Pre-existing differences in putative fertility signals give workers the
upper hand in ant reproductive hierarchies
, Nicolas Ch^
e Paris Est Cr
e Paris Diderot, CNRS, INRA, IRD, Institute of Ecology and Environmental ScienceseParis, IEES-
Paris, Paris, France
erimentale et Compar
e Paris 13, Villetaneuse, France
orio de Etologia Ecologia e Evoluç~
ao Dos Insetos Sociais, Departamento de Psicologia Experimental, Instituto de Psicologia, Universidade de S~
Received 22 January 2019
Initial acceptance 26 February 2019
Final acceptance 2 August 2019
MS. number: 19-00054R
In social groups, competition often gives rise to conﬂicts, which are regulated through a variety of
mechanisms. In several social insect species, the conﬂict for male production that takes place between
workers after queen loss, is regulated through the establishment of a reproductive hierarchy. A recent
study of Neoponera apicalis showed that workers differ in their fertility levels in the presence of the
queen and proposed that such idiosyncratic differences might inﬂuence access to the top of the hierarchy
after queen loss. In this study, we therefore sought to characterize the inﬂuence of the initial hetero-
geneity in ovarian development and its chemical and behavioural correlates on the establishment of
reproductive hierarchies among orphaned workers, which can only produce males. We monitored the
chemical proﬁle before and after hierarchy establishment in four groups of orphaned workers of
N. apicalis morph 6. The analysis of the cuticular proﬁles showed that tricosane (n-C
) was highly
correlated with ovarian development and could consequently act as a fertility signal in this ant. The
relative amount of tricosane on the cuticle, both before and after the establishment of the hierarchy, was
also correlated with the rank achieved within the hierarchy and with the expression of agonistic be-
haviours. Thus, our study experimentally shows that idiosyncratic differences in a putative fertility signal
(and therefore presumably in ovarian activity) between workers in the queen's presence reliably predict
the outcome of reproductive conﬂict after queen loss. We propose that this signal (together with an
increased agonistic motivation of the more fertile workers) could play a major role in the regulation of
dominance/submission behaviours, enabling the most fertile individuals to rapidly access top ranks and
monopolize reproduction, thereby maximizing the global reproductive success of all colony workers
while minimizing the costs associated with the expression of agonistic behaviour.
©2019 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Reproductive hierarchies often appear in social hymenopteran
species when the queen of the colony disappears or her repro-
ductive potential decreases. In most species, workers, although
they cannot mate, maintain an ability to develop their ovaries and
lay unfertilized male-destined eggs (Yagound, 2014). In these spe-
cies, the establishment of reproductive hierarchies through ritual-
ized agonistic interactions regulates the overt conﬂict for male
production (Heinze, Holldobler, &Peeters, 1994; Oliveira &
olldobler, 1990). An individual's rank stems from several factors
that are classically described as ‘intrinsic’and ‘extrinsic’and which
are intertwined in a network of feedback loops. Intrinsic traits refer
to the state of each individual (e.g. neuroendocrine titres, repro-
ductive status and motivation to ﬁght, as well as potential chemical
cuticular correlates) that determine its absolute ﬁghting ability (so-
called resource-holding power, or RHP, after Parker, 1974). Extrinsic
factors that play a role in establishing hierarchies include the effects
of past experiences (Rutte, Taborsky, &Brinkhof, 2006) and social
environment whose causal role is exerted through the modiﬁcation
of intrinsic factors, which in turn modify future experiences. These
inﬂuences are notably reﬂected in winnereloser effects where the
outcome of an encounter (victory or defeat) induces changes in the
neuroendocrine titres (Hsu, Earley, &Wolf, 2006), thus inﬂuencing
individual behaviour and the outcome of future encounters
(Dugatkin &Earley, 2004; Sasaki et al., 2016).
In ants, cuticular hydrocarbons (CHCs) are well known for indi-
cating colonial afﬁliation, but they also convey more subtle social
*Correspondence: R. Honorio, Sorbonne Universit
e Paris Est Cr
e Paris Diderot, CNRS, INRA, IRD, Institute of Ecology and Environmental
ScienceseParis, iEES-Paris, 75005, Paris, France.
E-mail address: email@example.com (R. Honorio).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/anbehav
0003-3472/©2019 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Animal Behaviour 157 (2019) 129e140
information about species, sex, caste, hierarchical status and repro-
ductive status (Greene &Gordon, 2003; Liebig, 2010), thus consti-
tuting unique individual chemical proﬁles. This chemical signal
results from quantitative or qualitative differences (or both) of
endogenousand exogenousorigins between one or morecompounds
across individuals, castes and colonies (d’Ettorre &Lenoir, 2010). The
signal can therefore allow the recognition of a congener's idiosyn-
cratic characteristics, and many studies have demonstrated the
involvement of CHCs in fertility and/or dominance signalling (Abril
et al., 2018; Holman et al., 2013, 2016; Smith et al., 2009, 2015). The
perception of the signal modiﬁes the behaviour of potential partners
(reviewed in Leonhardt, Menzel, Nehring, &Schmitt, 2016). When it
reﬂects RHP and/or fertility, the signal is thought to be honest, and
workers are accordingly expected to follow their own interests in
response to the signal and promote their inclusive ﬁtness (Keller &
Nonacs, 1993;Heinze &D’Ettorre, 2009). Thus, in the case of repro-
ductive hierarchies in a queenless colony, the most fertile worker
should be selected to access the top of the hierarchy and produce
males. Selection of the reproductive individuals stems from a ﬁne
balance between direct ﬁtness costs, indirect ﬁtness beneﬁts and
relatedness (Keller &Nonacs, 1993). Yagound, Blacher, Fresneau,
Poteaux, and Ch^
aline (2014) have shown that workers of a Neotrop-
ical ant species, Neoponera apica lis, can use CHCs as an index of rank in
workers' established reproductivehierarchies, the quantityof certain
compounds functioning as a reliable signal of both individual ovarian
development and social status.
Here, we studied the establishment of reproductive hierarchies
after queen loss in workers of N. apicalis. In this species, workers
cannot mate and therefore are unable to produce female progeny
(Fresneau, 1994). While queenright workers do not usually lay eggs,
they start producing males soon after being orphaned (Dietemann
&Peeters, 2000). Because there is no production of new workers,
which would care for the brood or adopt a new queen, males must
be produced rapidly after the queen's death or no nurses will be
available. Behavioural mechanisms exploiting interindividual dif-
ferences in queen presence for both reproductive physiology and
chemical signalling (Yagound et al., 2015) would allow the rapid
establishment of a reproductive hierarchy after queen loss and
hence meet the evolutionary pressure to rapidly solve the conﬂict
between workers over male production (Dietemann &Peeters,
2000). Namely, we propose that the most fertile workers could be
more motivated to enter the reproductive race, and that fertility
signalling could help resolve the ritualized agonistic encounters.
Such behavioural processes would ensure the most fertile workers
lead the hierarchy, thereby maximizing the inclusive ﬁtness of the
whole worker collective (Hamilton, 1964; Keller &Nonacs, 1993).
To test our hypothesis, we ﬁrst correlated variation in cuticular
proﬁles and ovarian development among workers to determine
which compound might be the putative fertility signal in N. apicalis
(Liebig, Peeters, Oldham, Markstadter, &Holldobler, 2000; Monnin,
Malusse, &Peeters, 1998; Yagound et al., 2015). We then jointly
monitored the development of this compound (as a noninvasive
proxy for ovarian development) and of ritualized agonistic behav-
iours by workers, from queen loss to the stabilization of the
reproductive hierarchy. We predicted that the workers most fertile
in the presence of the queen would be more active during the
establishment of the hierarchy and therefore would access the top
ranks and monopolize reproduction.
Neoponera apicalis is a common ant species in central-south
American tropical forests. We obtained collection permits (No
47615) from the Chico Mendes Institute for Conservation and
Biodiversity (ICMBio/SISBIO) from the Brazilian Ministry of the
Environment (MMA). Our experimental design in the laboratory
included the orphaning of four experimental groups of workers, the
labelling and behavioural observation of individual ant workers,
the monitoring of the cuticular compounds and the dissection of
workers to record ovary development. Ants were kept in artiﬁcial
nests which are commonly used in ant research and in which ants
do not show abnormal or stereotypical behaviour. The whole range
of expected behaviour was observed. Ants were manipulated with
soft forceps, which prevent any damage, and marked with paint,
which does not alter their behaviour in the long term. Ants were
killed by freezing before dissection. All these procedures were
conducted following the institutional guidelines of animal welfare
of both Brazil and France.
We collected 18 colonies of N. apicalis in Brazil in November
2016: eight queenless and six with fewer than 20 workers (1e56
workers, mean 22.1 workers per queenright colony, SD 17.5). The
fact that eight colonies were queenless thus suggests that queen-
lessness is common in this species. Comparison of hierarchy
establishment in the N. apicalis species complex showed that it
occurs earlier, and agonistic behaviour is more pronounced, in
monogynous species (Yagound, 2014). This suggests that an
increased chance of queenlessness selects for hierarchy establish-
ment mechanisms allowing quick conﬂict resolution. In this study,
we used four colonies: colony 1 was collected in Marituba, state of
W), and colonies 8, 18 and 20 in Santa
Barbara do Para (1
W). Cytochrome C oxidase I
sequence analysis revealed that our colonies belong to morph 6 of
N. apicalis (Yagound, Savarit, Fichaux, Poteaux, &Ch^
aline, n. d.).
Neoponera apicalis was divided into three morphospecies by
Delabie, Mariano, Mendes, Pompolo, and Fresneau (2008) based on
ﬁne morphological differences in this complex of cryptic species.
Ferreira, Poteaux, Delabie, Fresneau, and Rybak (2010) deﬁned
three additional morphs based on a set of morphological, acoustic,
chemical and genetic data. Yagound (2014) added a seventh morph.
Mackay and Mackay (2010) described morph 5 as Neoponera cooki,
but the original numeration is kept in order to be consistent. Col-
onies were harvested in mid-October 2016 and installed in the
laboratory a week later. The experiment started 2 months after
their installation. During this acclimation period, workers remained
with the queen. The ants were housed in plaster nests (18 x 14 cm)
connected to an external environment of the same size. They were
maintained at a temperature of 25 ±2
C, a relative humidity of
50 ±10% and a day:night cycle of 12:12 h. Each colony was fed
three times a week with an appleehoney mixture and thawed
crickets (Acheta domestica), as well as water ad libitum.
Based on the study by Yagound, Blacher, Chameron, and Ch^
(2012), which showed that workers close to the queen were the
ﬁrst to reproduce at the onset of hierarchical competition, we
assumed that the most fertile workers would stay next to the queen
within the nest. We selected and individually marked 20 workers in
the vicinity of the queen in each experimental colony. Each of these
workers received a number label glued on the thorax and two
coloured dots (Uni-ball marker). The chemical proﬁle of ants before
orphaning was extracted using SPME (see below). The individuals
were then released into the original nest. The following week, 15 of
these 20 selected workers were isolated and placed in another
artiﬁcial nest of the same type, to mimic an orphaning process. We
recorded agonistic interactions, that is, antennal boxing and bites,
in the nest (see Dominance Hierarchy below). Antennal boxing
consists of repeated and rapid strokes of one ant by another with
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140130
the antennae. This behaviour is typical of many ant species and is
often observed during the establishment of hierarchies. In estab-
lished hierarchies, dominant individuals perform antennal boxing
against subordinate individuals (Blacher, Lecoutey, Fresneau, &
Nowbahari, 2010; Monnin &Peeters, 1999; Yagound et al., 2014).
Biting occurs when the individual uses its mandibles to grip a part
of another individual's body. In most instances, biting was pro-
longed, thereby immobilizing the other individual with no
apparent damage or cuts, and we consider this behaviour ritualized
biting. On the 10th day of the experiment, workers were frozen for
later extraction of their chemical proﬁle after orphaning and
measurements of their ovarian development (Fig. 1). Of the initial
60 ants, 59 survived to this stage.
Extraction and Analysis of Chemical Proﬁles
The individuals’chemical proﬁles were analysed before they
were orphaned and after the establishment of the reproductive
hierarchy. The initial chemical proﬁle (before orphaning) was ob-
tained by solid-phase microextraction (Monnin et al., 1998). This
involved rubbing an SPME ﬁbre (polydimethylsiloxane 100
the ﬁrst segment of the abdomen for 2 min. The ﬁbre was then
desorbed in a Varian 3900 gas chromatograph with ﬂame ioniza-
tion detection (GC-FID). The carrier gas used was helium at 1 ml/
min, with hydrogen streams at 30 ml/min and air at 300 ml/min.
The programme was as follows: the initial temperature was 70
for 1 min, then it rose from 40
C/min for 4 min to 250
increased by 1
C/min for 8 min to 258
C and ﬁnally increased
C/min to 320
C and stabilized at 320
C for 3 min. The
temperature of the injector was maintained at 280
C and that of
the FID at 340
C. Proﬁles were extracted with the Varian system
control software Star Chromatography workstation version 6.2
(Varian, Palo Alto, CA, U.S.A.). The compounds were identiﬁed
based on their retention time (Appendix Table A1), comparing
them to standard hydrocarbons already identiﬁed in a gas chro-
matograph coupled to a mass spectrometer (GC-MS) as well as
chromatograms of Yagound (2014) for N. apicalis morph 6. The
advantage of this method was that it was not invasive; however, it
was time consuming and did not allow quantiﬁcation of
Owing to time constraints, the chemical proﬁle after orphaning
was obtained by a liquid phase extraction. The head and thorax of
each dissected ant were soaked in 200
l of pentane containing
l of compound n-C
(representing our internal standard) in a
vial tube for 5 min. The tube was then left to evaporate. After the
solution was completely evaporated, the sample was analysed by
GCeMS (Agilent A7890), by injecting 2
l of the extract
resuspended in 80
l of solvent (pentane), with electron impact
ionization at 70 eV. The carrier gas was helium at 1 ml/min. The
same analysis programme as above was applied. The chemical
proﬁles were integrated using the MSD ChemStation software
version E.02.01.1177 (Agilent Technologies Inc., Santa Clara, CA,
U.S.A.). The compounds were identiﬁed by comparing their reten-
tion time and spectra with already known compounds. The internal
standard allowed us to translate peak areas to absolute quantities
for the related compounds.
Antennal boxing and bites were recorded, together with the
identities of the interacting ants. The loser was the ant showing
submissive behaviour, that is, hunching or dodging. Twelve obser-
vation sessions were carried out (15 h total) per colony: two of 1.5 h
on the ﬁrst, second and third days after being orphaned, then two
of 1 h on the fourth and ﬁfth days and ﬁnally one of 1 h on the
eighth and 10th days. During these sessions, all boxing and bites
were recorded. Observations were made under red light to avoid
biasing the ants’behaviour in the interior of the nest (Depick
Fresneau, &Deneubourg, 2004).
The hierarchical rank of each worker in the orphaned colonies
was obtained using the ‘Glicko-rating’method, which is a dynamic
matched comparison model that calculates a score for each indi-
vidual, based on the outcome of each individual's interactions (vic-
tory or defeat; Glickman, 1999). From this score, a ranking can be
determined to deduce the hierarchy. The Glicko-rating algorithm
includes a positive constant ‘c’, which governs the size of the stan-
dard deviation over time. This constant is deﬁned by the user, an
increased value of ‘c’leading to a greater average deviation per in-
dividual over time. In our study, following the guidelines of
Glickman (1999; and see So, Franks, Lim, &Curley, 2015), we used a
value of 1 for ‘c’. We checked the impact of the ‘c’value on our results
by replicating the calculation over a range of 1e10. We obtained
similar results for the hierarchical rankings over the whole range.
Glicko-rating calculations were performed with the PlayerRatings
package v1.0 (Stephenson &Sonas, 2014) in R 3.4.1 (R Development
Core Team, 2017). Data were compiled in chronological order of
dyadic interactions. The same coefﬁcient was attributed to antennal
boxes and bites, so that in the calculation of the hierarchy the two
types of agonistic behaviours had the same power.
With a graduated 10binocular microscope, we measured the
ovarian development of the ants. The length of the three basal
Marking + initial profile
Final profile extraction
(GC-MS) + dissection
D1 D2 D3 D4 D5 D8 D10
Figure 1. Timeline of the experiment. The queen was removed from each colony 7 days after individuals were marked and the experiment ran for 10 days (D1 - D10). ‘Initial proﬁle’
corresponds to the workers' chemical proﬁle in the presence of the queen (determined by ﬂame ionization detection, GCeFID); ‘ﬁnal proﬁle’corresponds to the chemical proﬁle at
the end of the experiment (determined by gas chromatographyemass spectrometry, GCeMS). Agonistic interactions (antennal boxing and bite) were used to calculate the hier-
archical rank of the 15 orphaned individuals per colony.
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140 131
oocytes of the ovarioles of each ovary was measured. A fertility
index was calculated by summing the lengths of the six basal oo-
cytes (Yagound et al., 2014). We present this below as mean ±SD.
Establishment of the hierarchy
The distribution of the average number of agonistic interactions
per hour of observation was compared between colonies to
compare the dynamics of hierarchy establishment. For this, a two-
sample KolmogoroveSmirnov test was performed between each
pair of the four colonies. To compensate for multiple comparisons, P
values were then adjusted to P’values following Holm (1979).
The linearity ‘h’within our four colonies was calculated between
the 15 orphaned workers by the de Vries (1995) method using
software R (package compete, Curley, 2016).
To verify whether worker isolation led to the establishment of a
reproductive hierarchy, we investigated the link between the hi-
erarchical rank and fertility of individuals using Spearman corre-
lations for the 59 orphaned ants dissected at the end of the
Chemical data analysis
Although some intercolonial heterogeneity is expected in the
proportion of each compound in the cuticular proﬁles, a principal
coordinate analysis (PCo) and an analysis of similarity (ANOSIM)
were performed to verify whether our experimental colonies (59
workers) shared a similar chemotype, due tothe potential presence
of cryptic morphs, differing in chemical proﬁles. For this, we used
the PERMANOVAþfor PRIMER software (Anderson, Gorley, &
Clarke, 2008) using a Euclidean distance matrix calculated on
Using two different methods to extract the chemical proﬁles
was a potential source of methodological variability. To verify
whether our methods were reliable, we used Spearman correla-
tions to compare the proﬁles before and after orphaning using the
proportions of the major compounds, namely n-C
with a BonferronieHolm adjustment for multiple tests on the same
data set (Holm, 1979). Signiﬁcant correlations would indicate reli-
ability of the two methods (even if distinct methods can generate a
slight chemical distance between the proﬁles before and after
orphaning). This calculation could be done only for three colonies
(see Results for details). In addition, two individuals from colony 20
could not be included in these chemical analyses because of a
technical problem when acquiring the proﬁle before orphaning
(missing data). In colony 20 there were only 14 individuals because
a worker died during the experiment. We thus analysed a total of 42
workers. For these three colonies we also performed a Mantel test
(package ecodist in R) between the Euclidean distance matrix of the
square-root-transformed percentages of the chemical proﬁles
before and after orphaning to evaluate global concordance between
the two methods.
To identify the cuticular compound(s) potentially acting as a
fertility signal in our study, we used Spearman rank correlation (on
44 workers) to assess the relationship between the fertility index
measured at the end of the experiment and the ﬁnal quantities of
cuticular compounds. Once the putative fertility signal was iden-
tiﬁed, we also checked for correlations with the proportions before
orphaning to verify pre-existing heterogeneity between the
workers in the presence of the queen (42 workers). Pvalues were
adjusted to account for multiple testing of the same data (Holm,
Last, we investigated the presence of a correlation between the
putative fertility signal and the observed behaviour using
Spearman correlation. The deviation from the mean quantity of
fertility-related compound(s) (within each colony) was correlated
with the different behaviours expressed by each individual (42
workers). Using a mean deviation index allowed us to buffer the
effect of intercolonial variation in the quantity of compound.
Setting up Reproductive Hierarchies
Dynamics of agonistic behaviours
Despite some variability among colonies in the intensity of
agonistic displays (Fig. 2), the dynamics of agonistic behaviour did
not differ signiﬁcantly between them. No two by two comparison
between colonies was signiﬁcant (KolmogoroveSmirnov test with
BonferronieHolm correction: colony 1e8: D¼0.571, P’¼0.203;
colony 1e18: D¼0.571, P’¼0.203; colony 1e20: D¼0.571,
P’¼0.212; colony 8e18: D¼0.571, P’¼0.203; colony 8e20:
D¼0.571, P’¼0.203; colony 18e20: D¼0.429, P’¼0.575).
Agonistic interactions rose rapidly within the ﬁrst 2 days of being
orphaned and then returned to basal level.
Establishment of hierarchies
Hierarchies established in colonies 8, 18 and 20 had linearities of
h'¼0.52 (P¼0.001), h'¼0.66 (P<0.001) and h'¼0.65 (P<0.001),
per hour of observation
Figure 2. Number of agonistic interactions per hour of observation as a function of observation day during the 10 days after the queen was removed.
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140132
respectively. The hierarchy in colony 1 did not show a signiﬁcant
linearity (h'¼0.19, P¼0.48).
Rank and ovarian development
All but 10 of our 59 workers had activated ovaries. The average
ovarian development, measured at the end of the experiment, was
highest for colony 18 (4.16 ±2.59 mm), followed by colony 8
(3.72 ±3.10 mm), colony 1 (3.42 ±2.25 mm) and ﬁnally colony 20
(2.97 ±2.06 mm). Within each colony the fertility index of workers
was signiﬁcantly correlated with their hierarchical rank from the
ﬁrst day of being orphaned (Table 1).
Chemical Proﬁles, Fertility and Behaviours
Chemical analyses of proﬁles after orphaning
The chemical proﬁles were grouped by colony in the PCo and
with the ANOSIM, suggesting the existence of a characteristic
colonial signature (Appendix Fig. A1). Considering the average
chemical distance calculated between colonies, colony 18 was very
different from the others (0.75 on average with colony 18, against
0.20 between the other three colonies; Appendix Table A2).
The cuticular proﬁle of each ant was composed of 28e30 peaks
and included several series of n-alkanes, branched mono and
dimethyl-alkanes and alkenes, with carbon atom numbers ranging
from 19 to 33. The majority of compounds were linear alkanes and
alkenes. Consistent with the chemical distance results, colonies 1, 8
and 20 displayed a qualitatively distinct chemotype from colony 18
(Appendix Fig. A2). Colony 18 was thus excluded from correlation
analysis with the chemical proﬁles. The chemical proﬁles of the
workers from colony 18 were heterogeneous, some appearing
separated and others represented among the other colonies’pro-
ﬁles (Appendix Fig. A1). As several morphs of N. apicalis occur in the
collection area, this unusual result could thus be a consequence of a
chance hybridization between two morphs (i.e. a male from
another morph), which cannot be detected using nuclear DNA.
Although interesting, we have no additional means to explain this
Reliability between the two chemical extraction methods
The proportions of the three major compounds (n-C
) in the SPME samples analysed by GCeFID before orphaning
were signiﬁcantly correlated with those analysed by GCeMS after
orphaning (Table 2). The Mantel test between the two chemical
proﬁle matrices before and after orphaning were signiﬁcantly
correlated (P<0.001) with a Mantel value of 0.70, which, consid-
ering the potential variation due to fertility and environmental
changes between the two analyses, is sufﬁcient to validate the use
of the two methods. Initial and ﬁnal n-C
proportions were also
correlated (Spearman correlation: r
Correlation between chemical proﬁles and fertility
The amount of the alkane tricosane (n-C
) after orphaning was
signiﬁcantly correlated with the fertility index for each individual
(Spearman correlation: r
¼0.63, N¼44, P<0.001; Fig. 3a), as was
the proportion of n-C
in the chemical proﬁle before orphaning
(Spearman correlation: r
¼0.69, N¼42, P<0.001; Fig. 3b). This
compound was the component of the chemical proﬁle that best
correlated with ovarian development. Correlation values of the
other compounds are presented in Appendix Table A3.
Fertility and behaviour
We focused on n-C
which was the best correlated compound
with fertility. Both the number of ﬁghts won by an ant and the
number of interactions it was involved in were signiﬁcantly
correlated with the quantity of n-C
after orphaning (Table 3). The
same results were obtained when considering only the ﬁrst 2 days
of interaction. Last, behaviours at the beginning of the experiment
(ﬁrst 2 days) were highly correlated with all behaviours observed
during the whole 10 days of the experiment (Table 4), showing that
the hierarchy was established during the ﬁrst 48 h after queen loss.
Spearman correlations between an individual ant's fertility index and hierarchical
rank on the ﬁrst, second and 10th (ﬁnal) day of the experiment
Day 1 rank 0.3 0.03 59
Day 2 rank 0.39 0.006 59
Day 10 rank 0.44 0.002 59
To compensate for multiple comparisons, Pvalues were adjusted to P
following Holm (1979).
Spearman correlations between the proportions of the three main compounds
within individual ants’chemical proﬁles before and after queen removal
Cuticular hydrocarbons r
0.48 0.001 42
0.34 0.03 42
0.74 2.1e-8 42
To compensate for multiple comparisons, Pvalues were adjusted to P
following Holm (1979).
10 15 20 25 30 35
Quantity of n-C23 final (µg)
0.05 0.06 0.07 0.08 0.09 0.1 0.11
Proportion of n-C23 initial
Figure 3. Variation in the fertility index (summed lengths of the six basal oocytes in the ovary, mm) as a function of (a) the amount of tricosane in the ants' chemical proﬁle after
queen removal and (b) the proportion of tricosane in the proﬁle before queen removal.
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140 133
Our results conﬁrmed the rapidity of hierarchy establishment
over a period of 48 h after queen loss. The number of agonistic
behaviours decreased drastically after this period, which is typical
of a stabilized hierarchy. The cuticular proﬁles analysis showed
tricosane to be highly correlated with ovarian development,
therefore putatively acting as a fertility signal. Interestingly, the
relative amounts of tricosane on the cuticle both before and after
the establishment of the hierarchy were also correlated with (1) the
rank achieved within the hierarchy and (2) the frequency of the
agonistic behaviours displayed.
These results constitute the ﬁrst experimental evidence that
differences in ovarian activity (estimated by an indirect method)
between workers in the presence of the queen accurately predict
the outcome of the reproductive hierarchy, which is a consequence
of a tournament between workers. They also fully support our hy-
pothesis that physiological differences between workers are
mirrored in modulated motivations to ﬁght (Stevenson, Hofmann,
Schoch, &Schildberger, 2000). The outcome of the dominance/
submission could then be facilitated with chemical signalling
(Yagound et al., 2015).
Fertility signals have been identiﬁed in other morphs of
N. apicalis (Yagound, 2014), but not in morph 6. In three colonies
(the fourth having a different chemotype) tricosane was the
cuticular compound best correlated with fertility. Thus, tricosane is
the most probable fertility signal in these colonies. We used tri-
cosane as a proxy for the fertility signal, but we are aware that it
may also be part of a mixture of compounds used for fertility
recognition. This does not, however, change the conclusions of our
study. In his comparative study of fertility signalling in the
N. apicalis complex, Yagound (2014) found that an alkene was best
correlated with fertility in morph 6 (r
Yagound could analyse only one colony and he also found that
tricosane was correlated with ovarian development (r
P<0.01). This ﬁnding and the larger sample that we analysed
legitimize the interpretation of tricosane as a putative signal of
fertility for N. apicalis morph 6. Other compounds were also
correlated with fertility in our sample but tricosane appeared to be
the best correlated compound with both fertility and behavioural
parameters (the expression of agonistic behaviours and the social
ranks achieved) during the establishment of the hierarchy. This
consistency between physiological and behavioural data fulﬁls the
condition for tricosane to be an index of fertility. The correlation
between the putative fertility signal and the hierarchical ranks also
strengthens the idea of it being an honest signal (Heinze &
D’Ettorre, 2009). A reliable index of fertility allows appropriate
reproductive decision making, depending on individual interests in
terms of inclusive ﬁtness (Yagound, 2014). This signal would allow
workers to identify the best potential reproducer within the colony.
The initial heterogeneity between ants in queenright colonies
could be ampliﬁed during subsequent agonistic interactions. Idio-
syncratic variations could initially reﬂect the differences in
workers' ages affecting their physiological and hormonal states,
and hence their ovarian activation (Yagound et al., 2015). Workers
with an already partially active ovarian system would have a clear
advantage during the establishment of hierarchies. Lamba et al.
(2007) hypothesized that in other eusocial insects (wasps)
ﬁghting could be used not to exclude the other females from
dominance, but rather to speed up the development of the ovarian
system of the future reproductive (via an action on biogenic
amines) and so facilitate the monopolization of colony reproduc-
tion. Aggressive behaviours also lead to a decrease in juvenile
hormone titre (usually positively correlated with fertility) in sub-
ordinate individuals (Tibbetts, Fearon, Wong, Huang, &Tinghitella,
2018). Physical contact between workers in the ant Diacamma has
also been shown to affect dopamine secretion in the worker's brain
and to regulate reproduction inside the nest (Shimoji et al., 2017).
Agonistic interactions in Neoponera may thus impact ovarian
development through similar neuroendocrine changes.
Hierarchical status discrimination based on the putative fertility
signal can generate a linear hierarchy. Fertility signalling would be
involved in both the establishment (Yagound et al., 2015) and the
maintenance (Heinze, Stengl, &Sledge, 2002) of the reproductive
hierarchy. Agonistic interactions acting on the physiological and
hormonal secretions would reinforce the pre-existing differences in
fertility between individuals, and this would accelerate cooperation
within the nest. Subordinates would maximize their ﬁtness by
quickly resolving conﬂicts by reducing their ability to reproduce
(Tibbetts et al., 2018).
Interestingly, our results showed a strong correlation between
fertility and the number of ﬁghts an individual is involved in
(whatever the outcome). This result suggests two mutually
nonexclusive hypotheses. First, tricosane could be correlated with
both fertility and motivation to ﬁght and/or involvement in the
colony's hierarchy. Biogenic amines such as octopamine or dopa-
mine could possibly be involved in this process. Indeed, biogenic
amines mediate changes in dominance behaviour linked with
fertility in the ant Harpegnathos saltator (Penick, Brent, Dolezal, &
Liebig, 2014). Moreover, it has been demonstrated in the cricket
Gryllus bimaculatus that these bioamines are necessary to trigger
aggressive behaviour (Stevenson et al., 2000). Second, tricosane
Spearman correlations between the behaviours expressed after (ﬁnal n-C
) and before (initial n-C
) queen removal and the mean deviation in amount of n-C
individuals of the same colony
Final hierarchical rank 0.52 0.002 0.48 0.003 42
No. of ﬁghts won during days 1 and 2 0.46 0.008 0.53 0.002 42
Total no. of ﬁghts won 0.53 0.001 0.59 2.5e-4 42
Percentage of ﬁghts won during days 1 and 2 0.37 0.01 0.43 0.008 42
Total percentage of ﬁghts won 0.44 0.008 0.44 0.008 42
Fight number during days 1 and 2 0.45 0.008 0.51 0.002 42
Fight number total 0.5 0.002 0.59 2.2e-4 42
To compensate for multiple comparisons, Pvalues were then adjusted to P
values following Holm (1979).‘Final’and ‘total’correspond to the behaviours expressed from day 1
to day 10 of being orphaned. ‘Fight number’corresponds to the number of ﬁghts an individual was involved in. Data dispersions are presented in Fig. A3.
Spearman correlations between behaviours during days 1 and 2 combined and from
day 1 to day 10 of the experiment
Hierarchical rank 0.86 <2.2e-16 59
No. of ﬁghts won 0.96 <2.2e-16 59
Percentage of ﬁghts won 0.90 <2.2e-16 59
No. of ﬁghts 0.95 <2.2e-16 59
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140134
could act as a fertility signal and thus attract aggression from
competitors attempting to gain dominance. Such behaviour where
workers attack congeners that display fertility signals has been
shown, for example, in the context of worker policing in social
insects (ants: Hartmann, D’Ettorre, Jones, &Heinze, 2005; Monnin
&Peeters, 1999; Smith et al., 2009; bees: Visscher &Dukas, 1995;
wasps: Wenseleers, Toﬁlski, &Ratnieks, 2005). This mechanism
could thus ensure the fertility signal has similar functions in the
contexts of worker policing and establishment of the reproductive
hierarchy, namely regulating reproduction at the level of the
The fact that tricosane was also correlated with the percent-
age of ﬁghts won favours the ﬁrst explanation. Attacked in-
dividuals in the case of worker policing are indeed more likely to
be defeated (and their reproductive activity suppressed; Monnin
&Peeters, 1999), while highly motivated animals could have an
advantage in a tournament system. Tricosane could thus both
reﬂect ovarian development and be correlated with a network of
neuroendocrine activity that ensures ﬁghting motivation and,
maybe more generally, the ability to mobilize resources (RHP;
Parker, 1974). One mechanism ensuring the honesty of the
fertility signal (and its role in the reproductive hierarchy) could
be the strong links between the neuroendocrine networks
involved in the regulation of reproduction, agonistic behaviour
and ﬁghting abilities. A second mechanism could be that in-
dividuals ‘motivated’to ﬁght, but lacking the skills required to
occupy the top of the hierarchy, would be defeated by other
workers. This mechanism would be in line with theories pro-
posing that the costs (both physiological and social) of main-
taining a signal ensure its honesty (Zahavi, 1975; Heinze &
The loss of the queen probably lifts an inhibition for already
fertile individuals which very quickly start competing to reproduce.
The highest motivation for ﬁghting of these individuals probably
drives the expression of ritualized agonistic encounters within the
colony. The impact of social experience and especially
winnereloser effects would then help amplify the pre-existing
differences at the physiological (Oliveira, Silva, &Can
cognitive and behavioural (Hsu &Wolf, 2001; Rutte et al., 2006)
levels. The social system would then develop from the queenright
state, where all workers refrain from reproducing, to the estab-
lishment of the reproductive hierarchy based on self-organized
processes. After a short period of social perturbation with intense
ﬁghting behaviour, the social system stabilizes again with the se-
lection of a new reproductive individual and the disappearance of
To our knowledge, this is the ﬁrst study to monitor the devel-
opment of the chemical proﬁle from the queenright state to the
stabilization of a reproductive hierarchy by orphaned ant workers.
Our study supports the hypothesis that the pre-existing fertility
differences between individuals in the queenright condition predict
the destiny of workers in the reproductive hierarchy. The most
fertile workers reach the high ranks and produce males. The se-
lective pressures are strong after queen loss, with a short time
window for producing the last batch of reproductive ants
(Dietemann &Peeters, 2000). In response to these strong ecological
constraints, ants have developed a recognition system based on
cuticular hydrocarbons related to ovarian development and acting
as a fertility signal (Yagound et al., 2015). This fertility signal,
already perceptible in the presence of the queen, makes it possible
for workers to evaluate the interindividual differences and, subse-
quently, agonistic interactions help to establish and stabilize the
reproductive hierarchy (especially with winnereloser effects;
Chase, Tovey, Spangler-Martin, &Manfredonia, 2002). All these
mechanisms allow a quick resolution of the overt conﬂict and
ensure division of reproductive and ergonomic tasks inside
orphaned colonies to allow the production of males.
We thank Ronara De Souza Ferreira-Ch^
aline and R
tefarde for collecting ants, Chlo
e Leroy for chemical analyses,
eonard for genetic analyses and Paul Devienne
for technical assistance. Three anonymous referees provided
helpful comments on the manuscript. N.C. and S.C. received a
travel grant from Sorbonne Paris Cit
e (Excellence SPC-USP Br
N.C. was funded by the CNPq (chamada universal 458736/2014-7,
bolsa PQ (311790/2017-8) and CAPES (PROEX Psicologia Experi-
Abril, S., Diaz, M., Lenoir, A., Paris, C. I., Boulay, R., & Gomez, C. (2018). Cuticular
hydrocarbons correlate with queen reproductive status in native and invasive
Argentine ants (Linepithema humile, Mayr). PLoS One, 13(2), 1e17.
Anderson, M. J., Gorley, R. N., & Clarke, K. R. (2008). PERMANOVAþfor PRIMER: guide
to software and statistical methods. Plymouth, U.K: PRIMER-E Ltd.
Blacher, P., Lecoutey, E., Fresneau, D., & Nowbahari, E. (2010). Reproductive hierar-
chies and status discrimination in orphaned colonies of Pachycondyla apicalis
ants. Animal Behaviour, 79,99e105 .
Chase, I. D., Tovey, C., Spang ler-Martin, D. , & Manfredonia, M. (20 02). Indivi dual
hierarchies. Proceedings of the National Academy of Sciences, 99(8),
Curley, J. P. (2016). Compete: Analyzing social hierarchies: R package version 0.1.
Retrieved from https://www.rdocumentation.org/packages/compete.
Delabie, J. H. C., Mariano, C. S. F., Mendes, L. F., Pompolo, S. G., & Fresneau, D. (2008).
Problemas apontados por estudos morfol
ogicos e citogen
enero pachycondyla na regiaeo neotropical: o caso do complexo apicalis.In
E. F. Vilela, I. A. Santos, J. H. Schoereder, J. L. Neto, J. E. Serraeo, & L. A. O. Campos
(Eds.), Insetos sociais: da biologia
a aplicaçaeo(pp. 197e222). Viçosa, Brazil:
d'Ettorre, P., & Lenoir, A. (2010). Nestmate recognition. In L. Lach, C. L. Parr, &
K. L. Abbott (Eds.), Ant ecology (pp. 194e209). Oxford, U.K.: Oxford University
ere, S., Fresneau, D., & Deneubourg, J. L. (2004). The inﬂuence of red light on
the aggregation of two castes of the ant, Lasius niger.Journal of Insect Physiology,
Dietemann, V., & Peeters, C. (2000). Queen inﬂuence on the shift from trophic to
reproductive eggs laid by workers of the ponerine ant Pachycondyla apicalis.
Insectes Sociaux, 47(3), 223e228.
Dugatkin, L. A., & Earley, R. L. (2004). Individual recognition, dominance hierarchies
and winner and loser effects. Proceedings of the Royal Society B: Biological Sci-
ences, 271(1547), 1537e1540.
Ferreira, R. S., Poteaux, C., Delabie, J. H. C., Fresneau, D., & Rybak, F. (2010). Stridu-
lations reveal cryptic speciation in Neotropical sympatric ants. PLoS One, 5.
Fresneau, D. (1994). Biologie et comportement social d’une fourmi pon
eotropicale (Pachycondyla apicalis) [Unpublished Ph.D. thesis]. Villetaneuse,
France: University Paris XIII.
Glickman, M. E. (1999). Parameter estimation in large dynamic paired comparison
experiments. Journal of the Royal Statistical Society: Series C (Applied Statistics),
Greene, M. J., & Gordon, D. M. (2003). Social insects: Cuticular hydrocarbons inform
task decisions. Nature, 423(6935), 32e32.
Hamilton, W. D. (1964). The genetical evolution of social behaviour. I. Journal of
Theoretical Biology, 7(1), 17e52.
Hartmann, A., D'Ettorre, P., Jones, G. R., & Heinze, J. (2005). Fertility signalingeThe
proximate mechanism of worker policing in a clonal ant. Naturwissenschaften,
Heinze, J., & D'Ettorre, P. (2009). Honest and dishonest communication in social
Hymenoptera. Journal of Experimental Biology, 212(12), 1775e1779.
Heinze, J., Holldobler, B., & Peeters, C. (1994). Conﬂict and cooperation in ant so-
cieties. Naturwissenschaften, 81, 489e497.
Heinze, J., Stengl, B., & Sledge, M. F. (2002). Worker rank, reproductive status and
cuticular hydrocarbon signature in the ant, Pachycondyla cf. inversa.Behavioral
Ecology and Sociobiology, 52(1), 59e65.
Holm, S. (1979). A simple sequentially rejective multiple test procedure. Scandi-
navian Journal of Statistics, Theory and Applications, 6,65e70.
Holman, L., Hanley, B., & Millar, J. G. (2016). Highly speciﬁc responses to queen
pheromone in three Lasius ant species. Behavioral Ecology and Sociobiology,
Holman, L., Lanfear, R., & D'Ettorre, P. (2013). The evolution of queen pheromones in
the ant genus Lasius.Journal of Evolutionary Biology, 26(7), 1549e1558.
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140 135
Hsu, Y., Earley, R. L., & Wolf, L. L. (2006). Modulation of aggressive behaviour by
ﬁghting experience: Mechanisms and contest outcomes. Biological Reviews of
the Cambridge Philosophical Society, 59, 111e140.
Hsu, Y., & Wolf, L. (2001). The winner and loser effect: What ﬁghting behaviours are
inﬂuenced? Animal Behaviour, 61,777e786.
Keller, L., & Nonacs, P. (1993). The role of queen pheromones in social insects: Queen
control or queen signal? Animal Behaviour, 45, 787e794.
Lamba, S., Kazi, Y. C., Deshpande, S., Natesh, M., Bhadra, A., & Gadagkar, R. (2007).
A possible novel function of dominance behaviour in queen-less colonies of the
primitively eusocial wasp Ropalidia marginata.Behavioural Processes, 74(3),
Leonhardt, S. D., Menzel, F., Nehring, V., & Schmitt, T. (2016). Ecology and evolution
of communication in social insects. Cell, 164,1277e1287.
Liebig, J. (2010). Hydrocarbon proﬁles indicate fertility and dominance status in ant,
bee, and wasp colonies. In G. J. Blomquist, & A.-G. Bagni
eres (Eds.), Insect hy-
drocarbons: biology, biochemistry, and chemical ecology (pp. 254e281). Cam-
bridge, U.K.: Cambridge University Press.
Liebig, J., Peeters, C., Oldham, N. J., Markstadter, C., & Holldobler, B. (2000). Are
variations in cuticular hydrocarbons of queens and workers a reliable signal of
fertility in the ant Harpegnathos saltator?Proceedings of the National Academy of
Sciences, 97(8), 4124e4131.
Mackay, W.,P., & Mackay, E. E. (2010). The systematics and biology of the New World
ants of the genus Pachycondyla (Hymenoptera: Formicidae). Lewiston, ME: The
Edwin Mellen Press.
Monnin, T., Malusse, C., & Peeters, C. (1998). Solid-phase microextraction and
cuticular hydrocarbon differences related to reproductive activity in queenless
ant Dinoponera quadriceps.Journal of Chemical Ecology, 24(3), 473e490 .
Monnin, T., & Peeters, C. (1999). Dominance hierarchy and reproductive conﬂicts
among subordinates in a monogynous queenless ant. Behavioral Ecology, 10(3),
Oliveira, P. S., & H€
olldobler, B. (1990). Dominance orders in the ponerine ant.
Behavioral Ecology and Sociobiology, 385e393, 1990.
Oliveira, R. F., Silva, A., & Can
ario, A. V. M. (2009). Why do winners keep winning?
Androgen mediation of winner but not loser effects in cichlid ﬁsh. Proceedings
of the Royal Society B: Biological Sciences, 276(1665), 2249e2256.
Parker, G. A. (1974). Assessment strategy and the evolution of ﬁghting behaviour.
Journal of Theoretical Biology, 47(1), 223e243.
Penick, C. A., Brent, C. S., Dolezal, K., & Liebig, J. (2014). Neurohormonal changes
associated with ritualized combat and the formation of a reproductive hierar-
chy in the ant Harpegnathos saltator.Journal of Experimental Biology, 217(9),
Rutte, C., Taborsky, M., & Brinkhof, M. W. G. (20 06). What sets the odds of winning
and losing? Trends in Ecology &Evolution, 21(1), 16e21.
Sasaki, T., Penick, C. A., Shaffer, Z., Haight, K. L., Pratt, S. C., & Liebig, J. (2016).
A simple behavioral model predicts the emergence of complex animal hierar-
chies. The American Naturalist, 187(6), 765e775.
Shimoji, H., Aonuma, H., Miura, T., Tsuji, K., Sasaki, K., & Okada, Y. (2017). Queen
contact and among-worker interactions dually suppress worker brain
dopamine as a potential regulator of reproduction in an ant. Behavioral Ecology
and Sociobiology, 71(2).
Smith, A. A., H€
olldober, B., & Liebig, J. (2009). Cuticular hydrocarbons reliably
identify cheaters and allow enforcement of altruism in a social insect. Current
Biology, 19(1), 78e81.
Smith, A. A., Millar, J. G., & Suarez, A. V. (2015). A social insect fertility signal is
dependent on chemical context. Biology Letters, 11(1), 1e4.
So, N., Franks, B., Lim, S., & Curley, J. P. (2015). A social network approach reveals
associations between mouse social dominance and brain gene expression. PLoS
One, 10(7), 1e27.
Stephenson, A., & Sonas, J. (2014). PlayerRatings: Dynamic updating methods for
player ratings estimation. R package v1.0. Retrieved from https://cran.r-project.
Stevenson, P. A., Hofmann, H. A., Schoch, K., & Schildberger, K. (2000). The ﬁght and
ﬂight responses of crickets depleted of biogenic amines. Journal of Neurobiology,
Tibbetts, E. A., Fearon, M. L., Wong, E., Huang, Z. Y., & Tinghitella, R. M. (2018). Rapid
juvenile hormone downregulation in subordinate wasp queens facilitates stable
cooperation. In , 285.Proceedings of the Royal Society B: Biological Sciences, 1872.
Visscher, P. K., & Dukas, R. (1995). Honey bees recognize development of nestmates'
ovaries. Animal Behaviour, 49(2), 542e544.
de Vries, H. (1995). An improved test of linearity in dominance hierarchies con-
taining unknown or tied relationships. Animal Behaviour, 50(5), 1375e1389.
Wenseleers, T., Toﬁlski, A., & Ratnieks, F. L. W. (2005). Queen and worker policing in
the tree wasp Dolichovespula sylvestris.Behavioral Ecology and Sociobiology,
Yagound, B. (2014). Conﬂits, coop
eration et syst
emes de reconnaissance chez les
fourmis du complexe d’esp
eces Neoponera apicalis [Doctoral dissertation]. Ville-
taneuse, France: Universit
e Paris XIII.
Yagound, B., Blacher, P., Chameron, S., & Ch^
aline, N. (2012). Social context and
reproductive potential affect worker reproductive decisions in a eusocial insect.
PLoS One, 7(12), 1e7.
Yagound, B., Blacher, P., Fresneau, D., Poteaux, C., & Ch^
aline, N. (2014). Status
discrimination through fertility signalling allows ants to regulate reproductive
conﬂicts. Animal Behaviour, 93,25e35.
Yagound, B., Gouttefarde, R., Leroy, C., Belibel, R., Barbaud, C., Fresneau, D., et al.
(2015). Fertility signaling and partitioning of reproduction in the ant Neoponera
apicalis.Journal of Chemical Ecology, 41(6), 557e566.
Yagound B., Savarit F., Fichaux M., Poteaux C., &Ch^
aline N. (n.d.). Are chemical
signals of reproductive status conserved in the Neoponera apicalis (Hyme-
noptera: Formicidae) species complex? Manuscript in preparation.
Zahavi, A. (1975). Mate selectionea selection for a handicap. Journal of Theoretical
Biology, 53, 205e214.
PCO1 (76.8% of total variation)
PCO2 (7.2% of total variation)
PCO3 (3.7% of total variation)
Figure A1. Principal coordinate analysis (PCO) of the chemical proﬁles of the four colonies (based on the BrayeCurtis similarity matrix calculated with the square-root-transformed
proportions). N¼15 individuals in each colony.
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140136
2 000 000
4 000 000
6 000 000
8 000 000
5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5
2 000 000
4 000 000
6 000 000
8 000 000
5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5
Figure A2. Representative examples of the chemical proﬁles from (a) colony 1 (chemotype A) and (b) colony 18 (chemotype B).
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140 137
2 4 6 8 10 12 14
20 40 60 80 100 120 140
246810 12 14
0 50 100 150
020 40 60 80 100 120 140
Mean deviation per colony of n-C23
Hierarchical rank on day 10
No. of fights won on days 1 and 2
No. of fi
hts won on da
s 1 - 10
Figure A3. Plot of the data used in the Spearman correlations presented in Table 3. Correlations are shown between the recorded behaviours and the mean deviation of tricosane
) per colony after (left) and before (right) queen removal. (a) Hierarchical rank on the 10th day (the end of the experiment), (b) the number of ﬁghts won on days 1 and 2, (c)
the total number of ﬁghts won from day 1 to day 10, (d) the percentage of ﬁghts won on days 1 and 2, (e) the total percentage of ﬁghts won from day 1 to day 10, (f) the number of
ﬁghts individuals were involved in on days 1 and 2 and (g) the total number of ﬁghts individuals were involved in from day 1 to day 10.
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140138
%Fights won on days 1 and 2
%Fights won on days 1 - 10
No. of fights individual involved in on days 1 and 2
Mean deviation per colony of n-C23
050 100 150
050 100 150
No. of fi
hts individual involved in on da
s 1 - 10
Figure A3. (continued).
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140 139
Identiﬁcation of cuticular hydrocarbons (CHCs) on N. apicalis morph 6 for a moderately fertile individual (corresponding to chemotype A in Fig. A2a)
Peak Retention time Relative abundance Characteristic fragments CHC ID
1 5.876 ee n-C
2 6.532 0.05 268 n-C
3 6.915 0.08 282 n-C
4 7.265 0.7 294 C
5 7.399 14.85 296 n-C
6 7.563 0.06 140 196 295 9-MeC
7 7.694 0.07 70 267 295 4-MeC
8 7.785 1.41 308 C
9 7.906 0.97 310 n-C
10 8.139 0.07 169 182 309 11-MeC
11 8.523 62.42 322 C
12 8.633 11.13 324 n-C
13 8.847 0.19 168 196 323 11-MeC
14 8.962 0.16 85 252 281 323 5-MeC
15 9.167 0.09 336 C
16 9.371 0.07 338 n-C
17 10.135 0.99 350 C
18 10.392 1.4 352 n-C
19 11.606 0.14 366 n-C
20 12.756 0.04 378 C
21 13.168 2.45 380 n-C
22 14.374 0.12 394 n-C
23 14.824 0.33 365 393 2-MeC
24 14.918 0.06 406 C
25 15.046 0.83 408 n-C
26 15.225 0.24 168 196 252 281 407 11-16-diMeC
27 15.961 0.2 393 421 2-MeC
28 16.065 0.04 434 C
29 16.174 0.09 436 n-C
30 16.352 0.32 168 196 224 252 281 309 435 11-13-15-MeC
31 17.634 0.44 168 308 337 463 11-MeC
Analysis of similarity between the chemical proﬁles of the four colonies
Groups RP Permutations
1, 8 0.148 0.01 9999
1, 18 0.758 0.0001 9999
1, 20 0.208 0.0002 9999
8, 18 0.771 0.0001 9999
8, 20 0.281 0.0001 9999
18, 20 0.747 0.0001 9999
The global test of the analysis of similarity gives a global Rof 0.549 (P¼0.0001,
number of permutations ¼9999). Pairwise test results are given in the table.
Spearman correlations (with BonferronieHolm adjustment for multiple compari-
sons) between the fertility index and the compounds present in the chemical proﬁle
after orphaning of colonies 1, 8 and 20 (N¼44)
Cuticular hydrocarbons r
For colony 18, only the compound n-C
was correlated with fertility (r
15, P’¼0.057). To compensate for multiple comparisons, Pvalues were adjusted to
values following Holm (1979). Signiﬁcant values are highlighted in bold.
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140140