|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online March 28, 2008
Journal of Experimental Biology 211, 1249-1256 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.017301
Cuticular hydrocarbons as queen adoption cues in the invasive Argentine ant
Department of Entomology, North Carolina State University, Raleigh, NC 27695-7613, USA
* Author for correspondence (e-mail: jules_silverman{at}ncsu.edu)
Accepted 9 February 2008
 |
Summary |
|---|
 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Key words: Argentine ant, Linepithema humile, nestmate recognition, cuticular hydrocarbons, intraspecific aggression, non-nestmate queen adoption
|
INTRODUCTION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
; Vander Meer and
Morel, 1998). Nestmate recognition in social insects is adaptive
because workers obtain inclusive fitness benefits from aiding nestmates and
discriminating against non-nestmates, provided that nestmates are usually more
closely related to one another than to members of other nearby colonies
(Hölldobler, 1995).
Natural selection should favor the use of cues that optimize discrimination
because recognition errors – rejecting a desirable conspecific or
accepting an undesirable individual – lower the benefits expected from
kin interactions (Lehman and Perrin, 2002). Recognition cues in social insects
and other animals are primarily chemical in nature and perceived by olfaction
or contact chemoreception (Hölldobler
and Michener, 1980; Breed,
1998).
One model for nestmate recognition proposes that individuals discriminate
colony members from non-members by means of a phenotype matching mechanism in
which the phenotype of a newly encountered individual (actor) is compared with
the individual's (reactor) inner learned template
(Lacy and Sherman, 1983).
Phenotypic recognition cues must be reliable, and they originate from either
the environment (diet, nesting substrate), endogenous sources (genetically
determined), or both (Breed and Bennett,
1987; Vander Meer and Morel,
1998). The template represents a constantly changing memory
pattern of the colony's recognition cues, and the process of
cue–template matching guides a behavioral response, usually acceptance
or rejection of the encountered individual
(Reeve, 1989;
Gamboa et al., 1986). In
addition, recognition of phenotypic cues by allele matching
(recognition-allele mechanism), may also occur
(Keller and Ross, 1998). For
example, in fire ants, Gp-9 genotypic compatibility seems to regulate
queen identity and number via production of a distinct chemical label
and formation of a specific exclusionary template based on the allelic variant
possessed by workers and queens (Gotzek
and Ross, 2007).
Appropriate behavioral responses are guided by recognition decision rules
concerning the level of dissimilarity between the template and the phenotypic
cues of the encountered individual (Breed
and Bennett, 1987). Models for decision rules in recognition
include (i) an individualistic model in which individuals retain their own cue
integrity and score other individuals by comparison with themselves, accepting
them based on genotypic similarity; and (ii) a Gestalt model in which cue
transfer occurs among colony members resulting in a unique mixture of chemical
cues (colony `odor'), and individuals are classified as colony members based
upon the degree to which they possess the odour
(Crozier and Dix, 1979;
Getz, 1982;
Crozier, 1987). Also,
decisions may be made according to a recognition threshold so that if the
template–odor match is greater than a minimum similarity threshold (or
below a dissimilarity threshold) the individual is accepted and treated as
nestmate (Gamboa et al., 1986;
Reeve, 1989). Interaction
frequency with foreign conspecifics and the fitness consequences of accepting
or rejecting conspecifics may determine the optimal acceptance threshold
(Reeve, 1989); hence,
discrimination should vary according to the social and ecological context to
balance the fitness costs of accepting non-nestmates and rejecting nestmates.
Alternatively, a graded behavioral response depending on the degree of cue and
template similarity would suggest a non-threshold model
(Vander Meer and Morel,
1998).
In social insect species with large colonies, queens and workers seem to be
labeled by a more or less homogenous recognition odor, or colony gestalt
label, where each colony member bears a mixture of cues representative of the
variation among members of the colony
(Stuart, 1988;
Errard and Jallon, 1987). This
gestalt label is expected to prevail in polygynous ant species, although
extreme polygyny may limit the creation of unique labels, thereby reducing
intercolony variation. In addition, the presence of multiple nests within a
colony (polydomy) can potentially increase within-colony cue diversity leading
to a broader template, which may explain the reduced aggression toward alien
conspecific workers observed in polydomous ant colonies
(van Wilgenburg et al., 2006).
The lack of distinct intrinsic colony odors and a broader recognition template
may facilitate the formation of unicolonial populations in which colony
boundaries are weak or absent, although some odor differences arising from
extrinsic factors (e.g. the microenvironment) may still exist
(Hölldobler and Wilson,
1990).
Although various volatile and nonvolatile compounds have been demonstrated
as nestmate recognition cues, most frequently, long-chain cuticular
hydrocarbons (CHC) have been shown to serve this role in ants (e.g.
Lahav et al., 1999;
Thomas et al., 1999;
Boulay et al., 2000;
Liang and Silverman, 2000;
Ozaki et al., 2005;
Greene and Gordon, 2007),
wasps (e.g. Gamboa et al.,
1996; Dani et al.,
2001; Ruther et al.,
2002), and termites (e.g. Clèment and Bagnères,
1998). The sensory mechanism for detection of CHC in social insects is
unclear, however peripheral recognition of specific CHC blends (i.e. nestmate
versus non-nestmate) by specialized antenna sensillae may be achieved
by desensitization of gustatory receptor neurons to nestmate CHC blends
(Ozaki et al., 2005).
Introduced populations of the Argentine ant Linepithema humile
(Mayr), are highly polygynous, polydomous and unicolonial
(Newell and Barber, 1913;
Hölldobler and Wilson,
1990; Suarez et al.,
1999), and exhibit pronounced variation in intraspecific
aggression (Tsutsui et al.,
2000; Suarez et al.,
2002; Giraud et al.,
2002; Buczkowski et al.,
2004). These populations are, therefore, ideal models to examine
nestmate discrimination, and the effects of genetic similarity and social and
ecological context on behavioral thresholds
(Buczkowski and Silverman,
2005). Nestmate recognition in this widespread invasive species is
mediated by endogenous and exogenous CHC
(Suarez et al., 2002;
Liang and Silverman, 2000),
and because the contribution of environmentally derived cues to nestmate
recognition varies among introduced populations, it appears that phenology and
genotypic diversity affect the expression and perception of components of the
L. humile recognition system
(Buczkowski and Silverman,
2006). Therefore, examining variation in Argentine ant recognition
cue diversity and recognition threshold modulation may further our
understanding of recognition cue ontogeny, perception and action
thresholds.
We have recently demonstrated that unrelated L. humile colonies
from the southeastern United States selectively adopt foreign queens and fuse
(Vásquez and Silverman,
2008a; Vásquez and
Silverman, 2008b), thereby potentially altering colony genetic
composition and `eroding' non-nestmate discrimination. In unicolonial L.
humile populations that exhibit low variation in genetic-based
recognition cues, non-nestmates may be accepted if the template–cue
dissimilarity is below a rejection threshold
(Starks, 2003). Likewise, in
more genetically diverse populations, colonies with higher levels of
genetic-based recognition cue similarity may accept non-nestmates if the
template–cue match is below a dissimilarity threshold.
In this study, we investigated a possible mechanism underlying L.
humile non-nestmate queen acceptance by comparing queen CHC profiles
among colonies and examining the relationship between queen CHC profile
similarities and queen adoption rates in queenless and queenright host
colonies. We hypothesized that queen CHC similarities are correlated with, and
probably guide, behavioral interactions between queens and recipient workers.
We thus expected that the CHC profiles of adopted non-nestmate queens would be
more similar to host colony queens than the CHC profiles of non-adopted
queens. In addition, we examined the chemical profiles of adopted queens to
determine whether queens acquired non-nestmate CHC as a means of colony
integration. In L. humile, workers treated with prey-derived
hydrocarbons elicit nestmate worker aggression
(Liang and Silverman, 2000) as
do cotton balls dosed with extracted nestmate CHC that are supplemented with
n-alkanes (Greene and Gordon,
2007). Nevertheless, the effect of supplementing the CHC profiles
of live ants with non-nestmate CHC on worker aggression has yet to be tested.
To determine if queen CHC also modulate worker aggression and serve as
nestmate queen recognition cues, we applied purified non-nestmate CHC of
queens that were consistently attacked to live queens, recorded nestmate
worker behavior, and analyzed treated queen CHC. We expected that application
of naturally occurring non-nestmate queen CHC onto queens would also elicit
worker aggression.
|
MATERIALS AND METHODS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
). All colonies were maintained in soil-free,
FluonTM-coated trays (40x55x8 cm; AGC Chemicals American
Inc., Bayonne, NJ, USA). Nests were plastic Petri dishes (9 cm diameter)
filled with moist grooved Castone® (Dentsply International Inc., York, PA,
USA) dental plaster. Colonies were provided 25% sucrose solution, artificial
diet (Bhatkar and Whitcomb,
1970) ad libitum, hard-boiled egg once a week and a water
source. All colonies were maintained at 25±1°C and 50±15%
relative humidity, on a 12 h:12 h light:dark cycle. Source colonies from each
of the four locations containing ants not used to set-up the experimental
colonies were also maintained as described above.
Non-nestmate queen adoption assay and sampling of queen cuticular hydrocarbons
A non-nestmate Argentine ant queen was introduced into either queenless or
queenright CHH, COC, FOR and RTP experimental colonies, and worker behavior
towards the introduced queen was recorded for 24 h
(Vásquez and Silverman,
2008a). We established a total of 12 experimental colonies with
ants collected at each of the four locations in 2003; for each location there
were three colonies each with a different queen number (zero, one or six
queens), 100 pieces of brood, and 
3000 workers (1 g). Six queens from
each of the four source colonies were marked with a water-based paint for
identification, introduced individually into each queenless and queenright
(single and six-queen) experimental colony, and left in place for 24 h. The
response of the host workers toward the intruder queen was scored as 0 (no
aggressive response), 1 (physical attack) or 2 (intruder killed). Adoption was
regarded as having occurred if after 24 h, intruder queens were found in the
nest being tended by workers (response scored as 0). Data were analyzed as the
average recipient colony response score and as the percentage of queens
adopted. The adoption assay was replicated twice across time, and a total of
144 queens per source colony were tested in all six colony–pair
combinations.
We collected 10 queens from each of the source colonies used in this assay for CHC analysis. Queens were placed individually in glass vials and stored at –20°C until extraction, purification and analysis of cuticular lipids as described below. Queen CHC profiles were compared (see below) to determine queen CHC similarities between colonies, and to relate similarities of queen CHC to worker behavioral response and percentage queen adoption at 24 h.
To examine CHC profiles of queens before and after adoption, we developed, validated and employed a non-destructive CHC sampling method. A hexane-extracted air-dried cotton ball (2 mm diameter) held by a pair of hexane-rinsed forceps was gently stroked against a queen's abdomen for 3 min then stored in a glass vial at –20°C. We individually sampled nine queens per colony from the CHH, COC, FOR and RTP colony fragments collected in 2004. CHC profiles of queens sampled using the non-destructive method were compared with those of the 40 solvent-extracted source queens from the 24 h adoption assay.
We then introduced individual COC and CHH queens into queenless and
multiple queen FOR and RTP colonies. These queen/recipient colony combinations
(COC/FOR and CHH/RTP) were selected based on the high adoption rates observed
in the previous adoption experiment. Three queenless and three multiple-queen
(six queens) experimental colonies were established from FOR and RTP ants
collected in 2004, each with 100 pieces of brood, and 3000 workers.
Recipient colony response was recorded as previously described, and queens
surviving after 24 h were left in place for 2 weeks. Nestmate queen
introductions (FOR and RTP) were also performed. The assay was replicated four
times. We used the non-destructive CHC sampling method to examine CHC profiles
of nestmate and non-nestmate queens before and after adoption. We sampled all
queens tested (96) 24 h prior to introduction, and all queens adopted by
queenless colonies (42) 2 weeks after introduction. Because non-nestmate queen
adoption rates in queenright colonies were low, the few samples collected were
not included in the analysis. CHC profiles of adopted COC and CHH queens were
compared before and after introduction with those of FOR and RTP queens
adopted by their nestmate colonies, respectively, to determine if changes in
CHC occurred after adoption.
Application of non-nestmate queen cuticular hydrocarbons to queens: effects on nestmate worker aggression
To test if CHC are used as cues in L. humile nestmate queen
recognition we compared worker aggressive behavior towards nestmate queens
treated with purified nestmate and non-nestmate queen CHC. We selected the FOR
queen/RTP recipient colony combination based on the consistent rejection of
FOR queens by RTP recipient colonies. Three multiple-queen experimental
colonies (same brood and worker size as in queen adoption assay colonies) were
established from RTP source colonies collected in 2005. RTP queens were
treated with purified CHC extracts of FOR or RTP queens, or with hexane as
control. Purified CHC from six queens (cuticular lipid extraction and CHC
isolation procedures detailed below) were resuspended in 100 µl hexane,
applied to the inside surface of a 12x32 mm glass vial, and the solvent
allowed to evaporate. Three vials were coated per treatment and each vial was
used to treat three individual queens. Each queen was anesthetized by brief
exposure to CO2, placed individually in a treated vial, rotated
gently for 3 min, allowed 15–30 s to recover and then introduced to one
of three RTP multiple queen experimental colonies. Each colony received a
total of three queens per treatment. Worker behavior was scored as
non-aggressive (antennation, queen moving into nest without being attacked) or
aggressive (biting, pulling, lunging, gaster flexion) during a 3 min period by
an observer blinded to the type of treatment applied to queens and unfamiliar
with the hypothesis being tested. All tested queens were killed by freezing
(–20°C). CHC profiles of all queens were compared to determine if
they differed between treatments, and between attacked and non-attacked
queens.
Extraction, isolation and chemical analysis of cuticular hydrocarbons
Nonpolar cuticular lipids of thawed queens and cotton samples collected in
all behavioral assays were extracted by immersion in 1 ml hexane for 10 min,
followed by a brief second rinse in 100 µl hexane. Samples were lightly
shaken for the first and last 15–20 s of the immersion period. The
solvent was removed under a gentle stream of high purity N2, the
vial rinsed twice, each with 100 µl hexane, and the concentrated extract
(200 µl) was applied to a hexane-prewetted Pasteur pipette mini-column
filled with 500 mg of silica gel (100–200 mesh). The hydrocarbon
fraction was eluted with 6 ml hexane and the solvent was evaporated with
N2. Capillary gas chromatography (GC) was carried out using a
Hewlett-Packard (Rockeville, MD, USA) HP5890 gas chromatograph equipped with a
DB-XLB column (30 mx 0.25 mmx0.25 µm film thickness) for
analyses of CHC of source queens from the 24 h queen adoption assay, and a
DB-5 (30 mx0.25 mmx0.5 µm) for analyses of CHC of cotton
samples taken in the 2-week queen adoption assay and of queens treated with
non-nestmate and nestmate CHC. The change in columns was based on column
availability and the two columns gave identical results for split samples
(data not shown). Extracts were introduced into a split-splitless injector
operated at 300°C in splitless mode (2 min purge) and with a helium
carrier gas average linear velocity of 30 cm s–1. The oven
temperature was held at 80°C for 2 min, increased to 270°C at a rate
of 20°C min–1, then to 310°C at 3°C
min–1 and held at 310°C for 20 min. The flame-ionization
detector was operated at 310°C with nitrogen make-up gas at 30 ml
min–1. Whole queen extracts were resuspended in 20 µl
hexane, and 0.5 µl (0.025 queen equivalents) was injected. Cotton sample
extracts were resuspended in 4 µl of octane and 2 µl (0.5 queen
equivalents) were injected by an automatic injector. Quantitative data were
obtained by integrating the area under each peak and calculating its
percentage of the total CHC; only peaks with a mean percentage area across all
colonies of 1% or higher were used for data analysis. All selected peak areas
were standardized to 100%. The identity of discriminating peaks was determined
by matching L. humile n-alkanes with external hydrocarbon standards
(n-C23 – n-C36) and diagnostic peaks were confirmed by GC-MS with those
from previous studies (Liang et al.,
2001; de Biseau et al.,
2004). GC-MS analyses of queen cuticular hydrocarbons were
performed on a HP6890 GC equipped with a HP-5MS column (30 mx0.25
mmx0.25 µm film thickness), and connected to a HP5973A mass selective
detector. The injector was operated at 300°C in splitless mode with a
helium carrier gas average linear velocity of 45 cm s–1 (2
min purge). Data were recorded in electron ionization scan mode (25–550
m/z).
Statistical analyses
Data analyses were performed using SAS 9.1 statistical software
(SAS, 2004). Standardized
selected peak areas were transformed following Aitchison's formula:
Zij=ln[Yij/g(Yj)],
where Zij is the standardized peak area i, for
individual j, Yij is the peak area i for
individual j, and g(Yj) is the geometric
mean of all peaks for individual j. We performed a multivariate
analysis of variance (MANOVA) and tested the homogeneity of variance of these
transformed variables with Brown and Forsythe's test
(Brown and Forsythe, 1974)
using PROC GLM. We performed a stepwise discriminant analysis (stepwise DA) on
transformed variables that met the assumptions of homogeneity of variance in
MANOVA using PROC STEPDISC followed by DA on the selected peaks using PROC
CANDISC to determine whether the predefined groups (colonies or treatments)
could be discriminated on the basis of their chemical profiles. Pairwise
generalized square distances between groups and classification error rates
were calculated using PROC DISCRIM. Distances between group means (centroids)
were used as an estimate of the degree of CHC differentiation between colonies
or treatments.
Correlations between queen CHC similarities and recipient colony response and percentage non-nestmate queen adoption were performed using Pearson correlation coefficients (Pearson's r).
|
RESULTS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
=0.0004,
F=7.73, d.f.=69, 42.7, P<0.0001). The stepwise DA
selected 12 variables that clustered all queens according to their colony of
origin (Wilks' =0.0027 0.01, F=13.11, d.f.=36, 74.6,
P<0.0001) with function 1 (86.1% of variation) separating CHH and
RTP from both COC and FOR, and function 2 (9.5%) further separating CHH from
RTP and COC from FOR (Fig. 1A);
and all queens were correctly classified. Discriminating compounds selected in
the stepwise DA were identified as n-heptacosane (n-C27),
n-nonacosane (n-C29), 5-methylnonacosane (5-MeC29),
5-methyltriacontane (5-MeC30), 5-methylhentriacontane (5-MeC31),
hentriacontene (xC31:1), tritriacontene (xC33:1) and 5-methyltetratriacontane
5-MeC34, while four compounds remained unidentified.
|
|
Queen adoption in relation to similarities of queen cuticular hydrocarbon profiles
CHC similarities between colonies (distances between colony centroids) were
positively correlated with recipient colony response (0=queens adopted,
1=queens attacked, 2=queens killed) in queenless (Pearson's r=0.86,
P=0.0276) and single-queen host colonies (Pearson's r=0.90,
P=0.0154) with non-nestmate queens more likely to be attacked and
killed with increasing distances between queen CHC profiles
(Fig. 2). By contrast, we found
no association between CHC similarities and recipient response in multiple
queen colonies (Pearson's r=0.66, P=0.1504). Also, queen CHC
profile similarities between colonies were inversely associated with
non-nestmate queen adoption (percent) in queenless (Pearson's
r=–0.85, P=0.0329) and single queen colonies
(Pearson's r=–0.89, P=0.0154), but not in multiple
queen colonies (Pearson's r=–0.66, P=0.1503).
|
Non-destructive queen cuticular hydrocarbon sampling versus solvent extraction
Queens sampled using the non-destructive method were distinguished based on
32 transformed variables that differed among colonies according to MANOVA
(Wilks' <0.0001, F=8.80, d.f.=93, 9.9, P=0.0004)
with function 1 and function 2 explaining 80.4% and 13.9% of the total
variation. DA of CHC sampled by the non-destructive method showed that all
queens could be distinguished and correctly classified into their colony of
origin based on 13 variables selected by stepwise DA (Wilks' =0.0012,
F=13.95, d.f.=39, 62.9, P<0.0001), with function 1 (64.8%
of variation) separating COC and FOR from CHH and RTP, while function 2 (26.5%
of variation) distinguished RTP from CHH and COC from FOR
(Fig. 1B). Squared distances
between colony means obtained by DA of these 13 discriminating peaks followed
the same pattern as with solvent extraction
(Table 1), although they were
not associated with those obtained for queens from sources used in the 24 h
adoption assay and extracted by solvent (Pearson's r=0.78,
P=0.0689). Identified discriminating peaks by stepwise DA included
seven compounds selected when hexane-extracted queen CHC were analyzed
(n-C29, 5-MeC29, 5-MeC30, 5-MeC31, xC31:1, xC33:1, 5-MeC34), and
three unidentified compounds. These data show that similar results were
obtained with the non-destructive approach and with solvent extraction.
Changes in queen cuticular hydrocarbon profiles following adoption
FOR and RTP queens sampled 24 h before and 2 weeks after adoption by
queenless FOR colonies, and COC and CHH queens sampled 24 h before and 2 weeks
after adoption by queenless RTP colonies, were distinguished based on their
CHC profiles according to MANOVA performed on 32 variables (Wilks'
<0.0001, F=2.22, d.f.=217, 121.8, P<0.0001).
DA on nine variables selected by stepwise DA also showed that queens could be
differentiated (Wilks' =0.0101, F=4.41, d.f.=63, 220.13,
P<0.0001; Fig. 3).
COC and FOR queens could be distinguished based on CHC sampled before
adoption, but two COC queens were classified as FOR queens after adoption. FOR
queens were classified as a separate group after adoption by their nestmate
queenless workers. RTP and CHH queens were differentiated before and after
adoption. The distance between centroids for COC queens before and after
adoption (3.85) was not greater than for FOR queens before and after adoption
(9.87), suggesting that only slight changes in CHC profiles of COC queens were
detected. However, after adoption, FOR and COC queens were less dissimilar
than before adoption as indicated by a reduction in the distance between
centroids of these two colonies from 22.24 to 8.52, suggesting that queen CHC
changes after adoption may have produced more similar profiles. Similarly, the
distance between centroids decreased in CHH and RTP queens after adoption
(from 40.89 to 17.59). Overall, changes in queen CHC profiles after adoption
suggest a reduction in phenotypic cue dissimilarities between colonies.
However, a larger sample size and examining a greater number of colonies would
be needed to further support this trend.
|
Queens treated with non-nestmate queen cuticular hydrocarbons: chemical profiles and worker aggression
RTP queens treated with (i) nestmate RTP queen CHC, (ii) non-nestmate FOR
queen CHC, or (iii) hexane (control) could be distinguished by DA on four
variables (Wilks' =0.2277, F=5.48, d.f.=8, 40,
P=0.0001) selected out of 30 variables by stepwise DA, although these
groups could not be distinguished according to MANOVA performed on 24
variables (Wilks' =0.0007, F=1.61, d.f.=46, 2,
P=0.4582). The DA on four variables showed that function 1
(explaining 60.1% of variance) differentiated RTP queens treated with FOR-CHC
from queens treated with RTP-CHC and solvent control queens, and function 2
(explaining 39.9% of variance) indicated some differences in the CHC profiles
of RTP-treated and solvent control queens
(Fig. 4A). The DA correctly
classified 85.2% of the individuals. The four discriminating variables were
5-MeC29, 5-methyldotriacontane (5-MeC32), xC33:1 and one unidentified
compound.
|
=0.42,
F=5.47, d.f.=5, 20, P=0.0025) with 100% of the variance
explained by function 1 and 84.6% of the queens classified to the correct
group (Fig. 4B). These five
discriminating peaks were identified as n-C27, 5-MeC32, xC33:1 and
two unidentified compounds.
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
;
Greene and Gordon, 2007;
Torres et al., 2007), however,
behavioral and chemical data supporting the role of CHC in queen
discrimination have not been previously reported. We propose that over time,
adoption of non-nestmate queens may produce changes in colony genotypic
composition and in recognition cues among colony members. Moreover, these
changes could increase within-colony cue diversity and lead to recognition
template expansion with subsequent reduced aggression towards non-nestmates as
suggested for polygynous (Keller and
Passera, 1989; Vander Meer and
Morel, 1998) and polydomous
(van Wilgenburg et al., 2006)
ant species. Therefore, non-nestmate queen adoption in L. humile
could promote lower aggression towards genetically similar colonies, thereby
leading to more open colonies.
In queenless and single-queen colonies similarity of the CHC of introduced
and resident queens appeared to guide the responses of workers in the resident
colony (queens adopted, attacked or killed). Different worker responses to
various introduced queens suggests that workers discriminate among queens by
matching the cues from newly encountered queens with an internal nestmate
queen template, which may persist even in queenless colonies. However, the
lack of correlation between worker response towards introduced non-nestmate
queens and the degree of CHC profile similarity of introduced and resident
queens found in multiple queen colonies, suggests an influence of social
context on the acceptance threshold. It is possible that the slight
heterogeneity of CHC among multiple queens affects the stringency of the
workers' internal template, thus lowering the queen acceptance threshold, or
that subsets of workers tend predominantly one or two queens and form a narrow
template, thus rejecting new queens. Queen presence does not affect worker
aggression toward non-nestmate workers in L. humile
(Caldera and Holway, 2004);
however, queens do affect the aggressive response of workers to non-nestmate
queens in this species (Vásquez and
Silverman, 2008a). Similarly, queens influence worker aggressive
behavior in other ants (Vienne et al.,
1998; Provost,
1989; Boulay et al.,
2003). Therefore, it is possible that L. humile queen
pheromones, which influence other aspects of recognition, including aggression
towards female sexual larvae (Passera, 1995), may also affect nestmate
recognition at different levels. A flexible acceptance threshold may result
from differences in recognition context
(Reeve, 1989) and fluctuations
in the cost of recognition errors (Liebert
and Starks, 2004). For example, if a colony's survival is at high
risk, a reduction in the cost of accepting foreign conspecifics is expected
(Sudd and Franks, 1987). The
positive relationship between non-nestmate L. humile queen adoption
and queen CHC similarity may reflect this cost-benefit trade-off particularly
in queenless colonies. Studies on the fitness consequences of non-nestmate
queen adoption into queenless colonies should shed light on these colony-level
decision processes.
Non-destructive sampling of CHC allowed us not only to detect differences
in CHC profiles between colonies, but also revealed slight temporal changes in
these patterns. The CHC of adopted queens changed, but contrary to our
expectation, the profiles of adopted non-nestmate queens did not change more
than those of adopted nestmate queens. The CHC of Argentine ant queens are
dynamic, changing quantitatively and qualitatively in relation to queen
ovarian activity (de Biseau et al.,
2004). Queens have considerable amounts of monomethylalkanes
(5-MeC27 to 5-MeC34) and alkenes (C29:1, C31:1, C33:1)
(de Biseau et al., 2004),
whereas workers do not have these compounds (or have very low amounts) but
have dimethylalkanes and trimethylalkanes (diMe- and triMeC33, C35 and C37) as
major compounds (Liang et al.,
2001). These qualitative differences could result from selective
biosynthesis of CHC, with shorter and monomethylalkanes predominantly produced
by queens through enzymes that regulate the synthesis of hydrocarbons of
different chain length (Blomquist et al.,
1998), or by selective transfer of CHC from oenocytes, which
produce them, to the cuticle via lipophorin
(Schal et al., 2003). The
distinct CHC profiles of these two castes suggest a limited cue exchange
between queens and workers, and may partially explain the lack of more
pronounced CHC changes in adopted non-nestmate queens. Therefore, the subtle
profile changes observed in both nestmate and non-nestmate queens adopted by
queenless colonies could reflect physiological changes, although we cannot
rule out possible acquisition of CHC from host colony workers since queen CHC
between colonies were more similar after adoption. Hence, studies examining
CHC profiles of non-nestmate queens adopted by queenright colonies may reveal
that greater changes in queen recognition cues occur when host colony queens
and workers are present.
In our assays, queens acquired queen CHC mechanically from glass surfaces.
Similarly, when workers of this species were exposed to large quantities of
exogenous CHC they incorporated long-chain CHC (C35-C37) within the range of
their intrinsic CHC (Liang and Silverman,
2000). It is not known, however, whether in natural interactions
queens or workers would selectively acquire more queen or worker CHC. Transfer
of CHC between individuals of the same colony, between mixed species and in
dulotic and inquiline species is well documented
(Soroker et al., 1994;
Howard et al., 1980;
Vander Meer and Wojcik, 1982;
Kaib et al., 1993). However,
it has been suggested that unlike some other ant species in which colony odor
is derived from the queen (e.g. Carlin and
Hölldobler, 1986) or transferred from worker to queen
(Lahav et al., 1998), L.
humile represents an alternative model for colony odor formation since
reproductives and non-reproductives have very different CHC profiles
(de Biseau et al., 2004).
Therefore, L. humile colonies appear to lack a unified colony gestalt
odor and instead have two subsets of odors, queen-derived and worker-derived.
Thus, minor changes in queen CHC following adoption may reflect these presumed
caste-specific Gestalt. Similarly, in some other ants, queens appear not to be
important contributors to the colony Gestalt and have queen-specific profiles
(Boulay et al., 2003;
Dahbi and Lenoir 1998;
Dietemann et al., 2003).
Furthermore, individuals within a polydomous colony can differ in their cue
profiles due to incomplete CHC transfer
(van Wilgenburg et al., 2006),
suggesting not only the presence of subsets of colony odor but also the
formation of distinct templates within a colony. However, we cannot rule out
the possibility that a colony gestalt odor based on unknown compounds may
exist, and that CHC are used exclusively as caste signals and not as colony
recognition cues. Studies examining the role of these caste-specific CHC in
L. humile nestmate recognition would further support our suggested
model.
Worker aggression towards nestmate queens treated with non-nestmate queen CHC supports the view that CHC are important cues in nestmate queen recognition. Queens were distinguished based upon their CHC profiles, with most queens that were treated with non-nestmate CHC grouping together. The few queens treated with nestmate CHC or solvent control that were unexpectedly attacked were either more similar to the non-nestmate CHC-treated queens or less similar to the solvent control than other queens in the group, suggesting that the gentle rotation of ants in glass vials could have affected CHC profiles. For example, while some CHC could be acquired from the glass surface, native CHC could also be lost to the glass surface during this treatment procedure. Alternatively, physiological or behavioral variability among queens within a colony might have affected our results. These concerns could be addressed in future experiments by testing queens of known ages, by optimizing the time of exposure to minimize unintended CHC removal, by working with more inert substrates (e.g. silanized glass) or by direct application of precise CHC quantities to queens. Compounds that appear to be associated – at least statistically – with worker behavior in this assay were monomethyl alkanes and alkenes that are either absent or occur in considerably lower quantities in the CHC of workers.
We found that quantitative variation in queen CHC profiles reflects colony
identity, and direct manipulation of queen CHC affected aggression behavior in
L. humile workers. Additionally, our results indicate that not all
CHC but only a statistically derived subset of compounds, could mediate queen
discrimination; but whether all or only some of the CHC are indeed important
in nestmate recognition remains unknown. Interestingly, the subset of
hydrocarbons selected by DA in our experiments belong to at least two
structural classes, methyl-branched alkanes and n-alkenes, suggesting
that a mixture of CHC of different structural classes varying in their
relative proportions across colonies rather than a few compounds of a single
structural class may be used as nestmate recognition cues. Our results further
support the view that colony membership in L. humile is conveyed by a
mixture of structural classes as suggested by the finding that a mixture of
n-alkanes supplementing nestmate worker CHC profiles elicited high
aggression levels whereas no aggressive response was elicited when the mixture
of n-alkanes was presented alone
(Greene and Gordon, 2007). The
role of specific compounds or chemical classes as nestmate recognition cues
seems to differ considerably among social insects. For example,
methyl-branched alkanes, n-alkanes and an alkene and
n-alkane mixture have been shown to be important colony recognition
cues in wasps (Dani et al.,
1996; Gamboa et al.,
1996), whereas in ants, methyl-branched CHC (mono- and
dimethylalkanes and monomethylalkenes) are more colony-specific than
n-alkanes (Bonavita-Cougourdan et
al., 1987; Provost et al.,
1992; Astruc et al.,
2001; Lucas et al.,
2004), although dimethylalkanes seem not to be important in
nestmate recognition in Cataglyphis species
(Dahbi et al., 1996). We
cannot rule out that additional recognition-active compounds other than those
that seem to be linked to colony chemical profile specificity may also be
important. Therefore, chemical supplementation studies testing these
presumably important CHC structural classes or the compounds individually or
in mixtures, and at different concentrations, could corroborate our
findings.
Our combined behavioral and chemical data shed light on the dynamics and complexity of nestmate recognition in L. humile and suggest that interspecific variation in CHC and its perception may have colony-level consequences, e.g. the formation of more open colonies. Further investigation on recognition processes in this and other invasive ant species would enhance our understanding of the factors responsible for changes in their social organization and ecological success.
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Astruc, C., Malosse, C. and Errard, C. (2001). Lack of intraspecific aggression in the ant Tetramorium bicarinatum: a chemical hypothesis. J. Chem. Ecol. 27,1229 -1248.[CrossRef][Medline]
Bhatkar, A. and Whitcomb, W. H. (1970). Artificial diet for rearing various species of ants. Florida Entomol. 53,229 -232.[CrossRef]
Blomquist, G. J., Tillman, J. A., Mpuru, S. and Seybold, S. J. (1998). The cuticle and cuticular hydrocarbons of insects: structure, function, and biochemistry. In Pheromone Communication in Social Insects: Ants, Wasps, Bees, and Termites (ed. R. K. Vander Meer, M. D. Breed, K. E. Espelie and M. L. Winston), pp.34 -54. Boulder, CO: Westview Press.
Bonavita-Cougourdan, A., Clement, J. L. and Lange, C. (1987). Nestmate recognition: the role of cuticular hydrocarbons in the ant Camponotus vagus Scop. J. Entomol. Sci. 2,1 -10.
Boulay, R., Hefetz, A., Soroker, V. and Lenoir, A. (2000). Camponotus fellah colony integration: worker individuality necessitates frequent hydrocarbon exchanges. Anim. Behav. 59,1127 -1133.[CrossRef][Medline]
Boulay, R., Katzav-Gozansky, T., Vander Meer, R. K. and Hefetz, A. (2003). Colony insularity through queen control on worker social motivation in ants. Proc. R. Soc. Lond. B Biol. Sci. 270,971 -977.[Medline]
Breed, M. D. (1998). Chemical cues in kin recognition: criteria for identification, experimental approaches, and the honey bee as an example. In Pheromone Communication in Social Insects: Ants, Wasps, Bees, and Termites (ed. R. K. Vander Meer, M. D. Breed, K. E. Espelie and M. L. Winston), pp.57 -78. Boulder, CO: Westview Press.
Breed, M. D. and Bennett, B. (1987). Kin recognition in highly eusocial insects. In Kin Recognition in Animals (ed. D. J. C. Fletcher and C. D. Michener), pp.243 -285. Chichester, NY: J. Wiley & Sons.
Brown, M. B. and Forsythe, A. B. (1974). Robust tests for equality of variances. J. Amer. Stat. Soc. 69,364 -367.
Buczkowski, G. and Silverman, J. (2005). Context-dependent nestmate discrimination and the effect of action thresholds on exogenous cue recognition in the Argentine ant. Anim. Behav. 69,741 -749.[CrossRef]
Buczkowski, G. and Silverman, J. (2006). Geographical variation in Argentine ant aggression behaviour mediated by environmentally derived nestmate recognition cues. Anim. Behav. 71,327 -335.[CrossRef]
Buczkowski, G., Vargo, E. L. and Silverman, J. (2004). The diminutive supercolony: the Argentine ants of the southeastern United States. Mol. Ecol. 13,2235 -2242.[CrossRef][Medline]
Caldera, E. J. and Holway, D. A. (2004). Evidence that queens do not influence nestmate recognition in Argentine ants. Insectes Soc. 51,109 -112.[CrossRef]
Carlin, N. F. and Hölldobler, B. (1986). The kin recognition system of carpenter ants (Camponotus spp.). I. Hierarchical cues in small colonies. Behav. Ecol. Sociobiol. 19,123 -134.[CrossRef]
Crozier, R. H. (1987). Genetic aspects of kin recognition: concepts, models, and synthesis. In Kin Recognition in Animals (ed. D. J. C. Fletcher and C. D. Michener), pp.55 -73. Chichester, NY: J. Wiley & Sons.
Crozier, R. H. and Dix, M. W. (1979). Analysis of two genetic models for the innate components of colony odor in social Hymenoptera. Behav. Ecol. Sociobiol. 4, 217-224.[CrossRef]
Dahbi, A. and Lenoir, A. (1998). Queen and colony odour in the multiple nest ant species, Cataglyphis iberica (Hymenoptera, Formicidae). Insectes Soc. 45,301 -313.[CrossRef]
Dahbi, A., Cerda, X., Hefetz, A. and Lenoir, A. (1996). Social closure, aggressive behavior, and cuticular hydrocarbon profiles in the polydomous ant Cataglyphis iberica (Hymenoptera, Formicidae). J. Chem. Ecol. 22,2173 -2186.[CrossRef]
Dani, F. R., Fratini, S. and Turillazzi, S. (1996). Behavioural evidence for the involvement of Dufour's gland secretion in nestmate recognition in the social wasp Polistes dominulus (Hymenoptera:Vespidae). Behav. Ecol. Sociobiol. 38,311 -319.[CrossRef]
Dani, F. R., Jones, G. R., Destri, S., Spencer, S. H. and Turillazzi, S. (2001). Deciphering the recognition signature within the cuticular chemical profile of paper wasps. Anim. Behav. 62,165 -171.[CrossRef]
de Biseau, J. C., Passera, L., Daloze, D. and Aron, S. (2004). Ovarian activity correlates with extreme changes in cuticular hydrocarbon profile in the highly polygynous ant, Linepithema humile. J. Insect Physiol. 50,585 -593.[CrossRef][Medline]
Dietemann, V., Peeters, C., Liebig, J., Thivet, V. and
Hölldobler, B. (2003). Cuticular hydrocarbons mediate
discrimination of reproductives and nonreproductives in the ant Myrmecia
gulosa. Proc. Natl. Acad. Sci. USA
100,10341
-10346.
Errard, C. and Jallon, J. M. (1987). An investigation of the development of the chemical factors in ants intra-society recognition. In Chemistry and Biology of Social Insects: Proceedings of the Tenth International Congress of the International Union for the Study of Social Insects, Munich, 1986 (ed. J. Eder and H. Rembold), p. 478. Munich, Germany: Verlag J. Peperny.
Gamboa, G. J., Reeve, H. K., Ferguson, D. and Wacker, T. L. (1986). Nestmate recognition in social wasps: the origin and acquisition of recognition odours. Anim. Behav. 34,685 -695.[CrossRef]
Gamboa, G. J., Grudzier, T. A., Espelie, K. A. and Bura, E. (1996). Kin recognition in social wasps: combining behavioural and chemical evidence. Anim. Behav. 51,625 -629.[CrossRef]
Getz, W. (1982). An analysis of learned kin recognition in Hymenoptera. J. Theor. Biol. 99,585 -597.[CrossRef]
Giraud, T., Pedersen, J. S. and Keller, L.
(2002). Evolution of supercolonies: the Argentine ants of
southern Europe. Proc. Natl. Acad. Sci. USA
99,6075
-6079.
Gotzek, D. and Ross, K. G. (2007). Genetic regulation of colony social organization in fire ants: an integrative overview. Q. Rev. Biol. 82,201 -226.[CrossRef][Medline]
Greene, M. J. and Gordon, D. M. (2007).
Structural complexity of chemical recognition cues affects the perception of
group membership in the ants Linepithema humile and Aphaenogaster
cockerelli. J. Exp. Biol.
210,897
-905.
Hölldobler, B. (1995). The chemistry of
social regulation: multicomponent signals in ant societies. Proc.
Natl. Acad. Sci. USA 92,19
-22.
Hölldobler, B. and Michener, C. D. (1980). Mechanisms of identification and discrimination in social Hymenoptera. In Evolution of Social Behavior: Hypotheses and Empirical Tests (ed. H. Markl), pp. 35-58. Weinheim: Verlag Chemie.
Hölldobler, B. and Wilson, E. O. (1990). The Ants. Cambridge, MA: Harvard University Press.
Howard, R. W., McDaniel, C. A. and Blomquist, G. J.
(1980). Chemical mimicry as an integrating mechanism: cuticular
hydrocarbons of a termitophile and its host. Science
210,431
-433.
Kaib, M., Heinze, J. and Ortius, D. (1993). Cuticular hydrocarbon profiles in the slave-making ant Harpogoxenus sublaevis and its hosts. Naturwissenschaften 80,281 -285.[CrossRef]
Keller, L. and Passera, L. (1989). Influence of the number of queens on nestmate recognition and attractiveness of queens to workers in the Argentine ant, Iridomyrmex humilis (Mayr). Anim. Behav. 37,733 -740.[CrossRef]
Keller, L. and Ross, K. G. (1998). Selfish genes: a green beard in the red fire ant. Nature 394,573 -575.[CrossRef]
Lacy, R. and Sherman, P. W. (1983). Kin recognition by phenotype matching. Am. Nat. 121,489 -512.[CrossRef]
Lahav, S., Soroker, V., Vander Meer, R. K. and Hefetz, A. (1998). Nestmate recognition in the ant Cataglyphis niger: do queens matter? Behav. Ecol. Sociobiol. 43,203 -212.[CrossRef]
Lahav, S., Soroker, V., Hefetz, A. and Vander Meer, R. K. (1999). Direct behavioral evidence for hydrocarbons as ant recognition discriminators. Naturwissenschaften 86,246 -249.[CrossRef]
Lehmann, L. and Perrin, N. (2002). Altruism, dispersal and phenotype-matching kin recognition. Am. Nat. 159,451 -468.[CrossRef]
Liang, D. and Silverman, J. (2000). `You are what you eat': diet modifies cuticular hydrocarbons and nestmate recognition in the Argentine ant, Linepithema humile.Naturwissenschaften 87,412 -416.[CrossRef][Medline]
Liang, D., Blomquist, G. J. and Silverman, J. (2001). Hydrocarbon-released nestmate aggression in the Argentine ant, Linepithema humile, following encounters with insect prey. Comp. Biochem. Physiol. 129B,871 -882.[CrossRef][Medline]
Liebert, A. E. and Starks, P. T. (2004). The action component of recognition systems: a focus on the response. Ann. Zool. Fenn. 41,747 -764.
Lucas, C., Pho, D. B., Fresneau, D. and Jallon, J. M. (2004). Hydrocarbon circulation and colonial signature in Pachycondyla villosa. J. Insect Physiol. 50,595 -607.[CrossRef][Medline]
Newell, W. and Barber, T. C. (1913). The Argentine ant. US Department of Agriculture Bureau of Entomology Bulletin 122,1 -98.
Ozaki, M., Wada-Katsumata, A., Fujikawa, K., Iwasaki, M.,
Yokohari, F., Satoji, Y., Nisimura, T. and Yamaoka, R.
(2005). Ant nestmate and non-nestmate discrimination by a
chemosensory sensillum. Science
309,311
-314.
Passera, L., Aron, S. and Bach, D. (1995). Elimination of sexual brood in the Argentine ant Linepithema humile: queen effect and brood recognition. Entomol. Exp. Appl. 75,203 -212.[CrossRef]
Provost, E. (1989). Social environmental factors influencing mutual recognition of individual in the ant Leptothorax lichtensteini Bondr. (Hymenoptera: Formicidae). Behav. Processes 18,35 -39.[CrossRef]
Provost, E., Cerdan, P., Bagnères, A.-G., Morgan, E. D. and Rivière, G. (1992). Role of the queen in Messor barbarus colony signature. In Biology and Evolution of Social Insects (ed. J. Billen), pp.195 -202. Leuven: Leuven University Press.
Reeve, H. K. (1989). The evolution of conspecific acceptance thresholds. Am. Nat. 133,407 -435.[CrossRef]
Ruther, J., Sieben, S. and Burkhard, S. (2002). Nestmate recognition in social wasps: manipulation of hydrocarbon profiles induces aggression in the European hornet. Naturwissenschaften 89,111 -114.[CrossRef][Medline]
SAS (2004). SAS Online Document, Version 9. 1. 2. Cary, NC: SAS Institute.
Schal, C., Fan, Y. and Blomquist, G. J. (2003). Regulation of pheromone biosynthesis, transport, and emission in cockroaches. In Insect Pheromone Biochemistry and Molecular Biology (ed. G. J. Blomquist and R. Vogt), pp. 283-322. New York: Academic Press.
Soroker, V., Vienne, C. and Hefetz, A. (1994). The Postpharyngeal gland as a `Gestalt' organ for nestmate recognition in the ant Cataglyphis niger. Naturwissenschaften 81,510 -513.[CrossRef]
Starks, P. T. (2003). Selection for uniformity: xenophobia and invasion success. Trends Ecol. Evol. 18,159 -162.[CrossRef]
Stuart, R. J. (1988). Collective cues as a
basis for nest mate recognition in polygynous leptothoracine ants.
Proc. Natl. Acad. Sci. USA
85,4572
-4575.
Suarez, A. V., Tsutsui, N. D., Holway, D. A. and Case, T. J. (1999). Behavioral and genetic differentiation between native and introduced populations of the Argentine ant. Biol. Invasions 1,43 -53.[CrossRef]
Suarez, A. V., Holway, D. A., Liang, D. S., Tsutsui, N. D. and Case, T. J. (2002). Spatio-temporal patterns of intraspecific aggression in the invasive Argentine ant. Anim. Behav. 64,697 -708.[CrossRef]
Sudd, J. H. and Franks, N. R. (1987). The Behavioural Ecology of Ants. New York: Chapman & Hall.
Thomas, M., Parry, L., Allan, R. and Elgar, M. (1999). Geographic affinity, cuticular hydrocarbons and colony recognition in the Australian meat ant Iridomyrmex purpureus.Naturwissenschaften 86,87 -92.[CrossRef]
Torres, C. W., Brandt, M. and Tsutsui, N. D. (2007). The role of cuticular hydrocarbons as chemical cues for nestmate recognition in the invasive Argentine ant (Linepithema humile). Insectes Soc. 54,363 -373.[CrossRef]
Tsutsui, N. D., Suarez, A. V., Holway, D. A. and Case, T. J.
(2000). Reduced genetic variation and the success of an invasive
species. Proc. Natl. Acad. Sci. USA
97,5948
-5953.
Vander Meer, R. K. and Morel, L. (1998). Nestmate recognition in ants. In Pheromone Communication in Social Insects (ed. R. K. Vander Meer, M. Breed, M. Winston and K. E. Espelie), pp. 79-103. Boulder, CO: Westview Press.
Vander Meer, R. K. and Wojcik, D. P. (1982).
Chemical mimicry in the myrmecophilous beetle Myrmecaphodius
excavaticollis. Science 218,806
-808.
van Wilgenburg, E., Ryan, D., Morrison, P., Marriott, P. J. and Elgar, M. A. (2006). Nest- and colony-mate recognition in polydomous colonies of meat ants (Iridomyrmex purpureus). Naturwissenschaften 93,309 -314.[CrossRef][Medline]
Vásquez, G. M. and Silverman, J. (2008a). Queen acceptance and the complexity of nestmate discrimination in the Argentine ant. Behav. Ecol. Sociobiol. 62,537 -548.[CrossRef]
Vásquez, G. M. and Silverman, J. (2008b). Intraspecific aggression and colony fusion in the Argentine ant. Anim. Behav. 75,583 -593.[CrossRef]
Vienne, C., Errard, C. and Lenoir, A. (1998). Influence of the queen on worker behaviour and queen recognition behaviour in ants. Ethology 104,431 -446.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in JEB:
This article has been cited by other articles:
|
|
 |
|
  K. Phillips COLONIES ACCEPT QUEEN IF HYDROCARBON COAT IS RIGHT J. Exp. Biol., April 15, 2008; 211(8): i - ii. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
