Summary. Parasites represent significant challenges to social
insects. The high density, interaction rate and relatedness of
individuals within colonies are all predicted to make social
insect colonies particularly vulnerable to parasites. To cope
with this pressure, social insects have evolved a number of
defence mechanisms. These include the immune response,
which, aside from in bumblebees, has been relatively little
studied in social insects. Here we compare the immune re-
sponses of males and workers of the leaf-cutting ant Acro-
myrmex echinatior and examine the effect upon immuno-
competence of prior exposure to a virulent parasite. Males
have a far lower immune response than workers, suggesting
either haploid susceptibility or reduced investment in immu-
nity by the short-lived males. There was also significantly
less variation in the immune response of males than of work-
ers, which may be due to leaf-cutting ant workers being more
variable in age or more genetically diverse within colonies.
When exposed to the entomopathogenic fungus Metarhizium,
workers expressed a substantially reduced immune response
96 h after infection, suggesting that the immune system was
either depleted by having to respond to the Metarhizium infec-
tion or was depressed by the parasite. The results suggest 
that the immune response is a costly and limited process, but
further experiments are needed to distinguish between the
alternative explanations for the effects observed.
Key words: Encapsulation, immunocompetence, Metarhizium,
Acromyrmex, haploid susceptibility hypothesis.
Introduction
Social insect colonies are characterised by a dense aggrega-
tion of individuals that are generally highly related to one
another. These features facilitate the transmission of disease
and make social insect colonies particularly vulnerable to
parasites (Alexander, 1974; Schmid-Hempel, 1998; Booms-
ma et al., in press). Consequently, social insects have evolved
a number of specialised mechanisms to defend their colonies
against parasites, including grooming, antibiotic secretions
and hygienic behaviour (e.g. Kermarrec et al., 1986; Rosen-
gaus et al., 1998, 2000, 2004; Schmid-Hempel, 1998; Christie
et al., 2002; Hart and Ratnieks, 2001; 2002; Hughes et al.,
2002; Poulsen et al., 2002a,b; Turillazzi et al., 2004; Booms-
ma et al., in press). The effectiveness of these mechanisms is
such that rather than the group-living lifestyle being associ-
ated with increased susceptibility to disease, as is expected, it
may instead result in decreased susceptibility (Rosengaus et
al., 1998; Hughes et al., 2002; Shimizu and Yamaji, 2003). In
addition to these specialised defence mechanisms, social
insects also have the individual immune systems that are
found in most insects and a number of antibacterial peptides
have been identified from them (Casteels et al., 1990; 1993;
Casteelsjosson et al., 1993; Mackintosh et al., 1998; Taguchi
et al., 1998; Lamberty et al., 2001). The action of the immune
system has been intensively studied in the bumblebee Bom-
bus terrestris (reviewed in Schmid-Hempel, 2001), but there
have been relatively few investigations of the immune system
in other social insects (Rosengaus et al., 1999; Traniello et
al., 2002; Vainio et al., 2004). This is in spite of its probable
importance to the survival of individual social insects and
thus of their colonies.
In this study, we examine the immune response of Acro-
myrmex leaf-cutting ants (Hymenoptera: Formicidae: Attini)
by implanting a nylon filament into the haemolymph of the
ants and then measuring the resulting encapsulation response.
Insect. Soc. 52 (2005) 298?303
0020-1812/05/030298-06
DOI 10.1007/s00040-005-0809-x
? Birkh?user Verlag, Basel, 2005
Insectes Sociaux
Research article
Examination of the immune responses of males and workers 
of the leaf-cutting ant Acromyrmex echinatior and the effect of infection
B. Baer1, A. Krug1, J. J. Boomsma 1 and W. O. H. Hughes1, 2, *
1 Department of Population Biology, University of Copenhagen, Universitetsparken 15, Copenhagen 2100, Denmark, e-mail: bcbaer@bi.ku.dk,
andrea-krug@gmx.de, jjboomsma@bi.ku.dk
2 Present address: School of Biological Sciences, A12, University of Sydney, Sydney, N.S.W. 2006, Australia, e-mail: whughes@usyd.edu.au
Received 3 August 2004; revised 3 February 2005; accepted 2 March 2005.
* Author for correspondence.
Insect. Soc. Vol. 52, 2005 Research article 299
Materials and methods
Acromyrmex echinatior colonies were collected in Gamboa, Panama
and maintained in the laboratory at 25?C, 70% RH on a diet of bramble
leaves and rice. Adult males and large workers (head width 2.07 mm ?
0.015 mm) were sampled from six colonies (Ae33, Ae48, Ae109, Ae153,
Ae162 and Ae177). They were maintained in a container with an ad 
libitum supply of water and 10% sucrose water. We quantified im-
munocompetence by measuring the encapsulation response. This cellu-
lar response is a commonly used measure of immunity and involves
haemocytes attaching to a foreign particle, melanising and eventually
forming a capsule around it. We modified the protocol used previously
with bumblebees (Allander and Schmid-Hempel, 2000; Doums and
Schmid-Hempel, 2000; Baer and Schmid-Hempel, 2003a; Gerloff et al.,
2003), and utilised equipment designed for the artificial insemination of
bees (Baer and Schmid-Hempel, 2000). Ants were anaesthetized with
CO2 and held in place in a plastic holder. Two injection needles with
bent tips were used as hooks to stretch the individual?s sternites allow-
ing access to the intersegmental membranes. A needle was used to
pierce the intersegmental membrane between the second and the third
sternite and a nylon filament (0.13 ? 0.5 mm) was implanted into the
animal?s haemolymph. Ants were allowed to recover and were then
frozen 24 h later. Mortality during this procedure was low (<2%). The
implants were subsequently dissected out, mounted on microscope
slides with Eukitt, and photographed with a digital camera (Canon EOS
D30) connected to a Leica dissecting microscope. The degree of encap-
sulation was measured using imaging software (NIH Image Program)
by subtracting the mean grey value of the background from the mean
grey value of the melanised implant.
To examine the effect of prior exposure to a parasite upon the
immune response, we sampled large workers from each of ten colonies
of A. echinatior (Ae33, Ae48, Ae109, Ae132, Ae143, Ae153, Ae154,
Ae155, Ae168 and Ae177) and maintained them as above. As the exper-
imental parasite, we used strain KVL02-73 of the entomopathogenic
fungus Metarhizium anisopliae var. anisopliae. This strain was isolated
from the same site in Gamboa, Panama, as that from which the ant
colonies were collected (Hughes et al., 2004a), and it has previously
been shown to be highly pathogenic to A. echinatior (Hughes et al.,
2002; 2004b). We harvested spores from a freshly sporulating plate of
the fungus and used these to make a suspension of 1 ? 107 spores per ml
in 0.05% Triton-X. Spore viability was checked by plating the suspen-
sion on to agar plates (Lacey and Brooks, 1997), and was confirmed 
to be >95%. We treated half of the ants from each colony with 0.5 ml of
the spore suspension of the parasite, and the other half of the ants with
0.5 ml of a 0.05% Triton-X control solution. The ants were then main-
tained in individual pots with access to water and 10% sucrose water.
For each treatment and each colony, the encapsulation responses of a
third of the ants were measured immediately, and of the remaining thirds
at 48 h and 96 h after treatment. Implants were inserted at these times,
and the ants were then maintained for 24 h before being frozen. The
encapsulation response was quantified as described above.
Results
Comparison of the immune responses of males and workers
Encapsulation data were collected for a total of 131 large
workers and 102 males. The encapsulation response of males
(5.89 ? 0.53) was significantly lower than that of the workers
(19.9 ? 1.55; ANOVA, F1, 221 = 68.5, p < 0.001). This differ-
ence was present in all colonies, but the magnitude of it 
varied significantly between colonies (Fig. 1; F5, 221 = 4.10, 
p = 0.001), and colonies differed overall in their encapsula-
tion response (F5, 221 = 3.43, p = 0.005). The variation in the
encapsulation response was significantly greater between
Leaf-cutting ants have large long-lived colonies (H?lldobler
and Wilson, 1990), and exist in habitats that can contain a
high diversity of particular parasites (Hughes et al., 2004a).
Although much is known about their other defence mecha-
nisms against disease (Kermarrec et al., 1986; Jaccoud et al.,
1999; Bot et al., 2001, 2002; Hart and Ratnieks, 2001, 2002;
Hughes et al., 2002; Poulsen et al., 2002a,b), their immune
system has not previously been investigated. We set-out 
to determine two basic features of the immune response: 
1) whether males and workers differ in their encapsulation
response, and 2) whether the encapsulation response is either
increased or decreased by prior exposure to a parasite. 
Lower immunocompetence of males compared to females
has been found in two other social insect species (Bombus
terrestris: Gerloff et al., 2003; B. Baer and P. Schmid-Hempel,
unpubl.; Formica exsecta: Vainio et al., 2004;), and can be
expected for two reasons. In the Hymenoptera, males arise
from unfertilised eggs and are therefore haploid, whereas
females arise from fertilised eggs and are diploid. Hetero-
zygosity is often considered to improve an individual?s re-
sistance to disease, and it has been suggested that haploid
individuals, such as ant and bee males, will be less immuno-
competent than diploid females (the ?haploid susceptibility
hypothesis?; O?Donnell and Beshers, 2004). A non-mutually
exclusive alternative explanation derives from life-history
differences rather than ploidy. In leaf-cutting ants and most
social Hymenoptera, the males spend the vast majority of
their life within their natal colony and leave it only for a short
mating flight after which they die (Baer, 2003). Within their
natal colony males will be protected from parasites by the
colony?s workers, while the very short duration of the mating
flight means that any diseases that males contact there will
not impact upon their fitness. Female workers, in contrast,
spend a significant part of their lives foraging outside the
colony and therefore have a much higher lifetime exposure to
parasites than do males. Workers are likely to be selected to
be resistant to parasites both to maximise their individual 
value to their colony and to reduce the risk of them transmit-
ting diseases to nestmates. The immune system of insects is
costly (Kraaijeveld and Godfray, 1997; Fellowes et al., 1998;
Kraaijeveld et al., 2002; Rolff and Siva-Jothy, 2003; Schmid-
Hempel, 2003; 2005), and the much lower importance of 
diseases to males compared to workers means that males may
benefit by diverting resources from immunity to mating-
related traits (Rolff, 2002; Schmid-Hempel, 2005). 
The costly nature of immunity in insects also makes it
likely that there will be limitations on the number and level
of challenges that an individual can deal with at any one
time. In addition, many parasites can directly affect the im-
mune response by producing immunodepressant compounds
(Gillespie et al., 2000a; Schmid-Hempel, 2005). However,
evidence for an impact of parasites upon the immune response
is limited and conflicting. The encapsulation response of
bumblebees has been found to be reduced (Doums and
Schmid-Hempel, 2000), and unaffected (Allander and Schmid-
Hempel, 2000) by infection with the gut parasite Crithidia,
while the immune response of termites can be increased by
prior exposure to a parasite (Rosengaus et al., 1999).
300 B. Baer et al. Immunocompetence of leaf-cutting ants
large workers than between males (ANOVA using ranks of
standard deviations in encapsulation response as a dependent
variable, F1, 12 = 36.0, p = 0.002), but did not differ between
colonies (F5, 12 = 1.33, p = 0.380).
Effect of parasitic infection on immune response
The encapsulation response of large workers treated with
Metarhizium spores and those treated with the control solu-
tion did not differ immediately after treatment or 48 h later
(Fig. 2). However at 96 h after treatment ants exposed to the
Metarhizium parasite had a significantly lower encapsulation
response than those treated with the control solution (ANO-
VA, F2, 487 = 4.55, p = 0.011). There was also significant vari-
ation between colonies in the overall level of encapsulation
response (F9, 487 = 2.25, p = 0.018), but not in the way the
encapsulation response was affected by the two treatments
(F9, 487 = 0.58, p = 0.82).
Discussion
The leaf-cutting ant A. echinatior was found to have a clear
difference in immune response between males and workers,
with the former exhibiting a much lower encapsulation
response than the latter. Males have similarly been found to
have lower immunocompetence than females in both of the
other social insect species previously examined (Gerloff et
al., 2003; Vainio et al., 2004; B. Baer and P. Schmid-Hempel,
unpubl.), as well as in some other insects (Adamo et al.,
2001; Rolff, 2001, 2002; Siva-Jothy et al., 2001; Schmid-
Hempel, 2005). Although the data in our study do not allow
us to directly distinguish between haploid susceptibility and
life-history as explanations for low male immunocompe-
tence, there are two points that may shed light on the issue.
The first is that all male ants lack metapleural glands (H?ll-
dobler and Wilson, 1990). These glands are known to be
important in disease defence (Bot et al., 2002; Hughes et al.,
2002; Poulsen et al., 2002a), and are large and costly (Poulsen
et al., 2002a). Male ants have thus eliminated investment in
this costly disease defence mechanism, presumably because
disease resistance is less important to them than traits that
increase the number of matings they obtain. The reduced
immunocompetence of males might therefore be for the same
reason. Secondly, the differences in immunocompetence and
life-history between the males and workers of A. echinatior
and of the bumblebee Bombus terrestris exhibit an intriguing
pattern. Male leaf-cutting ants leave the protection of their
natal colony only for a brief nuptial flight after which they
die. There is therefore very little risk of diseases impacting
upon the fitness of male leaf-cutting ants and little that they
can gain by investing in immune defences. In contrast, male
bumblebees patrol mating territories or other colonies for
several weeks (Goulson, 2003). During this time the males
will be exposed to parasites and if infected may have reduced
lifespan and fitness. The benefit of being able to resist para-
sites is therefore similar for the males and workers of B. ter-
restris, whereas in A. echinatior the benefit is much lower for
males than for workers. In A. echinatior, the encapsulation
response of males was approximately four times lower than
that of workers, with the response of males being minimal. In
Bombus terrestris, the difference between males and workers
is much less with both having considerable immune respons-
es (Gerloff et al., 2003; B. Baer and P. Schmid-Hempel, un-
publ.). The lack of metapleural glands in male leaf-cutting ants
and the relative male-worker differences in the encapsulation
response of A. echinatior and B. terrestris, both suggest that
the low immunocompetence of male leaf-cutting ants may be
due to reduced investment in immunity by males rather than
haploid susceptibility. However, data from more species are
obviously needed to obtain a clearer comparative picture.
Figure 1. The mean (? s.e.) encapsulation response to a nylon filament
implanted in the haemolymph of males (clear bars) and workers (shaded
bars) from six colonies of A. echinatior. Sample sizes are presented as
numbers within bars
Figure 2. The mean (? s.e.) encapsulation response to a nylon filament
implanted in the haemolymph of A. echinatior workers treated with the
Metarhizium parasite (shaded bars) or a control solution (clear bars).
The encapsulation response of workers was examined either immediate-
ly, 48 h or 96 h after treatment. Sample sizes are presented as numbers
within bars
Insect. Soc. Vol. 52, 2005 Research article 301
et al., 2002), and the increased growth of an avirulent para-
site when coinfecting with Metarhizium has been attributed
to the immunodepressant activity of Metarhizium toxins
(Hughes and Boomsma, 2004b). The toxins are produced by
the hyphal bodies of Metarhizium and so the quantity of 
toxins produced will be positively correlated with parasite
growth. The reduced encapsulation response of workers 96 h
after infection could therefore be due to depletion or to tox-
in-mediated immunodepression and further work is needed
to distinguish these possibilities.
The immune response is a costly trait (Kraaijeveld and
Godfray, 1997; Fellowes et al., 1998; Kraaijeveld et al., 2002;
Rolff, 2002; Rolff and Siva-Jothy, 2003; Schmid-Hempel,
2003; 2005; Boomsma et al., 2005) and it is therefore to be
expected that there are trade-offs involved in the allocation of
resources to it. Investment will depend upon life-history and
the importance of being resistant to diseases, while the re-
sponse itself will be finite and may be depleted by multiple
challenges. The results of this study are suggestive of such
trade-offs occurring. 
Acknowledgements
We would like to thank Sylvia Mathiasen for technical assistance, the
Smithsonian Tropical Research Institute for providing facilities in 
Gamboa for the collection of ant colonies, and the Instituto Nacional de
Recursos Naturales Renovables for permission to collect and export the
ant colonies from Panama to Denmark. We are also grateful to Paul
Schmid-Hempel, Ross Crozier and two anonymous reviewers for com-
ments on previous versions of the manuscript. The work was funded by
a Carlsberg Foundation grant to W.O.H.H., a Danish Natural Science
Research Council grant to J.J.B and a Swiss Marie Curie Individual 
Fellowship (83EU-062441) to B.B.
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