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Behavioral Ecology
doi:10.1093/beheco/arr105
Advance Access publication 18 July 2011
Original Article
Cuticular hydrocarbons as potential kin
recognition cues in a subsocial spider
Lena Grinsted,
a,b
Trine Bilde,
b
and Patrizia d’Ettorre
a,c
a
Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15,
2100 København Ø, Denmark,
b
Ecology and Genetics, Department of Biological Sciences, Aarhus
University, Ny Munkegade 116, Building 1540, 8000 A
˚rhus C, Denmark, and
c
Laboratory of
Experimental and Comparative Ethology, University of Paris 13, 99 avenue J.-B. Cle´ment, 93430
Villetaneuse, France
In animal societies, recognition of group members and relatives is an important trait for the evolution and maintenance of social
behavior. In eusocial insects, nest mate recognition is based on cuticular hydrocarbons and allows colony members to reject
competitors and parasites. The study of recognition cues in subsocial species can provide insights into evolutionary pathways
leading to permanent sociality and kin-selected benefits of cooperation. In subsocial spiders, empirical evidence suggests the
existence of both kin recognition and benefits of cooperating with kin, whereas the cues for kin recognition have yet to be
identified. However, cuticular hydrocarbons have been proposed to be involved in regulation of tolerance and interattraction in
spider sociality. Here, we show that subsocial Stegodyphus lineatus spiderlings have cuticular hydrocarbon profiles that are sibling-
group specific, making cuticular hydrocarbons candidates for kin recognition cues. Our behavioral assays indicate that spider-
lings can discriminate between cuticular cues from kin and nonkin: In a choice set-up, spiderlings more often chose to reside
near cuticular chemical extracts of siblings compared with nonsiblings. Furthermore, we show that cuticular chemical compo-
sition changes during development, especially around the stage of dispersal, supporting the hypothesis that cuticular cues are
involved in regulating conspecific tolerance levels. Lastly, our results indicate that the potential kin recognition cues
might be branched alkanes that are influenced very little by rearing conditions and may be genetically determined. This
indicates that a specific group of cuticular chemicals, namely branched alkanes, could have evolved to act as social recognition
cues in both insects and spiders. Key words: chemical communication, cooperation, Eresidae, evolution of sociality, kin discrim-
ination. [Behav Ecol 22:1187–1194 (2011)]
INTRODUCTION
Social groups share valuable resources that can be exploited
both from the inside by selfish individuals and from the out-
side by competitors and parasites. To face the latter, recogni-
tion systems enabling discrimination of group members
from strangers have evolved and been exhaustively docu-
mented in many social animals (Fletcher and Michener
1987;Vander Meer and Morel 1998;Lenoir et al. 1999;Liebert
and Starks 2004). Insect societies, for instance, show complex
recognition abilities based on the perception of the variation
encoded in multicomponent chemical cues. Individuals be-
longing to the same colony share a common colony odor,
which usually consists of a specific blend of cuticular hydrocar-
bons whose proportions vary among colonies. Discrimination
occurs by comparing the chemical profile of an encountered
individual with the own colony odor, with dissimilarity leading
to rejection. The neural substrate responsible of information
coding has yet to be identified, although there is some evidence
that the process might be decentralized, that is, not necessarily
involving the higher brain centers (van Zweden and d’Ettorre
2010). Discrimination between nest mates and nonnest mates
allows social insects to share the benefits of cooperation only
with colony members, which are usually closely related (Howard
and Blomquist 2005;d’Ettorre and Lenoir 2010). Within
groups, individuals that exploit a common resource may be
prone to cheating through the ‘‘tragedy of the commons’’
(Hardin 1968), which results in a reduction in group fitness
(Rankin et al. 2007). This social dilemma can be resolved by
directing cooperation toward close kin because selfish acts
toward kin are costly owing to the loss of inclusive fitness that
could otherwise be realized through the propagation of
shared genes by related group members (Hamilton 1964).
Cooperation among relatives can evolve through the ability
to discriminate group members based on genetic relatedness
(Hamilton 1987).
Social animals that show preferential cooperation with kin
are ideal for studying the underlying mechanisms of kin recog-
nition, and the social spiders provide a good example. In con-
trast to the majority of spiders that are solitary hunters
intolerant of both hetero- and conspecifics, a few genera of spi-
ders have evolved cooperative group living characterized by
tolerance and interattraction (Kullmann 1972). These traits
require the ability to recognize, directly or indirectly, individ-
uals with whom to cooperate. Social spiders cooperate in
feeding, prey capture, and brood care but seem to lack re-
productive division of labor and overlapping generations
(Lubin and Bilde 2007), which are 2 key features characteriz-
ing eusocial societies. Spider sociality evolved by the subsocial
pathway, namely via extended maternal care and a prolonged
Address correspondence to L. Grinsted. E-mail: lena.grinsted
@biology.au.dk
Received 13 January 2011; revised 25 May 2011; accepted 25
May 2011.
The Author 2011. Published by Oxford University Press on behalf of
the International Society for Behavioral Ecology. All rights reserved.
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coexistence of spiderlings in the maternal nest where the
young cooperate in nest activities, such as foraging, before
dispersal (Avile´s 1997;Agnarsson et al. 2006).
Empirical evidence shows that subsocial spiders benefit from
cooperation among kin, and there is good evidence for kin dis-
crimination abilities. Within the genus Stegodyphus,Ruch et al.
(2009) found that subsocial S. tentoriicola young extracted
more mass from prey when feeding in groups of siblings com-
pared with nonsiblings, and Schneider and Bilde (2008)
found that subsocial Stegodyphus lineatus young foraging in
sibling-groups showed higher feeding efficiency and higher
individual weight gain compared with both familiar and un-
familiar nonsib groups. The use of cross-fostering in the latter
study suggests that kin-mediated benefits of cooperation re-
sulted from genetic similarity within the group and hence true
kin recognition, rather than through association with familiar
individuals. Indeed, kin recognition has been shown in the
permanently social spiders Diaea ergandros (Evans 1999) and
Delena cancerides (Beavis et al. 2007), as well as in the subsocial
S. lineatus (Bilde and Lubin 2001) in experiments where juve-
niles, when starved, showed higher levels of cannibalism
toward nonsiblings than toward siblings.
The recognition systems and cues that spiders use for kin
discrimination have not previously, to our knowledge, been
investigated. However, Pasquet et al. (1997) found that social
Anelosimus eximius showed quantitative differences in cuticu-
lar chemical profiles between colonies, suggesting that cutic-
ular chemical composition in social spiders may contain
some information of colony identity, despite a lack of obvi-
ous discrimination against noncolony members in this spe-
cies. Furthermore, a number of studies have suggested that
important traits in social spider evolution, namely tolerance
and interattraction of group members, may be based on cu-
ticular chemical cues, similar to the social insects. Kullmann
(1972) found that chemosensory perception alone allows
social Stegodyphus species to discriminate between prey and
conspecifics and suggested that tolerance is based on chem-
ical cues. Similarly, cuticular chemicals are thought to act as
cues in regulating agonistic versus tolerant behavior in Tege-
naria atrica (Trabalon et al. 1996,1998;Pourie and Trabalon
1999;Pourie et al. 2005). Young T. atrica stay for a while in
the maternal nest and are tolerated by their mother until
they disperse. The composition of cuticular compounds in
these spiders changes during development (Trabalon et al.
1996), and cuticular extracts from postdispersal solitary
young induce higher levels of aggression in adult females
compared with cuticular extracts from predispersal gregari-
ous young (Pourie et al. 2005). Hence, evolutionary changes
shaping the cuticular compound composition according to
age could be important steps in the evolution of spider soci-
ality, prolonging nonaggressive life stages by allowing recog-
nition of individuals that belong to the maternal nest
(Pourie and Trabalon 2001).
In the present study, we investigated the possible role of cu-
ticular hydrocarbons in communication and kin recognition
in the spider S. lineatus that shows adaptive kin discrimination
abilities (Bilde and Lubin 2001;Schneider and Bilde 2008).
Our aim was to address the following questions: 1) Can cu-
ticular hydrocarbons act as family-specific recognition cues in
cooperative predispersal spiderlings? 2) Do the cuticular hy-
drocarbon profiles of spiderlings change during develop-
ment, indicating that these chemical compounds could act
as cues in regulating tolerance levels? 3) Are cuticular chem-
ical profiles influenced by rearing conditions? Potential cues
for true kin recognition should be determined mostly by
genetic factors, independently of environmental factors, such
as rearing conditions. To address these questions, we com-
pared the cuticular hydrocarbon profiles of same-aged pre-
dispersal spiderlings from family groups of siblings and tested
whether families could be differentiated based on variation
in chemical profiles. In behavioral assays, we tested whether
spiderlings could discriminate between cuticular extracts of
siblings and nonsiblings and thus use cuticular chemicals as
recognition cues. We also characterized and compared chem-
ical profiles in 10-day interval age groups through the first 50
days of development, covering the predispersal and dispersal
stages of spiderlings. Finally, we tested whether spiderlings
bred in the laboratory showed cuticular chemical patterns
that differed from those of field-bred spiderlings to inves-
tigate environmental influence on cuticular hydrocarbon
composition.
MATERIALS AND METHODS
Study organism
The genus Stegodyphus (Eresidae) includes 18 subsocial and 3
social spider species (Kraus and Kraus 1988). Phylogenetic
evidence indicates that the social species represent 3 indepen-
dent events of sociogenesis, making Stegodyphus a prime genus
for the study of evolution of spider sociality (Kraus and Kraus
1988;Johannesen et al. 2007). The subsocial S. lineatus is
thought to resemble a transitional stage between solitary
and social spiders because it shows considerable behavioral
plasticity in social tendencies. This species is able to extend
the period of tolerance and cooperation under circumstances
of unlimited food availability or prevention from dispersal
(Schneider 1995).
Stegodyphus lineatus inhabit arid environments in Southern
Europe, Northern Africa, the Mediterranean islands, and
across to Kazakhstan and West Asia (Kraus and Kraus 1988).
In Israel, S. lineatus spiders are found in dry river beds where
they build capture webs and tubular retreats in low shrubs.
Males start maturing in March, females in April or May, and
the mating season and egg-laying period may extend into
June (Bilde et al. 2005). Eggs are laid approximately 4 weeks
after mating, and clutches of 40–140 eggs hatch after another
4 weeks. The mother spider feeds her offspring by regurgita-
tion, and after about 2 weeks of maternal care, spiderlings
consume their mother (matriphagy). Hereafter follows a pe-
riod of group living and cooperation in the natal nest for
approximately 4 weeks until juvenile dispersal is gradually
initiated (Kullmann 1972).
Collection and housing
In April and June 2008, mated S. lineatus females were col-
lected in Israel from 2 different populations located approx-
imately 70 km apart: near Lehavim (lat 3121.800#N, long 34
50.000#E) and near Kfar Adumim (lat 3149.000#N, long 35
21.200#E), which is a settlement located within the occupied
Palestinian territories, and young from these females were
used for behavioral assays.
In March 2009, juvenile and subadult S. lineatus of both
genders were collected in 2 different populations located
about 30 km apart, near the villages Lehavim and Har Amasa
(lat 3120.500#N, long 3507.100#E). When sexually mature,
females were mated to same population males in the laboratory
by placing a male with a female for 2 days, assuming mating
would take place (Schneider and Lubin 1996). The females
produced young that we henceforth refer to as ‘laboratory-bred’
young used for cuticular chemical analyses.
In June 2009, females with brood (eggs or hatched young)
were collected in Lehavim. The offspring of these females are
henceforth referred to as ‘field-bred’ young and these were
used for both chemical analyses and behavioral assays. (For
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a schematic overview of time and place of collections and
allocation of spiders for experiments, see Supplementary
Material.)
Each collected spider was kept in a plastic box (size in mm:
80 360 350) with a mesh on the side and provided with its
original tubular silk retreat. Boxes allowed for construction of
small prey capture webs. Spiders were kept at room tempera-
ture (25 65C) with a natural day/night light cycle, and
water was sprayed in the boxes twice a week. Spiders were
fed nymphs of small migratory locusts (Locusta migratoria)
and crickets (Acheta domestica) 3 times a week until spiderlings
emerged. After matriphagy (2 to 3 weeks after hatching), spi-
derlings were fed fruit flies (Drosophila melanogaster) twice
a week.
Chemical analysis
To examine whether cuticular chemical composition in
S. lineatus spiderlings varies sufficiently among genetic fami-
lies to contain potential cues for kin discrimination, we quan-
tified variation in cuticular hydrocarbon profiles among
sibling groups of same-aged spiderlings. Furthermore, we an-
alyzed the chemical profile of individual spiderlings at differ-
ent ages in order to characterize possible developmental
changes in compound composition at a temporal scale.
For the analysis of between-family variation of the chemical
profile, 6 field-bred spiderlings of the same age (30 62 days)
from each of 6 different families collected in 2009 were indi-
vidually sampled (36 individuals in total). At this age, matriph-
agy had usually occurred, and spiderlings were relatively mobile
yet still in their cooperative stage before dispersal. A family
refers to a group of siblings. In order to analyze developmental
changes in the chemical profile, 2 or 3 laboratory-bred spider-
lings from each of 5 different families were sampled at intervals
of 10 days, starting at the age of 10 days and ending when a nest
was depleted or at age 50 d at which age young have usually
dispersed (a total of 49 individuals). All same-aged spiderlings
were pooled into each age group in the subsequent statistical
analysis. This sampling method ensured representation of
chemical profiles from 5 discrete age groups covering the full
social cooperative period and the stage of dispersal.
All sampled spiderlings were freeze killed by placing them
in a 218 C freezer for 3 h. Spiders were then immersed
individually in clean glass vials (Supelco) containing 100 ll
of pentane (HPLC grade; Sigma-Aldrich, Chromasolv) for
5 min. The animal was removed, the solvent was allowed
to evaporate under a fume hood at room temperature, and
the total cuticular extract from each animal was stored at
218 C until chemical analysis. For analysis, the extract was
redissolved in 10 ll of pentane, and 4 ll of this mixture was
injected into an Agilent Technologies 6890N gas chromato-
graph (GC, capillary column: HP5MS 30 m 3250 lm30.25
lm; split–splitless injector; carrying gas: helium at 1 ml/min).
The initial temperature was 70 C and was increased at a rate
of 30 C/min to 230 C, then to 300 Cat4C/min, then to
320 Cat10C/min, and held for 6 min. The GC was coupled
to a 5975 Agilent Technologies Mass Spectrometer (MS, 70 eV
electron impact ionization). Chemical compounds were identi-
fied on the basis of their retention time (compared with stand-
ards) and by inspecting diagnostic ions in their mass spectra.
Behavioral assay
Stegodyphus lineatus spiderlings show no aggression or other
obvious discriminatory behavior toward nonsiblings in their
gregarious phase. Accordingly, choice tests rather than ag-
gression tests were conducted in order to examine whether
cuticular chemicals can be used as kin recognition cues by
S. lineatus spiderlings. We recorded where spiderlings settled
when they were placed individually in a T-like maze and pre-
sented with cuticular chemical extracts from siblings and non-
siblings. We expected spiderlings to be more attracted to
siblings (i.e., sibling chemical extract) than to unrelated con-
specifics due to the documented kin-mediated fitness benefits
in this species (Schneider and Bilde 2008).
Mazes were constructed from three 20-ml plastic vials (40 3
27 mm each) glued together constituting a middle compart-
ment with access to 2 choice compartments through holes
drilled in the plastic (see Supplementary Material for a dia-
gram of the maze). In each choice compartment of a clean
maze, we placed a clean pentane-washed cotton ball (diame-
ter 6–8 mm) to which we applied the cuticular chemical
extract of 4 siblings to one side and 4 nonsiblings to the
opposite side. Chemical extracts were made by washing 4
30 62 days old spiderlings in 200 ll of pentane for 5 min.
After application of chemical extracts, the solvent was
allowed to evaporate for 1 h, allowing only the cuticular
compounds to remain on the cotton balls. Hereafter, one
focal spiderling was placed in the middle compartment and
left for one night (from 17:30 61 h to the next morning at
10:00 61 h) after which settlement choice was recorded. Most
spiders settled directly on or in close proximity of a cotton ball.
Focal spiders were tested only once, at the age of 30 62d.
Similarly, cotton balls containing chemical extracts were used
only once. Mazes were washed in water and odorless detergent
before use to eliminate chemical cues from previous trials.
In October 2008, 32 spiderlings were tested in the laboratory
with a natural day/night light cycle. Nonsibling extracts orig-
inated from populations different from the focal individual
and were placed randomly in the right or left compartment
in each maze. Seven spiderlings did not make a choice (i.e.,
they were still residing in the middle compartment in the
morning) and were excluded from further analysis. In July
and August 2009, 29 spiderlings were tested using the same
apparatus but in a dark room to eliminate any other possible
cues, and nonsibling extracts, originating from the same pop-
ulation as the focal individual, were placed to the right in half
of the trials and to the left in the other half. All 29 spiderlings
made a choice in the tests performed in 2009.
Statistical analysis
The 2 data sets on behavioral assays were compared with Fish-
er’s exact test. Subsequently, we pooled the data sets and an-
alyzed for effect of kinship on settling choice using the
binomial test with the null hypothesis of equal preference
for sibling and nonsibling cuticular extract.
In order to analyze the chemical profile of spiderlings, 38
regularly occurring gas chromatography–mass spectrometry
peaks, representing identified hydrocarbons, were integrated
using Agilent Technologies ChemStation software. The nor-
malized peak areas within each profile were calculated accord-
ing to Aitchinson (1986) using the formula:
Zij ¼lnYij
gYj;
where Z
ij
is the transformed area of peak ifor individual j;Y
ij
is the area of peak ifor individual j; and g(Y
j
) is the geometric
mean of the areas of all peaks for individual j.
These normalized peak areas were used as variables in prin-
cipal component analyses (PCAs). The reduced number of var-
iables (principal components [PCs]) were used in subsequent
discriminant analyses (DAs), performed by STATIS TI CA 7. 1 (Stat-
Soft Inc.). For a more conservative test of chemical differences
between family groups, we performed a permutation test using
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the MASS package (Venables and Ripley 2002) in R (http://
www.R-project.org) according to Nehring et al. (2010).After
reducing the number of variables to 8 PCs, we randomly allo-
cated the individual profiles to 6 arbitrary groups in a DA
10 000 times. If the success rate (i.e., the percentage of cor-
rectly assigned samples to their group) from the original DA
was higher than the upper 95% confidence interval from the
permutation test, the family groups had significantly different
chemical profiles.
Chemical compounds that vary more between groups of
individuals than within groups can carry information about
group identity and hence may represent potential recognition
cues. In order to identify the best candidates for kin recogni-
tion cues, the diagnostic power (DP) of each GC–MS peak was
calculated in accordance with van Zweden et al. (2009). Peaks
with higher-than-average DP were considered as ‘‘high DP’’
compounds.
As a measure for chemical distance, squared Mahalanobis
distances (SMDs) between age groups were calculated to deter-
mine at which developmental stage (i.e., age interval), the
largest change in cuticular compound composition occurred.
The SMD measures normalized distances between points in
a multivariate space; it differs from the simple Euclidean dis-
tance by taking into account the covariance among the varia-
bles in calculating distances and by being independent of the
scale of measurements. A mixed-model analysis of variance
(ANOVA), followed by a post hoc Tukey Honestly Significant
Difference (HSD) test, was used to compare chemical changes
at different developmental stages, with age interval (e.g., 10–20
days or 20–30 days) as a fixed factor and family as a random
factor. Chemical distances between age groups (i.e., the chem-
ical changes for each age interval) were calculated as SMDs
from each individual sample to the centroid of the following
age group (10 days older) in order to determine which 10-day
age interval was associated with the largest change in cuticular
chemical composition. For instance, the SMD from age 20–30
days was calculated as the mean of the SMDs from each of the
samples from age group 20 days to the centroid of age group
30 days.
RESULTS
The cuticular chemical profile of S. lineatus was characterized by
38 regularly occurring peaks, which could be identified consis-
tently as hydrocarbons. These hydrocarbons belonged to 3 dif-
ferent classes: linear alkanes and mono- and di-methyl–branched
Figure 1
A gas chromatogram showing
the cuticular hydrocarbon pro-
file of a 30-day-old Stegodyphus
lineatus spiderling. The panel
shows peak identification and
the variation in chemical pro-
files between family groups ex-
pressed as the DP of each
compound.
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alkanes with chain lengths ranging between n-C
23
and n-C
35
(Figure 1). Unlike studies of the cuticular chemical profile of
solitary T. atrica (Trabalon et al. 1996;Pourie et al. 2005), we did
not find any fatty acids on the cuticle of S. lineatus young.
Between-family variation of the chemical profile and
behavioral assays
Field-bred spiderlings showed family-specific cuticular hydro-
carbon profiles (Figure 2). Chemical variation among families
was due to quantitative differences between chemical pro-
files, that is, differences in relative abundances of the same
set of compounds. A PCA produced 8 PCs with eigenvalues
greater than 1, accounting for 89.0% of the total variance.
A DA on the 8 PCs significantly differentiated the 6 families
(Wilks’ k¼0.0000009; F
40,103
¼60.4; P,0.00001) and 100%
of the samples were correctly assigned to their family (i.e.,
the success rate was 100%). The highest success rate from the
permutation test with random groups was 80.6% and the
mean 52.2%. The upper 95% confidence interval was a success
rate of 63.9%, confirming the statistical significance of our
100% success rate from the original DA using the real family
groups.
The DP of the different compounds ranged from 1.06 to
4.08, with a DP
average
¼1.88. All 16 peaks with high DP values
represented branched alkanes (with the exception of n-C
23
),
and all the 7 di-methyl alkanes present in the chemical profile
had high DP values. (A list of all compounds ranked accord-
ing to their DP is available as Supplementary Data.) The peak
with the highest DP was peak number 16 (Figure 1): a mixture
of 7,11-diMeC
27
and 3-MeC
27
(DP
peak 16
¼4.08).
Families could be significantly separated based on individual
cuticular hydrocarbon composition also for laboratory-bred
spiderlings. A DA significantly separated the families, correctly
assigning 100% of the samples to their family, even though
individuals of different ages were included for each family
(Wilks’ k¼0.00452; F
28,138
¼17.2; P,0.00001). The DP of
compounds were generally low, ranging from 0.15 to 0.39,
with a DP
average
¼0.23. However, the peak with the highest
DP was again peak number 16: 7,11-diMeC
27
and 3-MeC
27
(DP
peak 16
¼0.39), suggesting that these compounds in par-
ticular may contain information of family identity, indepen-
dent of age and rearing conditions of spiderlings.
The behavioral results indicated that spiderlings were able to
use the between-family variation in chemical profiles as cues for
discrimination between siblings and nonsiblings. There was no
difference between the 2 data sets from different years with
respect to settling choice (Fisher’s exact test, P¼0.395, de-
grees of freedom [df] ¼1); therefore we pooled the data. We
found a significant effect of kinship on choice in the mazes as
spiderlings preferred to reside near sibling cuticular extract
compared with nonsibling extract (binomial test; P¼0.0402,
df ¼1, N¼54, Figure 3).
Developmental changes of the chemical profile
The composition (i.e., relative abundances) of hydrocarbons
in the cuticular profile of spiderlings changed significantly
with age (see Supplementary Material for exact changes in
relative abundance for each compound according to age).
A PCA was conducted on 6–14 laboratory-bred individuals
from each of 5 age groups, from age 10 to 50 days (a total
of 49 individuals from 5 different families). The PCA pro-
duced 7 PCs with eigenvalues higher than 1, which together
explained 87.9% of the total variance. A subsequent DA
significantly separated the age groups (Wilks’ k¼0.0183;
F
28,138
¼10.1; P,0.00001) and correctly assigned 83.7% of
the samples to their age group (see Supplementary Material
for a graph of the DA).
Chemical distances between adjacent age groups, calculated
as Mahalanobis distances, differed significantly among age
intervals (mixed-model ANOVA; age interval [fixed effect]:
F¼38.03, P,0.0001, df ¼3; family [random effect]: Z¼
0.26, P¼0.397, N¼4). The chemical distance increased
substantially in age interval 40–50 days (see Supplementary
Material for a graph showing the chemical distances between
age groups). This age interval, 40–50 days, differed signifi-
cantly from each of the other 3 age intervals (Tukey HSD;
P,0.0001 for each of the 3 comparisons), whereas the 3
early age intervals (10–20, 20–30, and 30–40 days) did not
differ significantly from each other (P.0.05 for each of
the 3 comparisons).
Influence of rearing conditions on the chemical profile
Spiderlings bred in the laboratory could be significantly sepa-
rated from those bred in the field based on their cuticular hy-
drocarbon profiles (see Supplementary Material for a graph of
the PCA). Chemical data from 9 laboratory-bred 30-day
Figure 2
Between-family variation of the chemical profiles: a plot of the first 2
roots of the DA based on cuticular hydrocarbons from 30-day-old
field-bred spiderlings (36 individual chemical profiles in total). Each
symbol represents a family, that is, a group of siblings from the
Lehavim population. The percentage of the variance explained by
each root is given in parenthesis.
Figure 3
Behavioral assays: results from choice tests performed in 2008 (black)
and 2009 (gray), showing the number of spiderlings residing by
cuticular chemical extracts of siblings and nonsiblings.
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spiderlings from 3 different families (available from the ‘‘de-
velopmental chemical changes’’ assay) and 36 field-bred 30-
day spiderlings from 6 different families (from the ‘‘between-
family chemical variation’’ assay) were used for this analysis. A
PCA produced 7 PCs with eigenvalues higher than 1 that
together explained 87.5% of the total variance. The main
function (PC 1; explaining 44.1% of the variance) clearly sep-
arated the 2 groups. A subsequent DA significantly separated
the laboratory and field groups (Wilks’ k¼0.0240; F
7,37
¼
215; P,0.00001), assigning 100% correctly to their group.
Twenty peaks had high factor loadings on PC 1 (factor load-
ing .j0.70j) and these were all linear or mono-methyl alkanes.
The compounds with relatively high abundance in field-bred
young were all shorter hydrocarbons (chain lengths between
24 and 27 carbon atoms), whereas the compounds more
abundant on the laboratory-bred spiders were longer hydro-
carbons (chain lengths between 28 and 35 carbon atoms).
Only 5 of the 20 peaks that were important in separating field-
and laboratory-bred young were peaks that had high DP in
separating families (see between-family chemical variation re-
sults). All di-methyl alkanes (including peak number 16)
had low factor loadings on PC 1 (all factor loadings ,j0.52j)
in the analysis on rearing conditions. A list of all compounds
ranked according to their factor loadings is available in the
Supplementary Material.
DISCUSSION
Our study shows that young S. lineatus have complex cuticular
chemical profiles consisting mainly of long-chain hydrocar-
bons, namely linear and branched alkanes with 1 or 2 methyl
groups. We found that the cuticular chemical profiles of pre-
dispersal spiderlings varied significantly among sibling
groups, regardless of age and rearing conditions. This sug-
gests that cuticular hydrocarbon profiles carry information
about family identity and thus can potentially be used as cues
in kin recognition. Our behavioral results support this hypoth-
esis, indicating that spiderlings can discriminate between kin
and nonkin solely by means of cuticular hydrocarbon cues. We
also show that the cuticular hydrocarbon profiles of S. lineatus
spiderlings change significantly at the age of dispersal, which
supports the hypothesis that cuticular chemicals may act as
cues regulating the level of tolerance toward siblings in the
nest (Trabalon et al. 1996;Pourie and Trabalon 2001). Fur-
thermore, we show that spiderlings bred under different con-
ditions express significantly different hydrocarbon profiles,
yet the potential kin recognition cues identified by our anal-
ysis (a subset of branched alkanes) appear to be less affected
by environmental conditions, indicating that they might be
determined mostly by genetic factors. Thus, cuticular hydro-
carbon cues might have the potential to be used for genetic
kin recognition.
Group living and cooperation are associated with certain
costs, including having to share limited resources. In S. line-
atus, costs of feeding in groups involve decreased individual
growth rates due to competition for prey (Schneider 1995).
However, cooperating with genetic kin increases feeding effi-
ciency and growth rates, thereby decreasing costs of coopera-
tion (Schneider and Bilde 2008). Direct benefits of
cooperating with kin exerts a selective pressure on the ability
to distinguish between family and nonrelatives, and kin rec-
ognition has indeed been shown in S. lineatus in cannibalism
experiments (Bilde and Lubin 2001). Our study shows that
spiderlings have family-specific cuticular hydrocarbon pro-
files, that is, profiles specific enough for them to potentially
be used in recognition of siblings. Indeed, when given the
choice of cuticular chemical cues from siblings versus non-
siblings, spiderlings preferred to reside by sibling cues.
Hence, our study indicates that the cues S. lineatus uses for
kin recognition may be cuticular hydrocarbons. True kin rec-
ognition based on cuticular hydrocarbons has previously been
found in cockroaches (Blattella germanica) that discriminate
between sibling and nonsibling, independent of previous
social experience (Lihoreau and Rivault 2009), and chemical
cues may be involved in genetic kin recognition in the
primitively social sweat bees (Lassioglossum zephyrum) that ac-
cept unfamiliar bees in their nest at an increasing rate with
increasing genetic similarity (Greenberg 1979).
Arthropods have a large range of different cuticular hydro-
carbons present on their cuticle. The original purpose of these
hydrocarbons was to protect the animal from desiccation, but
a subset of these compounds have evolved to act as recognition
cues in inter- and intraspecific communication in different
arthropods (Gibbs et al. 1991;Lahav et al. 1999;Akino et al.
2004;Dani et al. 2005;d’Ettorre and Moore 2008). Because
cuticular hydrocarbons have been shown to play a role in
communication in both social and non-social insects, such
as fruit flies (Ferveur 2005), as well as in some spiders (Pourie
et al. 2005), the communication function of these chemicals
may be quite old. Nest mates recognition in social insects
based on cuticular hydrocarbons often relies on branched
alkanes and alkenes as recognition cues, whereas linear alka-
nes have little influence on nest mate recognition (Dani et al.
2001,2005;van Zweden and Dreier 2009). Interestingly, our
results indicate that this might be the case also in subsocial
spiders. We found that all di-methyl–branched alkanes pres-
ent on the cuticle of spiderlings had high DP in separating
families, as did several mono-methyl alkanes. Almost all linear
alkanes had low DP and therefore contained little information
of family identity. Although we do not have any direct exper-
imental evidence that branched alkanes play a key role in
communication, our data indicate that linear alkanes might
be less important in social recognition in S. lineatus, similarly
to what has been found in many social insects where alkenes
and branched alkanes, rather than linear alkanes, are impor-
tant recognition cues.
Spiders communicate with chemicals during many different
activities in life, such as identification of prey, brood (Pourie
et al. 2005), and sexual partners (Schulz and Toft 1993), and
possibly chemical cues play a role in behavioral transitions
like the one from gregarious to solitary living (Pourie and
Trabalon 2001). Our study shows that the cuticular hydrocar-
bon profile of S. lineatus changes significantly during the first
50 days of development. The relative proportions of longer
alkanes increased with age. Similarly, some ants produce
heavier (i.e., longer) hydrocarbons during specific seasons
(e.g., Camponotus aethiops in van Zweden and Dreier (2009)).
Heavier compounds are less volatile and have higher melting
points and are therefore produced more by some insects in
seasons or geographical areas that are dry and hot, in order to
provide better protection from desiccation (Gibbs et al. 1991).
Thus, the increase with age in the abundance of longer hydro-
carbons in S. lineatus could occur mainly to prepare the cuti-
cle for the risky dispersal stage, at which the spiders may be
exposed to different environmental conditions in their arid
habitat than those they have experienced in the maternal
nest.
Nevertheless, some of these long-chained compounds may
be involved in triggering the onset of developmentally specific
behaviors, such as matriphagy or dispersal, by acting as cues
for the regulation of tolerance levels and interattraction. In-
deed, we found a dramatic change in spiderlings’ cuticular
compound composition between age 40 and 50 days, where
dispersal often occurs. Hence, it is possible that developmen-
tal changes in cuticular compound composition play a role in
decreased acceptance and tolerance of nest mates, leading to
1192 Behavioral Ecology
at Aarhus Universitets Biblioteker / Aarhus University Libraries on October 25, 2011http://beheco.oxfordjournals.org/Downloaded from
dispersal of young, similar to in T. atrica (Trabalon et al.
1996). Evolutionary modifications of developmental changes
in chemical composition like these could have been impor-
tant in extending the cooperative stage of tolerance toward
siblings from temporary, in subsocial spiders, to permanent in
social spider species.
Both genetic and environmental factors can contribute to
the composition of the cuticular chemical profile of arthro-
pods, and diet, especially, can influence the ratios of com-
pounds in a chemical blend (Jutsum et al. 1979;Liang and
Silverman 2000;van Zweden et al. 2009). Thus, family-specific
cuticular hydrocarbon profiles may exist either simply because
siblings are exposed to a similar environment, because genetic
similarities between siblings are responsible for the cuticular
compound composition, or because of influence by both fac-
tors.
We examined the cuticular hydrocarbon profiles of laboratory-
bred spiderlings whose mothers had been mated in the labora-
tory and of spiderlings from broods collected in the field. Dur-
ing the first stages of life, young spiders are fed by maternal
regurgitation. The liquid food that the young from the field
ingested likely originated from a broad source of prey items
caught by the mother. In contrast, the mothers that were kept
in the laboratory (for a minimum of 2 months longer than the
freshly collected ones) had a uniform diet that could have influ-
enced the diet of their young. This differential diet, together
with other environmental factors, could be the cause of the clear
distinction between the cuticular profiles of field- and labora-
tory-bred spiderlings. Field-bred spiderlings had higher relative
proportions of short linear and mono-methyl–branched alkanes.
The di-methyl alkanes that were important in chemical separa-
tion of families, however, had very low power in separating spi-
derlings bred under the 2 different conditions (some branched
alkanes have been shown to be heritably synthesized in ants, see
van Zweden et al. 2010). This could indicate that the relative
amounts of the potential kin recognition cues in the chemical
profiles are little influenced by the environment and may there-
fore be determined more by genetic factors.
In conclusion, our results show that subsocial S. lineatus
young have complex cuticular hydrocarbon profiles that are
family specific throughout the social stage and change during
development especially at the age of dispersal. Cuticular
chemical profiles can thus carry information of family identity
and developmental stage of spiderlings. Our behavioral re-
sults indicate that spiderlings are able to use the information
encoded in the chemical profiles to recognize siblings. Bio-
assays accounting for genetic relatedness and familiarity are
now needed to investigate what our present results suggest;
that is, kin discrimination in S. lineatus as reported by Bilde
and Lubin (2001) and Schneider and Bilde (2008) may de-
pend on cuticular hydrocarbons as cues.
SUPPLEMENTARY MATERIAL
Supplementary material can be found at http://www.beheco.
oxfordjournals.org/.
FUNDING
This study was supported by the EU Marie Curie Excellence
Grant CODICES-EXT-CT-2004-014202 assigned to P.d’E.
We would like to thank Yael Lubin, Reut Tal and Cristina Tuni for help
with collection and housing of spiders. Thanks to Jelle van Zweden for
help with hydrocarbon identification, Volker Nehring for statistical as-
sistance and Luke Holman for feedback and comments on a previous
version of the manuscript, and the Centre for Social Evolution for a mo-
tivating work environment.
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