Appl. Entomol. Zool. 44 (1): 133–141 (2009) http://odokon.org/
Repellent effect on host Formica workers of queen Dufour’s gland secretion of the obligatory social parasite ant, Polyergus samurai (Hymenoptera: Formicidae)
Yousuke TSUNEOKA1,* and Toshiharu AKINO2 1 Faculty of Science, Ibaraki University; Mito, Ibaraki 310–8512, Japan 2 Chemical Ecology Laboratory, Kyoto Institute of Technology; Kyoto 606–8585, Japan (Received 9 July 2008; Accepted 29 September 2008)
Abstract Founding queens of the obligatory social parasite ant Polyergus samurai usurp the host Formica japonica colony. The queen secretes chemicals from its gaster when killing the host queen. At this time, the parasite queen is hardly at- tacked by host workers. We hypothesized that the queen’s secretion acts as a repellent to deter host workers’ aggres- sion during host queen killing. Gas chromatograph-mass spectrometry analyses confirmed that the chemical composi- tion of Dufour’s gland contents of P. samurai queens was almost identical to the chemicals remaining on the body sur- face of host Formica queens killed by them. Host F. japonica workers were used for bioassays. In an encounter with an intruder ant, the workers’ aggression to the intruder was reduced significantly when Dufour’s gland extract was ap- plied to the intruder. Treatment of workers with the gland extract significantly increased the receding behavior of nest- mates. The host workers receded from a glass dummy treated with the gland extract. The time necessary for a worker to get over a barricade applied with gland extract was significantly longer than that of controls. These results strongly suggest that Dufour’s gland secretion acts as a repellent allomone.
Key words: Repellent; social parasite; Dufour’s gland; Polyergus samurai; Formica japonica
2001), considered as relevant chemicals of nest- INTRODUCTION mate recognition in ants (Lahav et al., 1999; Akino In obligatory social parasite ants, queens and et al., 2004; Howard and Blomquist, 2005; Ozaki workers lack the ability to rear a brood, foraging, et al., 2005). and colony maintenance (Hölldobler and Wilson, Another strategy is the use of “chemical 1990; D’Ettorre and Heinze, 2001). The newly weapons.” Several offensive chemicals with which mated parasite queen of pirate ants invades a host a parasite can disrupt a host’s defense system have colony, kills the resident queen, and usurps the been reported: alarm (Lloyd et al., 1986), appease- colony. In this process, the parasite queen is re- ment (Topoff et al., 1988; Mori et al., 2000b), de- quired to break down host workers’ nestmate terrent (Martin et al., 2007) and repellent (D’Et- recognition, not only to deter aggression but also to torre et al., 2000; Ruano et al., 2005) substances. be adopted by host workers. For this purpose, at The genus Polyergus comprises five species, all least two different chemical strategies might be of which are obligatory social parasites on ants adopted by such social parasites. Chemical mim- of its related genus Formica. The usurpation icry is recognized as a major deception strategy processes have been described for two Polyergus used by various social parasites to break down species: P. breviceps (Topoff et al., 1988; Zimmerli nestmate recognition (Lenoir et al., 2001). During and Topoff, 1994) and P. rufescens (Mori et al., usurpation, the parasite queen acquires or biosyn- 1995; D’Ettorre et al., 1997; review in Mori et al., thesizes the cuticular hydrocarbons of the host 2001). Their behavioral sequences in usurpation queen (D’Ettorre and Errard, 1998; Johnson et al., were similar in many respects: Polyergus queens
*To whom correspondence should be addressed at: E-mail: [email protected] DOI: 10.1303/aez.2009.133
133 134 Y. TSUNEOKA and T. AKINO invade the host nest, kill the host Formica queens, equate amount of bloodworms, mealworms, or ter- and then continuously flex the gaster up and down. mites every 2 days. Accordingly, the aggression of host workers toward Separately, 10 dealated P. samurai queens were parasite queens decreases, and the reduction of collected during their nuptial flight season (mid- host worker aggression toward intruder ants ap- July) in Mito, Japan, 2007. plied with Dufour’s gland contents of a P. breviceps Usurpation experiment. One F. japonica queen queen was demonstrated (Topoff et al., 1988); and 30 workers were taken from each of the four therefore, parasite Dufour’s gland secretion is sug- small laboratory colonies. They were placed in a gested to serve as an appeasement allomone. Later, small plastic box (75 50 20 mm) on the day of this appeasement effect of the queen’s Dufour’s collecting P. samurai queens. The box had a hole gland was also suggested in P. rufescens (Mori et (5 mm diam.) in the lateral side; its upper side was al., 2000a). In contrast, D’Ettorre et al. (2000) re- transparent red. One P. samurai queen was placed ported that Dufour’s gland secretion of P. rufescens near the nest entrance an hour after Formica intro- queens acted as a repellent allomone to deter host duction. After 45-min observation, the killed host workers’ aggression. The effect of Dufour’s gland queens were taken from the nests for chemical secretion of the Polyergus queen is therefore con- analyses. This procedure was repeated four times troversial among researchers. using different lab colonies. This study investigated the role of the secretions Sample preparation. Six dealated P. samurai of queens of P. samurai, which is distributed queens were dissected to confirm their insemina- throughout eastern Asia (Japanese Ant Database tion soon after collection. The Dufour’s gland, poi- Group, 2003). The usurpation behavior of P. samu- son gland, pygidial gland and hindgut of the queens rai queens was recently reported in detail and they were dissected out in double-distilled water. In the were found to resemble other Polyergus queens same manner, four F. japonica queens were dis- (Tsuneoka, 2008). Most interestingly, P. samurai sected to obtain their Dufour’s glands. Each gland queens emitted a large amount of secretion toward was immersed immediately in 200 ml hexane for host F. japonica queens during killing. We tested 20 min. A quarter of each gland extract of P. samu- the hypothesis that P. samurai queens’ secretion rai queen was subjected to chemical analysis; the acts as a repellent to deter host workers’ aggression rest was concentrated to 15 ml by evaporation for during host queen killing. This study also con- the behavioral bioassay. firmed that most of the Dufour’s gland secretion of The F. japonica queens killed by P. samurai in P. samurai queens is emitted during host queen the usurpation experiments were immersed individ- killing, acting as a repellent allomone. ually in 1 ml of hexane for 5 min. Simultaneously, four F. japonica queens were immersed similarly in hexane. This enabled the extraction of compounds MATERIALS AND METHODS from the ants’ body surface, but there was some Ants. Four small queenright colonies of Formica unavoidable contamination from exocrine glands. japonica, which involved a few hundred workers Extracts of F. japonica queens were chro- each, were collected in Tsukuba, Ibaraki Prefec- matographed individually on approximately 0.8 g ture, Japan in 2005. In addition, 10 large queen- of silica gel (Wako-gel C-200; Wako Pure Chemi- right colonies, which involved more than 1,000 cal Industries Ltd., Japan). The hydrocarbons and workers, were collected in Ibaraki (five from Ishi- polar compounds were eluted successively with oka, two from Mito, and two from Tsukuba) and 10 ml of hexane and diethyl ether, respectively. In Yamanashi (one from Fujiyoshida) Prefectures, our preliminary analysis, the P. samurai queen Du- Japan during 2005–2007. They were reared sepa- four’s gland contents contained only polar com- rately in a laboratory using plastic containers pounds; therefore, only the polar fractions of F. (99 64.5 28 or 179 82 32 mm) as artificial japonica queens’ extracts were subjected to chemi- nests at 26 1°C in summer and 8 1°C in winter, cal analysis. 70 10% relative humidity, and 14L10D in sum- Effect of gland extract on aggression toward mer and 10L14D in winter. They were provided non-nestmate workers. We first tested the effects with aqueous honey water (1 : 1), water, and an ad- of the four gland extracts of P. samurai queens on Repellent Effect of Dufour’s Gland Secretion of Polyergus samurai 135 the aggression of F. japonica workers to non-nest- 2 min at most: the first response was recorded. Ob- mate workers. Non-treated workers and workers servations of the extract treatment and controls (no treated with hexane solvent were used as controls. and solvent treatments) were repeated for 10 One worker was marked on its hindleg using a colonies. Five replicates were set up per colony. marker (Paint Marker; Mitsubishipencil Co., Ltd., Repellent effect of Dufour’s gland extract on Japan) and gently placed in a glass Petri dish host workers. A F. japonica worker was placed (75 mm in diam. 15 mm in depth). Another gently in a Petri dish (60 mm diam. 7 mm high) worker from another colony was anesthetized on that had been divided into two by a filter paper wall ice for ca. 1 min; 0.5 ml of gland extract (1/40 (5 mm high). One minute before its introduction, gland equivalent) was applied to its head, thorax, 50 ml of Dufour’s gland extract (1/10 gland equiva- and gaster using a micro-syringe, and then the lent) was applied to the paper wall. The time for treated workers were gently placed into the Petri the worker to climb over the wall was recorded. dish. The marked worker was observed for 5 min. Observations of the extract treatment and controls Aggressive behavior toward the treated worker was (no and solvent treatments) were repeated for 10 scored according to the following aggression score colonies. (AS): 0: ignore or escape; 1: threat (open Gas chromatography and gas chromatogra- mandibles); 2: biting lasting for less than 15 s; 3: phy-mass spectrometry analysis. Dufour’s gland biting lasting for more than 15 s; 4: biting lasting extracts of queens of P. samurai and F. japonica, as for more than 60 s. Aggression experiments with well as the polar fraction of cuticular extract of F. five colony combinations were pursued for each japonica queens before and after being killed by P. treatment. Each colony was used for only one samurai queens, were applied to gas chromatogra- colony combination. Ten aggression tests with dif- phy (GC) analyses at 1/400, 1/40, and 1/100 ant ferent individuals were conducted per combination. equivalents, respectively. GC analyses were con- Effect of Dufour’s gland extract behavior to- ducted using a gas chromatograph (GC-14B; Shi- ward nestmate worker. The effects of Dufour’s madzu Corp.) equipped with a non-polar capillary gland secretion, which affected the aggressive be- column Rtx-1 (30 m length, 0.32 mm i.d., 0.25 mm havior of host workers significantly (see Results), film thickness; Restek Corp.) and a flame ioniza- were tested on nestmate workers’ behavior. Fifty F. tion detector. The injector was made in a splitless japonica workers from one colony (recipient ants) mode at 280°C for 1 min. The detector temperature were marked on their gaster using a marker and was set at 310°C. The oven temperature was kept moved to the nest box. A worker from the same at 50°C for 5 min, programmed at 10°C/min to colony was then treated with Dufour’s gland extract 300°C, and then kept at the final temperature for in the same manner as for the aggression test; the 15 min. Helium was used as a carrier gas at worker was then introduced into the nest box. The 120 kPa. Peak areas were computed using an inte- behavior of recipient ants toward treated ants was grator (C-R6A; Shimadzu Corp.) coupled with GC. observed for 20 min, and the behavior of the first Pooled Dufour’s gland extracts of the P. samurai 10 contacts was recorded. Observations of the ex- queen (1/20 ant equivalent) and extracts of the tract treatment and controls (no and solvent treat- killed host queen (1/5 ant equivalent) were pro- ments) were repeated for 10 colonies. vided for gas chromatography-mass spectrometry Response to a glass dummy. The response to a (GC-MS) analyses, which were performed using a glass dummy treated with Dufour’s gland extract gas chromatograph (GC-17; Shimadzu Corp.) in- was observed to confirm whether the Dufour’s terfaced to a QP5050 quadrupole mass detector in gland extract by itself affected host worker behav- EI mode with 70 eV, and operated with software ior. A glass bead (ca. 2 mm diam.) was applied (Class 5000 for Windows; Shimadzu Corp.). GC with 0.5 ml of Dufour’s gland extract (1/40 gland was performed under the same conditions. equivalent), and placed in the center of a Petri dish Statistical analysis. In the aggression test, the (60 mm diam. 7 mm high). A F. japonica worker summed AS per ant pair was fitted using a general- was gently placed in the Petri dish 1 min after treat- ized linear model (GLM) with gamma-Poisson dis- ing the glass dummy. The behavior of the F. japon- tribution using the log-transformed number of con- ica worker to the glass dummy was observed for tacts as an offset variable because the number of 136 Y. TSUNEOKA and T. AKINO contacts differed among ant pairs. The influences of factors on the AS were tested using the likeli- hood ratio (LR) test. The receding responses, indi- cating the repellent effect, of recipient ants to the treated nestmate ants and of introduced ants to the glass dummy were fitted using GLM with binomial distribution. The influence of factors was then tested using the LR test. Family-wise errors in mul- tiple analyses were corrected using Holm’s method (Sokal and Rohlf, 1990). Data analyses were car- ried out using R 2.5.1. (R Development Core Team, 2007). To compare the peak areas of common com- pounds between Dufour’s gland extracts and the polar fractions of killed F. japonica queens’ ex- Fig. 1. Effects of six treatments on host worker aggres- tracts, the peak areas of the Dufour’s gland extracts sion toward non-nestmate workers. One point and attached vertical lines represent the mean and SE in each combination were quadrupled. All peak areas were standardized of colonies (n 10 for each point). Horizontal bars represent as yij yij/zi, where yij is the peak area for peak i in the mean among the five combinations of colonies. Different sample j, and zi is the mean quadrupled peak area letters denote significant differences between treatments (LR for peak i of Dufour’s gland extracts. test with multiple corrections using Holm’s method).
RESULTS Effect of gland extract on host worker aggres- sion Aggressive behavior by F. japonica workers in most colony combinations was usually shown as threats and biting after antennation. Escape behav- ior was very rare (12 times among 1,579 observed contacts). Both the treatments and colony combina- tions significantly influenced the AS (treatments: df 5, c 2 34.51, p 0.001; colony combinations: df 4, c 2 19.44, p 0.001, [n 50 per treatment], Fig. 1). When workers were treated with Dufour’s gland extracts of P. samurai queens, they were usu- ally ignored or threatened, and only rarely suffered Fig. 2. Effects of Dufour’s gland on the response patterns continuous biting. The AS was significantly re- of host workers to nestmate ants. The means and SE of behav- duced by Dufour’s gland extract more than the ior frequency are shown (n 10). White bars indicate disre- others (LR test, p 0.01 in all comparisons of gard. Gray bars indicate grooming. Dark gray bars indicate Dufour’s gland vs. others, Fig. 1). startle response. Black bars indicate a receding response. Dif- ferent letters denote significant differences between treatments Effect of Dufour’s gland extract on behavior to- (LR test with multiple corrections by Holm’s method). ward nestmates The responses of recipient workers were shown applied with Dufour’s gland extract was receding as disregard, grooming, startled, or receding. For (mean SE: 5.7 0.45 [n 10], Fig. 2). The reced- control ants (non-treated and hexane-treated), dis- ing response disappeared in all trials 10 min after regard was the most frequent response among introduction. The receding response frequency the first 10 contacts (mean SE: 7.4 0.55 and among all responses was significantly different 6.5 0.76, respectively [n 10], Fig. 2). On the among treatments (df 2, c 2 77.966, p 0.001, other hand, the most frequent response to workers Fig. 2), and between that of Dufour’s gland and the Repellent Effect of Dufour’s Gland Secretion of Polyergus samurai 137 other treatment (no treatment vs. hexane, p 0.086; no treatment vs. Dufour’s gland, p 0.001; hexane vs. Dufour’s gland, p 0.001).
Response to a glass dummy treated with Du- four’s gland extract Usually, F. japonica workers disregarded no- treatment and hexane-treated glass dummies (no treatment, 46 of 50 responses; hexane, 47 of 50 re- sponses, Fig. 3), and showed aggression to a non- treated glass dummy only once. In Dufour’s gland extract treatment, 29 F. japonica workers receded from the treated glass dummies, 14 workers disre- garded them, and the remaining seven workers at- tacked them (Fig. 3). Five of seven workers which had attacked the glass dummy receded from them a few seconds after attacking (Fig. 3). The frequen- Fig. 3. Effects of Dufour’s gland on the response patterns of host workers to the glass dummy. The frequencies of re- cies of receding response differed significantly sponses by F. japonica workers are shown (n 50). White bars 2 among treatments (df 2, c 49.559, p 0.001, indicate disregard. Gray bars indicate receding response after Fig. 3) and between Dufour’s gland and the other aggressive behavior (see text). Dark gray bars indicate aggres- treatment (no treatment vs. hexane, p 1.000; no sion. Black bars indicate receding response. The receding re- treatment vs. Dufour’s gland, p 0.001; hexane vs. sponse was significantly greater in the Dufour’s gland treat- Dufour’s gland, p 0.001). ment (p 0.001).
Repellent effect of Dufour’s gland extract on host workers In two controls, F. japonica workers climbed over the paper wall soon after their introduc- tion (median [first and third quartiles]: no treat- ment, 12.5 [9.0–23.0] s, n 10; hexane, 18 [9.25– 26.75] s, n 10, Fig. 4). Treatment with Dufour’s gland extract markedly increased the period before climbing the wall (median [first and third quar- tiles]: 58 [30.5–120.5] s, n 10, Fig. 4). Multiple comparisons of Wilcoxon’s signed rank test (paired with the colony) revealed significant differences between Dufour’s gland treatment and the other treatment (no treatment vs. Dufour’s gland, p 0.017; hexane vs. Dufour’s gland, p 0.025). Fig. 4. Elapsed time before climbing the treated paper wall. Minimum and maximum (edge horizontal lines), first and GC and GC-MS analyses third quartiles (rectangle), and the median (middle horizontal GC profiles markedly differed between the intact line) are represented by a box plot (n 10). Only significant p F. japonica queen and the killed F. japonica queen values ( 0.05) analyzed by Wilcoxon’s signed rank test with (Fig. 5B, C). Most peaks detected in the intact F. multiple corrections by Holm’s method are shown. japonica queen had identical retention indexes to those in the Dufour’s gland of the F. japonica in Fig. 5). The killed F. japonica queen and Du- queen (Fig. 5A, B). Peaks 1, 6, 8–11, and 14–16 four’s gland of P. samurai shared peaks 1, 2 and were detected in killed F. japonica queens but in 6–16, of which the retention indexes and mass neither the intact F. japonica queen nor in F. japon- spectrum were mutually identical (Fig. 5, Table 1). ica queen’s Dufour’s gland (shown as vertical lines Among these common peaks, the mean values of 138 Y. TSUNEOKA and T. AKINO
Fig. 5. Comparison of gas-chromatographic profiles among Dufour’s gland extract of queen F. japonica (A) and P. samurai (D) and polar fractions of cuticular extract of queen F. japonica before (B) and after (C) being killed by P. samurai. Chromatograms A, B, C, and D were obtained at 1/40, 1/100, 1/100, and 1/400 ant equivalent of the respective samples. Vertical lines represent the peaks detected only in C and D. Letters above peaks are peak numbers shown in Table 1. BHT: dibutylhydroxytoluene. standardized peak areas of low-volatile compounds ters was dominant in Dufour’s gland secretion (ca. ( 300 molecular weight, peaks 10, 11 and 14–16) 90%, Table 1); the most abundant compound was from killed F. japonica queens were 0.72–0.96 decyl butyrate (mean SD: 63.84 4.13%, n 6). times those of Dufour’s gland extracts (Fig. 6). Eleven peaks in Dufour’s gland secretion of P. DISCUSSION samurai queen had ester-specific fragment ions (acetate at m/z 61; propionate at m/z 75; butyrate at The results of this study confirm the clear repel- m/z 89, Table 1). In both series of estimated ac- lent effect of the Dufour’s gland content of P. etates and butyrates, the differences of equivalent samurai queens on F. japonica workers. The gland chain length among compounds corresponded to extract significantly increased the frequency of the the number of carbons (Table 1). The series of es- receding behavior of F. japonica workers to both Repellent Effect of Dufour’s Gland Secretion of Polyergus samurai 139
Table1. Compounds in Dufour’s gland of P. samurai queen
Peak Estimated compound Mean proportion Molecular mass and diagnostic ions ECL No. name (% SD, n 6)
1 Octyl butyrate 200: 43, 56, 71, 83, 89, 112 13.59 5.44 13.74 2 Decyl acetate 200: 43, 55, 61, 70, 83, 97, 111, 140 4.81 2.10 13.94 3 Not identified —: 43, 69, 107, 136, 151 0.74 0.25 14.30 4 Nonyl butyrate 214: 43, 56, 71, 89, 126 0.30 0.05 14.73 5 Decyl propionate 214: 43, 57, 70, 75, 83, 97, 112, 140 0.28 0.08 14.86 6 Decyl butyrate 228: 43, 55, 71, 83, 89, 97, 111, 140 63.84 4.13 15.75 7 Dodecyl acetate 228: 43, 55, 61, 69, 83, 97, 111, 168 0.93 0.33 15.93 8 Dodecyl butyrate 256: 43, 55, 71, 83, 89, 97, 168 5.06 0.71 17.73 9 Not identified —: 41, 55, 69, 81, 93, 120, 133, 161 0.72 0.20 19.20 10 Not identified —: 43, 55, 69, 81, 93, 107, 121, 136, 332 2.41 0.78 22.74 11 Octadecenyl butyrate 338: 43, 55, 67, 82, 89, 96, 110, 250 0.97 0.37 23.45 12 Octadecyl butyrate 340: 43, 57, 71, 83, 89, 97, 252 0.39 0.13 23.73 13 Eicosyl acetate 340: 43, 57, 61, 69, 83, 97, 111, 281 0.37 0.17 23.92 14 Not identified —: 43, 55, 67, 71, 81, 96, 121, 336 0.40 0.23 24.26 15 Not identified —: 41, 55, 69, 81, 93, 107, 121, 135, 360 4.25 1.92 24.50 16 Eicosyl butyrate 368: 43, 57, 71, 83, 89, 97, 111, 125, 280 0.94 0.51 25.74
ECL: equivalent chain length.
workers’ aggression by Dufour’s gland secretion is a common strategy in Polyergus spp. This interpre- tation is supported by the response of “withdrawal” of host workers to a Pogonomyrmex worker treated with the Dufour’s gland of P. breviceps queen (Topoff et al., 1988). Decyl butyrate, reportedly a repellent substance of P. rufescens (D’Ettorre et al., 2000), constituted approximately 64% of the total contents of the Du- four’s gland of P. samurai, which possibly func- tions as the common repellent substance in the host subgenus Serviformica, at least in the two host species of F. cunicularia and F. japonica. Not only Fig. 6. Standardized peak areas of killed F. japonica decyl butyrate, but also eight other esters estimated queen (gray circle) and Dufour’s gland of P. samurai queen as the Dufour’s gland contents of P. samurai queen (open circle). Horizontal bars represent the means of standard- (Table 1) were common to those identified in P. ized peak areas of killed F. japonica queens. Only peaks de- tected exclusively from the killed F. japonica queen and Du- rufescens (D’Ettorre et al., 2000). This similarity four’s gland of P. samurai queen are shown. Peak numbers are of the chemical profiles among Polyergus queens identical to those in Fig. 5 and Table 1. and that of effects on the host among Serviformica workers might reflect phylogenetic relationships their nestmate workers and glass beads. It also sig- among parasites and among hosts. nificantly extended the time necessary for climbing The appeasement allomones suggested for other a treated filter paper wall. Such a repellent effect Polyergus spp. could be interpreted as repellent al- on the host worker enables parasite queens to deter lomones. The reduction of host workers’ aggres- aggression by host workers (D’Ettorre et al., 2000; sion by Dufour’s gland secretion of the parasite Ruano et al., 2005), and presumably contributes to queen has also been reported for P. breviceps para- successful colony usurpation. D’Ettorre et al. sitizing F. occulta (Topoff et al., 1988) and in P. (2000), who confirmed the same scenario in P. rufescens parasitizing F. cunicularia (Mori et al., rufescens, suggested that the reduction of host 2000a); gland secretion was interpreted as serving 140 Y. TSUNEOKA and T. AKINO as an “appeasement” allomone. According to asite might facilitate the adoption of the parasite Topoff and co-workers (1988), gland secretion when the host queen’s odor is incompletely ac- would imbue the parasite queen with an odor simi- quired. The parasite queens flexed their gasters lar to that of Formica, therefore reducing the ag- after killing the host queen (Topoff et al., 1988; gression of host workers until the parasite queen Mori et al., 1995; Tsuneoka, 2008); the function acquired Formica odorous chemicals. Johnson et and effects of this secretion-like behavior have al. (2001) described the Polyergus queen acquiring never been elucidated and require further study. the host queen’s cuticular hydrocarbons after Using repellent allomones is considered an usurpation, and suggested that the adoptive behav- adaptation of the parasite queen to avoid host ior of host workers is the consequence of this worker aggression during invasion of host colonies chemical mimicry (Johnson et al., 2001). This ap- in the pirate ant P. rufescens (D’Ettorre et al., pears to support the suggestion of Mori et al. 2000), Rossomyrmex minuchae (Ruano et al., (2000a) that gland secretion would work as an ap- 2005), and the social parasitic bumblebee Bombus peasement allomone against host workers, enabling norvegicus (Zimma et al., 2003). Behavioral obser- the Polyergus queen to deter the attacks of host vations suggest that the P. samurai queen uses Du- workers when invading. However, these phenom- four’s gland secretion from its gaster as a repellent ena can not be interpreted as appeasement effects allomone to deter host worker aggression during by only observing the response to intruders applied usurpation. Before she reaches the host queen, the with allomones, because some chemicals of the in- parasite queen bent her gaster toward the host truder, which normally act as to identify intruders, worker in an apparent defense against its continual could affect the reactions of recipient ants. In our P. biting; the worker eventually released the parasite samurai and F. japonica, the host F. japonica work- queen (Tsuneoka, 2008). ers apparently receded even from glass dummies P. samurai queens emit a copious amount of Du- treated with the gland extract of a P. samurai queen four’s gland contents, which contain repellent al- (Fig. 3); therefore, we considered that the gland se- lomone, when they kill the host queen (Figs. 5 and cretion of the P. samurai queen worked as repellent 6), suggesting that this secretion is used as a repel- allomone to host F. japonica workers. The observa- lent against host workers, principally to prevent tion that F. rufibarbis workers treated with the their interference during killing the host queen. gland of P. rufescens were eventually adopted by This is supported by laboratory observations that resident F. cunicularia workers (Mori et al., 2000a) soon after the fighting between queens had started, was not explainable using the interpretation that most host workers fled from the queens. Some host Dufour’s gland secretion has a repellent effect. workers in contact with both parasite and host However, that paper included no description of the queens during fighting showed receding behavior adopting behavior of F. rufibarbis workers; from (Y. T., unpublished observation). the experimental setup used for that study, it is dif- Using a repellent during host queen killing is at- ficult to determine whether it is the effect of an ap- tributed to the importance of killing the queen in peasement allomone. In the same species, D’Et- the usurpation. Host queen killing is known as an torre et al. (2000) showed that starved host workers important phase in the usurpation of the Polyergus refused to feed on honey applied with decyl bu- queen (Topoff et al., 1988; Tsuneoka, 2008; but see tyrate, the major compound of the queen’s Dufour’s Mori et al., 1995), and gaining time to acquire the gland. The effect that had been interpreted as host queen’s odor is expected to involve the use of appeasement should be reviewed to determine a repellent during host queen killing. In fact, the whether the effect is that of a repellent. queens of P. breviceps, P. rufescens, and P. samurai The repellent effect of the Polyergus queen was acquire (or biosynthesize) the cuticular hydrocar- demonstrated in this study. No phenomenon related bons of host queens during host queen killing to an appeasement allomone was found in usurpa- (D’Ettorre and Errard, 1998; Johnson et al., 2001; tion by the P. samurai queen; however, the possibil- Y. T., unpublished data). Successful chemical mim- ity exists that the parasite queen uses not merely a icry is expected to overcome host workers’ nest- repellent but also appeasement for host workers. mate recognition for a long time, and is expected to For instance, the appeasement allomone of the par- be the key factor for successful usurpation. There- Repellent Effect of Dufour’s Gland Secretion of Polyergus samurai 141 fore, a mechanism that keeps host workers from Lloyd, H. A., N. R. Schmuff and A. Hefetz (1986) Chemistry disturbing the parasite queen during host queen of the anal glands of Bothriomyrmex syrius Forel: olfac- killing would be favored. tory mimetism and temporary social parasitism. Comp. Biochem. Physiol. B 83: 71–73. ACKNOWLEDGEMENTS Martin, S. J., E. A. Jenner and F. P. Drijfhout (2007) Chemi- cal deterrent enables a socially parasitic ant to invade We are grateful to O. Kitade and anonymous reviewers for multiple hosts. Proc. R. Soc. B 274: 2717–2721. their valuable comments on this manuscript. We are also grate- Mori, A., P. D’Ettorre and F. L. Moli (1995) Host nest ful to S. Matsukawa for his helpful advice related to chemical usurpation and colony usurpation in the European ama- analysis, to S. Matsukawa, H. Yamamoto and S. Tatsuki for zon ant, Polyergus rufescens Latr. (Hymenoptera: Formi- their permission for use of their GC and GC-MS, and to Y. cidae). Insect. Soc. 42: 279–286. Tanaka and S. Nohara for their assistance of collection of ants. Mori, A., D. A. Grasso, R. Visicchio and F. L. Moli (2000a) This research was supported by the Fujiwara Natural History Colony founding in Polyergus rufescens: the role of the Foundation (2007). Dufour’s gland. Insect. Soc. 47: 7–10. Mori, A., R. Visicchio, M. F. Sledge, D. A. 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