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Symbiontmicrocosminanantsocietyandthe
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ImpactFactor:3.14·DOI:10.1016/j.anbehav.2008.05.010
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ANIMAL BEHAVIOUR, 2008, 76, 1477e1486
doi:10.1016/j.anbehav.2008.05.010
Available online at www.sciencedirect.com
Symbiont microcosm in an ant society and the diversity of
interspecific interactions
VOLKER WI TTE * , A N NETT E LEIN GÄ RTNER*, LEA ND RA S AB Aß* , ROSLI HA SH IM † & S U SA NN E FOI TZ IK *
*Department Biologie II, Ludwig-Maximilians Universität München (LMU)
yInstitute of Biological Sciences, University Malaya
(Received 9 July 2007; initial acceptance 1 September 2007;
final acceptance 13 May 2008; published online 3 September 2008; MS. number: 9448R)
Colonies of the ponerine army ant Leptogenys distinguenda are regularly inhabited by a highly diverse symbiont fauna including insects, spiders, mites, crustaceans and even molluscs. Each of these myrmecophiles
has adapted in a highly specific way to the lifestyle of its host. We studied this diverse myrmecophile fauna
of L. distinguenda as a new model for multispecies parasitism to gain a better understanding of fundamental
coevolutionary processes. Our study focused on behavioural and on chemical integration and exploitation
strategies of the different symbiont species. In addition, we examined potential counterstrategies of the
host ant. Myrmecophiles were studied both in large free-living L. distinguenda colonies and in more detail
in parts of colonies separated for observation. We found that at least five myrmecophile species imposed
cost on their host by exploiting its resources. Their impact varied considerably depending on both the type
of resources exploited and their abundance. Myrmecophile species were well integrated into host societies
either by chemical mimicry of host cuticular hydrocarbons or by remaining chemically insignificant, lacking most characteristic recognition cues. Despite these chemical integration strategies, host ants were able
to recognize and kill the alien intruders to various degrees. This important finding demonstrates that symbiont populations are actively counter-regulated by the host. By constructing a hosteparasite interaction
network, we finally suggest that host defences can maintain myrmecophile diversity by keeping parasite
populations small. This reduces interguild competition, comparable to topedown effects of predators
on lower trophic levels in ecological food webs.
Ó 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Keywords: army ant; chemical integration; community ecology; host resistance; hosteparasite interactions; Leptogenys
distinguenda; migration; transmission mode
Symbioses with other organisms are common and occur
with impressive diversity in ants. We use the term
symbiosis in its original meaning of organisms living in
close relationship, however, without any implication of
reciprocal positive or negative influences (Goff 1982).
Symbionts that live in close association with ants for considerable parts of their life cycle are also referred to as
Correspondence: V. Witte, Department Biologie II, Verhaltensökologie,
Ludwig-Maximilians-Universität München, Großhaderner Str. 2,
D - 82152 Planegg-Martinsried, Germany (email: [email protected].
uni-muenchen.de). R. Hashim is at the Institute of Biological Sciences,
Faculty of Science, University Malaya, 50603 Kuala Lumpur,
Malaysia.
0003e 3472/08/$34.00/0
myrmecophiles (Wilson 1971; Kistner 1982). Symbiotic
interactions with ants range from mutualism, for example
in the important antetrophobiont associations (Delabie
2001), to predation or parasitism as in the lycaenid butterfly larvae (Pierce et al. 2002). Myrmecophiles that are
highly integrated into the host colonies generally benefit
from the protected environment and, in addition, they
are frequently parasitic by using host resources (Hölldobler & Wilson 1990).
Parasitism is generally one of the most successful life
strategies known with estimates of the proportion of
parasites among eukaryotes ranging between 30 and
70%, depending on the analysis (de Meeûs & Renaud
2002). Moreover, hosteparasite associations also represent
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Ó 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
1478
ANIMAL BEHAVIOUR, 76, 5
excellent model systems for the understanding of coevolutionary and ecological processes (Anderson & May 1982;
Lafferty et al. 2006; Lambrechts et al. 2006). Since parasitism affects most organisms to some extent, including
humans, it has received intense scientific attention. Even
though systems involving numerous parasites are common, most previous studies have focused on one-hoste
one-parasite systems thereby neglecting much of the
existing complexity (Petneya & Andrews 1998; Pedersen
& Fenton 2006). Thus, it is important to consider entire
hosteparasite communities for a comprehensive functional understanding of parasitic interactions.
We chose the highly diverse myrmecophile community
of the host ant Leptogenys distinguenda as a model system
for multispecies parasitism. This Southeast Asian ponerine
ant reaches colony sizes of well over 50 000 individuals
and makes nocturnal army ant-type mass raids. Colonies
are regularly inhabited by a microcosm of various myrmecophiles, including species of the orders Araneae and Acariformes (Chelicerata), Collembola, Zygentoma, Coleoptera
and Diptera (Insecta), Malacostraca (Crustacea) and Pulmonata (Mollusca) (Ferrara et al. 1987; Wunderlich
1994; Witte et al. 1999, 2002; Witte 2001; Janssen & Witte
2003; Kistner et al. 2003). Most of these taxonomically diverse symbionts appear to be very well integrated into the
host society; however, these mechanisms of social integration are largely unknown. Generally, tight hostesymbiont
interactions imply highly specific adaptations. Specifically
in this system, symbionts must blend into the society
without being recognized and expelled. Furthermore,
they need to maintain contact with their host which is
particularly challenging in the highly mobile army ants.
The host ant L. distinguenda frequently shifts nest sites (average 1.5 days) over considerable distances (5e58 m, median 19; Maschwitz et al. 1989). While some symbionts
might be able to cover these distances on their own,
others are unable to achieve this because of taxon-specific
constraints. Finally, to complete their life cycles, symbionts need to reproduce successfully in host colonies or
they have to guarantee readoption into or transmission
between host colonies. Here again, host migratory behaviour imposes severe challenges.
The symbiont fauna of L. distinguenda can serve as
a hosteparasite model system that includes all general
levels of interaction (host defence system, parasite development and virulence, parasite transmission). Owing to
the phylogenetic constraints, each symbiont copes in a different way with the highly flexible raiding and emigration
behaviour of the host ant L. distinguenda. We investigated
whether universal integration strategies are used by the
various symbiont species or whether symbionts apply diverse, individual strategies. For example, chemical integration of alien organisms into social insect colonies can be
achieved with different strategies. Cuticular hydrocarbons
are mainly responsible for nestmate recognition in social
insects (Howard & Blomquist 2005) and various social parasites mimic the chemical profiles of their hosts either by
active biosynthesis of cuticular hydrocarbons or by acquiring host chemical profiles passively (Dettner & Liepert
1994; Lenoir et al. 2001). The term ‘chemical mimicry’ is
used here according to its original definition of ‘mimicry’
as an organism’s resemblance of another organism’s properties (Vane-Wright 1976). Host chemical profiles can also
change considerably in time (gestalt model; Hölldobler &
Wilson 1990). Consequently, intruders must be able to update their profiles regularly to match the changing chemical signature of their particular host colony over long
periods. Another strategy of chemical integration is the
suppression of recognition cues resulting in chemical
insignificance (Lenoir et al. 1999, 2001). In this study we
investigated which integration strategies the various
symbionts use.
Many army ants harbour complex symbiont communities (Gotwald 1995); however, they have not been studied
comprehensively because of methodological difficulties.
In contrast to most army ant species, the comparatively
small Leptogenys colonies can be captured with their associated myrmecophiles and maintained in the laboratory
for several weeks (Witte 2001). By observation of such colony fragments, we gained, for the first time, in vivo insights into the diverse symbiont fauna of an army ant.
The aim of this study was to use this methodological advance to analyse the symbiont fauna of L. distinguenda
comparatively with regard to interspecific interactions, including behavioural adaptations and chemical integration
mechanisms.
The construction of interaction networks, a technique
adopted from community ecology, was recently proposed
to facilitate the understanding of fundamental processes
in complex hosteparasite communities (Pedersen & Fenton 2006). We applied this approach to the diverse symbiont fauna of L. distinguenda. For this purpose, we analysed
in detail the occurrence of symbiont species, their individual behaviour, their ecological niches within host colonies
and the interrelations between host and symbiont species.
With this comprehensive approach we aimed to address
the following questions. (1) Which general patterns in
the composition of symbiont communities can be observed; do particular symbiont species dominate host colonies and do species co-occur regularly or do particular
species exclude others? (2) If the symbiont diversity is generally high, how is this diversity maintained under the assumption of direct competition for host resources? (3) Do
symbionts interact positively or negatively? (4) What are
the impacts of symbiont species on their host? Here we
present the first comparative overview of the diverse symbiont microcosm of an army ant species, and with these
data we discuss the questions raised above.
METHODS
Focal Species
Focal species were all symbionts inhabiting colonies of
the ponerine army ant L. distinguenda. Reports exist on
only five of at least nine symbiotic species; however, the
biology even of the described species is still poorly understood. Even a snail, Allopeas myrmekophilos, is found regularly in L. distinguenda colonies. Unable to follow the ants
actively, this gastropod manipulates host workers to transport it to new nest sites during emigrations (Witte et al.
WITTE ET AL.: SYMBIONT DIVERSITY IN AN ANT SOCIETY
observers over their entire duration (median 210 min,
N ¼ 14). Natural emigrations are conducted in a highly
organized manner with a single column of brood-carrying
ants heading one after the other to a new nest site. Thus,
observations of myrmecophiles should generally have
good reliability. Nevertheless, individuals can be overlooked, especially if they are small and/or riding on the
opposite side of the observer. Furthermore, all myrmecophile counts are probably slightly underestimated,
because emigrations were mostly in progress when
observation started so that unknown numbers of myrmecophiles could have been missed. Numbers were estimated
for myrmecophile species that were too small and/or
too abundant to be counted reliably. To observe whether
phoretic myrmecophiles were able to reattach themselves
to their hosts, we separated individuals experimentally
from natural emigration columns and reintroduced them
after 5 min (mites, N ¼ 8, woodlice, N ¼ 10; collembolans,
N ¼ 14; silverfish, N ¼ 25; ptiliid beetles, N ¼ 12).
Myrmecophiles and host ants were also collected with
an aspirator directly from emigration columns, including
darkly coloured adult workers, freshly hatched, lightly
coloured callow workers, larvae and pupae. Thus, sample
sets of symbionts and corresponding hosts originating
from the same colonies were gathered. Four sets of such
collections were used to construct laboratory colonies; for
six further sets plus two laboratory colonies we extracted
and analysed cuticular hydrocarbons (see below).
2002; Janssen & Witte 2003). A woodlouse, Exalloniscus
maschwitzi, in contrast, rides phoretically on pupae (Ferrara
et al. 1987), which are carried by migrating host workers.
A myrmecophilic spider, Gamasomorpha maschwitzi, participates actively in host emigrations (Wunderlich 1994;
Witte et al. 1999), and a staphylinid beetle, Trachydonia
leptogenophila, follows its host actively too, but always at
a distance (Kistner et al. 2003). Similar following behaviour can be observed for females of two phorid fly species,
Rhynchomicropteron necaphidiforme and Puliciphora rosei
(Witte 2001). All other myrmecophiles, including mites,
a silverfish, collembolans and ptiliid beetles are undetermined until now. During an extensive study on the communication system of L. distinguenda (Witte 2001), large
laboratory colonies were regularly maintained over several
months and numerous incidental observations of myrmecophiles were possible through transparent nest coverings. These observations are also included in the present
study together with additional extensive investigations
conducted at the field station in Malaysia in 2006 and
2007. Since several species are undetermined, for clarity
we refer to the myrmecophiles by their taxonomic group
or by abbreviations as coded in Table 1 rather than by
scientific names.
Field Observations
Field observations were carried out at the Field Studies
Centre of the University Malaya in Ulu Gombak, Malaysia
(03 19.47960 N, 101 45.16300 E, altitude 230 m), for 6e7 h
every night for 3 weeks each in March and September
2006 and in March 2007. Leptogenys raids were located
in the field and raiding columns followed back to the
nest sites. Nests were numbered, marked with tape and
checked regularly every 30 min for ongoing activities. If
nest relocations were encountered, emigration columns
were monitored for participating myrmecophiles by two
Laboratory Maintenance
Four laboratory colonies were assembled at the field
station in Ulu Gombak from all myrmecophiles and
a limited number of hosts collected from four different
L. distinguenda colonies. These colony fragments contained an unusually high myrmecophile/host worker ratio
to facilitate the observation of myrmecophile behaviour
Table 1. Summary of the occurrence and interactions of myrmecophilic species found in colonies (N ¼ 10) of Leptogenys distinguenda
Inhabited
Maximum
colonies (median) no.
(%)
per colony
Migration
mode
Preferred location
in nest
Symbiont taxon
Code
Scientific name
Oonopid spider
Sp
Gamasomorpha maschwitzi1
100
13 (3)
Unknown mites
Unknown collembolans
Mi
Co
Undetermined
Undetermined
100
100
>50*
>100*
Unknown silverfish
Sf
Undetermined
100
47 (14)
Staphylinid beetles
Ptiliid beetles
Phorid flies
St
Pt
Ph
80
50y
30y
4 (3.5)
>20*
11
Oniscid woodlice
Subulinid snail
Wl
Sn
Trachydonia leptogenophila2
Undetermined
Rhynchomicropteron
necaphidiforme &
Puliciphora rosei3
Exalloniscus maschwitzi4
Allopeas myrmekophilos5
Throughout nest, on
top of ants
Phoretic on pupae
At pupae
Phoretic on pupae & Throughout nest,
trail following
at pupae
Phoretic on pupae & Throughout nest, at
trail following
pupae, below ants
Trail following
Peripheral disposal sites
Phoretic on larvae
On brood
Trail following
Peripheral disposal sites
10
90
23
12 (3)
Phoretic on pupae
Carrying by workers
Trail following
At pupae
Throughout nest
For behavioural observations, reduced colony fragments were maintained at the field site, Sources: (1) Wunderlich (1994); (2) Kistner et al.
(2003); (3) R. Disney (personal communication); (4) Ferrara et al. (1987); (5) Janssen & Witte (2003).
*Estimated values.
yValues in the present study underrepresented according to previous collections (Witte 2001).
1479
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ANIMAL BEHAVIOUR, 76, 5
and anteguest interactions. Experimental colonies comprised between 70 and 130 adult workers and were housed
in clear plastic containers (20 14 cm and 1 cm high)
with an entrance 1 cm wide. The nest containers were
shaded with carton covers and placed on a moistened gypsum floor in a foraging arena (32 25 cm and 9 cm high).
Foraging arenas were covered with plastic lids when no
observations were carried out and the side walls were
treated with paraffin to prevent escape of the focal
animals.
Food Preferences
The four experimental colonies including their associated myrmecophiles were fed daily with dead insects,
which we caught in the field. We chose insects that we
had previously observed to be natural prey of L. distinguenda (mainly grasshoppers, crickets). Thus, the ants
and their myrmecophiles could choose between various
food items. After the offered prey was retrieved by host
ants into the laboratory nests, we were able to observe
the behaviour of the different myrmecophile species towards host prey (>26 h observation time). We studied dietary preferences of spiders, silverfish, staphylinids and
snails by isolating randomly chosen individuals in snap
lid vials and providing a potential food item to each individual for 24 h (N ¼ 10 each). These food items included
pieces of host prey (ca. 1 cm diameter), living and dead
host workers, and host brood (larvae and cocoons). In
a previous study, snails were offered living and dead plant
matter as well (Witte et al. 2002). We observed myrmecophile behaviour towards the food during the first hour of
isolation. Additionally, we noted the presence or absence
of food particles after 24 h. Food was considered accepted
if feeding was observed or if the resource was depleted
after 24 h.
Behavioural Observations
In our laboratory nests, we noted changes in nest
demography and myrmecophile numbers on a daily basis.
These changes in absolute numbers were considered in
data evaluation (see below). Locations of all myrmecophile individuals were recorded repeatedly by scan sampling during the activity period at night both before and
after feeding (N > 20 scans). Recorded locations included
within the nest chamber, at/on larval clusters, at/on
adults, at/on callows, at/in/on piles of pupae, outside the
nest chamber, at refuse sites. To reflect natural conditions,
nest structure and characteristic locations of symbionts
were observed additionally in entire rather than only in
reduced laboratory colonies, (N ¼ 3). The frequency of observations in a particular area compared to the average
across all areas was used as a measure of spatial preference.
Furthermore, all interactions of myrmecophiles and
host ants were monitored regularly during nightly ad
libitum observation periods in reduced observation colonies, with and without food present, summing to more
than 38 h over 29 days. Preliminary exchange experiments between two laboratory colonies were conducted
with adult workers to study nestmate recognition. Focal
species were introduced into alien colonies and all interactions monitored for at least 10 min.
Chemical Analysis
Cuticular hydrocarbons were extracted over 5 min in ca.
200 ml of pentane (HPLC grade) in 2 ml vials with PTFE
septum (Sigma-Aldrich, Chemie GmbH, Taufkirchen, Germany). For tiny myrmecophile species (<1.6 mm in
length; Mi, Co, Pt, Ph) we placed 10 individuals into
one vial to gain sufficient concentration. Chemical analyses were carried out at the LMU, Munich, Germany by
coupled gas chromatography and mass spectrometry
(GCeMS) on an Agilent Technologies 6890N GC and
5975 MSD equipped with a Restek Rxi-5MS column
(30 m length, 0.25 mm internal diameter, 0.25 mm film
thickness). Injection was splitless over 1.0 min at 280 C
under a pressure pulse of 16 psi for 0.5 min followed by
automatic flow control at 1.0 ml/min with helium as carrier gas. The oven program started isothermal at 120 C for
2 min and then increased rapidly by 25 C/min until
200 C, followed by a gentle temperature ramp of
4 C/min until 300 C and finally stayed isothermal
for 3 min. The transfer line was held constantly at 310 C.
A range of 50e500 atomic mass units was scanned after
an initial solvent delay of 3.8 min.
Data Evaluation
Since the odds of observing particular behavioural
patterns are influenced by the length of observation and
the number of potentially interacting animals, we standardized the behavioural data by dividing the number of
observations by the observed time span (min), and the
number of potentially interacting individuals present in
the nest. Nonparametric tests (ManneWhitney U tests)
were applied on standardized data using the XLStat package (Addinsoft U.S.A., New York, U.S.A.) with Microsoft
Excel 2003. Chemical data were evaluated for characteristic structure (1) between workers of different colonies and
(2) between within-nest groups of animals (comprising
workers, callows, larvae, pupae and symbiont species of
a particular host colony). To consider a GC peak characteristic for a particular within-nest group, 30% of the samples
belonging to this group had to show corresponding peaks.
The low value of 30% was used because sample sizes were
small for some myrmecophiles and we wanted to prevent
the exclusion of meaningful chemicals. All peaks that occurred only occasionally (in fewer than 30% of the samples of this group) were considered not consistent and
excluded from analysis. In addition, mass spectra were
compared with library spectra (Wiley7N Registry of mass
spectral data, J. Wiley, Chichester, U.K.) to identify and exclude contaminants. Only hydrocarbons with 21 or more
C atoms were included in the statistical evaluation, since
these represent characteristic insect cuticular compounds
(Carlson et al. 1998), whereas shorter hydrocarbons might
originate from exocrine glands. Samples lacking any characteristic peaks after the described filtering procedure were
WITTE ET AL.: SYMBIONT DIVERSITY IN AN ANT SOCIETY
not evaluated. Total peak area of the remaining substances
was fourth-root transformed and each sample was standardized to relative values of the largest peak. This standardization to maximum peak area is more robust when
comparing chemical profiles with large differences in
peak numbers, compared to standardization by total
peak area. Nonparametric statistical procedures robust to
data type and distribution (Clarke 1999) were applied using the Primer 6 package (Primer-E Ltd., Ivybridge, U.K.).
BrayeCurtis distances were calculated and then subjected
to the following statistical procedures. The similarity percentage (SIMPER) procedure (Clarke 1999) was used to calculate relative contributions of compounds to the
similarity of particular sample groups. Nonmetric multidimensional scaling (NMDS) was applied to visualize chemical distances. These were analysed further by hierarchical
cluster analysis (CA; with group average cluster mode)
and the results were combined with the NMDS plots to illustrate sample groups of particular similarity (15% and
50% similarity, respectively). Proximities between groups
of samples were tested in addition by analysis of similarities (ANOSIM; Clarke 1999).
RESULTS
Behavioural Integration
More than 22 h of 14 emigrations of 10 L. distinguenda
colonies were observed in the field (four colonies were observed repeatedly). The myrmecophile fauna of these 10
colonies is summarized in Table 1, including notes on
abundances and biological characteristics. While several
myrmecophile groups were detected in all sampled colonies, other groups were found less frequently. Phorid flies
were detected in only three and oniscid woodlice in only
one of the 10 sampled colonies. Maximum abundances of
myrmecophiles varied with species size. Species smaller
than 20% of the body size of adult ants (Co, Mi, Ph, Pt)
were more abundant than larger species (Sp, Wl, Sf, St,
Sn, with 30e60% of the ants’ body size; ManneWhitney
U test, two tailed: a ¼ 0.05, U ¼ 7020.22, N1 ¼ 32,
N2 ¼ 40, P < 0.001). In addition, completely different
modes of following host emigrations were found within
the symbiont fauna (summarized in Table 1).
(1) Trail following. Spiders (N ¼ 50), staphylinid beetles
(N ¼ 28) and phorid flies (N ¼ 23) always followed the
trails independently. Spiders always moved in close contact with the ants (0e5 cm) within the emigration column,
whereas beetles and flies followed the emigration column
at a considerable distance (1e5 m behind the last ant).
(2) Optionally phoretic. Silverfish (N ¼ 169) and collembolans (N ¼ 61) were mostly observed attached to pupae
(>70% and >90%, respectively), and thus were indirectly
carried by the ants. However, occasionally (<30% and
<10%, respectively) these myrmecophiles also actively followed the ant trails in between travelling ants. In these
cases, independently walking silverfish and collembolans
quickly took the opportunity to attach themselves to
one of the next pupae carried along by workers in the
emigration column (within 10e60 s, N ¼ 60).
(3) Obligatory phoretic. Woodlice (N ¼ 23), mites
(N > 20) and ptiliid beetles (N > 13) were always found
phoretic on pupae and larvae. These symbionts never travelled on their own. When separated experimentally, woodlice (N ¼ 10) and mites (N ¼ 8) were not able to regain their
hold of larvae or pupae being carried along and therefore
failed to reach the new nest site, whereas ptiliid beetles
(N ¼ 14) quickly reattached themselves within 1 min.
(4) Direct carrying. Snails (N > 38) were always carried
by worker ants in the typical way brood and prey are
transported.
Myrmecophiles were not only observed during emigration activities in the field. Three groups, collembolans,
staphylinid beetles and phorid flies, were also regularly
observed participating in raiding columns (N > 15 raids).
Symbionts collected from four L. distinguenda colonies
were used to set up laboratory nests for behavioural observations. Inside laboratory nests, symbionts were frequently observed at different characteristic locations
(summarized in Table 1).
(1) Throughout the nest. Spiders, collembolans, silverfish and snails were observed moving around freely within
the entire nest (each N > 20). Silverfish (N ¼ 64) and collembolans (N ¼ 91) were found mostly in contact with pupae (60% of observations each), while spiders regularly
climbed directly on top of motionless ants (N ¼ 245), including feeding workers or callows holding larvae in their
mandibles.
(2) In between pupae. Woodlice and mites were typically found moving around in between loosely piled up
pupae (>50% of observations, each N > 20).
(3) On brood. Ptiliid beetles were typically observed
phoretic on larvae and only rarely on pupae (48% of
observations, N > 30).
(4) Peripheral. Staphylinid beetles and phorid flies were
most frequently (>75%) observed at refuse sites, which
were typically located at the corners of the foraging arena
and outside the nest (each N > 20).
Leptogenys distinguenda workers were aggressive towards
staphylinid beetles. Aggression on the part of the host
ants was observed at every encounter with staphylinids.
Yet, the beetles were well able to defend themselves by
raising their abdominal tip, where a defensive gland is located. The staphylinid defences showed a clear repellent
effect on L. distinguenda workers. Less often, but repeatedly (N > 30), host workers snapped with their mandibles
at collembolans, which were, however, too quick to get
caught. Occasional aggression was observed in encounters
of host workers and silverfish (3.4 times/h) and even less
frequently towards spiders (0.4 times/h). As a result, all silverfish (N ¼ 40) were killed by host ants in our observation colonies. Aggression towards other myrmecophile
species was never observed.
Food Preference
Although feeding of symbionts was difficult to recognize unambiguously, we found clear indications for dietary preferences of most symbiont species (Table 2).
Spiders, silverfish, staphylinids and snails were observed
feeding on insect prey retrieved by ants, while phorid flies
1481
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ANIMAL BEHAVIOUR, 76, 5
Table 2. Accepted food items of Leptogenys distinguenda symbionts
in isolation experiments and during observations in laboratory
colonies
Accepted food items
Symbiont taxon
Isolation
Laboratory
experiments (N ) observations (N )
(N ¼ 15). Aliens were thoroughly inspected by antennating, and dominance behaviour was shown by resident
ants, including grabbing and holding down with the mandibles, on-top positioning and antennae boxing. Nevertheless, these behaviours ceased within 5 min so that
alien ants were always eventually accepted in the colony.
Chemical Integration
Oonopid spider
Unknown mites
Unknown collembolans
Unknown silverfish
Staphylinid beetles
? (10)
d
d
? (10)
Dead host
workers, host
larvae, host
prey (>10)
d
d
d
Host prey (10)
Ptiliid beetles
Phorid flies
Oniscid woodlice
Subulinid snail
Host prey (>10)
?
Host prey (>10)
Host prey (>10)
Refuse (>10)
?
Refuse (>10)
?
?
See Methods for details. N ¼ number of tests (isolation experiments)
or observations (laboratory colonies); ? ¼ no acceptance observed;
d ¼ not tested.
and staphylinid beetles appeared to feed on refuse. In addition, in isolation experiments the staphylinids consumed ant larvae and dead ant workers. Furthermore,
staphylinids showed cannibalism on one occasion when
three individuals were stored in the same container after
collection. Only one of the beetles and body parts of
two other beetles were found after 10 h. In addition,
only a single staphylinid beetle ever remained in our observation colonies, even if several were introduced initially (N ¼ 3).
Worker cuticular hydrocarbon profiles of eight different
colonies showed only slight colony-specific structure. Of
28 possible colony pairings, 20 were chemically different
based on worker profiles (ANOSIM: R < 0.2, N ¼ 5e10, dependent on colony, P < 0.05), but most differences were
marginal and are thus also not very obvious in an
NMDS plot (Fig. 1).
Among within-nest groups, workers and callows had
similar chemical signatures across all colonies and could
not be separated significantly by an ANOSIM (R ¼ 0.043,
N ¼ 95, P ¼ 0.092). Their profiles were composed to
a high degree of identical hydrocarbons in similar proportions (Table 3). Thus, 97% of the callows and 98% of
adults were sorted by cluster analysis into the same group
of 50% similarity (‘worker’ group ‘W’ in the NMDS plot in
Fig. 2). Profiles of larvae and even more so of pupae lacked
most of these hydrocarbons and they were consequently
sorted to 54% (larvae) and 64% (pupae), respectively,
into another group of 50% similarity (‘brood’ group ‘B’
in Fig. 2). The different myrmecophile species of L. distinguenda followed one or other of these patterns and were
hence sorted by cluster analysis mainly into group W, if
they resembled worker/callow profiles, or into group B, if
they largely lacked worker/callow cuticular compounds
(listed in Table 4).
Nestmate Recognition
Preliminary exchange experiments between colonies
revealed that conspecific workers from other colonies
were recognized as alien, but treated with low aggression
DISCUSSION
Our comparative study on the myrmecophile community
of L. distinguenda revealed distinct behavioural and
Colony
LD2
LD4
LD5
LD6
LD7
LD8
LD8B
LD9
2D Stress: 0.07
Figure 1. Nonmetric multidimensional scaling (NMDS) plot of worker cuticular hydrocarbon profiles of 58 worker ants from eight L. distinguenda colonies. LD2 and LD6 were laboratory colonies. LD8B was found in an area where LD8 was sampled previously. ‘Stress’ is a quality
measure of the NMDS.
WITTE ET AL.: SYMBIONT DIVERSITY IN AN ANT SOCIETY
Table 3. Main cuticular hydrocarbons and their relative contribution
to 90% chemical similarity of adult, callow, larvae and pupae profiles
according to a SIMPER analysis
Contribution to chemical
similarity (%)
Retention
time
14.7
17.5
17.7
17.9
18.0
21.0
21.1
24.3
Substance
Tricosane
Pentacosadiene
Pentacosene
Pentacosene
Pentacosane
Heptacosene
Heptacosene
Nonacosene
Sum
Adults Callows Larvae Pupae
16.28
14.83
13.09
7.58
8.85
12.19
7.98
11.44
15.68
16.23
13.27
6.2
8.04
11.88
7.36
12.64
75.78
6.86
3.93
d
d
d
d
5.78
96.25
d
d
d
d
d
d
d
92.24
91.3
92.34
96.25
See Methods for details.
chemical adaptations in the different symbiont taxa to
their army ant host. Several myrmecophiles had developed the ability to follow frequent nest emigrations independently, while others were optionally or obligatory
phoretic on ant brood or induced ant workers to carry
them directly. We also found clear differences in the degree of social integration, with some myrmecophile species never treated aggressively by host ants, while others
were frequently attacked or even killed.
Symbiont Chemical Integration
Obviously, two different strategies of circumventing the
host recognition system exist among the myrmecophile
Group
Ad
Wl
Ca
Dim
La
Mi
Pt
Pu
Ph
Sn
Sf
Sp
St
fauna of L. distinguenda. On the one hand, silverfish, spiders, ptiliid and staphylinid beetles clearly use chemical
mimicry by resembling cuticular hydrocarbon profiles of
L. distinguenda adults and callows. On the other hand,
woodlice, mites, phorid flies and snails lack most of the
host cuticular hydrocarbons and are in consequence
chemically widely insignificant, comparable to ant brood
(Lenoir et al. 1999, 2001). Nevertheless, small proportions
of certain hydrocarbons seem to spread passively depending on surface properties and area. In particular, tricosane,
the most abundant and shortest hydrocarbon in our analysis, was detectable in most of the samples. Spiders were
well accepted in L. distinguenda colonies although their
chemical profile deviated somewhat from that of their
host. In contrast, some staphylinid beetles matched the
host chemical signature very well, but were nevertheless
always treated aggressively. Possibly, slight differences in
the proportions of key hydrocarbons or other characteristic chemicals of the symbiont species itself, which were
not detected in this study, are responsible for these discrimination abilities. In summary, we gathered a good insight into general chemical integration mechanisms of
L. distinguenda myrmecophiles, although the fine tuning
in discrimination of myrmecophiles demands further investigation. Host aggression shows that there is selection
pressure for rejecting myrmecophiles, and, therefore, the
fine tuning of chemical integration is of great importance
for the social acceptance of intruders. None of the symbiont groups possessed group-specific surface chemicals in
sufficient amounts to be detected by our methods. It
seems conceivable that selection through host aggression
and rejection drives symbionts towards suppressing species-specific chemicals that could potentially uncover
their alien identity. In addition to a reduction of alien
Similarity
15%
50%
Group B
Group W
2D Stress: 0.05
Figure 2. Nonmetric multidimensional scaling (NMDS) plot of host and symbiont cuticular hydrocarbon profiles across eight L. distinguenda
colonies. For abbreviations see Tables 1 and 4. N ¼ 58 Ad, 37 Ca, 26 La, 11 Pu, 37 Sf, 20 Sp, 10 St, 3 Wl, 3 Sn, 1 Mi10 pooled, 1 Ph10 pooled,
1 Pt10 pooled and eight Leptogenys diminuta workers (Dim) from one colony as an outgroup. Similarity groups are indicated based on a hierarchical cluster analysis (see Methods): W ¼ worker similarity; B ¼ brood similarity. ‘Stress’ is a quality measure of the NMDS.
1483
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ANIMAL BEHAVIOUR, 76, 5
Table 4. Assignment of hosts and myrmecophiles to 50% similarity
groups W (worker) and B (brood) according to a hierarchical cluster
analysis based on chemical similarities
Group W (%) Group B Other
Adults
Callows
Silverfish
Spiders
Staphylinidae
Ptiliidae
Larvae
Pupae
Woodlice
Mites
Phoridae
Snails
Ad
Ca
Sf
Sp
St
Pt(10 pooled)
La
Pu
Wl
Mi(10 pooled)
Ph(10 pooled)
Sn
98
97
84
55
80
100
42
9
33
0
0
0
2
3
13
45
20
0
54
64
67
100
100
100
0
0
3
0
0
0
2
27
0
0
0
0
See Methods for details.
recognition cues, chemical integration can be further
enhanced by acquiring host-specific recognition cues.
Symbiont Maintenance of Host Contact
The majority of L. distinguenda symbionts are very well
adapted to maintain contact with their highly mobile
host. The different myrmecophile species had various
and highly elaborate strategies to participate in frequent
host emigrations. Actively following symbionts such as silverfish, spiders and collembolans perceive host pheromone trails (Witte 2001). Myrmecophiles of other ants
can also follow ant trails (Akre & Rettenmeyer 1966,
1968; Rettenmeyer & Akre 1968; Christian 1994). While
trail followers and optionally phoretic symbionts actively
maintain contact with their host, obligate phoretic or passively carried symbionts face the potential risk of being
separated during transport or of missing the onset of an
emigration and thus of being left behind in the old nest.
In fact, observations of laboratory nest emigrations
showed that this happened occasionally to snails and regularly to mites and ptiliid beetles (V. Witte, unpublished
data). Furthermore, owing to physical constraints, none
of these symbionts is able to cover significant distances
on its own and would not be able to reach a new nest
site independently. Frequent nest migrations can hence
be regarded as beneficial for the host to reduce symbiont
pressure, in particular because it should also negatively affect symbiont reproductive cycles. We do not have much
data on the life cycles of L. distinguenda myrmecophiles,
but reproduction clearly imposes severe challenges in respect of maintaining host contact. Several L. distinguenda
colonies were free of particular symbionts, in particular
woodlice, which suggest these myrmecophiles have difficulties maintaining contact with the host or in transmission between colonies.
Average emigration intervals of 1.5 days in L. distinguenda are probably too short for most animals to complete their reproductive cycle. This problem can be
solved by the following two strategies. First, myrmecophiles can become viviparous or come close to vivipary,
which has been reported for some L. distinguenda
symbionts (Ferrara et al. 1987; Witte et al. 1999; Janssen
& Witte 2003). In addition, symbiont offspring can attach
themselves to host brood to ensure passive transport, as
observed for the symbiotic silverfish in the present study.
As a second strategy, myrmecophiles can evolve efficient
host readoption mechanisms. This would allow quick reentry into another migrating L. distinguenda colony after
separation from a host colony. The second strategy demands host-independent survival of symbionts at least
over limited time spans and would automatically imply
horizontal transmission, which is generally beneficial for
symbionts. Yet, we have no evidence for the second
strategy.
Symbiont Biology and Impact
Our observations revealed that several myrmecophiles
including spiders, silverfish and collembolans feed directly
on host prey, and therefore generally have a negative
impact on host fitness. However, the abundances of larger
myrmecophiles (spiders, silverfish, woodlice, staphylinid
beetles) were rather low relative to the army ant colony
size. Thus, their overall impact on large host colonies must
remain negligible. Myrmecophiles that were more abundant were tiny compared to the body size of their hosts
(collembolans, ptiliid beetles, mites) so that their impact
should be minor as well. Of course this holds only as long
as the symbiont sizeeabundance relation remains at the
reported level. Costs for the host colony would rise
dramatically with increasing symbiont populations and
so does selection pressure for countermeasures. We observed, correspondingly, that L. distinguenda controls its
symbiont fauna by attacking and killing alien organisms
that are recognized inside the nests. Selection pressures
for recognition must be higher with increasing costs to
the host so that symbiont body size (e.g. silverfish), population number (e.g. collembolans) and impact (e.g. staphylinid beetles) are important parameters in the
evolutionary arm race. Most aggression was observed
against staphylinid beetles, which were expelled from
the nests and only followed nest emigrations at a distance.
These observations strongly suggest that the selection
pressure for recognition of staphylinid beetles is higher
than for other myrmecophiles. A reasonable cause for
this might be the potential threat of predation on host
brood if they entered the nest. Owing to high aggression
on the part of the host, staphylinid beetles apparently
avoid encounters by following emigration columns at
a distance and by occupying only peripheral niches of
the host colony.
Conclusion
The symbiont microcosm of L. distinguenda appears to
be a complex system of hostesymbiont interactions,
where parasite numbers and impact are potentially regulated through countermeasures of the host. The myrmecophile species of this ponerine army ant have evolved
specific integration strategies, which were clearly influenced by their respective phylogenetic constraints.
WITTE ET AL.: SYMBIONT DIVERSITY IN AN ANT SOCIETY
Symbiont adaptations are apparently counteracted by
host recognition and control abilities, presumably keeping
the overall fitness costs of myrmecophily low. Such coevolutionary interactions are common to hosteparasite systems; however, in L. distinguenda multiple species are
uniquely interrelated insofar as all are bound by the
same limits of host resource capacity. To illustrate this,
we constructed a simplified interaction network including
representative myrmecophile species for which sufficient
data were collected (Fig. 3). The fundamental difference
from other groups of multiparasite systems, such as
macro- or microparasites, is that most myrmecophilic parasites are confined to a single host colony and are unable
to switch to other colonies on demand. For most myrmecophile species, vertical transmission through colony budding is presumably more prevalent than horizontal
transmission between host colonies. Studies on different
hosteparasite systems clearly show that transmission
mode and rate are linked to the evolution of virulence,
in that low transmission rates and vertical transmission
will select for lower virulence (Anderson & May 1982;
Dunn & Smith 2001). Thus, it follows that those myrmecophile species, that have a low transmission rate or can
only spread through host colony reproduction will be
under selection to lower their impact.
The army ant symbiont fauna described here is a unique
model system, resembling natural ecosystems perhaps
better than other hosteparasite associations in which
parasitic individuals more frequently leave host ‘ecosystems’ and occupy new ones. Furthermore, the composition of these symbiont islands within L. distinguenda
colonies is rather simple compared to real ecosystems,
comprising only a small number of species and few trophic levels (Fig. 3). Consequently, interactions that might
also occur in larger ecosystems could be identified and
understood without great difficulties. The fact that interactions between different symbiont species are absent or
extremely rare seemed initially difficult to understand.
Competition should be intense between species using
the same resources. In ecological food webs, however,
predators can reduce population densities of lower trophic
levels, decreasing competition and facilitating coexistence
(Paine 1966; Shurin & Allen 2001; Chase et al. 2002).
Since aggression towards myrmecophiles seemed to be frequent in L. distinguenda, the hosts’ recognition and defence system probably has such an effect on symbiont
populations (Fig. 3). The host recognition and defence system seems to be also the most important level at which selection acts. Apparently, fitness benefits are highest if host
defence mechanisms work against the most detrimental
parasites. These are the staphylinid beetles in our example, which are predatory and which are consequently
attacked and expelled from ant nests. However, host
rejection behaviour does not only reduce symbiont abundance. The same mechanism can explain why we observe
characteristic abundances of symbionts in most colonies
and not different species dominating different host colonies, although many of them use host prey as a resource.
The system appears to be balanced through host control
by the synergistic effects of size, number and behaviour
of each symbiont and in consequence by its overall impact on host fitness (Fig. 3). Myrmecophiles can presumably be tolerated to some extent as long as their costs
are low. Increasing costs of specific myrmecophiles leads
to increasing selection on the host to reject them. As a result of this mechanism, symbiont diversity can be maintained, because each symbiont that becomes too costly
might be confronted with increasing host control. In conclusion, regarding the L. distinguenda symbiont community as a simply structured, enclosed ecosystem facilitates
Host defence
recognition
& rejection
Symbiont
regulation
P
I
Symbiont
community
Resources
used
Spider
Silverfish
Host prey
Collembolans
Organic
remains
Staphylinids
Host
brood
Figure 3. Hostesymbiont community interaction network of L. distinguenda (for simplicity reduced to four representative symbiotic species).
According to Pedersen & Fenton (2006) three trophic levels are defined in analogous to simple community ecological networks. The basal level
(primary producers) contains the resources used by symbionts. The intermediate level (primary consumers) includes the symbiont community.
Functional guilds are defined: I ¼ species inhabiting the nest interior; P ¼ species inhabiting the nest periphery. The top trophic level (top predators) is represented by the host and its ability to detect and destroy symbionts. Black arrows show interactions between components in the
network and boldness indicates their qualitative strength. Further explanations are given in the text.
1485
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ANIMAL BEHAVIOUR, 76, 5
the understanding of important regulatory processes. Similar approaches should generally contribute to an overall
understanding of multiple hosteparasite systems.
Acknowledgments
We are grateful for financial support from the DFG
(Deutsche Forschungsgemeinschaft); Project WI 2646/
3-1. Many thanks for assistance in the field to K. Staudt,
K. Ortner, S. Schreyer and A. Fenzl.
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