Journal of Chemical Ecology, Vol. 21, No. I I. 1995
CHEMICAL ANALYSIS OF STERNAL GLAND SECRETION OF PAPER WASP Polistes dominulus (CHRIST) AND ITS SOCIAL PARASITE Polistes sulcifer (ZIMMERMANN) (HYMENOPTERA: VESPIDAE)
FRANCESCA
R. D A N I , I'* E. D A V I D M O R G A N , 2 and STEFANO TURILLAZZI m
~Dipartimemo di Biologh~ Animale e Genetica University) di kTrenze Via Romana 17. 50125 Firenze. Italia 'D~TJartment ~/'Chemisto'. Keele Lhtiversity Sta.[Jbrdshire ST5 5BG. Fnghmd (Received January 3, 1995: accepted June 7. 1995) Abstract--The secretion or the tegumental glands of the fifth and sixth gastral sternites of P. ~hm#mdus and of its social parasite P. suh'~/'er was analyzed by gas chromatography-mass specmmaetry.Seven brag-chaincarboxylic acids have been identified: hexadecanoic, octadecadienoic and octadecenoic acids are the mai~r components of the ghmd secretion of P. dommMus and octadecenoic acid the main compounds of P..~uh'([~,r sternal gland secretion. The same carboxylic acids have been Ikmnd on the nesl pedicel. Key Words--Vespidae, Polistinac, PMistes. sternal glands, Ring-chain carboxylic acids.
INTRODUCTION In the independently founding polistine wasps (Polistes, M i s c h o o ' t t a r u s , B e h m ogaster, Par~q~olyhia, and many species o f R o p a l M i a ) the nest, consisting o f a single c o m b o f cells, is suspended from the substrate by a narrow pedicel or by a small number o f pedicels (Jeanne. 1975: Wenzel, 1991). As the pedicel constitutes the only access to the nest for invertebrate pedestrian predators (among which ants have been s h o w n to exert the highest predation pressure), this struc*To whom1correspondence should be addressed. 1709 tXIqS (~3311~15/I lOIJ 170q$I)7 501(I '
lye5 Plenum Publi~hin g Coqxwali~m
1710
DANI, MORGAN, AND TURILLAZZI
ture is advantageous for defending the immature brood contained in the nest. The effectiveness of the pedicel in the nest defense is increased by the wasps' application of a repellent substance by rubbing the pedicel with the abdomen (Jeanne, 1970; Marino Piccioli and Pardi, 1970; Corn, 1972; Hermann and Dirks, 1974; Turillazzi and Ugolini, 1979; Post and Jeanne, 1981; Kojima, 1983a,b; Raposo Filho, t989; Keeping, 1990). Gland cells situated on the gastral sternites, especially those on the anterior part of the two last sternites (see Downing, 1991; Jeanne, 1995), have been indicated as the source of the repellent substances. Nevertheless, some authors have hypothesized that the sternal glands have a role in the chemical communication of dominance between nestmates (West-Eberhard, 1982; Downing and Jeanne, 1983), and that the fifth and sixth gastral sternite glands may differ in their function (Post and Jeanne, 1980), In the three species of obligate and permanent social parasites among Polisles (P. suh'(fer, P. semenowi, P, alHmandibuhuis), females invade nests of other Polistes species and rely on the host workers for rearing their brood (Weyrauch, 1937). Soon after the invasion of the nest, the parasite intensively strokes the nest comb with the abdomen, often near the cell opening, Some hours after the invasion this behavior is no longer perlbm~ed (Turillazzi et al., 1990; Cervo el al., 1990). Indeed, during their cohabitation with the host, parasites rub the pedicel only rarely (Cervo, 1990). It has been hypothesized that the sternal glands of these social parasites may produce pheromones that control the host colony (Turillazzi et al., 1990: Borgioli and Delfino, 1994). Despite speculation about the role of the sternal glands, chemical analyses have been carried out only on the sixth gastral steruite glands of two new world species of Polistes, namely P, fi~scatus (Post et al., 1984) and P. annuhlris (Espelie and Hernlann, 1990). Methyl hexadecanoate has been identified in the secretion of the first species, while hexadecanoic and octadecenoic acids have been found in the second one. The aim of this work has been the analysis of the fifth and sixth gastral sternite gland secretions of foundresses and workers of the European species P. domimdus. We also analyzed the fifth and sixth gastral stemite gland secretions of P. stdcifer, one of the social parasites of P. dominulus: we wanted to determine if the sternal glands of this parasite produce a secretion similar to that of the host species.
METHODS AND MATERIALS
Polistes dominulus nests and foundresses were collected near Florence, Italy, during the month of May before the emergence of the first workers (number of nests = 4; number of foundresses = 2.5 +_ 0.58 (mean _+ SD), number
STERNAL GLAND SECRt-TION OF Polistes
171 I
of cells = 33.7 _+ 2.63, n u m b e r of pupae = 7 _+ 3.56) and again during the month o f June, when some workers had already e m e r g e d ( n u m b e r o f nest = 2: n u m b e r o f cells = 48.5 _+ 4.95). P. s u l c l f e r females (N = 3) that had invaded the nests o f P. d o m i n u l u s some days earlier were collected at the b e g i n n i n g o f s u m m e r on the pre-Alps near Verona in northern Italy. Once in the laboratory, nests and animals were frozen and maintained at - 2 0 ° C . All samples were chemically analyzed at the Chemistry D e p a r t m e n t of Keele University. The sternal glands were separated from cuticle under a binocular microscope using thin forceps and placed in glass capillaries (2 × 20 m m ) scaled at one end ( M o r g a n and W a d h a m s , 1972: Morgan, 1990). T h e samples prepared are reported in Table 1. As preliminary gas c h r o m a t o g r a p h y - m a s s spectrometry analysis of sternal glands had shown traces o f carboxylic acids in the satnples (but not the presence of methyl esters), acids were derivatizated to methyl esters to make them more suitable tk~r gas c h r o m a t o g r a p h y . An ethereal solution o f d i a z o m e t h a n e (Arndt, 1943) (20 p,l) was inserted into each capillary with a microliter syringe. After the solution made good contact with the tissue, the excess was evaporated with a stream o f nitrogen and the capillary' resealed in a small flame, The analyses were carried out by c o m b i n e d gas c h r o m a t o g r a p h y - m a s s spectmtnetry ( G C - M S ) on a Hewlett Packard 5890 gas c h r o m a t o g r a p h ctmnected to a 5970 Mass Selective Detector. The G C - M S unit was controlled by a H P 5 9 9 7 0 C C h e m S t a t i o n . The gas c h r o m a t o g r a p h was equipped with a fused silica capillary c o l u m n (12 m × 0.2 m i n i , coated with a 0 . 3 3 - # m - t h i c k film o f immobilized dimethylsiloxane. Helium was used as carrier gas. The mass spectrometer was set to scan m / : from 30 to 500.
TABLE I. SAMPLES ANALYSED"
Number of capillaries
Content of each capillary
Sixth sternal glands of one l'~undress of P. ~hmmudus Fifth sternal glands of one fimndrcss of P. domimdus Sixth sternal glands of three workers of P, dominulus Sixth sternal glands of one female of P. sulc(~.r Filth sternal glands of one female of P, sulci[er 3 mg of material taken near the cell openings of one preernergence nest (two different nests were used) 3 mg of pedicel material frorn one preernergence nest (two different nests were used) "Each capillary represents one analysis.
1712
DANI, MORGAN, AND TLIRILLAZZ1
A sealed capillary tube was placed in the iniection area of the gas chromatograph (Morgan and Wadhams, 1972: Morgan, 1990), which was heated to 220°C, and the capillary crushed after 2 min, at which time chromatography commenced. The initial oven temperature of 100°C was increased by 7°C/min to 290°C. Substances were identified by their mass spectra and retention times compared with standard samples of the same esters. The amount of each methyl ester was calculated by comparing the area of its peak with that given by a known amount of methyl octanoate. As two of the methyl esters (methyl octadecadienoate and methyl octadecenoate) elute in one peak (see below), we used the proportion between the area given by two diagnostic ions (m& 294 and m/:: 296, respectively) for calculating the amounts of these compounds. For samples containing the sixth sternal glands of P. domimdus foundresses, using this method, it was possible to estimate the amount of the two esters in only three of the live samples. RESULTS Polistes dominulus. The following carboxylic acids were identified (as their methyl esters) in the fifth and sixth sternite glands of the foundresses and in the sixth sternal glands of workers: dodecanoic (laurie), tetradecanoic (myristic), hexadecenoic (palmitoleic), hexadecanoic (palmitic), octadecadienoic (linoleic), octadecenoic (oleic), and octadecanoic (stearic) acids. Methyl octadecadienoate and methyl octadecenoate eluted as one peak (Figure IA). The average total quantity of carboxylic acids tound in P. domimdus foundress fifth and sixth sternal glands were 1.9 + 1.9 #g and 5.3 + 3.7 p.g, respectively, while an average of 6.6 p.g was found in the sixth sternal glands of the workers. In many samples, methyl dodecanoate and methyl tetradecanoate were only present in traces: sometimes also traces of an unidentified hydroxymethyl ester were found. As esters were not tbund in nonderivatizated samples, we are certain that these methyl esters indicate the presence in the tissues of the corresponding carboxylic acids. The amounts of the seven acids found in the fifth and sixth sternal glands of P. dominuhls foundresses and in the sixth sternal glands of workers are reported in Table 2. Octadecenoic acid was the main component extracted and identified for all three groups of samples. Octadecadienoic and hexadecanoic acids were present in similar percentages in the sixth sternite glands of workers and foundresses, while the quantity of octadecadienoic acid was higher than that of hexadecanoic acid in the fifth sternal glands of the foundresses. The same acids have been found on the nest carton. The total amount of carboxylic acids found on 3 mg of pedicel material is on average 5.5/,g, while an average of 2.0 #g was present in the same amount of nest bottom material.
STERNALGLANDSECRETIONOF Polistes
1713
P. sulcifer. Four acids found in P. dominulus were present in the sternal glands of P. sulcifer (Figure I B). The average quantity of carboxylic acids found in the sixth sternite glands was 1.5 + 1.2 /zg while only 157 + 79.2 ng was found on average in the glands of the fifth sternite. Table 2 shows the average quantity per female and the average percentage of each acid in the fifth and sixth sternal glands. DISCUSSION Our work confirms that, as already fbund by Espelie and Hermann (1990) in P. anntdatSs, the sternal glands of P. dominulus secrete carboxylic acids. In P. dominulus we found five acids in addition to those identified by Espelie and Hermann (1990). The presence of the same carboxylic acids in the sixth and fifth sternite glands of P. domimdus foundresses indicate that, as suggested (Hermann and Dirks, 1974; Tufillazzi, 1979), both glands may have the same function. The sixth sternite glands of P. &mfimdus workers contain the same carboxylic acids. Four of the same carboxylic acids are also present in the sixth and fifth stemite glands of the social parasite P. sulcifer. It seems likely that the function of the fifth and sixth sternite gland is similar for both foundress and workers of P. dominulus and for females of the social parasite P. sulc{/er. It does not seem that in the parasite P. sulcifer the sternal glands have assumed a different function with respect to nonparasite species. It is noteworthy that these acids are commonly found in the lipids of insects and higher animals, but the proportion of unsaturated acids (hexadecenoic, octadecadienoic, and octadecenoic) is much higher than in other animal lipids (Gunstone et al., 1986). Approximately 70% of the acids in the fifth sternal glands of P. dominulus foundresses are unsaturated, while 77 % of the acids in the sixth sternal glands of foundresses and 64% from workers are unsaturated. While there is less total secretion in the glands of P. sMc{fer, they are 88 % unsaturated in the fitch sternal glands and 80% unsaturated in the sixth sternal glands. The higher amount of carboxylic acids in the pedicel with respect to the comb material indicates that more of the carboxylic acids produced by the sternal glands are applied to the pedicel than to the comb. It must be stressed that the pedicel is enlarged by the wasps during nest growth and in our analyses we probably measured acids in the internal layers of the pedicel as well as on its surface. Bioassays are needed in order to deternline if the carboxylic acids have a repellent effect against ants. Unlike our results and those by Espelie and Hermann (1990), Post and coworkers (1984) found methyl esters in the sternal glands of P. fuscatus females; the reasons for this difference are unclear, and it would be interesting to repeat the analyses of the glandular secretion of this
1714
DANI, MORGAN, AND TURILLAZZI
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STERNAL GLAND SECRETION OF
1715
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26 (N = 51 58 (N = 5) 43 (N = 5) 293 IN = 5) 485 (N = 3) ~' 1652 (N = 3 / ' 116 (N = 5t
ng
8.93
64.05
11.75
13.91
1.04
1.30
0.59
of total
6.14
22.68
I 1,73
10.29
1.55
2.91
1.33
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77 IN = 31 260 (N = 3) 26(1 (N = 3) 859 IN = 3) 1547 (N = 31 1785 (N = 3) 545 (N = 3)
ng
8.65
42.22
22.16
16.54
4.79
4.81
0.80
% of total
3.45
19,73
15,86
1.00
0.20
1.33
1.40
SD of 9;
390
2523
1405
1410
271
393
195
ng
5.92
38.32
21.33
21.40
4. II
5.96
2.96
~,~ tff total
Average
workers
Sixth sternite glands of
O (N = 2) 0 tN = 2) 12 (N = 2) 126 ~N = 2) 1) (N = 2~ 1288 (N = 2) 112 (N = 2t
ng
7.86
80.06
0
1094
0.51
0
0
g of total
Average
0 IN = 2} 0 tN = 2) 0 (N = 21 14 ~N = 2) (I (N = 2) 132 (N = 2) II (N = 2)
ng
5.37
88.08
0
6.54
0
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Average
Fifth stcmite glands ol females
Sixth stcrnite shreds of I~:malcs
Poli,wes ~ulc(li'r
"For the sixth and fifth sternite glands o1 P. dominulus flmndresses, avera~ze quantity per individual, average of the percentages and standard deviation of the percentages of each carboxylic acids are given. For the workers, for which only one sample containing the sixth stemile glands of three individuals has been analyzed, the average quantity per workers and the percentage of each acids are given. For the sixth and fifth sternite glands of P, sulc(J~'r. average quantity per female and average of the percentages are given Ibr the carboxylic acids. ' O n l y in three samples of the sixth stemite glands was it possible to calculate the quantity of oleic and Iinoleic acids on the basis ~1 the diagnostic ions (see materials and methods.)
Octadecanoic
Octadecenoic
Octadecadienoic
Hexadecanoic
Hexadecenoic
Tetradecanoic
Dodecanoie
Acid
Average
timndresses
of foundmsses
Average
Fifth stcrnite glands of
Sixth stemite ghmds
Polistes domimdu,~
FEMALES"
TABLE 2. COMPOSITION OF STERNAL GLAND SI:(,-'REIIt)N OF Polistes domilllthls FOUNr)RESSIsS ANt) WORKERS AND {)F /'*. sItlcl]gr
N ~
-'t _~ c
> 2:
> z
O x~
K
> z
5
STERNAL GLAND SECRETION OF Polistes
1717
s p e c i e s by m e a n s o f G C - M S . T h e p r o d u c t i o n o f a n t r e p e l l e n t s s e e m s to be a c o m m o n f e a t u r e o f all i n d e p e n d e n t l y f o u n d i n g P o l i s t i n a e ( K o j i m a , 1992), a n d a l t h o u g h t h e sternal g l a n d s e c r e t i o n s h a v e b e e n i m p l i c a t e d in a n t r e p e l l e n c y , the c h e m i c a l n a t u r e o f t h e s e c r e t i o n f r o m the s t e r n a l g l a n d s h a s b e e n i n v e s t i g a t e d only in the g e n u s P o l i s t e s . I n d e e d , n o t h i n g is k n o w n a b o u t the c h e m i s t r y o f t h e s e c r e t i o n o f t h e s e g l a n d s in t h e s w a r m f o u n d i n g P o l i s t i n a e , w h e r e t h e y are t h o u g h t to be i n v o l v e d in the p r o d u c t i o n o f trail p h e r o m o n e s u s e d d u r i n g s w a r m ing ( J e a n n e et al., 1983). In the s u b f a m i l y V e s p i n a e , the f u n c t i o n o f the sternal g l a n d s is u n c e r t a i n ( L a n d o l t a n d A k r e , 1979) a n d o n l y r e c e n t l y M a r t i n (1992) h a s s h o w n that t h e fifth a n d s i x t h gastral s t e r n i t e g l a n d s o f t w o s p e c i e s o f V e s p a secrete a n t r e p e l l e n t s u b s t a n c e s . F u t u r e c o m p a r a t i v e s t u d i e s a w a i t f u r t h e r data on the c h e m i c a l n a t u r e o f the s e c r e t i o n o f t h e s e g l a n d s . 4cktlowh,dgmcnts--Wc wish to thank L. Cruz Lopez, R.R. do Nascimento, and N.J. Oldham Ior help and advice given during the laboratory work and R. Ccrvo for her comments on the manuscripl. The study was supported with funds from the Ministero della Pubblica Istruzione t6[19,: ).
REFERENCES ARNI)r, F. 1943. Diazomethanc. Or,t,,. Svnrh. Coll. 2: 165-167. B()RGlln I, C., and DFI.FIN¢), G. 1094. Class 111 glands of the VI abd¢mlinal sternilc are involved in parasitic relationships between Poli.~tes suh'il~'r and its host P. domhudus, p. 384. i~1 Les insectes sociaux. Proceedings, 12th Congress of the lnternal ional Union ti~r the Study of Social Insecls. Paris. Sorbonne. 21-27 August 1994. CFRV¢~. R. 1990. 11 parassitismo sociale nei Polisres. PhD disserlatitm. University of Florence, Italy. CI-RVO, R., LOREN/.I, M.C., and TURILI.-XZ71, S. 1990. Non-aggressive usurpation of the nest of Poh'stes biglumis bimacuhllus by the social parasite Sulcopolisles atrimamlibtdaris (Hymenoptera, Vespidae). hlsectes Sot'. 37:333-347. CORN. M.L. 1972. Notes on the biology of Po/istcs carlli/~,.r (Hymenoptera Vespidae) in Costa Rica and Colombia. Psyche 79: 150-157. D~WNING. H.A. 1991. The function and evolution of exocrine glands, pp. 540 56% in K.G. Ross and R.W. Matthews (eds.). The Social Biology of Wasps. Comstock, Ithaca, New York. DOWNING. H.A., and JEANNE, R L . 1983. Correlation of season and dominance status with activity of exocrine glands in Po/istes .[Jtscatus (Hyruenoptera: Vespidae). J. Karts. Entomol. Soc. 56:387-397. ESPkLIE, K.E., and HF.RMANN, H.R. 1990. Surface lipids of the social wasps Polistes amlularis (L.) and its nest and nest pedicel. J. Chem. Ecol. 16:1841-1852. GUNSfONt:, F D . , H,,,RWOOD,J.L., and PADLEY, F.B. 1986. The Lipid Handbook. Chapman and Hall, London. Ht~RMANN. H.R., and DIR~S, T.F. 1974. Sternal glands in polistine wasps: Morphology and associated behavior. J. Ga. Entomol. Sot'. 9: I-8. JE,~NNE, R.L. 1970. Chemical defense of brood by a social wasp. Science 168: 1465-1466. JEANNE, R.L. 1975. The adaptiveness of social wasp nest architecture. Qt. Rev. Biol. 50:267-287. JFANNE, R.L. 1995. The evolution of exocrine gland function in wasps, in S. Turillazzi and M.J. West-Eberhard (eds.). Natural History and Evolution of Paper Wasps. Oxlbrd University Press. In press.
1718
DANI, MORGAN, AND TURILLAZZI
JEANNI-., R.L- DOWNING. H.A- and POST. D.C. 1983. Morphology and function of sternal glands in polistine wasps t Hymenoptera: Vcspidac). Zoomorphoh,~y 103:149-164. KEFPING, M,G. 1990. Rubbing behavior and morphology of van der Vccht's gland in Bekmogaster petiolata (Hymenoptera: Vespidae). J. hlse('t Behav. 3:85-104. KOJIMA. J. 1983a. Occurrence of the rubbing behavior in a paper wasp, Parapol.~q~ia indica (Hymenoptem. VespidaeL Kono't't. 51 : 158-159. KOJIMA, J. 1983b. Defense of the pre-emergence colony against ants by means of a chemical barrier in Ropalidia fasciata (Hymenoptera. Vespidac). Jim. J. Ecol. 33:213-223. KOJIMA. J. 1992. The ant repellent function of the rubbing substance in an Old World polistine. Parapolyhia indica (Hymenopteva Vespidae). Ethol. EcoL EvM. 4:83-185. LA,,ar~oLr. P.J.. and AKRE, R D . 1979. Occurrence and location of exocrine glands in some social Vespidae (Hyruenoptera). Amt. Entomol. &,c. Am. 72: 141-148. MARIN¢) PICC'IOLL M.T,, and PARI)t. L. 1970. Studi sulla biologia di Beh,m~gaster (Hymenoptera, VespidaeL I. Sull'etogramma di Behmo,Vaster griseus (Fab). Monit. ZoM. hal. (&q~pl.). 3: 197-
225. MARTIN, S.J. 1992. Cohmy delcnce against ants m Vespa. lnsectcs Soc. 3'4:99 112. MOR(i,\N, E.D. 1990. Preparation of small scale samples Ironl insecls for chromatography, Amd. Chim..4eta 236:227-235. MORt-;.,,X, E.D., and WADHAMS. L.J. 1972. Gas chromatogruphy of w~lalile compounds in small samples of biological rualerials. J. Chr~,matogr. Sci. 10:528-529. POST. D.C., and Jl~.',',;'~l~. R.L, 1980. Morphology of the sternal glands of Poliste.~ .filscatus and P. camuk'nsis (Hymenoptera: Vespidae). P.~vcl/c 87:49 58. PosT, D.C., and JD',NXI-. R.L. 1981. Colony defense against ants by P~,listesfilscatus (Hymenop tera: Vespidae) in Wisconsin. J. Karts. Entomol. Soc. 54:599-615. POST, D.C., M¢~H~MEI), M.A.. C¢~t't'H_. H.C.. and JEANNE. R.L. 1984. Identification of ant repellent allomone produced by social wasp Polistesficscatus (Hyruenoptera, Vespidael. J. Chem. Ecol. 10:1799-1807. RAPOso FII_HO. J.R. t989. Comportan3ento de defizsa e agrega,4ao das cohmias de Mi.stq~ot3'tl.rus extinctus Zikan, 1935. BoLI. CB.G. 42:37-43, Tt'RILt.,,XZZl. S. 1979. Tegumental glands in the abdomen of some European Polistes (Hymenoptera Vespidae). Monit, ~ud. ltal. 13:67-7(1. Tt:RILLAZZt. S.. and UC;OLINI. A. 1979. Rubbing behaviour in some European Polistes IHyruenoptera Vespidae). Monit. Zool, ltal. 13:129-14 I. TURtLLAZZI, S., CERVO. R.. and CAVAI.LARI, I. 1990. Invasion of the nest of Polistes thmfinulus by the social parasite Suh'opolistes sulc~fer (Hymenopte~a, VespidaeL Ethology 84:47-59, Wt~:NZEL,J.W. 1991. Evolution of nest architecture, pp. 480-519, in K.G, Ross and R.W. Matthews {eds.). The Social Biology of Wasps. Comstock, Ithaca, New York. WEST-EBERtlARD. M.J. 1982. The nature and evolution of swarming in tropical social wasps {Vespidae, Polistinae, Polybiini), pp. 97-128, in P, Jaisson (ed.). Social Insects in the Tropics, Vol. I. University of Paris XIII Press, Paris. WEYR~,UCH, W. 1937. Zur Systematik und Biologie der Kuckuckswespen Psemhn'espa, Ps'eudovespuhl and PseudopMistes. Zool. Jahrb. (Syst). 70:243-290.
CHEMICAL ANALYSIS OF STERNAL GLAND SECRETION OF PAPER WASP Polistes dominulus (CHRIST) AND ITS SOCIAL PARASITE Polistes sulcifer (ZIMMERMANN) (HYMENOPTERA: VESPIDAE)
FRANCESCA
R. D A N I , I'* E. D A V I D M O R G A N , 2 and STEFANO TURILLAZZI m
~Dipartimemo di Biologh~ Animale e Genetica University) di kTrenze Via Romana 17. 50125 Firenze. Italia 'D~TJartment ~/'Chemisto'. Keele Lhtiversity Sta.[Jbrdshire ST5 5BG. Fnghmd (Received January 3, 1995: accepted June 7. 1995) Abstract--The secretion or the tegumental glands of the fifth and sixth gastral sternites of P. ~hm#mdus and of its social parasite P. suh'~/'er was analyzed by gas chromatography-mass specmmaetry.Seven brag-chaincarboxylic acids have been identified: hexadecanoic, octadecadienoic and octadecenoic acids are the mai~r components of the ghmd secretion of P. dommMus and octadecenoic acid the main compounds of P..~uh'([~,r sternal gland secretion. The same carboxylic acids have been Ikmnd on the nesl pedicel. Key Words--Vespidae, Polistinac, PMistes. sternal glands, Ring-chain carboxylic acids.
INTRODUCTION In the independently founding polistine wasps (Polistes, M i s c h o o ' t t a r u s , B e h m ogaster, Par~q~olyhia, and many species o f R o p a l M i a ) the nest, consisting o f a single c o m b o f cells, is suspended from the substrate by a narrow pedicel or by a small number o f pedicels (Jeanne. 1975: Wenzel, 1991). As the pedicel constitutes the only access to the nest for invertebrate pedestrian predators (among which ants have been s h o w n to exert the highest predation pressure), this struc*To whom1correspondence should be addressed. 1709 tXIqS (~3311~15/I lOIJ 170q$I)7 501(I '
lye5 Plenum Publi~hin g Coqxwali~m
1710
DANI, MORGAN, AND TURILLAZZI
ture is advantageous for defending the immature brood contained in the nest. The effectiveness of the pedicel in the nest defense is increased by the wasps' application of a repellent substance by rubbing the pedicel with the abdomen (Jeanne, 1970; Marino Piccioli and Pardi, 1970; Corn, 1972; Hermann and Dirks, 1974; Turillazzi and Ugolini, 1979; Post and Jeanne, 1981; Kojima, 1983a,b; Raposo Filho, t989; Keeping, 1990). Gland cells situated on the gastral sternites, especially those on the anterior part of the two last sternites (see Downing, 1991; Jeanne, 1995), have been indicated as the source of the repellent substances. Nevertheless, some authors have hypothesized that the sternal glands have a role in the chemical communication of dominance between nestmates (West-Eberhard, 1982; Downing and Jeanne, 1983), and that the fifth and sixth gastral sternite glands may differ in their function (Post and Jeanne, 1980), In the three species of obligate and permanent social parasites among Polisles (P. suh'(fer, P. semenowi, P, alHmandibuhuis), females invade nests of other Polistes species and rely on the host workers for rearing their brood (Weyrauch, 1937). Soon after the invasion of the nest, the parasite intensively strokes the nest comb with the abdomen, often near the cell opening, Some hours after the invasion this behavior is no longer perlbm~ed (Turillazzi et al., 1990; Cervo el al., 1990). Indeed, during their cohabitation with the host, parasites rub the pedicel only rarely (Cervo, 1990). It has been hypothesized that the sternal glands of these social parasites may produce pheromones that control the host colony (Turillazzi et al., 1990: Borgioli and Delfino, 1994). Despite speculation about the role of the sternal glands, chemical analyses have been carried out only on the sixth gastral steruite glands of two new world species of Polistes, namely P, fi~scatus (Post et al., 1984) and P. annuhlris (Espelie and Hernlann, 1990). Methyl hexadecanoate has been identified in the secretion of the first species, while hexadecanoic and octadecenoic acids have been found in the second one. The aim of this work has been the analysis of the fifth and sixth gastral sternite gland secretions of foundresses and workers of the European species P. domimdus. We also analyzed the fifth and sixth gastral stemite gland secretions of P. stdcifer, one of the social parasites of P. dominulus: we wanted to determine if the sternal glands of this parasite produce a secretion similar to that of the host species.
METHODS AND MATERIALS
Polistes dominulus nests and foundresses were collected near Florence, Italy, during the month of May before the emergence of the first workers (number of nests = 4; number of foundresses = 2.5 +_ 0.58 (mean _+ SD), number
STERNAL GLAND SECRt-TION OF Polistes
171 I
of cells = 33.7 _+ 2.63, n u m b e r of pupae = 7 _+ 3.56) and again during the month o f June, when some workers had already e m e r g e d ( n u m b e r o f nest = 2: n u m b e r o f cells = 48.5 _+ 4.95). P. s u l c l f e r females (N = 3) that had invaded the nests o f P. d o m i n u l u s some days earlier were collected at the b e g i n n i n g o f s u m m e r on the pre-Alps near Verona in northern Italy. Once in the laboratory, nests and animals were frozen and maintained at - 2 0 ° C . All samples were chemically analyzed at the Chemistry D e p a r t m e n t of Keele University. The sternal glands were separated from cuticle under a binocular microscope using thin forceps and placed in glass capillaries (2 × 20 m m ) scaled at one end ( M o r g a n and W a d h a m s , 1972: Morgan, 1990). T h e samples prepared are reported in Table 1. As preliminary gas c h r o m a t o g r a p h y - m a s s spectrometry analysis of sternal glands had shown traces o f carboxylic acids in the satnples (but not the presence of methyl esters), acids were derivatizated to methyl esters to make them more suitable tk~r gas c h r o m a t o g r a p h y . An ethereal solution o f d i a z o m e t h a n e (Arndt, 1943) (20 p,l) was inserted into each capillary with a microliter syringe. After the solution made good contact with the tissue, the excess was evaporated with a stream o f nitrogen and the capillary' resealed in a small flame, The analyses were carried out by c o m b i n e d gas c h r o m a t o g r a p h y - m a s s spectmtnetry ( G C - M S ) on a Hewlett Packard 5890 gas c h r o m a t o g r a p h ctmnected to a 5970 Mass Selective Detector. The G C - M S unit was controlled by a H P 5 9 9 7 0 C C h e m S t a t i o n . The gas c h r o m a t o g r a p h was equipped with a fused silica capillary c o l u m n (12 m × 0.2 m i n i , coated with a 0 . 3 3 - # m - t h i c k film o f immobilized dimethylsiloxane. Helium was used as carrier gas. The mass spectrometer was set to scan m / : from 30 to 500.
TABLE I. SAMPLES ANALYSED"
Number of capillaries
Content of each capillary
Sixth sternal glands of one l'~undress of P. ~hmmudus Fifth sternal glands of one fimndrcss of P. domimdus Sixth sternal glands of three workers of P, dominulus Sixth sternal glands of one female of P. sulc(~.r Filth sternal glands of one female of P, sulci[er 3 mg of material taken near the cell openings of one preernergence nest (two different nests were used) 3 mg of pedicel material frorn one preernergence nest (two different nests were used) "Each capillary represents one analysis.
1712
DANI, MORGAN, AND TLIRILLAZZ1
A sealed capillary tube was placed in the iniection area of the gas chromatograph (Morgan and Wadhams, 1972: Morgan, 1990), which was heated to 220°C, and the capillary crushed after 2 min, at which time chromatography commenced. The initial oven temperature of 100°C was increased by 7°C/min to 290°C. Substances were identified by their mass spectra and retention times compared with standard samples of the same esters. The amount of each methyl ester was calculated by comparing the area of its peak with that given by a known amount of methyl octanoate. As two of the methyl esters (methyl octadecadienoate and methyl octadecenoate) elute in one peak (see below), we used the proportion between the area given by two diagnostic ions (m& 294 and m/:: 296, respectively) for calculating the amounts of these compounds. For samples containing the sixth sternal glands of P. domimdus foundresses, using this method, it was possible to estimate the amount of the two esters in only three of the live samples. RESULTS Polistes dominulus. The following carboxylic acids were identified (as their methyl esters) in the fifth and sixth sternite glands of the foundresses and in the sixth sternal glands of workers: dodecanoic (laurie), tetradecanoic (myristic), hexadecenoic (palmitoleic), hexadecanoic (palmitic), octadecadienoic (linoleic), octadecenoic (oleic), and octadecanoic (stearic) acids. Methyl octadecadienoate and methyl octadecenoate eluted as one peak (Figure IA). The average total quantity of carboxylic acids tound in P. domimdus foundress fifth and sixth sternal glands were 1.9 + 1.9 #g and 5.3 + 3.7 p.g, respectively, while an average of 6.6 p.g was found in the sixth sternal glands of the workers. In many samples, methyl dodecanoate and methyl tetradecanoate were only present in traces: sometimes also traces of an unidentified hydroxymethyl ester were found. As esters were not tbund in nonderivatizated samples, we are certain that these methyl esters indicate the presence in the tissues of the corresponding carboxylic acids. The amounts of the seven acids found in the fifth and sixth sternal glands of P. dominuhls foundresses and in the sixth sternal glands of workers are reported in Table 2. Octadecenoic acid was the main component extracted and identified for all three groups of samples. Octadecadienoic and hexadecanoic acids were present in similar percentages in the sixth sternite glands of workers and foundresses, while the quantity of octadecadienoic acid was higher than that of hexadecanoic acid in the fifth sternal glands of the foundresses. The same acids have been found on the nest carton. The total amount of carboxylic acids found on 3 mg of pedicel material is on average 5.5/,g, while an average of 2.0 #g was present in the same amount of nest bottom material.
STERNALGLANDSECRETIONOF Polistes
1713
P. sulcifer. Four acids found in P. dominulus were present in the sternal glands of P. sulcifer (Figure I B). The average quantity of carboxylic acids found in the sixth sternite glands was 1.5 + 1.2 /zg while only 157 + 79.2 ng was found on average in the glands of the fifth sternite. Table 2 shows the average quantity per female and the average percentage of each acid in the fifth and sixth sternal glands. DISCUSSION Our work confirms that, as already fbund by Espelie and Hermann (1990) in P. anntdatSs, the sternal glands of P. dominulus secrete carboxylic acids. In P. dominulus we found five acids in addition to those identified by Espelie and Hermann (1990). The presence of the same carboxylic acids in the sixth and fifth sternite glands of P. domimdus foundresses indicate that, as suggested (Hermann and Dirks, 1974; Tufillazzi, 1979), both glands may have the same function. The sixth sternite glands of P. &mfimdus workers contain the same carboxylic acids. Four of the same carboxylic acids are also present in the sixth and fifth stemite glands of the social parasite P. sulcifer. It seems likely that the function of the fifth and sixth sternite gland is similar for both foundress and workers of P. dominulus and for females of the social parasite P. sulc{/er. It does not seem that in the parasite P. sulcifer the sternal glands have assumed a different function with respect to nonparasite species. It is noteworthy that these acids are commonly found in the lipids of insects and higher animals, but the proportion of unsaturated acids (hexadecenoic, octadecadienoic, and octadecenoic) is much higher than in other animal lipids (Gunstone et al., 1986). Approximately 70% of the acids in the fifth sternal glands of P. dominulus foundresses are unsaturated, while 77 % of the acids in the sixth sternal glands of foundresses and 64% from workers are unsaturated. While there is less total secretion in the glands of P. sMc{fer, they are 88 % unsaturated in the fitch sternal glands and 80% unsaturated in the sixth sternal glands. The higher amount of carboxylic acids in the pedicel with respect to the comb material indicates that more of the carboxylic acids produced by the sternal glands are applied to the pedicel than to the comb. It must be stressed that the pedicel is enlarged by the wasps during nest growth and in our analyses we probably measured acids in the internal layers of the pedicel as well as on its surface. Bioassays are needed in order to deternline if the carboxylic acids have a repellent effect against ants. Unlike our results and those by Espelie and Hermann (1990), Post and coworkers (1984) found methyl esters in the sternal glands of P. fuscatus females; the reasons for this difference are unclear, and it would be interesting to repeat the analyses of the glandular secretion of this
1714
DANI, MORGAN, AND TURILLAZZI
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STERNAL GLAND SECRETION OF
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26 (N = 51 58 (N = 5) 43 (N = 5) 293 IN = 5) 485 (N = 3) ~' 1652 (N = 3 / ' 116 (N = 5t
ng
8.93
64.05
11.75
13.91
1.04
1.30
0.59
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6.14
22.68
I 1,73
10.29
1.55
2.91
1.33
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77 IN = 31 260 (N = 3) 26(1 (N = 3) 859 IN = 3) 1547 (N = 31 1785 (N = 3) 545 (N = 3)
ng
8.65
42.22
22.16
16.54
4.79
4.81
0.80
% of total
3.45
19,73
15,86
1.00
0.20
1.33
1.40
SD of 9;
390
2523
1405
1410
271
393
195
ng
5.92
38.32
21.33
21.40
4. II
5.96
2.96
~,~ tff total
Average
workers
Sixth sternite glands of
O (N = 2) 0 tN = 2) 12 (N = 2) 126 ~N = 2) 1) (N = 2~ 1288 (N = 2) 112 (N = 2t
ng
7.86
80.06
0
1094
0.51
0
0
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Average
0 IN = 2} 0 tN = 2) 0 (N = 21 14 ~N = 2) (I (N = 2) 132 (N = 2) II (N = 2)
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5.37
88.08
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6.54
0
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Average
Fifth stcmite glands ol females
Sixth stcrnite shreds of I~:malcs
Poli,wes ~ulc(li'r
"For the sixth and fifth sternite glands o1 P. dominulus flmndresses, avera~ze quantity per individual, average of the percentages and standard deviation of the percentages of each carboxylic acids are given. For the workers, for which only one sample containing the sixth stemile glands of three individuals has been analyzed, the average quantity per workers and the percentage of each acids are given. For the sixth and fifth sternite glands of P, sulc(J~'r. average quantity per female and average of the percentages are given Ibr the carboxylic acids. ' O n l y in three samples of the sixth stemite glands was it possible to calculate the quantity of oleic and Iinoleic acids on the basis ~1 the diagnostic ions (see materials and methods.)
Octadecanoic
Octadecenoic
Octadecadienoic
Hexadecanoic
Hexadecenoic
Tetradecanoic
Dodecanoie
Acid
Average
timndresses
of foundmsses
Average
Fifth stcrnite glands of
Sixth stemite ghmds
Polistes domimdu,~
FEMALES"
TABLE 2. COMPOSITION OF STERNAL GLAND SI:(,-'REIIt)N OF Polistes domilllthls FOUNr)RESSIsS ANt) WORKERS AND {)F /'*. sItlcl]gr
N ~
-'t _~ c
> 2:
> z
O x~
K
> z
5
STERNAL GLAND SECRETION OF Polistes
1717
s p e c i e s by m e a n s o f G C - M S . T h e p r o d u c t i o n o f a n t r e p e l l e n t s s e e m s to be a c o m m o n f e a t u r e o f all i n d e p e n d e n t l y f o u n d i n g P o l i s t i n a e ( K o j i m a , 1992), a n d a l t h o u g h t h e sternal g l a n d s e c r e t i o n s h a v e b e e n i m p l i c a t e d in a n t r e p e l l e n c y , the c h e m i c a l n a t u r e o f t h e s e c r e t i o n f r o m the s t e r n a l g l a n d s h a s b e e n i n v e s t i g a t e d only in the g e n u s P o l i s t e s . I n d e e d , n o t h i n g is k n o w n a b o u t the c h e m i s t r y o f t h e s e c r e t i o n o f t h e s e g l a n d s in t h e s w a r m f o u n d i n g P o l i s t i n a e , w h e r e t h e y are t h o u g h t to be i n v o l v e d in the p r o d u c t i o n o f trail p h e r o m o n e s u s e d d u r i n g s w a r m ing ( J e a n n e et al., 1983). In the s u b f a m i l y V e s p i n a e , the f u n c t i o n o f the sternal g l a n d s is u n c e r t a i n ( L a n d o l t a n d A k r e , 1979) a n d o n l y r e c e n t l y M a r t i n (1992) h a s s h o w n that t h e fifth a n d s i x t h gastral s t e r n i t e g l a n d s o f t w o s p e c i e s o f V e s p a secrete a n t r e p e l l e n t s u b s t a n c e s . F u t u r e c o m p a r a t i v e s t u d i e s a w a i t f u r t h e r data on the c h e m i c a l n a t u r e o f the s e c r e t i o n o f t h e s e g l a n d s . 4cktlowh,dgmcnts--Wc wish to thank L. Cruz Lopez, R.R. do Nascimento, and N.J. Oldham Ior help and advice given during the laboratory work and R. Ccrvo for her comments on the manuscripl. The study was supported with funds from the Ministero della Pubblica Istruzione t6[19,: ).
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1718
DANI, MORGAN, AND TURILLAZZI
JEANNI-., R.L- DOWNING. H.A- and POST. D.C. 1983. Morphology and function of sternal glands in polistine wasps t Hymenoptera: Vcspidac). Zoomorphoh,~y 103:149-164. KEFPING, M,G. 1990. Rubbing behavior and morphology of van der Vccht's gland in Bekmogaster petiolata (Hymenoptera: Vespidae). J. hlse('t Behav. 3:85-104. KOJIMA. J. 1983a. Occurrence of the rubbing behavior in a paper wasp, Parapol.~q~ia indica (Hymenoptem. VespidaeL Kono't't. 51 : 158-159. KOJIMA, J. 1983b. Defense of the pre-emergence colony against ants by means of a chemical barrier in Ropalidia fasciata (Hymenoptera. Vespidac). Jim. J. Ecol. 33:213-223. KOJIMA. J. 1992. The ant repellent function of the rubbing substance in an Old World polistine. Parapolyhia indica (Hymenopteva Vespidae). Ethol. EcoL EvM. 4:83-185. LA,,ar~oLr. P.J.. and AKRE, R D . 1979. Occurrence and location of exocrine glands in some social Vespidae (Hyruenoptera). Amt. Entomol. &,c. Am. 72: 141-148. MARIN¢) PICC'IOLL M.T,, and PARI)t. L. 1970. Studi sulla biologia di Beh,m~gaster (Hymenoptera, VespidaeL I. Sull'etogramma di Behmo,Vaster griseus (Fab). Monit. ZoM. hal. (&q~pl.). 3: 197-
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