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Pre-existing differences in putative fertility signals give workers the upper hand in ant reproductive hierarchies

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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 23) 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 behaviours. 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.
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Pre-existing differences in putative fertility signals give workers the
upper hand in ant reproductive hierarchies
Romain Honorio
a
,
b
,
*
, Nicolas Ch^
aline
c
,St
ephane Chameron
b
a
Sorbonne Universit
e, Universit
e Paris Est Cr
eteil, Universit
e Paris Diderot, CNRS, INRA, IRD, Institute of Ecology and Environmental ScienceseParis, IEES-
Paris, Paris, France
b
Laboratoire
Ethologie Exp
erimentale et Compar
ee, Universit
e Paris 13, Villetaneuse, France
c
Laborat
orio de Etologia Ecologia e Evoluç~
ao Dos Insetos Sociais, Departamento de Psicologia Experimental, Instituto de Psicologia, Universidade de S~
ao
Paulo, Butant~
a, Brazil
article info
Article history:
Received 22 January 2019
Initial acceptance 26 February 2019
Final acceptance 2 August 2019
MS. number: 19-00054R
Keywords:
dominance behaviour
fertility signalling
idiosyncratic difference
ponerine ants
reproductive hierarchy
In social groups, competition often gives rise to conicts, which are regulated through a variety of
mechanisms. In several social insect species, the conict 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 inuence access to the top of the hierarchy
after queen loss. In this study, we therefore sought to characterize the inuence 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 prole before and after hierarchy establishment in four groups of orphaned workers of
N. apicalis morph 6. The analysis of the cuticular proles showed that tricosane (n-C
23
) 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 conict 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 conict for male
production (Heinze, Holldobler, &Peeters, 1994; Oliveira &
H
olldobler, 1990). An individual's rank stems from several factors
that are classically described as intrinsicand extrinsicand 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 ght, as well as potential chemical
cuticular correlates) that determine its absolute ghting 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 modication
of intrinsic factors, which in turn modify future experiences. These
inuences are notably reected in winnereloser effects where the
outcome of an encounter (victory or defeat) induces changes in the
neuroendocrine titres (Hsu, Earley, &Wolf, 2006), thus inuencing
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 afliation, but they also convey more subtle social
*Correspondence: R. Honorio, Sorbonne Universit
e, Universit
e Paris Est Cr
eteil,
Universit
e Paris Diderot, CNRS, INRA, IRD, Institute of Ecology and Environmental
ScienceseParis, iEES-Paris, 75005, Paris, France.
E-mail address: romain.honorio@sorbonne-universite.fr (R. Honorio).
Contents lists available at ScienceDirect
Animal Behaviour
journal homepage: www.elsevier.com/locate/anbehav
https://doi.org/10.1016/j.anbehav.2019.09.007
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 proles. This chemical signal
results from quantitative or qualitative differences (or both) of
endogenousand exogenousorigins between one or morecompounds
across individuals, castes and colonies (dEttorre &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 modies the behaviour of potential partners
(reviewed in Leonhardt, Menzel, Nehring, &Schmitt, 2016). When it
reects 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 tness (Keller &
Nonacs, 1993;Heinze &DEttorre, 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 ne
balance between direct tness costs, indirect tness benets 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 conict
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 tness of the
whole worker collective (Hamilton, 1964; Keller &Nonacs, 1993).
To test our hypothesis, we rst correlated variation in cuticular
proles 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.
METHODS
Ethical Note
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 articial
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.
Ants
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 conict resolution. In this study,
we used four colonies: colony 1 was collected in Marituba, state of
Para (1
21
0
18
00
S, 48
20
0
21
00
W), and colonies 8, 18 and 20 in Santa
Barbara do Para (1
13
0
36
00
S, 48
17
0
43
00
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
ne morphological differences in this complex of cryptic species.
Ferreira, Poteaux, Delabie, Fresneau, and Rybak (2010) dened
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^
aline
(2012), which showed that workers close to the queen were the
rst 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 prole 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
articial 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 prole 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 Proles
The individualschemical proles were analysed before they
were orphaned and after the establishment of the reproductive
hierarchy. The initial chemical prole (before orphaning) was ob-
tained by solid-phase microextraction (Monnin et al., 1998). This
involved rubbing an SPME bre (polydimethylsiloxane 100
m
m) on
the rst segment of the abdomen for 2 min. The bre was then
desorbed in a Varian 3900 gas chromatograph with ame 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
C
for 1 min, then it rose from 40
C/min for 4 min to 250
C, then
increased by 1
C/min for 8 min to 258
C and nally increased
from 40
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. Proles were extracted with the Varian system
control software Star Chromatography workstation version 6.2
(Varian, Palo Alto, CA, U.S.A.). The compounds were identied
based on their retention time (Appendix Table A1), comparing
them to standard hydrocarbons already identied 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 quantication of
compounds.
Owing to time constraints, the chemical prole after orphaning
was obtained by a liquid phase extraction. The head and thorax of
each dissected ant were soaked in 200
m
l of pentane containing
4 ng/
m
l of compound n-C
17
(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
m
l of the extract
resuspended in 80
m
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
proles were integrated using the MSD ChemStation software
version E.02.01.1177 (Agilent Technologies Inc., Santa Clara, CA,
U.S.A.). The compounds were identied 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.
Dominance Hierarchy
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 rst, second and third days after being orphaned, then two
of 1 h on the fourth and fth days and nally 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 antsbehaviour in the interior of the nest (Depick
ere,
Fresneau, &Deneubourg, 2004).
The hierarchical rank of each worker in the orphaned colonies
was obtained using the Glicko-ratingmethod, 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 dened by the user, an
increased value of cleading 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 cvalue 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 coefcient 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.
Fertility Measurement
With a graduated 10binocular microscope, we measured the
ovarian development of the ants. The length of the three basal
Marking + initial profile
extraction (GC-FID)
20 selected
individuals
15 orphaned
individuals
Agonistic
interactions
(hierarchical rank)
Individuals
killed
Final profile extraction
(GC-MS) + dissection
(ovarian development)
Queenright Queenless
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 prole
corresponds to the workers' chemical prole in the presence of the queen (determined by ame ionization detection, GCeFID); nal prolecorresponds to the chemical prole 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.
Statistical Analysis
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 Pvalues following Holm (1979).
The linearity hwithin 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
experiment.
Chemical data analysis
Although some intercolonial heterogeneity is expected in the
proportion of each compound in the cuticular proles, 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 proles. For this, we used
the PERMANOVAþfor PRIMER software (Anderson, Gorley, &
Clarke, 2008) using a Euclidean distance matrix calculated on
square-root-transformed percentages.
Using two different methods to extract the chemical proles
was a potential source of methodological variability. To verify
whether our methods were reliable, we used Spearman correla-
tions to compare the proles before and after orphaning using the
proportions of the major compounds, namely n-C
21
,C
23:1
and n-C
23
,
with a BonferronieHolm adjustment for multiple tests on the same
data set (Holm, 1979). Signicant correlations would indicate reli-
ability of the two methods (even if distinct methods can generate a
slight chemical distance between the proles 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 prole 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 proles
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 nal quantities of
cuticular compounds. Once the putative fertility signal was iden-
tied, 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,
1979).
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.
RESULTS
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 signicantly between them. No two by two comparison
between colonies was signicant (KolmogoroveSmirnov test with
BonferronieHolm correction: colony 1e8: D¼0.571, P¼0.203;
colony 1e18: D¼0.571, P¼0.203; colony 1e20: D¼0.571,
P¼0.212; colony 8e18: D¼0.571, P¼0.203; colony 8e20:
D¼0.571, P¼0.203; colony 18e20: D¼0.429, P¼0.575).
Agonistic interactions rose rapidly within the rst 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),
0
20
40
60
80
120
12345810
Da
y
Agonistic interactions
per hour of observation
Colony
1
8
18
20
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 signicant
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 nally colony 20
(2.97 ±2.06 mm). Within each colony the fertility index of workers
was signicantly correlated with their hierarchical rank from the
rst day of being orphaned (Table 1).
Chemical Proles, Fertility and Behaviours
Chemical analyses of proles after orphaning
The chemical proles 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 prole 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 proles. The chemical proles of the
workers from colony 18 were heterogeneous, some appearing
separated and others represented among the other coloniespro-
les (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
discrepant chemotype.
Reliability between the two chemical extraction methods
The proportions of the three major compounds (n-C
21
,C
23:1
and
n-C
23
) in the SPME samples analysed by GCeFID before orphaning
were signicantly correlated with those analysed by GCeMS after
orphaning (Table 2). The Mantel test between the two chemical
prole matrices before and after orphaning were signicantly
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 sufcient to validate the use
of the two methods. Initial and nal n-C
23
proportions were also
correlated (Spearman correlation: r
s
¼0.75, P<0.001).
Correlation between chemical proles and fertility
The amount of the alkane tricosane (n-C
23
) after orphaning was
signicantly correlated with the fertility index for each individual
(Spearman correlation: r
s
¼0.63, N¼44, P<0.001; Fig. 3a), as was
the proportion of n-C
23
in the chemical prole before orphaning
(Spearman correlation: r
s
¼0.69, N¼42, P<0.001; Fig. 3b). This
compound was the component of the chemical prole 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
23
which was the best correlated compound
with fertility. Both the number of ghts won by an ant and the
number of interactions it was involved in were signicantly
correlated with the quantity of n-C
23
after orphaning (Table 3). The
same results were obtained when considering only the rst 2 days
of interaction. Last, behaviours at the beginning of the experiment
(rst 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 rst 48 h after queen loss.
Table 1
Spearman correlations between an individual ant's fertility index and hierarchical
rank on the rst, second and 10th (nal) day of the experiment
r
S
P
0
N
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
0
values
following Holm (1979).
Table 2
Spearman correlations between the proportions of the three main compounds
within individual antschemical proles before and after queen removal
Cuticular hydrocarbons r
S
P
0
N
n-C
21
0.48 0.001 42
C
23:1
0.34 0.03 42
n-C
23
0.74 2.1e-8 42
To compensate for multiple comparisons, Pvalues were adjusted to P
0
values
following Holm (1979).
0
2
4
6
8
0
2
4
6
8
10 15 20 25 30 35
Fertility (mm)
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
(a) (b)
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 prole after
queen removal and (b) the proportion of tricosane in the prole before queen removal.
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140 133
DISCUSSION
Our results conrmed 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 proles 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 rst 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 ght (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 identied 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
s
¼0.75, P<10
-4
). However,
Yagound could analyse only one colony and he also found that
tricosane was correlated with ovarian development (r
s
¼0.51,
P<0.01). This nding 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 fulls 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 &
DEttorre, 2009). A reliable index of fertility allows appropriate
reproductive decision making, depending on individual interests in
terms of inclusive tness (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 amplied during subsequent agonistic interactions. Idio-
syncratic variations could initially reect 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)
ghting 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 tness by
quickly resolving conicts by reducing their ability to reproduce
(Tibbetts et al., 2018).
Interestingly, our results showed a strong correlation between
fertility and the number of ghts 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 ght 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
Table 3
Spearman correlations between the behaviours expressed after (nal n-C
23
) and before (initial n-C
23
) queen removal and the mean deviation in amount of n-C
23
between
individuals of the same colony
r
S
(nal n-C
23
)P
0
r
S
(initial n-C
23
)P
0
N
Final hierarchical rank 0.52 0.002 0.48 0.003 42
No. of ghts won during days 1 and 2 0.46 0.008 0.53 0.002 42
Total no. of ghts won 0.53 0.001 0.59 2.5e-4 42
Percentage of ghts won during days 1 and 2 0.37 0.01 0.43 0.008 42
Total percentage of ghts 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
0
values following Holm (1979).Finaland totalcorrespond to the behaviours expressed from day 1
to day 10 of being orphaned. Fight numbercorresponds to the number of ghts an individual was involved in. Data dispersions are presented in Fig. A3.
Table 4
Spearman correlations between behaviours during days 1 and 2 combined and from
day 1 to day 10 of the experiment
r
S
PN
Hierarchical rank 0.86 <2.2e-16 59
No. of ghts won 0.96 <2.2e-16 59
Percentage of ghts won 0.90 <2.2e-16 59
No. of ghts 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, DEttorre, Jones, &Heinze, 2005; Monnin
&Peeters, 1999; Smith et al., 2009; bees: Visscher &Dukas, 1995;
wasps: Wenseleers, Tolski, &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
worker collective.
The fact that tricosane was also correlated with the percent-
age of ghts won favours the rst 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
reect ovarian development and be correlated with a network of
neuroendocrine activity that ensures ghting 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 ghting abilities. A second mechanism could be that in-
dividuals motivatedto ght, 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 &
DEttorre, 2009).
The loss of the queen probably lifts an inhibition for already
fertile individuals which very quickly start competing to reproduce.
The highest motivation for ghting 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
ario, 2009),
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
ghting behaviour, the social system stabilizes again with the se-
lection of a new reproductive individual and the disappearance of
agonistic interactions.
To our knowledge, this is the rst study to monitor the devel-
opment of the chemical prole 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 conict and
ensure division of reproductive and ergonomic tasks inside
orphaned colonies to allow the production of males.
Acknowledgments
We thank Ronara De Souza Ferreira-Ch^
aline and R
emi Gout-
tefarde for collecting ants, Chlo
e Leroy for chemical analyses,
Chantal Poteaux-L
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
esil).
N.C. was funded by the CNPq (chamada universal 458736/2014-7,
bolsa PQ (311790/2017-8) and CAPES (PROEX Psicologia Experi-
mental 2016/1964).
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Appendix
1
0
–1
–2
–1
0
1
2
3
4–1
0
1
PCO1 (76.8% of total variation)
PCO2 (7.2% of total variation)
PCO3 (3.7% of total variation)
Colon
y
1
8
18
20
Figure A1. Principal coordinate analysis (PCO) of the chemical proles 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
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4 000 000
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1e+07
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2.6e+07
2.8e+07
3e+07
3.2e+07
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3.6e+07
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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
Time
Abundance
(a)
(b)
Figure A2. Representative examples of the chemical proles from (a) colony 1 (chemotype A) and (b) colony 18 (chemotype B).
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140 137
–10
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–0.03
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2 4 6 8 10 12 14
–10
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020 40 60 80 100 120 140
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Mean deviation per colony of n-C23
Hierarchical rank on day 10
No. of fights won on days 1 and 2
No. of fi
g
hts won on da
y
s 1 - 10
(a)
(b)
(c)
After Before
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
(n-C
23
) 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 ghts won on days 1 and 2, (c)
the total number of ghts won from day 1 to day 10, (d) the percentage of ghts won on days 1 and 2, (e) the total percentage of ghts won from day 1 to day 10, (f) the number of
ghts individuals were involved in on days 1 and 2 and (g) the total number of ghts individuals were involved in from day 1 to day 10.
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140138
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–5
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%Fights won on days 1 and 2
(d)
–10
–5
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–0.03
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–0.01
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%Fights won on days 1 - 10
(e)
–10
–5
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–0.01
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0.02
0.03
No. of fights individual involved in on days 1 and 2
(f)
100
806040
200 100
806040
200
100806040200 100806040200
150100500 150100500
Mean deviation per colony of n-C23
–10
–5
0
5
10
15
–0.03
–0.02
–0.01
0
0.01
0.02
0.03
050 100 150
050 100 150
No. of fi
g
hts individual involved in on da
y
s 1 - 10
(g)
Figure A3. (continued).
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140 139
Table A1
Identication 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
17
(internal standard)
2 6.532 0.05 268 n-C
19
3 6.915 0.08 282 n-C
20
4 7.265 0.7 294 C
21:1
5 7.399 14.85 296 n-C
21
6 7.563 0.06 140 196 295 9-MeC
21
7 7.694 0.07 70 267 295 4-MeC
21
8 7.785 1.41 308 C
22:1
9 7.906 0.97 310 n-C
22
10 8.139 0.07 169 182 309 11-MeC
22
11 8.523 62.42 322 C
23:2
þC
23:1
12 8.633 11.13 324 n-C
23
13 8.847 0.19 168 196 323 11-MeC
23
14 8.962 0.16 85 252 281 323 5-MeC
23
15 9.167 0.09 336 C
24:1
16 9.371 0.07 338 n-C
24
17 10.135 0.99 350 C
25:1
18 10.392 1.4 352 n-C
25
19 11.606 0.14 366 n-C
26
20 12.756 0.04 378 C
27:1
21 13.168 2.45 380 n-C
27
22 14.374 0.12 394 n-C
28
23 14.824 0.33 365 393 2-MeC
28
24 14.918 0.06 406 C
29:1
25 15.046 0.83 408 n-C
29
26 15.225 0.24 168 196 252 281 407 11-16-diMeC
28
27 15.961 0.2 393 421 2-MeC
30
28 16.065 0.04 434 C
31:1
29 16.174 0.09 436 n-C
31
30 16.352 0.32 168 196 224 252 281 309 435 11-13-15-MeC
31
31 17.634 0.44 168 308 337 463 11-MeC
33
Table A2
Analysis of similarity between the chemical proles 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.
Table A3
Spearman correlations (with BonferronieHolm adjustment for multiple compari-
sons) between the fertility index and the compounds present in the chemical prole
after orphaning of colonies 1, 8 and 20 (N¼44)
Cuticular hydrocarbons r
S
P
0
n-C
19
¡0.51 0.009
n-C
20
0.15 1
C
21:1
0.23 1
n-C
21
0.18 1
9-MeC
21
0.28 1
4-MeC
21
0.44 0.05
C
22:1
0.07 1
n-C
22
0.48 0.023
11-MeC
22
0.41 0.14
C
23:2
þC
23:1
0.27 1
n-C
23
0.63 1.5e-4
11-MeC
23
0.07 1
5-MeC
23
0.61 2.9e-4
C
24:1
0.34 0.42
n-C
24
0.3 1
C
25:1
0.45 0.05
n-C
25
0.48 0.03
n-C
26
0.27 1
C
27:1
0.01 1
n-C
27
0.41 0.13
n-C
28
0.16 1
2-MeC
28
0.08 1
C
29:1
0.22 1
n-C
29
0.24 1
11-16-diMeC
28
0.12 1
2-MeC
30
0.05 1
C
31:1
0.11 1
n-C
31
0.22 1
11-13-15-MeC
31
0.05 1
11-MeC
33
0.06 1
For colony 18, only the compound n-C
27
was correlated with fertility (r
S
¼0.5, N¼
15, P¼0.057). To compensate for multiple comparisons, Pvalues were adjusted to
P
0
values following Holm (1979). Signicant values are highlighted in bold.
R. Honorio et al. / Animal Behaviour 157 (2019) 129e140140
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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.