate immunity are
hemocyte concentration and phenoloxidase activity.
Hemocytes are defensive blood cells (similar to white
blood cells in verbebrates) that assist in the recognition,
phagocytosis, and encapsulation of diverse microbial
pathogens (Rolff and Reynolds, 2009). Phenoloxidase is
a key enzyme in the melanization pathway that helps to
wall off parasites, producing a dark capsule around
foreign material (e.g., Christensen et al., 2005). Prior
work shows that these defenses can be energetically
costly to insects, resulting in elevated metabolic rates
and trading off against reproduction (e.g., Freitak et al.,
2003; Jacot et al., 2005; Zuk and Stoehr, 2002).
Monarch butterflies Danaus plexippus offer a useful
system to examine the relationships between migration
physiology, infectious disease, and immune defenses.
Each fall, monarchs in eastern North America migrate
up to 3,000 km from the northern U.S. and southern
Canada to overwintering sites in central Mexico, where
they spend over 4 months in reproductive diapause
(Brower, 1996). Like many migratory species (Kent and
Rankin, 2001; McWilliams et al., 2004), fall-migrating
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monarchs fuel their journey and overwintering period
by accumulating lipids as they migrate, foraging on
nectar and converting sugars into lipid reserves while
they travel southward (Brower et al., 2006; Gibo and
McCurdy, 1993). In Mexico, monarchs rely heavily on
stored lipids for winter survival (Alonso-Mejia et al.,
1997). Monarchs break diapause in March and migrate
north to recolonize the southern U.S. (Malcolm et al.,
1993). Given the role of lipid reserves in migration
physiology, measures of lipids can be treated as a proxy
for energetic investment in migration and overwintering.
Monarchs are commonly infected by the specialist
protozoan Ophryocystis elektroscirrha (OE; McLaughlin and Myers, 1970; Leong et al., 1997a). OE is transmitted when infected adult butterflies scatter dormant
parasite spores onto milkweed leaves, especially during
oviposition. Larvae ingest the spores, parasites replicate
within larval and pupal tissues, and adult butterflies
emerge with millions of dormant spores on the outsides
of their bodies (McLaughlin and Myers, 1970; Leong et
al., 1997b; de Roode et al., 2007). Adults can transfer
small numbers of spores externally to other adult monarchs during close contact (de Roode et al., 2009;
Vickerman et al., 1999), but spores must be ingested by
a larva to cause a new infection. While no further parasite replication occurs at the monarch adult stage, infected adults suffer from decreased body size, eclosion
success, lifespan, and flight performance (Altizer and
Oberhauser, 1999; Bradley and Altizer, 2005). Infected
monarchs could also suffer from reduced migration
success: Eastern North American monarchs show
markedly lower OE prevalence at overwintering sites
after fall migration than at summer-breeding sites before
fall migration (Bartel et al., 2011). Further, OE prevalence also appears to decline again after spring migration, suggesting infected monarchs experience higher
mortality during both the fall and spring journeys
(Bartel et al., 2011). Monarchs show evidence for
qualitative and quantitative resistance to OE strains (de
Roode and Altizer, 2010; Lefèvre et al., 2011) and larval
immune defenses appear to correlate negatively with
adult parasite loads following experimental inoculations
(Altizer S, unpubl. data).
Here, we focused on wild fall-migrating monarchs
sampled in eastern North America to investigate relationships between lipid reserves, immune measures, and
parasite infection. We predicted that monarchs infected
with OE would have reduced lipid reserves compared to
uninfected monarchs (Fig. 1), because OE infection
might reduce monarchs’ ability to acquire or convert
SATTERFIELD DA et al.: Monarch lipid reserves and immune defense in migrating monarchs
nectar into lipids and might deplete the lipid reserves
monarchs carry over from the larval to the adult stage.
We also predicted that greater investment in lipid reserves (needed to fuel migration and overwintering)
would diminish the pool of resources available for immune defenses (Fig. 1), causing a negative relationship
between immune measures and lipid resources in migrating monarchs. Alternatively, higher-quality individuals might be able to invest in both lipid reserves and
immune defense, resulting in either a positive or no relationship between immune measures and lipid resources.
1
Materials and Methods
1.1
Butterfly sources, infection status, and morphometric data
Between Sept.–Nov. 2010, we caught wild fall-migrating monarchs at 6 sites in Clarke and Oconee Counties, GA, USA. Adult monarchs were captured using an
aerial net between 0900 and 1600 hr. Following capture,
monarchs were stored individually in glassine envelopes
and held at 14ºC for up to 8 hr prior to sampling. Wing
condition, which qualitatively reflects age or distance
traveled, was recorded as the sum of ordinal-scale val-
Fig. 1
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ues for wing damage (0–4, based on the number of
wings with evidence of tears or other physical damage)
and wing wear (1–5, based on the level of scale loss
across wings, following Cockrell et al., 1993 and described at www.monarchlab.org). Wet mass was recorded to the nearest mg.
We initially used a non-destructive and highly sensitive method to assess individual infection status by
pressing a clear adhesive sticker (1.27cm2) on the adult
abdomen (described in Altizer et al., 2000). OE spores
on the sticker (Fig. 1) were then counted at 63X magnification. Samples with >100 spores were considered to
be heavily infected, as previous laboratory work showed
that larvae that ingested even a single OE spore could
experience high levels of OE replication and suffer from
pathogenic effects; these monarchs consistently produced sticker samples with > 100 OE spores (de Roode
et al., 2007). Samples with 1–100 spores were not used
in further analysis as these individuals likely acquired
dormant spores through horizontal transfer from other
adults and thus did not suffer from internal parasite replication (e.g., Altizer et al., 2004; de Roode et al., 2009).
We retained all heavily infected monarchs and an
Predicted relationships between lipid reserves, immune defense, and infection in fall-migrating monarchs
We expected to find a negative relationship between lipid reserves (top right: lipids as pale yellow fat bodies dissected from monarch abdomen) and
immune defenses (top left: hemocytes and phenoloxidase activity). We also predicted that monarchs infected with a protozoan parasite (top center:
OE spores from monarch abdomen, 400X) would have lower lipid reserves than healthy individuals due to depletion of host resources by parasites,
and that infection status might also affect measures of immune defense.
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additional set of uninfected monarchs (0 spores detected,
hereafter ‘healthy’) from across the same collection
dates for further analysis.
For all monarchs, we used Adobe Photoshop to
measure average wing area from digital scans of both
forewings following Davis et al. (2005; 2007). Forewing area was included in analyses described below, as
this measure has been found to be a predictor of migration success (reviewed in Altizer and Davis, 2010) and
larger wings can indicate more abundant larval resources (Boggs and Freeman, 2005). We collected
hemolymph for immune assays as described below and
froze monarchs at -12ºC prior to quantifying parasite
load and total lipid mass. To quantify parasite loads for
heavily infected monarchs, abdomens were vortexed at
high speed in 5 mL H2O for 5 mins. We estimated the
total number of spores on the adult abdomen using a
hemocytometer as described in de Roode et al. (2007).
1.2 Immune measurements: Hemocyte counts and
phenoloxidase activity
We quantified two measures of innate immunity,
hemocyte counts and phenoloxidase (PO) activity. Although these measures in adult monarchs do not affect
OE replication (which occurs only at the larval and pupal stages; McLaughlin and Myers, 1970), these measures are important for wound healing and defenses
against other pathogens such as opportunistic bacteria
(Rolff and Reynolds, 2009; Beckage, 2008). Within 8
hours post-capture, we used a 25-gauge needle to prick
the dorsal sinus between the second and third segments
of the abdomen and collected up to 2–10 µL of clear
hemolymph (average = 4.9 µL ± 2.7 SD). For hemocyte
counts, 2 µL of hemolymph was rapidly diluted 1:10 in
sterile Pringle’s Saline [1x in 1L dD H20: 9.0g NaCl,
0.2g KCl, 0.2 g CaCl, 4.0 g dextrose] and loaded into
Kova® glasstic hemocytometer slides. We performed
hemocyte counts on two replicate chambers per sample
and calculated the average number of hemocytes per µL
(hereafter, ‘hemocyte concentration’ Fig. 1). For PO
activity assays, an additional 6 to 8 μL of hemolymph
(if available) per individual was mixed 1:1 with ice cold
Pringle’s saline and loaded into a microcentrifuge tube.
Samples were stored at -80 C for up to 6 weeks prior to
running PO assays. A total of 10 µL of sample was
loaded into 96-well plates with 190 µL assay buffer [in
dD H20: 50 mM Na2PO4 monobasic monohydrate adjusted to 6.5 pH, 2 mM dopamine, and Micrococcus
luteus cell wall elicitor at 3% total volume]. We measured absorbance at 490 nm every 27 seconds at 30 C for
300 measures (total time: 02:14:33) using a Biotek mi-
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croplate reader. We recorded the final absorbance of
each sample to estimate the rate of melanization (Hall et
al., 1995; Barnes and Siva-Jothy, 2000). Prior work in
our lab showed that final absorbance in monarchs correlates strongly with the slope of the kinetic curve (absorbance per hr) during linear phase (r = 0.946; n = 206;
Altizer S, unpubl. data). PO assays were run twice for
samples with additional available hemolymph, and the
highest final absorbance of the two assays per sample
was used for analysis.
1.3 Lipid assays
We quantified total lipid mass in monarchs (Fig. 1)
following the protocols of Alonso-Mejia et al. (1997)
and Brower et al. (2006) with small modifications.
Briefly, monarchs were dried for ≥48 h at -50ºC in a
freeze dryer, dry mass was recorded to the nearest mg,
and the entire butterfly was placed into a 10 mL scintillation vial and crushed by placing the sample on a vortex at high speed and using a glass rod to homogenize
the dry tissue for 3 min. Three mL of petroleum ether
was added and grinding continued for an additional 3
min. The sample was transferred to a 25-mL vial, to
which another 7 mL of petroleum ether was added (for a
total of 10 mL), vortexed for 10 sec, and placed for 30
minutes in a 35ºC water bath while vortexing for 10 sec
every 10 min. The tube was then centrifuged for 10 min
at 1000 rpm (Dynac centrifuge) and the supernatant was
pipetted into a pre-weighed aluminum pan. Another 20
mL of petroleum ether was added to the centrifuge tube
and the extraction procedure was repeated. Pans were
allowed to air dry in a fume hood for at least 2 days to
evaporate the petroleum ether and the extracted total
lipid mass was then measured to the nearest mg. We
also calculated the ratio of total lipid mass to dry mass
(hereafter, ‘lipid proportion’) and lean mass (dry mass
minus lipid mass) for analysis.
1.4 Statistical analysis
Data were analyzed in SPSS version 20.0 (2011).
Response data for total lipid mass, lipid proportion, PO
activity, hemocyte concentration, and parasite load were
log10-transformed to normalize error variance. Date of
capture was treated as a categorical measure by assigning monarchs to one of three migration phases, following the approach of Gibo and McCurdy (1993) and
Brower et al. (2006), based on two-week intervals: early
(Sept. 28–Oct. 11), middle (Oct. 12–Oct. 25; peak of
migration), and late (Oct. 26–Nov. 10). Initial analysis
used Pearson correlations to investigate relationships
between total lipid mass, lipid proportion, lean mass,
average forewing area, PO activity, hemocyte concen-
SATTERFIELD DA et al.: Monarch lipid reserves and immune defense in migrating monarchs
tration, parasite load, and dry mass.
We used generalized linear models (GLM) to investigate the following: (1) Predictors of immune measures
(i.e., PO activity and hemocyte concentration) and lipid
mass measures (i.e., lipid proportion, total lipid mass,
and lean mass) were examined separately by treating
the following as independent variables: sex, infection
status, and migration phase as fixed factors and average
forewing area and wing condition as covariates. Maximal models included all two-way interactions except
those involving wing condition, owing to low variance
in this measure. The maximal model for PO activity also
excluded interactions between fixed factors to avoid low
sample sizes (n<5) within some categories. (2) To explicitly test how quantitative parasite load influenced
predictors of immunity and lipids, we repeated tests described in analysis 1 using data only for infected monarchs. As before, we treated total lipid mass, lipid proportion, lean mass, and hemocyte concentration as
separate dependent variables and examined the following independent variables: sex and migration phase as
fixed factors and average forewing area, wing condition,
and parasite load as covariates. Due to low sample sizes,
PO activity was not evaluated as a dependent variable,
and interaction effects were excluded from maximal
models. (3) The relationship between immune and lipid
measures was examined by treating total lipid mass,
lipid proportion, and lean mass as separate dependent
variables and immune measures (PO activity and
hemocyte concentration) as independent variables.
Maximal models also included variables found to be
significant predictors of lipid measures in the first set of
GLMs (including migration phase, forewing area, and
infection status) and all two-way interactions between
these predictors and each immune measure.
For all GLMs, we explored model fit and conducted
model simplification following Crawley (2002), based
on Akaike information criteria corrected for small sample sizes (AICc). We report results of the final models
including only main effects and interaction terms that
explained substantial variation in each response variable.
In cases where multiple comparisons were performed
using two or more measures to test a similar hypothesis
(i.e., multiple measures of immunity or lipid mass), we
controlled for multiple comparisons using Bonferroni
corrections (making α=0.025).
2
Results
Of all the monarchs we sampled in Georgia in fall
2010 (n=216), 14.8% were infected with OE. We re-
397
tained 25 infected monarchs (8 females and 17 males)
and 49 uninfected monarchs (16 females and 33 males)
for further analysis. Monarchs had a mean forewing
length of 51.48 mm (± 2.27 SD) and mean wet mass of
49.09 mg (± 7.62 SD). The average parasite load for
infected monarchs (log10-transformed) was 4.90 (± 0.50
SD), which is within the range reported in prior studies
(e.g., deRoode et al., 2008). Females were palpated for
spermatophores but none were detected, indicating they
were likely in reproductive diapause. We captured more
monarchs in the middle migration phase (n=39, including 12 females and 27 males) relative to the early phase
(n=17, including 5 females and 12 males) and late phase
(n=18, including 7 females and 11 males). We obtained
sufficient volumes of hemolymph to conduct both
hemocyte counts and PO assays from 40 monarchs and
to conduct hemocyte counts alone from an additional 20
monarchs. Across all monarch samples, correlation tests
revealed that PO activity decreased with lipid proportion (r=-0.337, P=0.034) and increased with lean mass
(r =0.351, P =0.027). The two measures of immune
defense (PO assays and hemocyte counts) were not significantly correlated. Total lipid mass was positively
correlated with lipid proportion (r =0.972, P <0.001),
lean mass (r =0.335, P=0.004), and dry mass (r =0.707,
P <0.001). Other measures of size were also positively
correlated; for example, lean mass increased with dry
mass (r =0.885, P <0.001) and with average wing area
(r=0.283, P =0.015).
2.1 Multivariate predictors of lipid and lean mass
GLM analysis showed that lipid proportion increased
with migration phase (Fig. 2A; F2,72=4.456, P=0.015)
and also depended on the phase*forewing area interaction (F2,72=4.291, P=0.018). In particular, monarchs
with larger wings had higher lipid proportions in the
early and middle phases but lower lipid proportions in
the late phase. The final model for lipid proportion did
not include any main effects or interactions involving
sex, infection status, or wing condition. Although total
lipid mass (in mg) increased with migration phase (Fig.
2), the GLM analysis for this variable did not reveal any
significant predictors. Lean mass was significantly
lower for infected relative to healthy monarchs (Fig. 3;
F 1,72=6.498, P =0.013) and increased significantly with
average wing area (F1,72=8.241, P =0.005).
Among infected monarchs (n=25), lipid proportion
significantly increased with migration phase (F2,24=
4.515, P=0.023). Lipid proportion was highest for monarchs with better wing condition (condition categories
1–3), although this trend was NS (F1,24=2.836, P=0.107).
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Fig. 2 (A) Total lipid mass (mean mg + SEM, white bars,
left y-axis) and lipid proportion (mean lipid mass/dry mass
+SEM, gray bars, right y-axis) across three migration
phases: early (Sept. 28–Oct. 11), middle (Oct. 12–Oct. 25),
and late (Oct. 26–Nov. 10). (B) Phenoloxidase (PO) activity
(mean final maximal absorbance + SEM, white bars, left
y-axis) and average hemocyte concentration (mean hemocytes x1000/µL +SEM; gray bars, right y-axis) across the
same three migration phases
Asterisks note significant differences at P<0.025 between monarchs in
the early compared to the middle migration phases, using univariate
analysis of variance and LSD post-hoc tests.
Fig. 3 Lean mass (mean mg + SEM) of uninfected and
infected monarchs
Lean mass was significantly lower for infected (n=25) compared to
uninfected (n=49) fall-migrating monarchs (F1,72=6.498, P=0.013), as
indicated by asterisks.
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Quantitative parasite load and other independent variables were not significant predictors of lipid proportion.
GLM analyses for total lipid mass and lean mass among
infected monarchs did not reveal any significant predictors.
2.2 Multivariate predictors of immune measures
GLM analysis showed that the relationship between
hemocyte concentration and infection status was opposite for males and females: Among females, uninfected
monarchs had more hemocytes, and among males, infected monarchs had more hemocytes (sex*infection
status: F1,59=6.588, P=0.013). Neither sex nor infection
status alone were significant predictors of hemocyte
concentration when tested as main effects. Although
hemocyte concentration decreased with migration phase
(Fig. 2B), this variable was not retained in our final
model. Likewise, PO activity declined with migration
phase (Fig. 2B), but this effect was only nearly significant (F2,40=3.424, P=0.043). No other predictors for PO
activity were significant. For analyses constrained to the
subset of infected monarchs, hemocyte concentration
had no significant predictors and PO activity was not
analyzed due to low sample sizes.
2.3 Relationships between lipid and lean mass and
immune measures
GLM analysis showed that lipid proportion declined
with PO activity (Fig. 4) in an analysis that also included hemocyte concentration and variables significant
in earlier tests (phase, average wing area, and interactions between immune measures and phase), but this
trend was only nearly significant (F1,39=4.862, P=0.034).
No other variables were retained in the final model for
lipid proportion. Total lipid mass was also analyzed as a
Fig. 4 Correlation between lipid proportion (lipid mass/
dry mass) and phenoloxidase (PO) activity
Lipid proportion and PO activity are negatively associated for infected
monarchs (triangles, dotted line; r=-0.369, P=0.263, R2=0.138, n=11),
uninfected monarchs (circles, solid line; r=-0.357, P=0.058, R2=0.128,
n=29) and across all monarchs (r=-0.335, P=0.034, R2=0.113, n =40).
SATTERFIELD DA et al.: Monarch lipid reserves and immune defense in migrating monarchs
dependent variable, but no significant predictors were
found. GLM analysis of lean mass showed that this
measure increased with PO activity, however this effect
was NS (F1,39=5.325, P =0.027).
3 Discussion
Results from this study provide weak evidence for a
trade-off between energy reserves and immune defense
in fall-migrating monarchs. In particular, lipid measures
were higher but immune measures were lower for monarchs sampled later in the fall migration, suggesting that
these two measures changed in opposite directions over
time. More importantly, lipid proportion correlated negatively with one measure of immune defense (phenoloxidase activity), although this relationship did not
reach significance in a multivariate analysis, and lipids
did not covary with hemocyte concentration. Moreover,
lipid measures were not significantly lower for monarchs infected by OE (or with higher quantitative parasite loads), suggesting that previously-demonstrated
costs of parasitism for monarch migration (e.g., Bradley
and Altizer, 2005; Bartel et al., 2011) are probably
caused by factors other than inadequate lipid reserves.
Monarchs sampled earlier in the fall migration had
higher levels of immune defense compared to migrants
sampled later in the season, however this effect was not
significant at the α=0.025 level. Still, this temporal decline in immunity is consistent with some prior work on
insect immunity. For example, Krams et al. (2011)
found that male water striders entering overwintering
diapause early in the season displayed greater encapsulation responses than male water striders entering diapause later in the season. One explanation for temporal
changes in PO activity reported here is that monarchs
could down-regulate immunity as the fall migration
progresses, in response to energetic constraints or
scarcer nectar resources. However, it is important to
note that our sampling scheme did not follow individual
cohorts over time during the migration, but rather sampled different groups of individuals passing through the
same location. As an alternative explanation, the decline
in PO from early to late migrants could reflect a positive
relationship between migratory performance and PO
activity, such that the most robust monarchs (with
higher levels of PO defenses) migrated faster and passed
through our study sites earlier in the migration (Davis et
al., 2012; Davis et al., 2007). In support of this idea, our
results showed a positive association between PO and
lean mass (largely comprised of wing and muscle tissue),
indicating that early migrants may have been in better
399
condition overall than late migrants. In either scenario,
monarchs with lower PO activity could be more susceptible to pathogen infection and might experience slower
wound healing as a result of lower PO activity.
The negative correlation between lipid proportion
and PO activity among migrating monarchs is consistent
with the idea that measures of immunity could decline
when resources are limited and other activities are demanding (reviewed in Norris and Evans, 2000). However, this negative relationship was weak. Further, there
were no associations between lipid proportion and cellular immunity (hemocyte concentration). In contrast to
the results reported here, other studies have found that
animals in better condition had both higher lipid measures and higher immune defense, as reported in male
damselflies, for which individuals with more lipid reserves (and that were better competitors for mates) also
had higher encapsulation responses (Koskimäki et al.,
2004; Contreras-Garduño et al., 2006).
Because OE infection has been shown to reduce
monarch body size (especially mass at eclosion), longevity, and flight performance in captive monarchs
(e.g., Altizer and Oberhauser, 1999; Bradley and Altizer,
2005; de Roode et al., 2007), we predicted that OE infection would reduce the ability of wild monarchs to
acquire or convert nectar into lipid reserves during migration or would erode lipid reserves carried over from
the larval and pupal stages. However, our results
showed that lipid measures for infected monarchs were
not significantly different from healthy monarchs, and
lipids did not correlate negatively with parasite load.
This suggests that previously-documented negative effects of OE infection on monarch flight performance
and migration success (Bradley and Altizer, 2005;
Bartel et al., 2011; Altizer et al., 2000) were not caused
by the differential ability of infected monarchs to acquire or maintain energy reserves needed to fuel
long-distance flight. Alternatively, the absence of association between lipids and infection reported here could
be caused by poor survival of heavily infected monarchs,
such that by the time monarchs reached our sampling
sites in Georgia, those suffering from the most intense
parasite infections would have already been removed
from the population. In support of the latter idea, the
average parasite load found here was lower but within
the range of parasite loads reported from experimental
infections of captive monarchs (de Roode et al., 2008;
de Roode and Altizer, 2010).
Interestingly, lean mass was significantly lower among
infected monarchs compared to healthy monarchs. This
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finding probably indicates lower muscle mass, rather
than wing mass, because wing area was similar for infected and healthy monarchs in our study (t =-0.530,
P=0.598). Lean mass measures reported here (mean =
162.7 mg ± 21.8 SD) were comparable to that of 7 other
studies measuring lean mass in eastern North American
monarchs (range of means 104–184 mg; summarized in
Alonso-Mejia et al., 1997). Although our study cannot
determine the direct cause of this difference in lean
mass, future work could examine whether flight differences previously reported for healthy and infected monarchs (Bradley and Altizer, 2005) are explained by differences in flight muscle and flight metabolism, as opposed to lipids or other morphometric constraints.
Immune measures (PO or hemocyte concentration)
did not depend on infection status as a main effect, but
hemocyte concentration did depend on the interaction
between infection status and sex. Specifically, infected
males had significantly higher hemocyte concentration
than healthy males, whereas infected and healthy females had similar hemocyte measures. A nearly identical pattern was observed in a previous study (Lindsey
and Altizer, 2008) examining larval hemocyte concentration. As Lindsey and Altizer suggest, one possibility
is that these results reflect lower levels of baseline immunity in males, such that males increase immune defenses in response to infection but invest little otherwise.
Females, by comparison, might invest in higher levels
of baseline immunity, perhaps because they experience
a higher cost of infection, and thus greater benefits for
investing more heavily in baseline immune defenses.
Evidence for greater investment or benefits of baseline
immunity in females has been previously reported in
some insects (reviewed in Zuk and Stoehr, 2002).
Lipid measures in monarchs sampled here were lowest early in the migration period and peaked in the middle phase. A similar temporal pattern was noted by Gibo
and McCurdy (1993), who sampled lipids in migrating
monarchs over 8 weeks in southern Ontario, and found
that lipids peaked in the middle phase of migration and
declined among the latest migrants. This prior study
suggested that results could be due to temporal changes
in nectar availability and to different cohorts of monarchs passing through the area, assuming that the earliest migrants have not yet had time to accumulate high
levels of lipids (e.g., Alonso-Mejia et al., 1997; Brower,
2006) and that the last migrants are stragglers in poorer
condition. Our lipid mass measures (mean = 25.5 mg ±
15.3 SD) are similar to those collected by Brower (un-
Vol. 59
No. 3
published data referenced in Alonso-Mejia et al., 1997)
from Georgia fall-migrating monarchs (n=46) in November 1980 and to those collected by Beall (1948)
from Louisiana late-fall migrants. It is important to note
that fall-migrating monarchs are thought to accumulate
a substantial portion of their lipid reserves once they
reach Texas en route to Mexico (Brower et al., 2006).
Thus, investigating the relationship between lipid reserves and immune measures in an area such as Texas
where lipids rapidly accumulate might help to identify
mechanisms that influence lipid-immune defense tradeoffs.
In sum, the allocation of resources during migration
and overwintering is expected to be under strong selection. This could be especially true for migrating insects
that engage in reproductive diapause and have not yet
reproduced (Rankin and Burchsted, 1992), as selection
is typically strongest on pre-reproductive life stages
(Medawar, 1952). On one hand, immune defense might
increase animal survival during migration and overwintering by conferring protection from novel pathogens.
At the same time, these defenses can also be metabolically costly (Schmid-Hempel, 2005). Thus, the relationship between seasonal behaviors and immunity is
not always straightforward, and likely depends on resource availability and the potential risk of infection
(Buehler et al., 2008). To better understand the links
between migration and immunity, and implications for
infectious disease risk, further monitoring of immune
changes over seasonal periods of breeding, migration,
and overwintering is needed across taxa (Hawley and
Altizer, 2011). In addition, experimental studies are
needed to tease apart how immune defenses respond to
the effects of (i) pre-migratory status, (ii) energetic demands of movement, and (iii) resource availability, and
thus to better elucidate the mechanisms that might generate synergy or conflict between investment in migration and immune defenses.
Acknowledgements We thank M. Maudsley and M. Weathers for conducting PO assays and assisting with monarch capture, hemocyte counts, and parasite load quantification; A.
Davis for analysis of morphometric wing data; L. Brower, T.
Maddox and the UGA Analytical Chemistry Lab for guidance
and resources for lipid extractions, and the Altizer lab and two
anonymous reviewers for comments on previous drafts of the
manuscript. Funding for this project was provided by the National Science Foundation (grant DEB-0643831 to S.A. and a
Graduate Research Fellowship to D.S.) and the UGA Integrated Life Science Program to D.S.
SATTERFIELD DA et al.: Monarch lipid reserves and immune defense in migrating monarchs
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