Journal of Insect Physiology 47 (2001) 1013–1020 www.elsevier.com/locate/jinsphys
Nestmate recognition in Parischnogaster striatula (Hymenoptera Stenogastrinae), visual and olfactory recognition cues P. Zanetti a, F.R. Dani a, S. Destri a, D. Fanelli a, A. Massolo b, G. Moneti c, G. Pieraccini c, S. Turillazzi a, d,* b
a Dipartimento di Biologia Animale e Genetica dell’Universita` di Firenze. Via Romana 17, 50129 Firenze, Italy Ethology and Behavioural Ecology Group, Dipartimento di Biologia Evolutiva dell’Universita` di Siena, Via Mattioli 4. 53100 Siena, Italy c Centro Interdipartimentale di Spettrometria di Massa dell’Universita` di Firenze, Viale G. Pieraccini, 5000 Firenze, Italy d Centro di Studio per la Faunistica ed Ecologia Tropicali del C.N.R., via Romana 17, 50125 Firenze, Italy
Received 5 September 2000; received in revised form 29 January 2001; accepted 30 January 2001
Abstract The recognition of nestmates from alien individuals is a well known phenomenon in social insects. In the stenogastrine wasp Parischnogaster striatula, we investigated the ability of females to recognize nestmates and the cues on which such recognition is based. Recognition of nestmates was observed in naturally occurring interactions between wasps approaching a nest and the resident females on that nest. This recognition was confirmed in experiments in which nestmates or alien conspecifics were presented to resident females. In naturally occurring interactions, nestmates generally approach their nest with a direct flight, while aliens usually hover in front of the nest before landing. In experiments in which the presented wasps were placed close to the nest in a direct manner, antennation of the presented wasp generally occurred, indicating that chemical cues are involved. Experiments in which dead alien individuals, previously washed in hexane, and then reapplied with extracts were recognized by colonies giving further evidence that chemical cues mediate nestmate recognition. Epicuticular lipids, known to be nestmate recognition cues in social insects, were chemically analysed by GC-MS for 44 P. striatula females from two different populations (13 different colonies). Discriminant analysis was performed on the data for the lipid mixture composition. The discriminant model showed that, in the samples from these two populations, 68.2% and 81.9% of the specimens could be correctly assigned to their colony. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Social wasp; Stenogastrine; Epicuticular lipids; Recognition pheromones
1. Introduction In social insect colonies, nestmate recognition, i.e. the ability of an individual to recognise members of its own colony from aliens, is critical in preventing non-colony members from exploiting colony resources. Moreover, since workers usually do not reproduce but work to rear the offspring of reproductive nestmates, nestmate recognition decreases the likelihood of fitness losses due to alien reproductive individuals, such as intra- and interspecific social parasites. All studies aimed at identifying the cues used for nestmate recognition in social insects agree on the impor-
* Corresponding author. E-mail address:
[email protected] (S. Turillazzi).
tance of the chemicals present on the cuticle. In fact, in several species the composition of the lipid mixture on the cuticle is more similar among individuals belonging to the same colony rather than among alien individuals (see Lorenzi et al., 1996). Although studies aimed at identifying the chemicals involved in recognition in social wasps first appeared in the early nineties (cf. Singer et al., 1998), nestmate recognition capacity and its olfactory basis had already been demonstrated for some species of the genus Polistes in the early eighties (cf. Gamboa, 1996). In the last decade, epicuticular recognition pheromones have been investigated in several vespine species and, among Polistinae, in several species of Polistes species (cf. Singer et al., 1998). There have been few studies, however, of nestmate recognition in the Stenogastrinae. This subfamily,
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present in South East Asian and Papuan regions, exhibits a primitive eusocial organisation and includes species with small colonies and varying degrees of complexity in division of labour among females (Turillazzi, 1991). Morphological and behavioural differences between this group and the other eusocial vespid subfamilies (Polistinae and Vespinae) are so striking that Stenogastrinae were first considered to be a completely separate taxon (cf. Turillazzi, 1991). Later, Carpenter (1981, 1988), demonstrated their phylogenetic proximity to Polistinae and Vespinae. Although the results of a recent analysis of mitochondrial DNA (Schmitz and Moritz, 1998) seem to support the opinions of previous authors, Carpenter and Starr (2000), recently stated that the “combination of Schmitz and Moritz’s molecular data with morphological characters supports vespid monophyly”. Behavioural observations of several stenogastrine species (Samuel, 1986; Turillazzi et al., 1997; CosterLongman, 1998) and experimental evidence (Cervo et al., 1996) show that colony members easily recognise and attack wasps from other colonies approaching their nest. Lack of aggression towards alien individuals landing on the nest is sometimes observed in three species that nest in clusters, Liostenogaster flavolineata, L. vechtii and Parischnogaster alternata (Samuel, 1986; Turillazzi et al., 1997). Nestmate recognition capacity in such a primitively eusocial group is not surprising considering that kin recognition is also a widespread phenomenon in several groups of non-social organisms (Hepper, 1986; Fletcher and Michener, 1987). Experimental evidence (Cervo et al., submitted) shows that recognition can be based on chemical cues. Nevertheless, there is currently no information in the literature about the chemistry of epicuticular lipids of any Stenogastrine species, nor their possible role as nestmate recognition pheromones. Parischnogaster striatula lives in South East Asia. Colonies very seldom contain more than three females and some males. The nests are implanted on thread-like substrata and have a particular rope-like architecture, with cells twisting and opening in a central tube. Usual nesting sites are sheltered niches along forest roads or trails, or under the vaults of caves and human buildings where thread-like suspensions are present. Among the Stenogastrinae, P. striatula is one of the least known species in terms of social behaviour. In the present study we investigated if nestmate recognition occurs in this species. We performed observations (in the field) of the reactions by resident females towards conspecifics attempting to land on their nests, as well as experiments in which conspecifics (alive, dead and hexane-washed individuals) were presented to resident females. We also analysed the cuticular lipids of P. striatula females from 13 colonies by means of gas chromatography coupled with mass spectrometry. The compositional data were
subjected to univariate and multivariate statistical analyses to test if individuals belonging to different colonies could be separated on the basis of the epicuticular lipid composition.
2. Materials and methods 2.1. Behavioural observations and nestmate recognition experiments Field studies were carried out at Genting Sempah (abbreviated as GS) in the Malaysian Peninsular (610 m a.s.l., Pahang State) in February and March 2000. Members of 11 different colonies were individually marked with a colour code. Using a camcorder we videotaped the behaviour of the animals on the nests for a total of 71 hours. In the laboratory, we noted the responses of resident wasps towards conspecifics landing or attempting to land on the nest. In order to compare the data and to reduce the problem of non-independent observations (unavoidable for unmarked alien individuals), we approached the phenomenon from the colony point of view. When considering the approaches by nestmates and by alien individuals separately, each colony was scored as “directed approached” or “hovering approached” depending on whether it had more direct or more hovering landings, (in the case of equality or uncertainty, a score of 0.5 was attributed). Similarly, the same was performed for reactions to nestmates and aliens, scoring a colony as “aggressive” or “pacific”. The reaction of each colony was classified as aggressive if the resident females bit, attempted to sting, repeatedly attacked or fought with the approaching female. If none of these behaviours were observed, the reaction of the colony was considered pacific. In nestmate recognition experiments, one nestmate female and one alien conspecific female were presented (alternately, and approximately one hour apart) to the resident females of 11 colonies in the same manner as described by Cervo et al. (1996). All experiments were conducted over a period of 10 days, between 10 am and 6 pm, and were suspended in case of rain. Live, immobilised wasps were held in front of the nest at a distance of 1–2 cm. The reactions of resident females were videotaped and the tapes observed in the laboratory a few weeks later. The person watching the videotapes was unaware if aliens or nestmates were presented in the experiments he/she was observing. The behaviour of the wasps was observed for one minute after a first clear contact between one resident and the presented female. Differences in number of bites received from resident individuals by presented alien and nestmate females were analysed with the Wilcoxon signed rank test (Siegel and Castellan, 1989), so that each colony was
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compared for its own reaction when facing aliens and nestmates. In a second set of experiments performed (November 2000) on ten colonies at Genting Sempah, dead alien females (collected approximately 100 km away and killed by freezing) were presented, in the same manner as above, to the resident females. These alien individuals were then washed in 0.5 ml of hexane for 5 hours and the extract stored in a freezer. These same specimens were then re-washed in hexane for 10 more hours. After drying, they were presented to the same colonies. Half an hour later they were reapplied with the 5-hour extract, dried for 15 min, and then re-presented to the colonies. As for the first set of experiments, the number of bites received by the presented lure from the resident individuals were counted. Even though the ideal experimental foresees a similar set of experiments to be performed on a nestmate female (Breed, 1998), we were unable to do this, owing to the low number of colonies with more than two adult females present during the daytime.
acetonitrile as the reagent gas as described by Moneti et al. (1996, 1997b). The relative abundance of compounds was determined by integrating the peaks on the chromatograms obtained with a Varian Star 3400CX gas chromatograph equipped with a flame ion detector, and with an hydrogen generator which supplied both the gas carrier and gas for the detector. The system was managed by the Varian Star Chromatography Workstation software. A Restek Rtx 5-MS column (30 m×0.25 mm, 95% polyidimethylsiloxane and 5% phenyl, phase thickness 0.5 µm) was installed on both gas chromatographs. Furthermore, in both cases, the same temperature programme was used: the initial temperature of 150°C was maintained for 2 min and then raised by 5°C min⫺1 until 200°C, then by 2°C min⫺1 until 260°C and, finally by 10°C min⫺1 until 310°C. This temperature was maintained for 13 min.
2.2. Specimen collection for chemical analysis
2.4.1. Behavioural observations and nestmate recognition experiments The Wilcoxon signed rank test was used to compare aggressive and pacific reactions towards nestmates and aliens in the presentation experiments (Siegel and Castellan, 1989). The Fisher exact test was used to compare frequencies of reaction to hovering and direct flight approaches (Sokal and Rohlf, 1995).
Forty-four females belonging to 13 of the more populated colonies were collected from two localities in the Malaysian Peninsula. Twenty two specimens from six colonies (mean=3.7 females, SE=0.88) were collected at Genting Sempah and 22 specimens from seven colonies (mean=3.1 females, SE=0.57) at Bukit Fraser (abbreviated as BF; 1300 m a.s.l., Pahang State). The specimens were frozen shortly after collection. Subsequently, the fifth gastral segment of each specimen (including both sternite and tergite and cleared of internal tissues) was removed and inserted in a soft glass capillary tube (2×30 mm) which was then sealed in a flame as described by Morgan (1990). 2.3. Gas chromatography and mass spectrometry In Florence, the capillaries were inserted in a glass vial (2 ml) and broken. Each vial was then immediately closed and heated in an aluminium heater block at 170°C. Headspace sampling was then performed using a solid phase microextraction (SPME) fiber covered with a 7 µm thick polydimethylsiloxane film (SUPELCO). The fiber was inserted into the vial (kept at 170°C) through the cap and then exposed in the headspace for 10 min as described by Moneti et al. (1997a). For gas chromatographic-mass spectrometric analyses the fiber was inserted in the injection port of a HP5890 II series gas-chromatograph coupled to a HP 5971A quadropole mass spectrometer. In addition, a Varian Saturn 2000 Ion Trap mass spectrometer coupled to a Star 3400CX gaschromatograph was used to identify double bond positions in unsaturated hydrocarbons and alcohols, using
2.4. Statistical analyses
2.4.2. Chemical analysis The areas of the identified compounds were transformed by: Ln∗(Ap/GmAp) where Ap is the area of the peak and GmAp is the geometric mean of the areas of the peaks in each specimen. This transformation is recommended when compositional data have to be subjected to multivariate statistical analyses (Reyment, 1989). The transformed data were analysed with the Statistical Package for Social Sciences (SPSS 8.0). Principal Components Analysis (PCA) was performed to identify subsets (principal components, PCs) of strongly correlated variables (chemical concentrations) to be subsequently subjected to Discriminant Function Analysis (DFA). Only those chemicals whose degree of association was higher than 0.8 were subjected to PCA (as suggested by Norusˇ is, 1992), while the others were excluded from the PCA and then included in the DFA as independent variables. Varimax rotation was used in the PCA to render the interpretation of the results easier. Since preliminary analyses revealed large differences in the composition of cuticular lipids in the two populations (PC1 and 14 out of the 18 original variables were significantly different; Student’s t test, P⬍0.05), further
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statistical analyses were performed separately on the two population datasets. The Kruskall–Wallis test was used to identify which variables (PCs and concentrations of single chemicals) were significantly different (P⬍0.05) among colonies belonging to the same populations. Discriminant Function Analysis was performed to evaluate the existence of synergistic effects of the PCs and the original variables excluded from the PCA on the assignment of each individual to its own colony. The PCs and the excluded original variables were entered simultaneously into the model. Contributions of the different DFs to the model were evaluated through the study of ⌳, i.e. the product of each univariate DF Wilk’s ⌳. The validity of the classification was based on the Bayes method, taking 0.7 as the threshold posterior probability for an acceptable assignment to a given group (Norusˇis, 1992). To evaluate the contribution of each independent variable entered in the Discriminant Function Analysis to the model, we calculated the Pearson’s correlation coefficients between the DF scores and the PCs scores and between the DF scores and the concentration values for the compounds excluded from the PCA. Because of limitations due to the small number of specimens in each colony, the DFA was performed more with an explorative aim rather than an inferential one.
3. Results 3.1. Behavioural observations and nestmate recognition experiments A strong difference was noted in the way wasps approached a nest, depending on whether they belonged to the nest or not. Generally, wasps approached their own nests with a direct flight. In contrast, individuals were found to hover in front of alien nests before landing on them. This behavioural difference was noticed both for females (74 direct landings and 5 hoverings observed for 15 females approaching their own nest; 0 direct landings and 18 hoverings observed for females approaching alien nests) and males (24 direct landings and 2 hoverings observed for 12 males approaching their own nests; 8 direct landings and 4 hoverings for males approaching alien nests). The approach and landing of a conspecific on the nest generally aroused the attention of the resident females; if inside the nest, females usually came out on to the surface. In almost all the cases when the resident females responded to an approaching female or male nestmate (in total, 18 and 15 respectively), a non-aggressive behaviour was observed (49 pacific reactions and 0 aggressive reactions towards nestmate females; 12 pacific reactions and 6 aggressive reactions towards nestmate males). In contrast, resident wasps behaved aggressively towards alien individuals or were alarmed
(6 aggressive responses, 13 alarms and 0 pacific interactions towards alien females; 10 aggressive responses, 1 alarm and 0 pacific interactions towards alien males). Direct approach vs hovering (Table 1) was used significantly more by female nestmates (10 vs 0) than by aliens (0 vs 8; Fisher Test, P=0.0002). A significant difference was also found between alien females and males (3.5 vs 0.5; P=0.02, Fisher Test), suggesting that males tended to directly approach alien nests more often than females. Colonies showed significantly more aggressive reactions (Table 2) towards alien females (4 vs 0) and males (4 vs 0) than towards nestmate females (0 vs 7; P=0.003, Fisher Test) and males (1.5 vs 5.5; P=0.015, Fisher Test). In nestmate recognition experiments, the bites received in 11 colonies by the presented nestmate females were much less than those received by the alien females (on average 14.81 and 2.81 respectively, N=11, Z=⫺2.601, P⬍0.001, Wilcoxon signed rank test). In “removal and replacement” experiments, we observed a very aggressive reaction towards dead alien conspecifics in the ten colonies tested (average number of bites received=22.7). The same lures washed in hexane, however, received much fewer bites (on average 6.9; Z=⫺2.1, P=0.036, Wilcoxon signed rank test) but subsequently received a higher number of bites after these had been reapplied with extracts (on average 11.7; Z=⫺2.807, P=0.005, Wilcoxon signed rank test). Differences in number of bites received by the not-manipulated and reapplied lures were not significant (Wilcoxon signed rank test, Z=⫺1.784, NS). 3.2. Cuticular lipids composition and discrimination of colonies Five classes of compounds were detected on the cuticle of Parischnogaster striatula: linear alkanes, monoenes, dienes, saturated and unsaturated primary alcohols. Branched alkanes were completely absent. Monoenes made up 54.15% and n-alkanes 45.21% of the epicuticular volatile mixture. The most common compounds were monoenes with double bonds in positions 9- and 7(Table 3). Ten of the identified compounds, present in trace amounts in only some of the specimens, were not considered in the statistics because the area values obtained for their peaks were not considered reliable. The PCA analysis produced the following principal components (6,9-C23:2, 1-eicosanol and 7-C25:1 were excluded from the analysis because of their low communality index): — PC1 correlated mainly with unsaturated hydrocarbons (9-C23:1, 7-C23:1, 7-C21:1, 9-C24:1) and with two alkanes (n-C23 and n-C22); it can be defined as the “component of middle and short chain alkenes”;
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Table 1 Direct and hovering approached colonies of P. striatula by nestmate and alien conspecifics (see Section 2) Colonies approached
Direct Hover
Nestmates
Aliens
Females
Males
Total
Females
Males
Total
10 0
6 1
16 1
0 8
3.5 0.5
3.5 8.5
Table 2 Colonies reacting aggressively and peacefully to nestmate and alien conspecifics landing on nests Colony reaction
Pacific Aggress.
Nestmates Females
Males
Total
Aliens Females
Males
Total
7 0
5.5 1.5
12.5 1.5
0 4
0 4
0 8
Table 3 Means and standard deviations of the volatile compounds found on the cuticle of P. striatula females (N=44)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Compoundsa
% Average
SD
9-heneicosene (9-C21:1)* 7-heneicosene (7-C21:1) 1-octadecanol (1-C18OH)* n-heneicosane (n-C21) 9-docosene (9-C22:1)* 7-docosene (7-C22:1)* n-docosane (n-C22) 6,9-tricosadiene (6,9-C23:2) 9-tricosene (9-C23:1) 7-tricosene (7-C23:1) 1-eicosanol (1-C20OH) n-tricosane (n-C23) 9-tetracosene (9-C24:1) 8-tetracosene (8-C24:1)* 7-tetracosene (7-C24:1)* n-tetracosane (n-C24) 9-pentacosene (9-C25:1) 7-pentacosene (7-C25:1) 1-docosanol (1-C22OH)* n-pentacosane (n-C25) n-hexacosane (n-C26) 9-heptacosene (9-C27:1) 7-heptacosene (7-C27:1)* n-heptacosane (n-C27) n-octacosane (n-C28) 9-nonacosene (9-C29:1)* 17-hexacosen-1-Ol (17-C26:1OH)* n-nonacosane (n-C29)
0.117 1.021 0.083 15.005 0.885 0.473 0.932 0.359 33.686 15.625 0.364 18.275 0.271 0.081 0.072 0.228 2.961 0.537 0.072 1.361 0.306 0.502 0.073 4.179 0.166 0.275 0.291 1.800
0.121 1.156 0.184 4.577 1.774 0.487 0.291 0.232 18.367 15.551 0.441 9.704 0.124 0.178 0.134 0.662 2.163 0.458 0.327 0.958 0.446 0.367 0.100 3.087 0.162 0.324 0.523 2.165
a *
Compounds not included in the statistical analyses.
— PC2, associated mainly with n-C27, n-C28 and nC29; the “component of the long chain alkanes”; — PC3, associated mainly with n-C24, n-C25 and nC26; the “component of the middle-long chain alkanes”.
— PC4, is correlated with 9-C27:1 and 9-C25:1 and with n-C21; the “component of the middle-long chain alkenes”. The four PCs explained 87.73% of the total variance. Factor loadings of the variables on the first four PCs are reported in Table 4. In both populations, colonies differed significantly for PC2 (Kruskall–Wallis test: GS P=0.015, BF P=0.042). Furthermore 7-C25:1 and PC3 were were also significantly different among colonies in the Genting Sempah population (P=0.046 and P=0.008 respectively). For the BF population, the DFA correctly attributed 68.2% of the individuals to their own colonies. Two other specimens were correctly assigned but with a posterior probability lower than 0.7; four out of the five misclassified specimens were correctly assigned in the second highest probability group. The first and the Table 4 Factor loadings after varimax rotation Variables
PC1
9-C23:1 9-C24:1 7-C23:1 n-C23 7-C21:1 n-C22 n-C29 n-C28 n-C27 n-C26 n-C24 n-C25 9-C27:1 n-C21 9-C25:1
0.916 0.872 ⫺0.811 0.810 ⫺0.800 0.661
PC2
PC3
PC4
0.523 0.916 0.883 0.761 0.873 0.867 0.735 ⫺0.566
⫺0.820 0.668 ⫺0.656
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Table 5 Discriminant functions for the BF (Bukit Fraser) samples: principal statistics Function
Eigenvalue
% of variance
Canonical correlation
1 2 3
4.186 2.257 1.390
47.4 25.5 15.7
0.898 0.832 0.763
second DFs explained 47.4% and 25.5% of the total variance respectively (Table 5). The difference between colonies was significant (⌳0=0.11; c2=62.5; P=0.02) if all discriminant functions were considered together, but not significant (⌳1=0.06; c2=39.5; P=0.11) when DF1 was removed. DF1 was correlated most strongly with 7-C25:1 (r=0.45, P⬍0.05), while DF2 was correlated more strongly with PC2 (r=0.72, P⬍0.001). For the GS population, 81.9% of specimens were correctly attributed to their colonies; another specimen was assigned to its own colony but with a posterior probability lower than 0.7 and all three misclassified females were correctly attributed in the second highest probability group. Together DF1 and DF2s explained 88.3% of the total variance, with the former explaining 70.3% (Table 6). The difference between colonies was still significant after removal of DF1 (⌳1=0.80; c2=36.64; P=0.047), but not significant when also DF2 was also removed (⌳2=0.29; c2=17.76; P=0.27). Among the variables with statistically significant correlations, DF1 was correlated most strongly with PC3 (r=0.935, P⬍0.001) and PC1 (r=0.445, P⬍0.05), DF2 with PC2 (r=0.720, P⬍0.001).
4. Discussion As reported for other stenogastrine species, Parischnogaster striatula discriminates between nestmates and alien conspecifics. Discrimination experiments similar to those described here have been performed on P. jacobsonii and on two cluster-nesting species, Liostenogaster flavolineata and L. vechtii (Cervo et al., 1996). Like P. jacobsonii, P. striatula rarely fails to discriminate between nestmates and alien conspecifics. In contrast, Table 6 Discriminant functions for the GS (Genting Sempah) samples: principal statistics Function
Eigenvalue
% of variance
Canonical Correlation
1 2 3
10.437 2.677 1.217
70.3 18.0 8.2
0.955 0.853 0.741
both L. flavolineata and L. vechtii often allow alien conspecifics to land onto the nest. P. alternata, another cluster-nesting species, showed a similar tendency in naturally occurring interactions (Turillazzi et al., 1997). Other authors (Samuel, 1986; Turillazzi et al., 1997; Coster-Longman, 1998) have reported that colonies in clusters experience continuous landing attempts by alien individuals. As discussed elsewhere (Cervo et al., submitted) this may lead to a lower level of guarding by resident females, and consequently to a higher risk of colony usurpation and immature brood destruction, which may counterbalance the advantages that these species obtain from nesting in clusters (Coster-Longman, 1998). Although visual cues are generally not taken into consideration as recognition cues in social insects, our study confirms the observation by West-Eberhard (1969) in Polistes fuscatus, that the direct or hesitant manner in which conspecifics approach a nest could determine the first response by resident females towards the landing individuals. More reliable information based on olfactory cues could then be acquired through direct contact and antennation with the individuals once landed, behaviours often observed in naturally occurring interactions and in our experiments. Experiments performed with hexane-washed and extract-reapplied females bring further support for this claim that chemical cues are important in recognition. Furthermore, when considering the nature of these cues, the compositional data suggest a possible implication of cuticular lipids. As already stated, colony size strongly limited the elaboration of a multivariate model of discrimination between colonies based on the lipid mixture composition of the various individuals. Despite this limitation, the discriminant models obtained for the two populations appear to be satisfactory. Thus in this species, epicuticular lipid profiles tend to be more similar among nestmates than among alien individuals. This is consistent with results found in several other social insects. However, final confirmation of the hypothesis that cuticular hydrocarbons are used as recognition pheromones in discrimination between nestmates and aliens, must come from bioassays in which cuticular lipids are altered (for chemical supplementation experiments, see Breed, 1998). Among social wasps for which the cuticular lipid composition has been reported in the literature, Parischnogaster striatula is the only one lacking of branched alkanes. Analyses of three other Stenogastrine species in our laboratory have also revealed the absence of branched alkanes in Eustenogaster fraterna (Sledge, pers. comm.) and Liostenogaster flavolineata (Cervo, et al., submitted), while methyl alkanes are abundant in L. vechtii and P. mellyi (Sledge, pers. comm.). The abundance of monoenes is a recurrent characteristic in many of these species. Because of the high percentage of
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monoenes, and the absence of branched alkanes found in some species, the hover wasps differ remarkably from all the Polistes species analysed to date. The latter, in fact, possess small amounts of alkenes, while methyl alkanes form a high percentage of the compounds present, both in number and in quantity (Bonavita-Cougourdan et al., 1991; Lorenzi et al., 1997; Singer et al., 1998). Among the Vespinaes, Vespa crabro and Dolichovespula maculata present a high percentage of monoenes, and methyl alkanes are present both in these two species and in the other vespine wasps analysed (Butts et al., 1991). With regard to hydrocarbon chain length, Stenogastrinae tend to have the shortest chains, while the longest chains are found in Polistes. Another feature of P. striatula and of several other Stenogastrine species (Cervo et al., submitted., Sledge et al., pers. comm.) is the presence of long chain alcohols. In fact, although fatty alcohols have been found in cuticular lipids of other insects (see Buckner, 1993), only one free fatty alcohol has only ever been reported in social wasps (in Polybioides tabidus; Dani et al., 1997). Acknowledgements We thank Dr. Matthew Sledge (University of Firenze), Dr. Angelo Fortunato (Rice University, Houston, Texas) and Dr. Rosli Hashim (Inst. of Biological Sciences, (University of Malaya, Kuala Lumpur, Malaysia) for their help in Malaysia and Mr. Henry Barlow who welcomed us on his property. The study has been supported with funds from the Italian M.U.R.S.T. (PRIN “Organizzazione sociale e spazio temporale del comportamento”), University of Firenze and the Italian C.N.R. References Bonavita-Cougourdan, A., The´ raulaz, G., Bagne`res, A.G., Roux, M., Pratte, M., Provost, E., Cle´ ment, J.L., 1991. Cuticular hydrocarbons, social organization and ovarian development in a polistine wasp: Polistes dominulus Christ. Comparative Biochemistry and Physiology 100B, 667–680. Breed, M.D., 1998. Chemical cues in kin recognition: criteria for identification, experimental approaches, and the honey bee as an example. In: Vandermeer, R.K., Breed, M.D., Winston, M.L., Espelie, K.E. (Eds.), Pheromones Communication in Social Insects. Westview Press, Boulder, pp. 57–78. Buckner, J.L., 1993. Cuticular polar lipids in insects. In: StanleySamuelson, D.W., Nelson, D.R. (Eds.), Insect Lipids, Chemistry, Biochemistry and Biology. University of Nebraska Press, Lincoln, pp. 227–270. Butts, D.P., Espelie, K.E., Hermann, H.R., 1991. Cuticular hydrocarbons of four species of social wasps in the subfamily Vespinae: Vespa crabro L., Dolichovespula maculata (L.), Vespula squamosa (Drury), and Vespula maculifrons (Buysson). Comparative Biochemistry and Physiology 99B, 87–91. Carpenter, J.M., 1981. The phylogenetic relationships and natural classification of the Vespoidea (Hymenoptera). Systematic Entomology 7, 11–38.
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