Behaviour and chemical signature of pre-hibernating females of Polistes dominulus infected by the strepsipteran Xenos vesparum

Abstract

Polistes dominulus are social wasps which are the host of the strepsipteran endoparasite Xenos vesparum. In the hibernating phase, unparasitized and parasitized wasps leave natal nests and aggregate together in sheltered quarters. In aggregations, wasps are socially active, and some individuals perform helping behaviour. Here we investigated if castrated parasitized wasps perform worker tasks in mixed aggregations. Moreover, by gas chromatography and mass spectrometry, we examined the cuticular hydrocarbons of unparasitized and parasitized wasps to evaluate if the infection alters the composition of cuticular waxes that are recognition cues in social insects. In clusters, infected females do not perform helping behaviour and they are less active than unparasitized wasps. Cuticular hydrocarbons are slightly differentiated between unparasitized and parasitized wasps but, generally, unparasitized wasps are more similar to wasps infected by Xenos females compared to wasps infected by Xenos males. Wasps infected by Xenos males do not usually survive the winter. This chemical similarity is probably a consequence of the similar physiological condition of unparasitized and female-affected Polistes wasps. At this stage, it is difficult to affirm whether these modifications are a true parasite manipulation or a consequence of infection.

Full text

Behaviour and chemical signature of pre-hibernating
females of Polistes dominulus infected by the strepsipteran
Xenos vesparum
L. DAPPORTO
1
*, A. CINI
1
, E. PALAGI
1
, M. MORELLI
1
, A. SIMONTI
1
and S. TURILLAZZI
2
,
3
1
Centro Interdipartimentale Museo di Storia Naturale e del Territorio dell’Universita
`
di Pisa, Via Roma 79,
56011 Calci (PI), Italy
2
Dipartimento di Biologia Animale e Genetica ‘‘ Leo Pardi’’, Universita
`
di Firenze via Romana 17 50125 Firenze , Italy
3
Centro Interdipartimentale di servizi di Spettrometria di Massa (CISM) University of Firenze, Viale G. Pieraccini 6,
50139 Firenze, Italy
(Received 8 July 2006; revised 30 September 2006; accepted 2 October 2006; first published online 23 November 2006)
SUMMARY
Polistes dominulus are social wasps which are the host of the strepsipteran endoparasite Xenos vesparum. In the hibernating
phase, unparasitized and parasitized wasps leave natal nests and aggregate together in sheltered quarters. In aggregations,
wasps are socially active, and some individuals perform helping behaviour. Here we investigated if castrated parasitized
wasps perform worker tasks in mixed aggregations. Moreover, by gas chromatography and mass spectrometry, we
examined the cuticular hydrocarbons of unparasitized and parasitized wasps to evaluate if the infection alters the com-
position of cuticular waxes that are recognition cues in social insects. In clusters, infected females do not perform helping
behaviour and they are less active than unparasitized wasps. Cuticular hydrocarbons are slightly differentiated between
unparasitized and parasitized wasps but, generally, unparasitized wasps are more similar to wasps infected by Xenos
females compared to wasps infected by Xenos males. Wasps infected by Xenos males do not usually survive the winter. This
chemical similarity is probably a consequence of the similar physiological condition of unparasitized and female-affected
Polistes wasps. At this stage, it is difficult to affirm whether these modifications are a true parasite manipulation or a
consequence of infection.
Key words: Polistes dominulus, Xenos vesparum, hibernation, behaviour, cuticular hydrocarbons.
INTRODUCTION
Social animals are particularly prone to parasite
infections. Indeed group living may favour parasite
infection; consequently, parasitism is considered
as one of the most important costs of group living
(Schmid-Hempel, 1998). Several authors have
predicted that the costs linked to parasite trans-
mission may modify social systems (Freeland, 1976;
Hamilton, 1987; Schmid-Hempel, 1998). On the
other hand, O’Donnell (1997) hypothesized that
under certain conditions, parasites can favour the
expression of social behaviour in their hosts. Indeed
several parasites are known to reduce or disrupt host
fecundity (Strambi and Strambi, 1973; O’Donnell,
1997); in social species, where there is severe intra-
colony competition for reproduction, reduced re-
productive potential induced by parasites in some
individuals may promote a non-competitive division
of labour.
Polistes dominulus (Christ) is a social wasp which is
a host of the strepsipteran parasites Xenos vesparum
(Rossi). These wasps have an annual colony cycle.
In spring, mated queens initiate new colonies, often
cooperatively. When polygynous colonies are
founded, a linear hierarchy is established by agonistic
interactions successively maintained by ritualized
dominance behaviour (Pardi, 1942, 1946). In the
summer, reproductive individuals (males and future
foundresses) emerge, mate, and leave natal nests.
After abandoning natal nests, several species of
polistine wasps spend the non-nesting phase (winter
in temperate climates) aggregated in clusters. In the
Northern Hemisphere, the aggregating stage starts
in September-November (pre-hibernating stage)
when wasps, often belonging to different colonies,
aggregate in sheltered quarters (Pardi, 1942 ; Starks
2003; Dapporto and Palagi, 2007). In the spring,
wasps terminate diapa use and found new colonies.
Strepsiptera are obligate endoparasites of various
insects (Kathirithamby, 1989). These parasites ex-
hibit extreme sexual dimorphism: the short-living
* Corresponding author: Centro Interdipartimentale
Museo di Storia Naturale e del Territorio dell’Universita
`
di Pisa, Via Roma 79, 56011 Calci (PI), Italy. Tel:
+390502212969/963. Fax: +390502212975. E-mail:
leondap@katamail.com
545
Parasitology (2007), 134, 545–552. f 2006 Cambridge University Press
doi:10.1017/S0031182006001739 Printed in the United Kingdom

(usually less than 5 h) winged adult males and the
first instar larvae ar e the only free-living stages,
whereas the neotenic, larviform, adult females are
permanently parasitic. Infection by X. vesparum
begins with the entry of the first instar larva (tri-
ungulin) into a P. dominulus larva. The female
extrudes its cephalothorax and becomes a neotenic
adult; conversely, the male pupates, emerges and
flies off to search for a female. Females are vivipar-
ous, and the triungulin larvae emerge via a brood
canal which opens in the cephalothorax. Colony
infection occurs after a wasp parasitized by a gravid
female X. ve sparum releases triungulins on flowers
after which the triungulins are transported to the
nest via foraging wasps. Moreover, in spring, in-
fected wasps can land on temporarily undefended
nests and triungulins directly infect the host larvae
(Hughes et al. 2003). Only X. vesparum females
overwinter (inside their hosts). Hosts carrying empty
male puparia usually die before spring (Hughe s
et al. 2004 a).
As X. vesparum induce sterility in thei r Polistes
hosts (Strambi and Strambi, 1973), infection could
result in a host division of labour (O’Donnell, 1997).
However, foundresses affected by female X. ves-
parum do not found colonies. Although parasitized
females usually rest close to nests, they do not join
colonies and do not participate to any colony task
(Beani et al. 2004). Similarly, workers infected by
male and female X. vesparum do not eng age in typical
worker tasks on the natal nest. Indeed infected
workers and future foundresses leave natal nests in
the first week after emergence, forming precocious
extranidal aggregations. Hughes et al. (2004b)
suggested that the formation of such aberrant ag-
gregations is an example of manipulation in order
to facilitate X. vesparum encounters and mating.
The unparasitized future foundresses leaving natal
nests at the end of the summer enter parasitized wasp
aggregations resulting in mixed groups. As autumn
proceeds, aggregations are composed by a rising
number of unparasitized future fo undresses (Hughes
et al. 2004b). As parasitized females leave natal nests
and do not engag e successively in nest foundation,
the autumn and winter aggregations are the only
phase where unparasitized and infected wasps live
together for a relatively long time.
In pre-hibernating aggregations female P. dom-
inulus wasps exhibited most of the rank-dependent
behaviours (ritualized dominance and food re quest
behaviours, RDB; attacks, ATT; trophallaxis , TRP)
(Dapporto et al. 2005 a, 2006). The frequency of
these early behaviours reflects the physical and
physiological characteristics of pre-hibernating
wasps and their capability to become the alpha
female in spring (Dapporto et al. 2006). A few wasps
helped the others in aggregations by performing
external foraging and providing food to the cluster
mates; helpers generally die before spring (Dapporto
et al. 2005a). We examined infertile parasitized
wasps to determine whether they perform helping
behaviour in aggregations more frequently than
unparasitized females, as predicted by O’Donnell
(1997). In alternative hypotheses the infection could
not have observable effects on wasp behaviour in
clusters or could lower the frequency of altruistic and
other energy-wasting interactions.
According to Hughes et al. (2004b), parasitized
females are not attacked on colony by nestmates
after their emergence, probably because they are not
infecting. On the other hand, also in spring, during
the first colony phase, when parasitized females are
strongly infective, they release triungulins while
resting beside nests without being a target of ag-
gression by resident wasps at least in captivity (Beani
et al. 2004). In Polistes wasps cuticular hydrocarbons
(CHCs) play a pivotal role in recognition (Lorenzi
et al. 1996; Gamboa, 2004). The composition of
cuticular mixtures is characteristic for species, sex,
colony and population and also depends on social
and physiological status (Bonavita-Cougourdan
et al. 1991 ; Dapporto et al. 2004a, 2005b). Since
parasitized females display strongly altered physi-
ology and behaviour (Strambi and Strambi, 1973 ;
Strambi et al. 1982 ; Beani et al. 2004; Hughes et al.
2004b; Beani, 2007), they may also present alter-
ations in CHC composition that could be used by
conspecifics to recognize them. On the other hand,
if female parasites alter wasp physiology very little
to favour overwintering, no great differences in
CHC composition would be expected between un-
parasitized and parasitized females.
MATERIALS AND METHODS
Subjects and housing
In September, 2005, at the beginning of the pre-
hibernating stage, we collected 62 Polistes dominulus
females from Reggello (Tuscany, Italy) from an
aggregation partially exposed to sunlight. As aggre-
gating wasps belong to several neighbouring nests
(Starks, 2003), a cluster can be considered a rep-
resentative unit of a population. Five females died
during the first days of observation and we removed
them from the analyses. The studied aggregatio n was
then composed of 41 unparasitized females and 16
females parasitized by Xenos vesparum (11 by male
and 5 by female parasites). We marked the wasps on
the wings with enamel paint using a different colour
combination for each individual. We caged them in
containers (50r50r50 cm) consisting of a large
wood frame closed by 3 glass and 2 net sides. The net
sides avoided overheating in the cages. The corners
of the large frame offered appropriate shelters for
the wasps. The containers were placed in a garden
where the animals re ceived direct sunlight in the
early afternoon. This setting was similar in terms of
L. Dapporto and others 546

temperature, humidity and light exposure to those
of the site from which the aggregation was collected.
The wasps were supplied with water and sugar lumps
at the centre of the cage.
Behavioural observations
We examined the 7 behavioural items recorded in
wasp aggregations by D apporto et al. (2006) i.e.
ritualized dominance behaviour (RDB) performed
and received, attacks (ATT) performed and received,
trophallaxis (TRP) obtained and given, and foraging
on the sugar (FOOD). In RDB, the dominant wasp
climbs on and antennates the subordinate wasp,
often seeking for food by mouth-to-mouth contact.
TRP occurs when one wasp gives liquid food to
another. ATT includes lunges, bites, aggressive
mounts, chases, falling fights, and stings. We re-
corded behaviour, actor, and receiver by the ‘all
occurrences’ sampling method and we used the scan
sampling method at 5 min intervals to record the
individuals foraging on the sugar (Altmann, 1974).
We observed wasps from 10.00 a.m to 04.0 0 p.m
between September 5 and October 2 during warm
and sunny days.
Chemical analyses
All females were sampled for cuticular hydrocarbons
using pieces of filter paper (5r10 mm
2
) with a pro-
cedure similar to that used by Dapporto et al.
(2004a). Filter paper was held with dissecting
forceps and gently rubbed on the wasp’s thoracic
scutum for 30 sec. The filter paper was then placed
directly onto a clean aluminium sheet. Epicuticular
compounds were extracted from the filter paper in
300 ml of pentane for 10 min. The solution was dried
in a nitrogen stream and re-suspended in 25 mlof
heptane for gas chromatography-mas s spectrometry
(GC-MS) analysis. We injected 2 ml of solution
into a Hewlett Packard (Palo Alto, California) 5890A
gas chromatograph coupled with an HP 5971A
mass selective detector. A fused silica capillary
column coated with 5% diphenyl-95 % dimethyl
polysiloxane (Rtx-5MS, 30 mr0
.
25 mmr0
.
5 mm;
Restek, Bellefonte, Pa.) was used in the GC analysis.
The injector port and transfer line were set at
280 xC and the carrier gas was helium (at 12 psi).
The temperature protocol was: 70–150 xC at a rate
of 30 xC/min (held for 5 min), and 150–310 xCat
5 xC/min (held for 11.3 min). Analyses were per-
formed in splitless mode. Cuticular compounds
were identified on the basis of their mass spectra
produced by electron impact ionization (70 eV).
Statistical analysis
Possible correlations among the frequencies of the
behavioural items were examined using principal
components analysis (PCA). We retained PCs with
eigenvalues of more than 1. Bartlett’s test was used
to check for homogeneity of variances and Kaiser–
Meyer–Olkin (KMO) was used to measure sampling
adequacy. We varimax-rotated the components. We
used Kruskall Wallis test to search for differences
in the frequencies of single behaviours between
unparasitized females and females affected by male
and female X. vesparum. In cases of significant
difference, we employed the multiple comparison
tests (post-hoc test) to determine which pairs of wasp
classes differed significantly (Siegel and Castellan,
1988).
For the analysis of chemical data, the areas of each
peak (representing one or more compounds) of the
epicuticular gas chroma togram of each wasp were
transformed into percentages and statistically
analysed using the Kruskall Wallis test and Step-
wise Discriminant Analysis with SPSS
1
9.05 for
Windows. We used the Kruskall Wallis test to
search for differences in the percentages of single
chemicals between unparasitized females and
females affected by male and female X. vesparum.
Stepwise Discriminant Analysis (DA) was used to
determine whether pre-defined groups and categor-
ies (unparasitized females and females affected by
male and female X. vesparum) could be discriminated
on the basis of a variable data set (in this case
cuticular compounds). By stepwise analysis of vari-
ables it was possible to identify a reduc ed set of
compounds particularly important for discrimi-
nation among groups.
RESULTS
Behavioural data
We collected 22 hours by ‘all occurrences’ obser-
vations. Prin cipal compon ent analysis extracted
7 PCs (KMO=0
.
782, Bartlett’s test P< 0
.
001) from
the 7 behavioural vari ables. PC1 and PC2 were the
only ones that had eigenvalues greater than 1 (3
.
05
and 1
.
34). PC1 (explained variance=44
.
45% after
rotation) was positively represented by ATT and
RDB received, TRP given, and FOOD while PC2
(expl. var.=24
.
77% after rotation) was positively
represented by RDB performed and TRP obtaine d
(Table 1), thus confirming the results of Dapporto
et al. (2005a, 2006).
Regression values of PC1 and PC2 for unpara-
sitized and parasitized females are shown in Fig. 1.
It is possible to highlight the presence of some
wasps that showed high values on PC1; these in-
dividuals (helpers) spent a long time on the sugar,
and when they returned to the aggregations, other
gynes solicited food from them and sometimes ob-
tained it (Dappo rto et al. 2005 a). Conversely, other
individuals with high PC2 values were characterized
by high frequency of RDB performed and they
Behaviour and cuticular hydrocarbons of parasitized Polistes wasps 547

obtained food more frequently. Values for PC1 and
PC2 were low for parasitized females. They do not
perform helping behaviour and they show low fre-
quencies of dominance and food request interactions
(Fig. 1).
Frequencies of RDB and ATT performed were
lower in wasps parasitized by male and female Xenos
vesparum (Kruskall Wallis n1 = 41, n2=11, n3=5,
RDB performed: chi-square=16
.
851, P<0
.
001;
ATT performed: chi-square=9
.
131, P=0
.
010;
post-hoc tests revealed no differences in AT T per-
formed between any classes of wasps; conversely, for
RDB performed, both wasps parasitized by males
and females differed from unparasitized females but
they did not differ from each other, Fig. 2). There
were no differences for the remaining interactions
and food foraging (Kruskall Wallis n1=41, n2=11,
n3=5, TRP performed : chi-square=2
.
538, P=
0
.
281; TRP obtaine d : chi-square=2
.
538, P=0
.
281;
ATT received : chi-square=0
.
079, P=0
.
961; RDB
received: chi-square=2
.
046, P=0
.
359; FOOD : chi-
square=3
.
391, P=0
.
183; Figs 3 and 4).
Chemical analysis
Two GC-MS analyses (on 1 unparasitized female
and 1 female affected by male Strepsiptera) failed
and were removed from the statistical analysis.
Table 1. PCA loadings for the two rotated
components
(PC1 explaining 44
.
45% of variance after rotation is posi-
tively represented by attacks performed and received, ri-
tualized dominance behaviour received, trophallaxis given,
and food foraging. PC2 explaining 24
.
77% of variance after
rotation is positively represented by attacks performed,
ritualized dominance behaviour performed and tro-
phallaxis obtained.)
Behaviour
Component
PC1
(44
.
45%
expl. var.)
PC2
(24
.
77%
expl. var)
Attacks performed (ATTP) 0
.
523 0
.
591
Attacks received (ATTR) 0
.
846 x0
.
105
Ritualized Dominance behaviour
performed (RDBP)
0
.
130 0
.
820
Ritualized Dominance behaviour
received (RDBR)
0
.
907 0
.
154
Trophallaxis obtained (TRPO) 0
.
003 0
.
755
Trophallaxis given (TRPG) 0
.
819 0
.
191
Food foraging (FOOD) 0
.
782 0
.
268
PC2 (24.77% expl. var)
3210-1-2
PC1 (44.45% expl. var.)
5
4
3
2
1
0
-1
-2
Fig. 1. PCA regression factor values. PC1 represents
trophallaxis given and foraging activity (helping
behaviour). PC2 represents ritualized dominance
behaviour (RDB), and trophallaxis obtained. Both PCs
were low in parasitized females. Circles – unparasitized
wasps; empty triangles – male-parasitized wasps ; black
triangles – female-parasitized wasps.
51141N
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
FMH
Hourly frequencies of RDB performed
*
*
Fig. 2. Hourly frequency of RDB performed by
unparasitized wasps (H) and wasps parasitized by male
(M) and female (F) Xenos vesparum.
51141
N
FMH
Hourly frequencies of TRP given
1.2
1.0
0.8
0.6
0.4
0.2
0.0
n.s.
Fig. 3. Hourly frequency of trophallaxis given in
unparasitized wasps (H) and wasps parasitized by male
(M) and female (F) parasites. The outliers in the
unparasitized female group are helpers.
L. Dapporto and others 548

We detected a total of 25 cuticular hydrocarbons.
The cuticular mixture in hibernating P. dominulus
was dominated by a series of linear, mono-methyl
branched, dimethyl-branched saturated and linear
unsaturated hydrocarbons with chains ranging from
26 to 35 carbon atoms. The chemical diversification
among unparasitized and parasitized females was
weak. Indee d, DA assigned only 83
.
3% of cases to
their correct group (Function 1 : Wilks l=0
.
367,
P<0
.
001; Function 2: Wilks l=0
.
652, P<0
.
001,
explaining 100 % of variance), and there was no clear
separation between groups (Fig. 5). One alkene
(n-C
31
:
1
), 1 alkane (n-C
31
), and 1 mono-methyl
branched comp ound (7-meC
31
) were responsi ble for
the discrimination.
By examining differences in percentages of indi-
vidual compounds, there were 5 peaks with signifi-
cant differences among different wasp classes
(Kruskall Wa llis n1=39, n2=10, n3=5 in each case:
n-C
26
: chi-square=10
.
00, P =0
.
007; n-C
28
: chi-
square=10
.
813, P=0
.
004; n-C
29
:
1
: chi-square=
8
.
952, P =0
.
011; n-C
31
:
1
: chi-square=13
.
831, P=
0
.
001; 7-meC
31
: chi-square=13
.
076, P=0
.
001).
Post-hoc comparisons revealed that for 3 of these
compounds (n-C
28
,
n-C
29
:
1
,
n-C
31
:
1
), there were no
significant difference s between unparasitized wasps
and wasps parasitized by female Xenos, but wasps
affected by male parasites were different from the
other two classes (Figs 6, 7 and 8). Regarding n-C
26
and 7-meC
31
,
we found significant differences only
between unparasitized and male-parasitized wasps.
DISCUSSION
Although parasitized females showed behavioural
modifications, they did not eng age in helping be-
haviour in clusters. This result does not support
O’Donnell (1997), who predicted that infected in-
dividuals would specialize in worker-like helping
behaviour. Infected females performed less domi-
nance and attack interactions than unparasitized
individuals, but foraged and obtained food from
helpers with comparable frequencies. The mechan-
ism by which the behavioural modifications occurre d
is probably very simple, i.e. a direct consequence of
castration resulting in ovary reduction. Aggressive-
ness in wasps depends mainly on juvenile hormone
(JH) secreted by the corpora allata and on ecdyster-
oids secreted by the ovaries ; domina nt wasps have
larger ovaries and corpora allata (Ro
¨
seler, 1991), and
the application of JH and/or ecdysteroids induces
increasingly aggressive behaviour (dominance and
51141N
8
7
6
5
4
3
2
1
0
-1
FM
H
Hourly frequencies of FOOD
n.s.
Fig. 4. Hourly frequency of foraging for food by
unparasitized wasps (H) and wasps parasitized by male
(M) and female (F) parasites.
Function 1
43210-1-2-3
Function 2
5
4
3
2
1
0
-1
-2
-3
Fig. 5. Discriminant Analysis of the CHC composition
of 39 unparasitized Polistes females, 10 Polistes females
parasitized by Xenos males and 5 Polistes females
parasitized by Xenos females. Circles – unparasitized
wasps; empty triangles – male-parasitized wasps ; black
triangles – female-parasitized wasps.
51039N
5.0
4.0
3.0
2.0
1.0
0.0
FMH
Percentage of n-C28
*
*
Fig. 6. Percentage of n-C28 in the cuticular samples of
unparasitized wasps (H) and wasps parasitized by male
(M) and female (F) parasites.
Behaviour and cuticular hydrocarbons of parasitized Polistes wasps 549

aggression). In aggregations, wasps with a high RDB
frequency had larger ovaries than the other wasps
(Dapporto et al. 2006).
Regarding parasitism, there is a heated debate
about the host behavioural modification hypothesis.
Indeed, it is difficult to ascertain whether a
phenotypic change in the host is really adaptive for
parasites or hosts, or whether it is a pathological
effect (Poulin, 1995, 2000; Thomas et al. 2005).
Behavioural modifications increasing host and para-
site fitness are well known in bumblebees affected
by canopid flies (Poulin, 1992; Muller and Schmid-
Hempel, 1993 ; Muller, 1994). In the first days after
infection, bumblebees spend the night and a large
part of the day outsid e the nest to retard the paras ite’s
development (Muller and Schmid-Hempel, 1993)
and keep the infection away from kin (Poulin, 1992).
On the other hand, a few days before emergence, the
parasite induces digging behaviour in the host to
maximize the possibilities of overwintering as a
buried pupa (Muller, 1994). Xenos vesparum para-
sites manipulate the behaviour of infected Polistes
dominulus females by inducing them to leave the
maternal nest and form precocious aberrant ag-
gregations (Hughes et al. 2004 b). However, in view
of the lack of further information about the Xenos-
Polistes system, it is impossible to fully understand
whether the behavioural modifications we found
should be considered parasite manipulation, host
defence or simple effe cts of pathology.
Nevertheless, we tried to evaluate the potential
benefits of the altered behaviour of parasitized wasps
for hosts and parasites. In fact, parasitized females
are de facto ‘ dead’ from a reproductive point of view.
They do not obtain either direct fitness (females
parasitized by males die before spring, females
parasitized by females do not found nests) or indirect
fitness (they leave the natal nest early, do not help
in aggregations, and do not join spring colonies as
subordinates). On the other hand, the ‘ inactive
strategy’ could favour the parasite since it may in-
crease survival of the host over winter by avoiding
energy-wasting behaviours (helpers die before
spring). This strategy could also favour parasites by
allowing the infected females to climb onto a nest
in spring. In this phase of the colony cycle, before
the occurrence of pupae, foundations are very
unstable (Reeve, 1991; Starks, 2001; Dapporto et al.
2004b), and although only 1 female usually starts
the construction of the nest, other individuals may
join later. However, usurpations are frequent and
alien foundresses often aggressively steal nests. A
less aggressive, parasitized foundress may be readily
accepted by resident females, permitting the para-
site to release triungulins dire ctly on the nest. These
hypotheses, however, remain highly speculative.
The different physiological modifications induced
by Xenos males and females reveal a clear manipu-
lative pattern. Indeed, although wasps parasitized
by Xenos females are also castrated (Strambi and
Strambi, 1973), they survive winter along with
unparasitized foundresses. Moreover, Beani (2007)
found that fat bodies are reduced only in wasps with
male parasites. These observations suggest that
Xenos females do not alter some physiological
characteristics of their host so that they can over-
winter.
Intriguingly, this similarity matches the differ-
ences in CHCs that we found. The cuticular
hydrocarbon composition differed slightly between
unparasitized and Xenos-female parasitized wasps.
Parasitized and unparasitized females were at-
tacked and dominated with comparable frequencies,
suggesting that the former are accepted in clusters.
We do not know if wasps are unable to perceive the
slight differences in CHC composition induced by
the parasite infection or if they detect the presence
of the infected females but accept them in the
51039N
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
FMH
Percentage of n-C29:1
*
*
Fig. 7. Percentage of n-C29 : 1 in the cuticular samples
of unparasitized wasps (H) and wasps parasitized by male
(M) and female (F) parasites.
51039N
8.0
6.0
4.0
2.0
0.0
FMH
Percentage of n-C31:1
**
Fig. 8. Percentage of n-C31 : 1 in the cuticular samples of
unparasitized wasps (H) and wasps parasitized by male
(M) and female (F) parasites.
L. Dapporto and others 550

aggregation. Cluster formation is advantageous for
wasps, and unparasitized females probably would
not benefit from driving off their temporarily benign
cluster mates (production of triungulins and infec-
tion will start 6 months la ter in spring). However,
if parasitized females are actually recognized, the
behaviour of helpers that give food with comparable
frequency to parasitized and unparasitized females
is difficult to explain. Since parasitized females will
not reproduce in the following spring, helper efforts
toward them cannot provide any increase in indirect
fitness.
In conclusion, it is premature to decide whether
behavioural and bi ochemical modifications of ag-
gregating Polistes females caused by Xenos should
be considered true parasite manipulation. However,
by castrating their hosts, Xenos may obtain a co-
incidental advantage (Thomas et al. 2005) by putting
the wasps in a low social condition, which may in-
crease the likelihood of engaging in energy-wasting
behaviours in autumn and the possibility of being
accepted as a nest-joiner in spring. Similarly, the
slight modifications of CHC composition may favour
Xenos females, which could remain unnoticed in
autumnal clusters and on spring nests when direct
infection occurs. Also in this case, the similarity
between unparasitized and Xenos-female infected
wasps may not be parasite manipulation ; the CHC
composition could simply reflect the similarity in
some physiological characteristics (e.g. well devel-
oped, fat bodies) typical of unparas itized hibernating
wasps.
This study was inspired by laB he bivB saz ‘‘ Live hidden’’, a
quotation from Epicurus IV Century BC. We thank Laura
Beani and two anonymous referees for reviewing the
manuscript. We thank Lambrusco di Sorbara for clarifying
some difficult questions. This study was funded by the
Universities of Pisa and Florence. All the experimental
procedures conformed to Italian law.
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