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Chemical Signature and Reproductive Status in the Facultatively Polygynous ant Pachycondyla Verenae

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In insects, cuticular hydrocarbons (CHCs) generally are used as cues and signals for within colony processes, such as signaling reproductive status, and between colony processes, such as colony membership. We examined CHC profiles of the facultatively polygynous ant Pachycondyla verenae in order to identify chemical signals of reproductive queens within colonies containing many gynes. Colonies of P. verenae, belonging to two different members of a complex of morphospecies, were collected from three geographic localities within South America. We also tested whether CHC profiles differed between geographic localities and morphospecies. We found three alkenes, two isomers of pentacosene and heptacosene, which were more abundant in CHC profiles of reproductive queens of this morphospecies complex. When we tested whether these differences were consistent across geographic localities, we found the abundance of these alkenes differed according to morphospecies, with the isomers of pentacosene being more abundant in queens from morph one, and heptacosene being more abundant in queens from morph two. Our study has given further insight into the mechanisms behind maintenance of reproductive dominance, and has demonstrated that chemical signatures associated with reproductive status in Pachycondyla verenae are not conserved within this species complex.
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Chemical Signature and Reproductive Status
in the Facultatively Polygynous ant
Pachycondyla Verenae
Sophie E. F. Evison &Ronara S. Ferreira &
Patrizia DEttorre &Dominique Fresneau &
Chantal Poteaux
Received: 12 April 2012 / Revised: 23 September 2012 / Accepted: 2 October 2012
#Springer Science+Business Media New York 2012
Abstract In insects, cuticular hydrocarbons (CHCs) gener-
ally are used as cues and signals for within colony process-
es, such as signaling reproductive status, and between
colony processes, such as colony membership. We exam-
ined CHC profiles of the facultatively polygynous ant
Pachycondyla verenae in order to identify chemical signals
of reproductive queens within colonies containing many
gynes. Colonies of P. verenae, belonging to two different
members of a complex of morphospecies, were collected
from three geographic localities within South America. We
also tested whether CHC profiles differed between geo-
graphic localities and morphospecies. We found three
alkenes, two isomers of pentacosene and heptacosene,
which were more abundant in CHC profiles of reproductive
queens of this morphospecies complex. When we tested
whether these differences were consistent across geographic
localities, we found the abundance of these alkenes differed
according to morphospecies, with the isomers of pentaco-
sene being more abundant in queens from morph one, and
heptacosene being more abundant in queens from morph
two. Our study has given further insight into the mecha-
nisms behind maintenance of reproductive dominance, and
has demonstrated that chemical signatures associated with
reproductive status in Pachycondyla verenae are not con-
served within this species complex.
Keywords Fertility signal .Cuticular hydrocarbons .
Morphospecies .Chemotaxonomy
Introduction
Animals living in societies need efficient communication
systems to coordinate social interactions and protect group
resources from competitors and parasites. Ants and other
social insects have evolved advanced societies, which are
protected by efficient recognition systems based on chemi-
cal cues (reviewed by dEttorre and Lenoir, 2010). Indeed,
social insects live in a world of odors; over 100 exocrine
glands that send a multitude of chemical messages regulat-
ing colony life and reproduction have been characterized in
social insects (Billen, 2004). Discrimination between friends
and foes (nestmates and non-nestmates) is assured by
colony-specific body odor, a mixture of cuticular hydro-
carbons (CHCs), the main cues used in social insect nest-
mate recognition (dEttorre and Lenoir, 2010). These cues
also play an important role in the processes of within-colony
recognition, such as signaling of social status, queen fertil-
ity, and task allocation (Greene and Gordon, 2003; Smith et
al., 2009; van Zweden, 2010).
Typically, social insect colonies contain two castes: re-
productive queens and non-reproductive workers. The term
gynerefers to potential queens that are unmated or repro-
ductively inhibited, whereas queenrefers to a reigning
Electronic supplementary material The online version of this article
(doi:10.1007/s10886-012-0195-6) contains supplementary material,
which is available to authorized users.
S. E. F. Evison :R. S. Ferreira :P. D Ettorre :D. Fresneau :
C. Poteaux
Laboratoire dEthologie Expérimentale et Comparée, EA 4443,
Université Paris 13, 99 avenue J.-B. Clément,
93430 Villetaneuse, France
S. E. F. Evison (*)
Institute of Integrative and Comparative Biology,
Faculty of Biological Sciences, University of Leeds,
Leeds LS2 9JT, UK
e-mail: s.evison@hotmail.co.uk
J Chem Ecol
DOI 10.1007/s10886-012-0195-6
mated gyne (Michener, 1974). Although colony odor gen-
erally is uniform, in several ant species it has been shown
that reproductive individuals have CHC profiles distinct
from non-reproductives. This may also co-vary with ovarian
activation and productivity, suggesting that CHCs signal
caste and/or reproductive potential (Liebig, 2010). In several
species of social insects, a single colony may contain more
than one queen (polygynous colonies). Despite the many
potential benefits of polygyny (see Keller and Reeve, 1994
for a review), the costs predominantly involve conflicts over
reproduction (Bourke, 2011). Such conflicts generally are
regulated through pheromones and/or policing behaviors;
i.e., overt or ritualized aggression and egg cannibalism that
prevent worker reproduction (Ratnieks et al., 2006). Fertility
signals are, predominantly, a reliable indicator of ovarian
development and are honest cues for colony members (van
Zweden, 2010). They are pheromones indicating reproduc-
tive status, and are thought to be taxonomically widespread.
However, empirical studies of their effects are limited.
Commonly, especially in large colonies of social insects,
queen pheromones act to suppress the activation of the
ovaries of workers and gynes (Keller and Nonacs, 1993;
Heinze and dEttorre, 2009). Physiological effects on work-
er reproduction by a queen-produced pheromone only have
been demonstrated experimentally in the honey bee, a single
species of ant, and a termite (Slessor et al.,2005; Holman et
al., 2010; Matsuuraa et al., 2010).
The neotropical ant species Pachycondyla verenae is a
primitive species of ant showing a set of traits that makes it
particularly interesting for studying chemical signaling,
within and between colonies. Colonies of this species are
small, consisting of about 40 (Gobin et al.,2003) to 200
workers (Fresneau, 1984), making it possible for a single, or
small number of individuals, to control all nestmates in the
colony. Thus, fertile individuals could utilize either physical
intimidation or pheromones, or a combination of the two, to
exert control over workers. Pachycondyla verenae is facul-
tatively polygynous (Fresneau, 1984); nests are usually mo-
nogynous, but some polygynous nests have been found. The
reproductive caste is slightly morphologically distinct from
the non-reproductive worker caste. We collected mature
nests from three distinct populations, and found that, within
each population, some colonies exhibited high levels of
polygyny for a species of this genus. Moreover, P. verenae
has genetically, morphologically, and ecologically distinct
morphs that can exist in sympatry (Delabie et al,2008), and
belongs to a complex of closely related species (the P.
apicalis species complex; Ferreira et al, 2010). Thus, there
may be a generic mechanism for signaling fertility by re-
productive individuals, thereby reducing conflicts, that is
common to entire groups of these primitive ant species.
Here, we used a combination of behavioral, chemical,
and genetic analyses to assess the levels of effective
polygyny within colonies, in order to determine if truly
reproductive queens can be distinguished chemically from
gynes and workers (i.e., if they exhibit reproductive status
with chemical signals). We also examined whether the
chemical signatures of reproductives remain constant
throughout geographic locality and morphospecies. We
expected CHC profiles to differ according to morphospe-
cies, as these two morphs live in distinct habitats, which
should influence their CHC profiles; however, it is not
known how this ties into signaling of reproductive status.
Since individual CHC profiles of ants have a genetic basis
(Lockey, 1991), the use of a chemical approach to assess
differences between morphospecies is particularly valuable
when sibling or cryptic species are involved (Howard,
1993).
Methods and Materials
Study Animals and Field Collection Polygynous colonies of
P. verenae were collected from three different areas in South
America: a) from the grounds of The Universidade Federal
de Viçosa (UFV) in the state of Minas Gerais, eastern Brazil;
b) from the Centro de Pesquisas do Cacau (CEPLAC
ERJOH) from Belém in the state of Pará, northern Brazil;
c) from the Kerrenroch forest, Petit Saut, French Guiana.
According to Delabie et al. (2008), at Belém and French
Guiana, morph one should be found, and at Viçosa, morph
two. Entire colonies were collected and kept under standard
conditions in the laboratory in France (26± 2 °C, 65 %
humidity, 12:12 hr L:D photoperiod). We studied a total of
12 colonies (3 from Belém, 4 from French Guiana, and 5
from Viçosa) with queen (and gyne) numbers upon collec-
tion ranging from 2 to 18. Colonies were kept in plaster
nests, each consisting of a chamber covered with a glass seal
and red Plexiglas to allow observations, connected to a
foraging area (18 × 14 cm). Colonies were fed twice weekly
with crickets and a honey/apple mixture, and provided with
water ad libitum.
Behavioral Assays We determined the identities of truly
reproductive individuals (queens) within polygynous colo-
nies through behavioral comparisons with non-reproductive
gynes so that we could efficiently and non-destructively
assess CHC profiles. Each putative reproductive female
(gyne), individually marked with a small plastic, numbered
marker attached with glue, was observed for a total of 6 hr
(divided into daily 20-min. continuous observations) per
colony. We also observed queens in monogynous colonies
so as to identify a series of typical queen behaviors, which
aided us in identifying true queens within polygynous col-
onies. Behaviors such as egg laying or policing, brood care,
and foraging were assessed. In polygynous ponerine ants,
J Chem Ecol
queens typically establish a dominance hierarchy during
colony foundation, with dominant queens staying in the nest
and caring for brood, and subordinate queens foraging
(Dreier and dEttorre, 2009).
Analyses of CHCs CHCs of individual workers were sam-
pled non-destructively by Solid Phase Micro Extraction
(SPME), using a Supelco 7 um polydimethylsiloxane-
coated fiber. Seven workers and either all or a selected
sample (if numerous) of gynes, including the putative
queens identified by behavioral observation, were sampled
from colonies from each of the regions: five colonies from
Viçosa, 3 from Belém, and 4 from French Guiana. Live ants
were immobilized with clean soft forceps, and the SPME
fiber carefully rubbed against the first and second segments
of the gaster and thorax of each individual ant for 3 min. The
SPME fiber then was desorbed in the injection port of an
Agilent 7890A gas-chromatograph, equipped with an HP-
5MS capillary column (30 m × 250 μm, 0.25 μm thickness)
and a split-splitless injector, coupled to a 5975 Agilent Mass
Spectrometer operated at 70 eV in the electron impact ion-
ization mode. The carrier gas was helium at 1 ml.min
1
. The
column oven was programmed as follows: an initial hold of
1 min at 70 °C, to 260 °C at 30 °C.min
1
, to 280 °C at 1 °
C.min
1
, and then to 320 °C at 10 °C.min
1
(held for 5 min).
Forty CHCs, present in all individuals, were selected for
analysis (Table 2; Supplementary data Table S2). These
compounds were identified on the basis of mass spectra
and their retention times, compared to standard linear hydro-
carbons. The areas of the 40 peaks were integrated by the
Agilent Chemstation software, and the relative proportion of
each peak calculated.
Physiological Status of Reproductives Gynes that died dur-
ing experiments were collected and stored at 23 °C.
Subsequent to the experiments, entire colonies were killed
by freezing, and all remaining gynes, along with the dead
gynes, were dissected under a stereomicroscope to assess
ovarian development, by counting chorionated basal
oocytes (i.e., reproductive eggs).
Molecular Procedures and Genetic Analyses To confirm
that our populations belonged to the assumed morphospe-
cies, we sequenced a part of the mitochondrial DNA. The
DNA of several workers from colonies of Belém and Viçosa
was extracted from ethanol-preserved tissues (head and
thorax) using a standard Chelex 10 % procedure. We used
sequences already known for workers from French Guiana
(9 individuals from 5 colonies; HM770113 to HM770121).
Variation was assayed by the sequencing of a portion of
mtDNA of cytochrome b (cyt b, <700 bp), according to the
procedures described in Ferreira et al. (2010). Sequence
analyses were edited and aligned using the default settings
of Clustal X (Thompson et al., 1997) and checked visually.
Average intra- and inter-morph genetic divergences were
calculated using the Kimura 2-parameter distance (Kimura,
1980), with the MEGA4 program (Tamura et al., 2007).
In order to identify the reproductive queens whose off-
spring were raised by colony members, 1624 workers (per
monogynous/polygynous colony, respectively) and all po-
tential queens were genotyped. DNA was extracted from the
head and thorax in 500 μl 10 % Chelex® solution and 20 μl
proteinase K (10 mg/ml), and diluted 5-fold. We used eight
variable microsatellite loci, according to procedures in
Evison et al. (2010), with two modifications: for primers
2111 and 4053, the forward and reverse primer concentra-
tion was 0.5 μM; for primer 2056, a touchdown program
(annealing temperature decreasing at each PCR cycle from
62 to 58°, then 21 cycles at 58 °C). The labeled products
were mixed with internal size marker GeneScanLiz500
(Applied Biosystems, Foster City, CA, USA) and run on an
automated ABi 3100 Sequencer (Applied Biosystems).
Fragment length was scored using the freeware application
Peak Scannerv1.0 (Applied Biosystems).
Statistical Analyses The amount of genetic variation at each
locus was quantified by calculating the allelic richness (N
A
)
and unbiased expected heterozygosities with FSTAT 2.9.3
(Goudet, 2001;tableS1). The mating structure of the popula-
tion was estimated as the inbreeding coefficient, F(Weir and
Cockerham, 1984;tableS1). Genetic differentiation was ana-
lyzed over pairs of nests by genotypic differentiation through
F-statistics, using the program GenoDive (Meirmans and
Tienderen, 2004). Identities of actively reproducing queens
present, or previously present in colonies, and their mating
frequencies were inferred from genotypes.
The relative proportions of each of the 40 CHCs identi-
fied as common to every individual analyzed were arcsine
transformed to normalize the data, then standardized by
subtracting the mean and dividing by the variance of the
data set. Linear discriminant analyses (LDA) were per-
formed using the chemical variables from the standardized
data set to determine whether predefined groups (either
caste, as identified through consistency of behavioral obser-
vations, ovarian activation, and genetic analyses) could be
discriminated on the basis of their cuticular profiles.
Statistical significance between these groups was tested by
WilksMANOVA. We also performed principal compo-
nents analysis (PCA) to explore the sources of variation
within the data set in order to identify key CHC compounds
responsible for discrimination of the different castes, geo-
graphic localities, and morphospecies. Following this, we
used linear mixed-effects models to confirm which specific
compounds had the most influence in signaling reproductive
status, geographic locality, and morphospecies. Here, we
treated each CHC as a separate treatment and used its
J Chem Ecol
normalized relative amount as the response variable in our
model to look at the interaction between CHC and caste or
geographic locality. We then checked to see if these effects
were consistent between geographic localities (and morpho-
species). We fitted individual, nested within colony (and
nested within geographic locality when modelling caste),
as the random term. All tests were performed using the
program R (R Development Core Team, 2012).
In order to assess any potential associations among di-
verging CHC profiles, genetic relatedness, and geographic
locality, we performed Mantel correlation tests (Mantel,
1967), based on 999 random permutations using the pro-
gram GenoDive (Meirmans and Tienderen, 2004). Here, we
correlated matrices of average inter-colony chemical (using
pairwise Euclidean distances), geographic (using the natural
log of physical distance between nests in metres), and ge-
netic (using pairwise Fst) distances.
Results
Behavioral Observations In every colony, one or more
gynes exhibited typicalqueen behaviors. Each of the re-
productive queens we identified exhibited all of the follow-
ing behaviors: quivering abdomen, lack of foraging, little
or no aggression toward workers and other gynes, and egg
laying. Conversely, non-reproductive gynes behaved in a
similar manner to workers, exhibiting foraging, aggressive
and agonistic behaviors, mainly involving antennal boxing,
both symmetrical and asymmetrical (Denis et al., 2008), and
occasional incidences of biting. Two thirds (8/12) of our
colonies were functionally monogynous, with the remaining
colonies effectively polygynous, with 24 queens per colo-
ny. Polygyny occurred only in colonies from French Guiana
and Viçosa (Table 1). These behavioral observations
allowed us to sample the CHC profiles of putative queens
and gynes non-destructively, with more confidence of their
status prior to dissection and genetic analysis.
Physiological Status of Reproductives When queens and
gynes were dissected, we found that the queens identified
by behavioral observations sometimes did not correspond
with their state of ovarian activation, so the effective queen
number increased or decreased in certain cases (Table 1).
We found activated ovaries in up to six gynes in colonies
from Belem, even though all colonies were identified as
monogynous by behavioral observations. Generally, we
found activated ovaries in only one gyne in colonies from
French Guiana, although in one colony we found five,
whereas in colony FG23 we found none with activated
ovaries. In colonies from Viçosa, we found up to three gynes
with activated ovaries, which almost exactly matched our
behavioral observations.
Genetic Analyses MtDNA sequences confirmed the morph
status of our colonies; populations of P. verenae from
French Guiana and Belém belonged to morph one, and
populations from Viçosa belonged to morph two (accession
numbers from JQ819734 to JQ819742). Average inter-
morph genetic divergence was 0.046±0.008, corresponding
to 24 variable sites (20 transitions and 4 transversions).
Genetic differentiation also was possible through microsa-
tellite loci, with specific alleles or differences in allelic
frequencies observed between localities (Supplementary
Table S1). Fst values between the three localities were high
and significant (mean00.4± 0.13).
Microsatellite data showed five out of twelve nests were
effectively monogynous. Two polygynous nests, with 35
Table 1 Number of Pachycon-
dyla verenae queens identified
by the three different methodol-
ogies (all queens were identified
as monandrous except the
queens from UFV7), and the
numbers of queens, gynes and
workers whose cuticular hydro-
carbon profiles were analyzed by
linear discriminant analyses
(LDA)
Number of queens identified by Number of individuals in LDA
Colony Behavior Dissections Genetics Queens Gynes Workers
Bel5 1 1 1 1 1 7
Bel6 1 3 3 1 8 7
Bel13 1 6 5 1 1 7
FG12 1 1 1 3 8 7
FG13 4 5 5107
FG17 1 1 1 1 10 7
FG23 1 0 1 0 8 7
UFV1 4 3 2 2 0 7
UFV2 1 1 2 1 0 7
UFV3 1 1 1 1 0 7
UFV7 3 3 2 (polyandrous) 1 0 7
UFV10 2 2 4137
Total group size 14 39 84
J Chem Ecol
queens, were from Belém, one polygynous nest, with >4
queens, was from French Guiana, and two polygynous
nests, with 24 queens, were from Viçosa (Table 1). It was
apparent that only one or two males per colony mated all the
reproductive queens. Additionally, the data showed that all
gynes within these colonies were re-adopted daughter
queens.
Chemical Discrimination of Fertile vs. Non-fertile
Individuals We discriminated pre-assigned groups of repro-
ductive (fertile queens, as identified through consistency of
behavioral observations, physiological status, and genetic
analyses) and non-reproductive individuals (non-fertile
gynes, identified as above, and workers) by a chemical
approach. LDA, performed on the relative amounts of all
40 CHCs, discriminated between queens, gynes and work-
ers (Wilksλ< 0.05, F04.297, df080, 190, P<0.001;
Fig. 1). We then investigated what compounds are important
in signaling reproductive status through the use of several
exploratory techniques. PCA showed heavy loadings of
variance within the dataset on several compounds, and our
linear mixed-effects model showed an interaction between
CHC and caste (F
78, 5187
02.57, P<0.001), and allowed us
to identify which of the CHCs showed differences between
relative amounts of gyne, queen, and worker compounds.
These were attributable to four different alkenes: tricosene
(C
23:1
), two isomers of pentacosene (C
25:1a
and C
25:1b
), and
heptacosene (C
27:1
) (Table 2). However, the relative quanti-
ties of these four compounds in the three castes were not
consistent across localities (Fig. 2; Supplementary Table
S2). Ants from Belém and French Guiana showed no differ-
ences in the abundance of tricosene between castes, but
queens from Belém had more of the pentacosene isomer
C
25:1a
(t
93
03.79, P<0.001), and queens from French Guiana
had more of the isomer C
25:1b
(t
165
03.89, P<0.001).
Furthermore, gynes from French Guiana had higher levels
of pentacosane isomer C
25:1a
(t
165
03.11, P00.002). Ants
from Viçosa had higher levels of heptacosene in queens
(t
122
05.76, P<0.001), while tricosene was higher in gynes
(t
122
08.33, P<0.001).
Discrimination of Geographic Origin and Morph We dis-
criminated the three geographic locations that the ant colo-
nies were collected from using the relative amounts of all 40
CHCs (Fig. 3;Wilksλ< 0.05, F024.349, df080, 220, P<
0.001), as well as the two different morphs the ants belong
to (Wil ksλ00.874, F019.167, df040, 110, P<0.001).
PCA showed numerous compounds to be responsible for
variation in the dataset, and, along with the linear mixed-
effects model, we found that 21 out of 40 (52.5 %) of the
CHCs analyzed differed among geographic locations,
whereas only 8 out of 40 (20 %) differed between morphs
(Table 2).
Association Between Variation of the Cuticular Profile
and Genetic and Geographic Distance We found corre-
lations between the chemical Euclidean distances and both
genetic (Mantel test; r00.436, P00.003) and geographic
(Mantel test; r00.485, P00.001) distances, as well as a
correlation between genetic and geographic distance
(Mantel test; r00.847, P00.001).
Discussion
We demonstrated a chemical distinction between fertile and
non-fertile individuals within colonies of Pachycondyla ver-
enae, as well as a chemical distinction among colonies from
divergent geographic populations and morphospecies. Our
chemical analyses showed that reproductive queens, non-
reproductive gynes, and workers are discriminated by their
respective CHC profiles. This supports previous research on
this group of ants, and in several other ant species (Monnin,
2006; Peeters and Liebig, 2009; Liebig, 2010). In addition,
we showed that there are chemical differences among
queens, gynes, and workers, and that the compounds re-
sponsible for these differences differ between morphs and
among populations from different localities. Four alkenes
were important in distinguishing castes chemically: trico-
sene (C
23: 1
), two isomers of pentacosene (C
25:1a
and
C
25:1b
), and heptacosene (C
27: 1
). Only the pentacosene
isomers and heptacosene were consistently higher in
Fig. 1 Linear discriminant analysis showing chemical distance be-
tween queens (Q), gynes (G), and workers (W) of Pachycondyla
verenae. Each caste group is discriminated (Wilksλ<0.05,F0
4.297, df080, 190, P< 0.001). Axes show linear discriminator (LD) 1
and 2
J Chem Ecol
reproductive queens than in non-reproductive gynes and
workers; tricosene was lower in reproductives only in
Viçosa. Although the two pentacosene isomers were impor-
tant in discriminating reproductive individuals from Belém
and French Guiana, the reproductive chemical signatures
differed most distinctly between morph one (ants from
Belém and French Guiana, in which the two isomers of
pentacosene were more important) and morph two (ants
from Viçosa, in which heptacosene was more important).
CHCs that co-vary with fertility are seldom the same across
species (Peeters and Liebig, 2009). The differences we
found mirrored our results from the Mantel tests that showed
genetic divergence correlates with both CHC profile differ-
ences and geographic distance.
Ants are the best-studied group with respect to differ-
ences in CHC profiles relating to reproduction, with 21
Table 2 Cuticular hydrocarbons
included in the chemical
analyses, and their statistical
significance in discriminating
caste, geographic locality or
morph of Pachycondyla
verenae.NS0not
significant
Hydrocarbon Effect on caste Effect on region Effect on morph
C
21 :1a
NS NS NS
C
21 :1b
NS NS NS
C
21
NS t
5187
08.619; P<0.001 t
5226
04.435; P<0.001
9-, 11-MeC
21
NS NS NS
3-MeC
21
NS NS NS
C
22 :1
NS t
5187
02.144; P00.032 NS
C
22
NS t
5187
02.935; P00.003 NS
10-, 12-MeC
22
NS NS NS
C
23 :1
t
5187
02.195; P00.028 t
5187
025.47; P<0.001 t
5226
02.831; P00.005
C
23
NS t
5187
02.332; P00.019 NS
9-, 11-MeC
23
NS NS NS
7-MeC
23
NS NS NS
5-MeC
23
NS NS NS
3-MeC
23
NS NS NS
C
24 :1
NS NS NS
C
24
NS NS NS
9-,10-,11-MeC
24
NS NS NS
C
25 :1
t
5187
02.069; P00.039 t
5187
09.594; P<0.001 NS
C
25 :1
t
5187
03.886; P<0.001 t
5187
03.208; P00.001 t
5226
03.822; P<0.001
C
25
NS t
5187
05.786; P<0.001 t
5226
04.010; P<0.001
9-, 11-, 13-MeC
25
NS t
5187
05.196; P<0.001 t
5226
03.340; P<0.001
5- , 11-MeC
25
NS NS NS
9,13-diMeC
25
NS NS NS
C
26 :1
NS t
5187
02.979; P00.003 NS
C
26
NS NS NS
11-, 12-, 13-MeC
26
NS NS NS
C
27 :1
NS t
5187
011.72; P<0.001 t
5226
05.798; P<0.001
C
27 :1
t
5187
03.055; P00.002 t
5187
04.188; P<0.001 NS
C
27
NS t
5187
03.021; P00.003 NS
9-, 11-, 13-MeC
27
NS t
5187
03.477; P<0.001 NS
5-MeC
27
NS NS NS
5,15-, 5,17-diMeC
27
NS NS NS
C
28 :1
NS t
5187
02.208; P00.027 NS
C
28
NS NS NS
4-MeC
28
NS t
5187
02.461; P00.014 NS
C
29 :1
NS t
5187
07.401; P<0.001 t
5226
02.697; P00.007
C
29 :1
NS t
5187
03.439; P<0.001 NS
C
29
NS t
5187
04.573; P<0.001 NS
11-, 13-, 15- MeC
29
NS t
5187
04.864; P<0.001 t
5226
02.146; P00.020
C
31 :1
NS t
5187
03.069; P00.002 NS
J Chem Ecol
species from 16 genera having been studied. These studies
have identified a range of different compounds as important
in signaling reproductive status (Peeters and Liebig, 2009;
Liebig, 2010). Considering the chemical signatures of repro-
ductives identified in this and other studies, and how these
signatures vary, it is unlikely that a single, universal com-
pound signals fertility in hymenopteran groups such as
Pachycondyla. Rather, it is more likely that quantitative
differences in cuticular profiles are important in identifying
fertile individuals; such differences correlate with degrees of
fertility in both queens and workers in several species of ant
(Peeters and Liebig, 2009). This is consistent with the quan-
titative, rather than qualitative, differences in CHC profiles
we found in P. verenae among queens, gynes, and workers
in this study.
We also found differences in the composition of ant
CHCs among the different geographic locations. Ants from
the different areas belong to different morphs (Delabie et al.,
2008). Our results show that populations of P. verenae from
French Guiana and Belém belong to morph one, while
populations from Viçosa belong to morph two. The LDA
was unable to distinguish ants within morph one on the LD2
axis, as indicated in Fig. 3by the symbols B and G being on
the same level, highlighting their closer similarity in CHC
chemistry to each other than to ants of morph two. This also
corroborates our findings in regard to differences in the
chemical signature of reproductives. The physical differen-
ces between morphs are fairly subtle, and are based on
morphological variations in bristle structures, petioles, legs,
and antennae (Delabie et al., 2008; Ferreira, pers. comm.).
However, they represent distinct ecological traits with dif-
fering habitats and nesting behaviors: morph one nests in
1.0 0.2 0.4 0.6 0.8 1.0
Relative Quantity
1.00.20.40.60.8
C23: 1 C25:1a C25:1b C27: 1
Cuticular Chemical
0.0 0.2 0.4 0.6 0.8
a
b
c
Fig. 2 Mean± s.e. of relative amounts of specific cuticular hydrocar-
bons of gynes (black bars), queens (grey bars) and workers (white
bars)ofPachycondyla verenae ants from a) Belém b) French Guiana,
and c) Viçosa
-6 -4 -2 0 2 4
-4 -2 0 2 4 6
LD1 (63.43%)
LD2 (36.57%)
B
B
B
B
B
B
BB
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
BB
B
B
B
B
B
B
B
B
BB
B
B
G
G
G
G
G
G
GG
G
G
G
G
G
G
G
G
G
GG
G
G
G
G
G
G
G
G
G
G
G
G
G
GG
G
G
G
G
G
GG
GG
G
GG
G
G
G
G
G
GG
G
G
G
G
G
G
G
V
V
V
V
V
V
V
V
VV
V
V
V
VV
VV
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
VV
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
Fig. 3 Linear discriminant analysis showing chemical distance be-
tween Pachycondyla verenae ants from the three different geographic
locations: Belém (B), French Guiana (G), and Viçosa (V). Each geo-
graphic group is discriminated (Wilksλ< 0.05, F024.349, df080,
220, P<0.001). Axes show linear discriminator (LD) 1 and 2
J Chem Ecol
rotting tree trunks or fruits on the ground, while morph two
nests on the ground in open arid environments (Ferreira,
pers. comm.). Therefore, it is not surprising that the respec-
tive CHC profiles are fairly distinct, considering that a
primary role of CHCs is considered to be the prevention of
desiccation (Blum, 1996).
Our three different methods of queen assessment
revealed variable effective queen numbers in our colo-
nies. This likely occurred because the three methods
probably sample a colony population differently. The
behavioral observations take a snapshotof the current
kin structure of the colony, whereas the genetic analyses
probably also included daughters of queens that may
have died or left the colony (Kellner et al.,2007). The
dissections may have sampled the current kin structure as
well as the past or future structure (i.e., effectively a
combination of the other two methods), depending on
the interaction between behavioral control of reproduction
and the chemical inhibition of ovarian activation. These
differences in effective queen numbers among the assess-
ment methods requires further study and may help dis-
entangle how chemical signals may be exhibited by, and
have effects upon, current members of the colony, as
well as how the route to reproductive dominance is
initially achieved. Chemical signals can act as phero-
mones in two ways: as a stimulus leading to a prompt
behavioral response by an individual (releaser effect) or,
indirectly, by stimulating hormone secretion, which can
result in physiological changes (primer effect) (Wyatt,
2003). However, only in three species of insects have
specific chemical compounds (or blends) signaling fertil-
ity been found to have such effects (Slessor et al.,2005;
Le Conte and Hefetz, 2008;Holmanetal.,2010;
Matsuuraa et al., 2010). The specific effects of the com-
pounds we identified as associated with reproductive
status need to be investigated further before concluding
whether they function as pheromones or not.
Our overall findings suggest that reproductive conflicts in
this complex of morphospecies of ant may be controlled
through pheromonal inhibition by the chemical signature of
actively laying queens. Our findings also highlight the im-
portance of an integrated approach to assessing signaling
of reproductive status. Using a chemotaxonomic approach
is particularly valuable for identification of sibling or
cryptic species (Howard, 1993), such as the complex of
morphospecies that P. verenae belongs to (Wild, 2005).
As only three geographic localities were investigated in
this study, more extensive sampling is needed to confirm
the validity of our conclusion. However, it appears that
signaling of reproductive status in Pachycondyla verane
queens is an important aspect resolving reproductive con-
flicts in polygynous colonies, which is not highly con-
served between morphospecies.
Acknowledgements We thank Paul Devienne and Marie Claire
Malherbe for technical assistance, Serge Aron and Laurent Grumiau
for assistance in molecular work, and all the people who helped with
ant collecting. We also thank the Fyssen Foundation who funded this
work, and the project PRONEX (Rede Multidisciplinar de Estudos
sobre Formigas Poneromorfas do Brasil) for ant collection in Brazil.
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J Chem Ecol
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Inclusive fitness theory explains how helpers reproduce indirectly via the breeders they help. The inclusive fitness helpers get depends on their relatedness to the breeder(s), colony productivity and fertility of the breeder(s). It is therefore critical for workers to assess breeder fertility. There is strong evidence that, in wasps, bees and ants, the cuticular hydrocarbon (CHC) profiles of breeders are a signal of fertility. Chemical and behavioural evidence suggests that linear alkanes are not involved in communication, whereas methyl-branched alkanes and alkenes may constitute, or at least contribute to, the fertility signal. The correlation between CHCs and reproduction is well established, as well as the fact that CHCs are detected and that workers react accordingly. However, whether CHC profiles are honest is yet to be demonstrated. Hormonal and genetic studies, such as inactivating genes regulating the production of alkenes, are promising approaches to investigate the honesty of CHC profiles
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The social organization of insect colonies indicates the importance of information that is usually not needed in solitary insects. Information about the presence and fertility of a queen strongly affects worker behavior and colony organization. Reproductive competition in colonies requires the correct assessment of each others' rank. All of this information about fertility status and/or dominance status can be encoded in the cuticular hydrocarbon profile of members of ant, wasp, and bee colonies. Understanding variations in these hydrocarbon profiles, their composition, and relation to fertility is key to the further understanding of the major property of eusocial insects, reproductive division of labor. Cuticular hydrocarbons are part of the lipid layer of the insect cuticle that protects from desiccation (Lockey, 1988) and are thus present in basically every social insect (see Chapter 6). Insects have the sensory apparatus to detect these profiles. So it is not surprising that they utilize variations in hydrocarbon profiles between individuals within and between species to detect various properties in other individuals, such as species identity, gender, colony membership (Howard and Blomquist, 1982, 2005; and various chapters in Part II of this book). In this chapter I will review the evidence indicating that hydrocarbon profiles are also used in colonies of ants, bees, and wasps for the regulation of reproduction. I will especially focus on patterns of variation in hydrocarbon profiles on the cuticle and the eggs in relation to fertility differences, which has not been done in such detail in previous reviews (Heinze, 2004; Monnin, 2006; Hefetz, 2007; Le Conte and Hefetz, 2008; Peeters and Liebig, 2009).
Article
Semiochemicals, the pheromones and allomones, have been detected in arthropod species in six orders. Multifunctional pheromones have been especially characteristic of the queens of eusocial species. Compounds such as the queen substance of the honey bee Apis mellifera possess unrelated primer and releaser functions for the workers and act as a sex attractant for drones. Females of other hymenopterous species exploit the secretions of sting-associated glands as sex pheromones. A variety of nonhymenopterous species have adapted components in diverse defensive secretions to function as sex pheromones. The alarm pheromones of many arthropods are also used as defensive allomones, activity inhibitors, cryptic alarm pheromones, aggregative attractants, robbing agents, digging agents, trail pheromones, and antimicrobial agents. Some of these compounds also possess highly distinctive roles, eg functioning as lethal attractants for prey or, in the aquatic milieu, cuticular wetting agents. -from Author
Chapter
The ability to recognize group members is a key characteristic of social life. Ants are typically very efficient in recognizing non-group members and they aggressively reject them in order to protect their colonies. There are a range of different recognition mechanisms including prior association, phenotype matching, and recognition alleles. The concept of kin recognition should be considered different from that of nestmate recognition. Most of the available studies address the nestmate recognition level, namely the discrimination of nestmates from non-nestmates, independently of actual relatedness. Indirect and direct evidence identify long-chain cuticular hydrocarbons as the best candidates to act as recognition cues in ants, even if other chemical substances could also play a role, at least in some ant species. The relative importance of genetic and environmental factors on the expression and variation of the cuticular hydrocarbon profile vary among species and is linked to life history strategies.
Article
Several hypotheses have been put forward to explain the adaptive significance of interspecific variation in mating frequencies by eusocial hymenopteran queens. Four of these hypotheses assert that polyandry is advantageous to queens because of the resultant increase in genetic variability within colonies (referred to as the "GV" hypotheses). Here we compare the frequency of polyandry between monogynous (single-queen) and polygynous (multiple-queen) ant species to test the hypotheses that (1) multiple mating functions primarily to increase intracolonial genetic variability, and (2) mating has costs (such as increased energetic losses or risk of predation or venereal disease). If one of the GV hypotheses is true and mating is costly, the frequency of polyandry should be lower in polygynous species (in which the presence of multiple queens results in low relatedness among workers) than in monogynous species. As predicted by the GV hypotheses, polyandry is less common among polygynous than among monogynous species. Furthermore, it seems that the causal relationship underlying this association is that the number of matings by queens depends on the number of queens present in the colony (rather than the number of queens being influenced by the number of matings), which also supports the GV hypotheses together with the assumption that mating has costs.