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Genetic Distance and Age Affect the Cuticular Chemical Profiles
of the Clonal Ant Cerapachys biroi
Serafino Teseo &Emmanuel Lecoutey &Daniel J. C. Kronauer &
Abraham Hefetz &Alain Lenoir &Pierre Jaisson &Nicolas Châline
Received: 15 February 2014 /Revised: 27 March 2014 / Accepted: 7 April 2014
#Springer Science+Business Media New York 2014
Abstract Although cuticular hydrocarbons (CHCs) have
received much attention from biologists because of their
important role in insect communication, few studies have
addressed the chemical ecology of clonal species of eusocial
insects. In this study we investigated whether and how differ-
ences in CHCs relate to the genetics and reproductive dynam-
ics of the parthenogenetic ant Cerapachys biroi.Wecollected
individuals of different ages and subcastes from several colo-
nies belonging to four clonal lineages, and analyzed their
cuticular chemical signature. CHCs varied according to colo-
nies and clonal lineages in two independent data sets, and
correlations were found between genetic and chemical dis-
tances between colonies. This supports the results of previous
research showing that C. biroi workers discriminate between
nestmates and non-nestmates, especially when they belong to
different clonal lineages. In C. biroi, the production of indi-
viduals of a morphological subcaste specialized in reproduc-
tion is inversely proportional to colony-level fertility. As
chemical signatures usually correlate with fertility and repro-
ductive activity in social Hymenoptera, we asked whether
CHCs could function as fertility-signaling primer pheromones
determining larval subcaste fate in C. biroi.Interestingly,and
contrary to findings for several other ant species, fertility and
reproductive activity showed no correlation with chemical
signatures, suggesting the absence of fertility related CHCs.
This implies that other cues are responsible for subcaste
differentiation in this species.
Keywords Social insects .Ants .Cerapachys biroi .
Cuticular hydrocarbons .Pheromones .Fertility signaling .
Biological invasions .Clonality
Introduction
In social Hymenoptera, the hydrocarbons present on the sur-
face of the cuticle (cuticular hydrocarbons or CHCs) play an
essential role in social communication (Howard and
Blomquist 2005), while their primary function is to limit
desiccation and infiltration of microorganisms (Gibbs 2002;
Gibbs and Crockett 1998;Martinetal.2009). Much research
on social evolution and behavior, where insect societies are
S. Teseo (*):E. Lecoutey :P. Jaisson :N. Châline
Laboratoire d’Ethologie Expérimentale et Comparée, EA4443,
Université Paris 13, Sorbonne Paris Cité, 99 avenue J.B. Clément,
Villetaneuse 93430, France
e-mail: teseo@leec.univ-paris13.fr
S. Teseo
Laboratoire Evolution, Génomes et Spéciation, CNRS-UPR 9034,
91198 Gif-sur-Yvette, Cedex, France
D. J. C. Kronauer
Laboratory of Insect Social Evolution, The Rockefeller University,
1230 York Avenue, New York, NY 10065, USA
D. J. C. Kronauer
Museum of Comparative Zoology, Harvard University, 26 Oxford
Street, Cambridge, MA 02138, USA
A. Hefetz
Department of Zoology, George S. Wise Facultyof Life Sciences, Tel
Aviv University, Ramat-Aviv, Israel
A. Lenoir
UMR-CNRS 7261, Faculté des Sciences et Techniques, Université
François Rabelais, Tours 37200, France
N. Châline
Departamento de Biologia, FCLRP, Universidade de São Paulo
(USP), Av. Bandeirantes, 3900, 14040-901 Ribeirão Preto - SP,
Brazil
N. Châline
Department of Experimental Psychology, Institute of Psychology,
University of São Paulo, Av. Prof. Mello Moraes, 1721, Cidade
Universitária, São Paulo 05508-030, Brazil
JChemEcol
DOI 10.1007/s10886-014-0428-y
extensively used as model systems, has focused on the evo-
lution and the functional role of CHC variability. A significant
amount of work has been conducted to understand how chem-
ical signatures can both enable colony-level coherence and
bear information about caste, age, and reproductive status of
individuals within colonies (Denis et al. 2006,Ichinoseand
Lenoir 2009; Le Conte and Hefetz 2008;Liebigetal.2000;
Monnin 2006; Smith et al. 2008). From this perspective,
clonal hymenopteran species have received little attention,
despite their potential importance in the understanding of
these issues. Genetic homogeneity within colonies allows
experimental control over the genotype of individuals, and
clonal species can, therefore, be used to tease apart the effects
of genetics versus environment or caste (either behavioral or
morphological) on chemical signatures. In the cerapachyine
ant Cerapachys biroi, females lack a spermatheca, i.e., they
cannot be inseminated, and reproduce via thelytokous parthe-
nogenesis (Tsuji and Yamauchi 1995). Male production oc-
curs exceedingly rarely in laboratory colonies (Kronauer et al.
2012). Even though several clonal lineages have been found
in C. biroi (Kronauer et al. 2012), as far as we know, natural
colonies are always monoclonal, i.e., all the individuals in a
colony belong to the same clonal lineage with intra-colonial
relatedness =0.99 (Kronauer et al. 2013; Oxley et al. 2014). A
recent study has shown that individuals are able to discrimi-
nate between nestmates and non-nestmates, especially when
they are of different clonal origin (Kronauer et al. 2013). As
social recognition in ants is based on the divergence of CHC
profiles, we explored the inter-colonial variability of chemical
signatures in C. biroi and the relationship between genetic and
chemical distances between colonies from the same or differ-
ent clonal lineages.
The colonies of C. biroi include two worker subcastes that
differ in morphology, behavior and fertility levels, referred to
as high and low reproductive individuals, or HRIs and LRIs
(Teseo et al. 2013,2014). These are also called intercastes and
workers by Ravary and Jaisson (2004) and ergatoid queens
and workers by Lecoutey et al. (2011). Low reproductive
individuals (LRIs) have two ovarioles and lay eggs exclusive-
ly during their first four-five months of life, during which they
also provide care to the developing brood. After this stage,
they become nonreproductive foragers (Ravary and Jaisson
2004). High reproductiveindividuals (HRIs), which constitute
approximately 5 % of the individuals in a colony, show low
activity levels and produce up to eight eggs during each
reproductive phase, having four to six ovarioles in total
(Ravary and Jaisson 2004). These individuals probably re-
main fertile for a much longer time compared with LRIs. From
a functional perspective, colonies therefore comprise two
groups: old LRIs that behave as reproductively-inactive for-
agers, as well as young LRIs and HRIs of all ages that act as
nurses and are fertile. In C. biroi, the production of HRIs is
regulated via a feedback system based on the actual fertility
level of the colony (Lecoutey et al. 2011). The more fertile a
colony is, the greater its proportion of HRIs and young LRIs,
and the less HRIs it produces, and vice versa.Insocial
Hymenoptera, CHC signatures often are correlated with fer-
tility, and are used by reproducers to signal their presence and
reproductive status (reviewed by Monnin 2006). As a previ-
ous study on C. biroi indicated that the HRI regulation system
is not based on volatile chemical signals (Lecoutey et al.
2011), we hypothesized that non-volatile, cuticular chemical
cues could signal fertility and/or reproductive status. We fur-
ther hypothesized that these cues act as primer pheromones
that inhibit the HRI developmental trajectory. Cerapachys
biroi colonies undergo stereotypic reproductive cycles in
which they alternate between a reproductive and a foraging
phase (Ravary and Jaisson 2002,2004;Ravaryetal.2006). In
the reproductive phase, all individuals aggregate while a new
batch of eggs is laid by fertile individuals, whereas in the
foraging phase, young individuals and HRIs remain inside
the nest chamber while older LRIs forage for prey. We ana-
lyzed the CHC profiles of HRIs, the most fertile individuals,
throughout the reproductive cycle, in order to assess whether
the phasic ovarian activity of egg-layers (Teseo et al. 2013)
correlated with changes in cuticular profiles. A reproductive
activity-related change in chemical signatures could possibly
play a role in the feedback regulation of HRI production. We
thus compared intranidal (more fertile) workers and foragers
(less fertile workers) in the foraging phase, when the two types
of individuals can easily be distinguished on the basis
of their behavior. This allowed us to assess whether
chemical differences between both groups could be
involved in this fertility-related cue.
Methods and Materials
Colonies The geographic origins, clonal lineages, and
collection dates of the C. biroi colonies used in this
study are listed in Table 1. These were kept in the
laboratory under constant conditions of 27 °C, ~70 %
relative humidity, and 12 h L:12 h D photoperiod. Nests
weremadeof~30x30x10cmplasticboxeswith
Fluon®-coated edges, with a ca. 2.5 cm thick floor
made of plaster of Paris. A single nest chamber was
dug in the center of the nest and covered with a red
glass sheet. Colonies were fed twice a week with live
pupae of the ant Aphaenogaster senilis during foraging
phases.
Genetic Analyses Prior to chemical analyses, one individual
of each of ten colonies (Table 1) was genotyped at 30 micro-
satellite loci and sequenced for two mitochondrial gene frag-
ments (658 bp of cytochrome oxidase I and 575 bp of cyto-
chrome oxidase II) to determine the clonal origin of each
JChemEcol
colony (data reported in Kronauer et al. 2012). Ten individuals
then were randomly collected from each colony and geno-
typed at 17 polymorphic microsatellite loci (CKPWC,
D6CNC, B3KAG, EFOHK, D3N3P, ESOCS, E27C5,
B8PND, DK371, ESI77, ETWBP, EH2OX, ESA52, E324Z,
ED6BM, EPCI6, D8EP1) as in Kronauer et al. 2012,inorder
to estimate the inter-individual relatedness within our colonies
and to assure that the initially selected worker was represen-
tative of the genetic makeup of the colony. The software
GenClone 2.0 (Arnaud-Haond and Belkhir 2007) was used
to assign individuals to multilocus genotypes (MLGs) based
on these 17 microsatellite loci. Multilocus genotypes (MLGs)
were used to produce matrices containing pair-wise Euclidean
genetic distances between individuals of the same clonal
lineage. Based on these matrices, we calculated the average
pairwise relatedness within colonies as r=(A-D)/A,whereA
is the maximum possible allele distance given the markers,
and Dis the average observed allele distance between indi-
viduals in a given colony (Kronauer et al. 2013). Given that
C. biroi reproduces asexually and that the studied populations
show very low clonal diversity (Kronauer et al. 2012), we did
not perform standard calculations of pairwise regression relat-
edness (e.g., Queller and Goodnight 1989).
Chemical Analyses Initially, a set of one hundred individuals
was used for the identification of the CHC peaks of C. biroi.
Fifty individuals were randomly collected in the foraging area
and fifty individuals in the nest chamber of one colony (T5,
clonal lineage A, Table 1). Ants were frozen and pooled, and
their CHCs were extracted in 2 ml pentane during 1 h. The
extract was analyzed with a VGM250Q GC/MS equipped
with a DB-5 column (30 m x 0.32 mm x 0.25 μm, J & W,
Agilent Technologies, PaloAlto, CA, USA). Helium was used
as carrier gas, with a 28.57 cm/s flow. The column tempera-
ture was held at 150 °C during 2 min, then was increased to
300 °C at 5 °C/min, and finally held at 300 °C for 30 min. The
injection port was maintained at 200 °C. The MS detector was
a Fisons MD 800 (Foremost Equipment, Rochester, NY,
USA) set at 70 eV. This experimental step was conducted as
a first exploration of the chemical signature of C. biroi.We
used a temperature program to maximize the definition of the
part of the spectrum including CHC peaks, thus facilitating
their identification.
Origin and Preparation of Samples
We used two further independent sets of individuals to explore
different aspects of the chemical signature of C. biroi. Set 1
included foragers that show low fertility levels on average,
and intranidal workers with higher fertility on average, col-
lected at the beginning of the foraging phase from different
colonies belonging to different clonal lineages. Set 2 included
HRIs collected throughout the complete colony reproductive
cycle from different colonies from different clonal lineages.
Analyzing individuals from set 1 allowed exploration of the
CHC variability within and between colonies and clonal lin-
eages, thereby highlighting CHC-related differences on the
basis of the behavioral subcastes of individuals and their
clonal origin. Set 2 individuals were used to investigate
whether and how chemical signatures correlate with the
changes in reproductive activity related to the biphasic colony
cycle of C. biroi.
Set 1 For each colony, 80 callow LRIs, 40 foraging LRIs
and 40 HRIs were collected 5 d after the beginning of the
foraging phase without previous feeding. Since ants do not
forage and eat during the reproductive phase, which lasts
around 18 d, the collected individuals had not eaten during
at least the previous 23 d. This helped minimize any diet-
dependent influences on the chemical profiles. All collected
individuals were killed by freezing, except for 40 callow LRIs
per colony that were placed in separate nests with larvae for
one colony cycle of around 34 d. These individuals then were
collected after the beginning of the following foraging phase
for chemical analyses, and were considered as young fertile
intranidal LRIs. Thus, four morphological/behavioral subcastes
Tabl e 1 Colonies used in this
study Colony Clonal lineage Origin Field collection date Used in Set
J1 A (MLL1) Java, Indonesia 2005 1, 2
O4 A (MLL1) Okinawa, Japan 2006 1
O5 A (MLL1) Okinawa, Japan 2006 1,2
T5 A (MLL1) Taiwan 2001 1,2
O6 B (MLL4) Okinawa, Japan 2006 1
T1 B (MLL4) Taiwan 1997 1,2
T3 B (MLL4) Taiwan 2000 1,2
C10 C (MLL6) Okinawa, Japan 2008 1
C9 C (MLL6) Okinawa, Japan 2008 1
T4 D (MLL3) Taiwan 2001 1,2
JChemEcol
were analyzed for each colony: 1) five-7-d-old callow LRIs
with ovaries not yet functional, 2) intranidal fertile LRIs
(nurses), 1–1.5 mo-old, 3) foragers (reproductively inactive
LRIs older than 4 months), and 4) HRIs, fertile and at least 1
mo-old. For each of ten colonies and each of these four
categories, five groups of eight individuals were pooled and
extractedduring1hin200μl of pentane containing 10 μl/l n-
tetradecane and 15 μl/l n-tetracosane as internal standards.
Cuticular hydrocarbon components were identified on the
basis of their Retention Indices relative to those of n-alkanes
and their mass spectral fragmentation patterns.
Chemical analyses (200 samples in total) were carried out
using a Varian GC 3900 gas chromatograph equipped with a
VF-5 ms column (30 m x 0.32 mm x 0.25 μmfilmthickness;
Varian). The GC injection port was set to 220 °C and the flame
ionization detector at 300 °C. The column temperature was
held at 60 °C during 2 min, then was increased to 300 °C at
10 °C/min, and finally held at 300 °C for 10 min. Helium was
used as carrier gas at 1 ml/min.
Set 2 To evaluate possible variation during the two
phases of the colony cycle, five HRIs were collected
every third day throughout one colony cycle from six
colonies (three colonies from A, two from B, and one
for D, total of 375 HRIs). We chose HRIs to be sure
that the analyzed individuals were fertile, thus able to
lay eggs and showing, if present, fertility-related com-
ponents in their cuticular profiles. Cuticular washes
were prepared by immersing single ants for 10 min in
20 μl of a pentane solution with n-triacontane (10 ng/ml)
as internal standard. We used a lower quantity of sol-
vent because ants were extracted individually and not in
groups of eight, and in this way we increased the
concentration of CHC’s in the samples. Aliquots of each
extract (2 μl) were injected manually onto an Agilent
Technologies 7890A Gas Chromatography System con-
nected to an Agilent Technologies 5975C mass spec-
trometer. The GC column was an HP- 5MS capillary
column (30 m×250 μm, 0.25 μm thickness; Agilent),
and the oven temperature was kept at 70 °C for 1 min,
increasedat30°C/minto260°C,thenat5°C/minto
300 °C, then at 20 °C/min to 320 °C, and held for
3 min. This method was aimed at minimizing the time
of each run by focusing on the part of the spectrum
including the CHC peaks identified previously.
Statistical Analyses
Eighteen peaks corresponding to CHCs appeared in all
samples of Set 1, while only 16 peaks appeared persis-
tently in Set 2. We, thus, used 18 peaks for the statis-
tical analyses of Set 1 and 16 peaks for the statistical
analyses of Set 2 (details in Table 2and Fig. 1). The
relative concentrations of the compounds used for the
discriminant analyses were transformed into proportions
of the total, and then imported in the software PRIMER
(Clarke and Gorley 2006) with the PERMANOVA
+
add-
on package. Data were square root normalized and
transformed in matrices of Euclidean inter-individual
distances prior to statistical analyses. We used
PERMANOVA tests in order to include random factors
in our statistical design. This allowed taking the inter-
colony and inter-subcaste variability into account, while
pooling individuals for statistical tests.
Set 1 CHC profiles were analyzed using a PERMANOVA
design including three factors: Subcaste (four levels
(HRIs, callow, young and old LRIs), fixed), Clone (four
levels (A, B, C, D), fixed), and Colony (ten levels
(Table 1), random, nested within Clone). This test was
aimed at investigating the subcaste-related variability in
chemical signatures within colonies, and testing whether
differences could be found in relation to the clonal origin
of the 10 colonies we included in the study. Pvalues
were obtained using 999 permutations of residuals. In
order to test for differences in individual CHC com-
pounds among morphological and behavioral subcastes,
we conducted separate Linear Mixed Model analyses on
each CHC peak in the software STATISTICA. We im-
plemented the subcaste as fixed factor and the colony
nested within the clone as random factor.
Set 2 CHC profiles were analyzed with a
PERMANOVA design using the factors Phase (two
levels (reproductive or foraging phase), fixed), Clone
(fixed) and Colony (six levels (Table 1), random,
nested within Clone). The test was designed to inves-
tigate the influence of reproductive state of fertile
individuals (HRIs) on chemical signatures and to un-
derstand whether and what type of inter-clonal differ-
ences could be found in the chemical signatures of the
analyzed individuals. Pvalues were obtained using 999
permutations of residuals.
Chemical and Genetic Distances In order to assess any po-
tential associations between CHC profiles and genetic re-
latedness, we performed Mantel correlation tests (Mantel
1967) based on 9999 random permutations using the soft-
ware GenoDive (Meirmans and Tienderen 2004). We cor-
related matrices containing chemical Euclidean pairwise
distances between colony centroids, obtained in the soft-
ware PRIMER (Clarke and Gorley 2006) from the square-
root transformed areas of CHC peaks, and genetic dis-
tances between colonies. Euclidean genetic distances were
obtained with the software GenoDive based on 30 nuclear
microsatellite loci analyzed for 10 individuals (one for
each colony).
JChemEcol
Results
Genetic Analyses The 10 individuals (one per colony) se-
quenced for two mitochondrial DNA fragments and 30
nuclear microsatellite loci belonged to four previously de-
scribed asexual lineages from Okinawa and Taiwan (MLL1,
MLL4, MLL6, and MLL3 in Kronauer et al. 2012,referred
to as A, B, C, and D, respectively, in this study (see
Tab le 1)). Four individuals had A genotypes, three had B
genotypes, two had C genotypes, and one had the D
genotype. Based on the 17 polymorphic microsatellite loci
analyzed for ten individuals per colony, we detected a single
clonal lineage in each colony. On average, we detected 1.75
multilocus genotypes (MLGs; usually differing by a single
allele across all 17 loci) per colony for the eight colonies
belonging to clonal lineages A, B, and D. As predicted,
average pairwise relatedness within colonies was extremely
high (r=0.985 on average). Similar results have been report-
ed previously for our two colonies from clonal lineage C
(Kronauer et al. 2013). This confirms that colonies in our
Tabl e 2 The 19 cuticular hydrocarbon peaksused in the present study and their identifications using Retention Indices and mass spectral fragmentation
patterns
Peak # Retention time (min) Retention Index Identification MS Diagnostic ions
1 22.42 2300 C23 324
2 25.26 2500 C25 352
3 25.77 2534 11+13-me-C25 168, 224, 196, 351
4 26.22 2564 2-me-C25 323, 351
5 26.38 2575 3-me-C25 57, 337, 309, 351
6 26.76 2600 C26 366
7 27.27 2632 11+ 12 +13-me-C26 168, 238, 182, 224, 196, 210, 365
8 27.73 2662 2-me-C26 337, 365
9 28.34 2700 C27 380
10 28.85 2734 11+13-me-C27 168, 252, 196, 224, 379
11 29.38 2769 11,15-dime-C27 168/9, 196/7, 239, 267, 393
12 29.51 2778 4,15-dime-C27 71, 365, 239, 196, 365, 393
13 29.85 2800 C28 394
14 29.94 2806 3,11-dime-C27 182, 252, 379
15 30.34 2832 13+14-me-C28 196, 238, 210, 224, 393
16 30.76 2859 11,15-dime-C28 168, 211, 239, 281, 407
17 31.38 2900 C29 408
18 31.85 2931 13+15-me-C29 196, 252, 224, 407
19 32.27 2958 13,17-dime-C29 196, 267, 421
C23
C25
11+1 3-me -C2 5
2-me-C25
3-me-C25
C26
C28
11+12+13-me-C26
2-me-C26
C27
11+13-me-C27
3,11-dime-C27
13+14-me-C28
11,1 5-d ime-
C29
13+15-me-C29
13,17-dime-C29
23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00
1e+07
1.5e+07
2e+07
2.5e+07
3e+07
3.5e+07
4e+07
4.5e+07
5e+07
5.5e+07
6e+07
5000000
Retention time
Abundance
11,13-dime-C27
4,15-dime-C27
Fig. 1 The 19 cuticular
hydrocarbon peaks used in the
present study. We analyzed 18
peaks for Set 1, i.e. all the peaks
shown except for 3-methyl
pentacosane, and 16 peaks for Set
2, i.e. all the peaks shown except
for tricosane, 13+ 15-methyl
nonacosane and 13,17-dimethyl
nonacosane
JChemEcol
study were genetically homogeneous, and shows that the
genotype of a single individual per colony reliably repre-
sents the genetic makeup of that colony.
Chemical Analyses
Identification of CHC’sNineteen peaks were detected in GC/
MS analyses of CHC’sasshowninTable2and Fig. 1.
Several peaks contained more than one compound but
were considered as one for the analyses. We analyzed
18 peaks for Set 1, i.e., all the peaks shown except for
3-methyl pentacosane. We analyzed 16 peaks for Set 2,
i.e., all the peaks shown except for tricosane, 13+15-
methyl nonacosane, and 13,17-dimethyl nonacosane.
Set 1 We found significant differences between experimental
groups for all the factors included in our PERMANOVA
analysis, and for their interactions (Table 3a). Interestingly,
we found significant differences among C. biroi subcastes
(PERMANOVA, Pseudo F=4.574, df=3, P=0.001, Fig. 2a).
A subsequent pairwise PERMANOVA revealed that signifi-
cant differences were present exclusively between callow
LRIs and HRIs, between callow LRIs and young LRIs, and
between callow LRIs and old LRIs (P=0.001, P=0.004 and
P=0.015, respectively). This suggests that callow LRIs bear
chemical signatures distinct from all the other groups, while
the remaining three groups are identical in their CHC profiles.
These results are supported by an additional Linear Mixed
Model analysis in which we compared the four morphological
and behavioral subcastes for each individual CHC peak
(Table 4).
We also found significant differences between colonies of
different clonal lineages (PERMANOVA, Pseudo F=
5.1442, df=3, P=0.009, Fig. 2b, Table 3a). While clones A,
B, and D were not significantly different from one another
(pairwise PERMANOVA, all P> 0.05), clone C was signifi-
cantly different from clones A, B, and D (pairwise
PERMANOVA, all P< 0.001). A positive correlation was
found between chemical and genetic distances between colo-
nies (Mantel test, r=0.58, P< 0.001). Within clonal lineages,
the analyses including the factor subcaste and the inter-
action between the factors subcaste and colony revealed
significant differences between experimental groups for
all clones (Table 3b).
Set 2 We found significant differences between experimental
groups for some of the factors included in the PERMANOVA
analysis, and for some of their interactions (Table 3c). No
significant differences were found between the chemical signa-
tures of fertile individuals in different phases of the colony
cycle, i.e., there was no influence of reproductive status
(activity or inactivity) on chemical signatures (PERMANOVA,
Pseudo F=0.1.9648, df =1, P=0.234, Fig. 2c,Table3c). No
differences among groups were found for the interaction be-
tween the clonal lineage and the phase of the colony cycle
(PERMANOVA, Pseudo F =1.9648, df=1, P=0.198,
Tabl e 3 Results of the PERMANOVA analyses on the cuticular signa-
tures of individuals. (*: P<0.05; **: P<0.01; ***: P<0.001; NS:not
significant). Random factors are given in italics (a). Set 1, comparisons
among subcastes and clones; (b). Set 1, comparisons among colonies
within the same clone; (c). Set 2, comparisons of the HRI signatures
between the reproductive and foraging phase; (d). Set 2, comparisons
among groups of HRIs collected every third day throughout a colony
cycle; (e). Set 2, comparisons among clones)
df Pseudo-FSignificance
a.
subcaste 3 4.574 ***
clone 3 5.1442 **
colony(clone) 6 47.357 ***
subcaste*clone 9 2.3327 **
colony(clone)*subcaste 18 11.498 ***
b. Clone A
colony 3 46.202 ***
subcaste 3 74.685 ***
colony*subcaste 9 15.581 ***
Clone B
colony 2 63.55 ***
subcaste 3 28.954 ***
colony*subcaste 6 10.554 ***
Clone C
colony 1 3.4732 **
subcaste 3 27.464 ***
colony*subcaste 3 5.3728 ***
c.
clone 213.655 *
phase 1 1.9648 NS
colony(clone) 3 20.694 ***
clone*phase 2 2.1743 NS
phase*colony(clone) 3 3.7517 **
d.
clone 212.963 *
collection day 11 0.85237 NS
colony(clone) 3 26.968 ***
clone*collection day 22 0.95215 NS
Collection day*colony(clone)333.177 ***
e.
clone 2 13.655 *
phase 1 1.9648 NS
colony(clone) 3 20.694 ***
clone*phase 2 2.1743 NS
phase*colony(clone) 3 3.7517 **
JChemEcol
Tab le 3c). In addition, no significant differences were found
between cuticular signatures among any of the groups of HRIs
collected every third day during a complete colony cycle
(PERMANOVA, random factor: colony nested in clones; fixed
factors: clones and group of HRIs collected the same day;
Pseudo F=0.8524, df =11, P= 0.6; complete results are shown
in Table 3d; pairwise PERMANOVA, all P>0.089). The dif-
ferent clones showed significant differences in their chemical
signature (PERMANOVA, Pseudo F =13.655, df=1,
P=0.023, Fig. 2d; complete results in Table 3e). How-
ever, the correlation between genetic and chemical
distances was marginally non-significant (Mantel test,
9999 permutations, r=0.7, P=0.064).
Discussion
Within-Colony CHC Variability Our chemical analyses re-
vealed a high degree of homogeneity in cuticular signatures
within C. biroi colonies, with only callow LRIs being different
from all the other groups we considered. However, this find-
ing is probably due to the fact that often in social insects the
cuticular profile of recently eclosed individuals is not
completely matured, and as a result they are chemically dif-
ferent from older nestmates (Breed et al. 2004;Ichinoseand
Lenoir 2009; Teseo et al. 2013).
In many species of social Hymenoptera, CHC profiles signal
fertility and/or reproductive activity (see Monnin 2006),
which allows non-reproductive individuals to perceive the
presence of reproductives and refrain from egg-laying
(Holman et al. 2010; Le Conte and Hefetz 2008; Slessor
et al. 2005; ) ensuring a balance between reproductive and
ergonomic colony function. In some ant species where all
nestmates are able to mate and produce female offspring,
fertility-related cuticular hydrocarbons serve to maintain
colony-level reproductive dominance hierarchies (Cuvillier-
Hot et al. 2004;Heinzeetal.2002;Monnin2006;Monnin
et al. 1998;MonninandPeeters1999; Peeters et al. 1999). In
the ponerine ant Platythyrea punctata, where individuals
are able to produce female brood asexually, fertility and
dominance signaling via cuticular signatures maintains a
single reproductive individual per colony (Hartmann et al.
2003,2005). Cerapachys biroi is different in its colony-
level reproductive dynamics and social structure, in that all
individuals can reproduce (Ravary and Jaisson 2002), and
there are no reproductive dominance hierarchies. Cuticular
signatures, thus, are not expected to bear fertility signals
related to dominance or inducing nestmates to refrain from
laying eggs. It seems improbable that the colony-level
regulation of reproduction relies on cuticular signals relat-
ed to individual fertility or reproductive activity. However,
HRI production in C. biroi varies depending on the aver-
age colony-level fertility, implying the existence of some
Fig. 2 Principal Coordinates Analyses on CHC data Sets 1 and 2 (a) and (b) samples of Set 1 labeled by subcaste and by clone, respectively; (c) and (d)
samples of Set 2 labeled by clone and phase of colony cycle, respectively
JChemEcol
Tabl e 4 Set 1. Post hoc comparisons among subcastes for each individ-
ual CHC peak (Linear Mixed Model, Fisher’s LSD test; *: P< 0.05; **:
P<0.01; ***: P<0.001). The peak of 13+ 15-methyl nonacosane was
excluded because it did not show significant changes in the overall
comparison. LRI =Low reproductive individuals; HRI=high reproduc-
tive individuals
Callow LRIs HRIs Foragers Nurses Callow LRIs HRIs Foragers Nurses
1. Tricosane 2. Pentacosane
Callow LRIs - *** * *** Callow LRIs - *** *** *
HRIs - - 0.060 0.833 HRIs - - 0.933 ***
Foragers - - - 0.095 Foragers - - - ***
Nurses - - - - Nurses - - - -
3. 11+13-Methyl pentacosane 4. 2-Methyl pentacosane
Callow LRIs - *** *** *** Callow LRIs - ** *** *
HRIs - - *** *** HRIs - - 0.054 0.464
Foragers - - - 0.193 Foragers - - - **
Nurses - - - Nurses - - - -
5. Hexacosane 6. 10+ 12-Methyl hexacosane
Callow LRIs - *** *** *** Callow LRIs - *** *** ***
HRIs - - 0.684 0.229 HRIs - - 0.298 0.276
Foragers - - - 0.421 Foragers - - - *
Nurses - - - - Nurses - - - -
7. 2-Methyl hexacosane 8.Heptacosane
Callow LRIs - *** *** *** Callow LRIs - *** *** ***
HRIs - - ** 0.780 HRIs - - 0.208 *
Foragers - - - *** Foragers - - - ***
Nurses - - - - Nurses - - - -
9. 11+13-Methylheptacosane 10. 11+15 Methylheptacosane
Callow LRIs - 0.645 ** * Callow LRIs - *** *** ***
HRIs - - * 0.098 HRIs - - 0.599 *
Foragers - - - 0.359 Foragers - - - **
Nurses - - - - Nurses - - - -
11. 5, 11-Dimethylheptacosane 12. Octacosane
Callow LRIs - *** 0.051 * Callow LRIs - ** *** *
HRIs - - * * HRIs - - 0.085 0.615
Foragers - - - 0.559 Foragers - - - *
Nurses - - - - Nurses - - - -
13. 10-Methyloctacosane 14. 12-Methyloctacosane
Callow LRIs - 0.483 0.842 * Callow LRIs - *** *** ***
HRIs - - 0.615 0.066 HRIs - - 0.065 0.572
Foragers - - - * Foragers - - - 0.200
Nurses - - - - Nurses - - - -
15. 14+16-Methyl octacosane 16. Nonacosane
Callow LRIs - *** *** *** Callow LRIs - ** *** ***
HRIs - - 0.521 0.436 HRIs - - 0.070 0.160
Foragers - - - 0.155 Foragers - - - 0.688
Nurses - - - - Nurses - - - -
17. 13,17-Dimethyl nonacosane
Callow LRIs - *** *** ***
HRIs - - * 0.103
Foragers - - - 0.437
Nurses - - - -
JChemEcol
regulation acting on larval fate (Lecoutey et al. 2011). The
lack of correlation between fertility levels and cuticular
signatures suggests that the regulation of HRI development
is either non-chemical or does not depend on signals
derived from CHCs. One possibility is that a non-CHC,
non-volatile chemical signal that is present on the cuticular
surface or secreted from other glandular sources is in-
volved. Moreover, the quality of the food that may be
admixed with some glandular products of adults can direct
larval development toward different pathways, which un-
derlies caste determination in many social Hymenoptera
(Hölldobler and Wilson 1990;Wheeler1986,1991). Prim-
er pheromones transmitted from adults to larvae via direct
contact during parental care might play a complementary
role in caste differentiation. Indeed, workers regularly per-
form a peculiar behavior during brood care that consists of
licking the developing larvae ventrally under the head
capsule (Lecoutey, personal observations). Further studies
on the mandibular secretions of nurses are needed to
investigate whether they play a role in larval differentia-
tion in C. biroi.
Subcaste differentiation might also exclusively rely on the
quantity of food available to larvae. Quantitative differences
in food intake during pre-imaginal stages have major effects
on development in insects, and give rise not only to differ-
ences in adult size, but also differential expression of adult
polyphenisms (Emlen 1994; Hunt and Simmons 1997;
Moczek and Emlen 2000). Adult C. biroi could quantitatively
limit larval feeding in several ways, e.g., by actively keeping
larvae away from prey items within the nest, or simply by
competing with larvae for food. HRI production is inversely
proportional to the proportion of fertile individuals in a colo-
ny, and fertile individuals might need a higher quantityof food
in order to produce eggs. Thus, the more fertile individuals are
present in a colony, the less food might be available for larvae,
which possibly limits HRI production. Other types of influ-
ences on larval fate, such as mechanical stress on developing
larvae due to biting from adults (Brian 1973; Pennick and
Liebig 2012), also could be involved. Observations on the
behavior of adult individuals towards larvae during the forag-
ing phase will clarify the proximate factors determining sub-
caste differentiation in C. biroi.
CHC Variability Among Clones Our study shows that the
colony-level chemical signatures of C. biroi vary according
to the clonal lineage, with chemical distances between colo-
nies growing with genetic distances. The fact that we observed
only a marginally non-significant correlation between genetic
and chemical distances for colonies of Set 2 was probably due
to the low number of colonies included in that analysis (N=6).
Colonies belonging to the same clonal lineage also show some
variability in their cuticular signature, even though the chem-
ical distances among those are lower than distances among
colonies from different clones. Overall, our findings support
the results of a previous study on the invasive C. biroi popu-
lation in Okinawa, where individuals were able to discrimi-
nate between nestmates and non-nestmates, especially when
non-nestmates belonged to unrelated asexual lineages
(Kronauer et al. 2013). According to our results, chemical
signatures might be the proximate cues indicating genetic
dissimilarity between interacting individuals, which in turn
might prevent fusions between unrelated colonies. This might
be a reason that explains why natural C. biroi colonies have
been found to be exclusively monoclonal (Kronauer et al.
2013). However, given that the putative native range of
C. biroi remains still largely unexplored, it cannot be excluded
that in natural populations different clones mix in chimeric
colonies.
Invasive populations of ants are likely to originate via the
introduction of few individuals, i.e., population bottlenecks
which most of the time produce a strong founder effect
(Tsutsui and Suarez 2003). As a result, invasive populations
are overall genetically less diverse than native populations.
This can result in individuals from different colonies
displaying similar cuticular hydrocarbon profiles, potentially
leading to a loss of aggression even between non-nestmates
(Tsutsui and Suarez 2003). This loss of aggression is thought
to promote the formation of supercolonies (Giraud et al.
2002). For example, many invasive species forming
supercolonies exhibit negligible levels of between-colony ag-
gression, even between individuals taken from nests separated
by several kilometers (Blight et al. 2012;Drescheretal.2010;
Giraud et al. 2002). Cerapachys biroi is invasive (Kronauer
et al. 2012,2013; Wetterer et al. 2012), but to our knowledge it
does not form supercolonies. This might in part be due to the
maintenance of non-nestmate discrimination between colo-
nies from different clonal lineages (Kronauer et al. 2013).
Acknowledgments We thank Paul Devienne and Marjorie Labédan for
technical support with ant colonies at LEEC, and Chloé Leroy for
technical assistance with gas chromatography–mass spectrometry. This
work was supported by a Ph.D. research grant from the French Ministry
of Research to S.T., as well as a Junior Fellowship from the Harvard
Society of Fellows and a Milton Fund Award to D.J.C.K. All coauthors
have agreed on the contents of the manuscript.
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