Journal of Chemical Ecology, Vol. 9, No. 11, 1983
MANDIBULAR GLANDS OF STINGLESS BEES (HYMENOPTERA: APIDAE): Chemical Analysis of Their Contents and Biological Function in Two Species of Melipona
B.H. S M I T H 1 a n d D . W . R O U B I K 2 1Department o[" Entomology, University of Kansas Lawrence, Kansas 66045 Smtthsoman Tropical Research Institute P.O. Box 2072, Balboa, Republic of Panama 2
9
.
(Received November 24, 1981; revised January 14, 1983) Abstract Workers of Melipona fasciata and M. interrupta triplaridis respond to their respective mandibular gland extracts with alarm recruitment and defensive behavior. Workers rapidly exit from the nest entrance, land on an intruding object, and bite with the mandibles while vibrating the flight muscles. These behaviors are accompanied by the release of the contents of the mandibular glands. Colonies of both species exhibited greater response to their own mandibular gland extracts than to those of other stingless bee species. Chemical analysis identified 2-heptanol as the major component in hexane extracts of each species. Undecane was a constituent of both species; skatole and nerol were identified only in extracts of M. i. triplaridis. Key Words--Melipona, Hymenoptera, Apidae, mandibular glands, 2heptanol, skatole, nerol, undeeane, alarm response, stingless bees. INTRODUCTION A m a j o r fi~nction of a l a r m r e c r u i t m e n t p h e r o m o n e s of social insects is the release of d ef en s i v e b e h a v i o r in the p r o x i m i t y of the nest (Wilson, 1971; B l u m and Brand, 1972; P a r r y and M o r g a n , 1979). Volatile c o m p o u n d s of biting and stinging H y m e n o p t e r a are released f r o m glands associated with the sting a p p a r a t u s or m a n d i b l e s of w o r k e r s ( B a r o n i U r b a n i , 1979). G r o u p s of w o r k e r s , m a n y times those specialized f o r nest defense, r e s p o n d to the release o f a l a r m p h e r o m o n e s near the c o l o n y by l e av i n g the nest, after which a d d i t i o n a l stimuli m a y cause t h e m to a t t a c k an i n t r u d i n g object (Gary, 1974; 1465 0098-0331 / 83/1100-1465503.00/0 9 1983 Plenum Publishing Corporation
1466
SMITH AND ROUBIK
Blum, 1979); in some species, however, workers are less inclined toward leaving the nest (Johnson and Weimer, 1982). Among the highly social bees, i.e., the honeybees (Apinae) and the stingless honeybees (Meliponinae), alarm pheromones have been identified for all species of Apis and for several meliponine species of the tribe Trigonini (Blum, 1966, 1979; Keeping et al., 1982). Here we present the first description of possible alarm substances in the genus Melipona and of the inter- and intraspecific alarm recruitment responses to natural extracts. We also consider evolutionary reasons for species-specific responses to substances used in nest defense. METHODS AND MATERIALS
Studies with natural colonies of Melipona fasciata and M. interrupta triplaridis (Cockerell) near Gamboa, Panama, indicated pronounced response of workers to colony disturbance (Roubik, in preparation). One colony of each species was used as a source of mandibular glands and in testing colony response to the resultant extracts. For bioassay, eight workers of each Melipona species were taken from the nests; the glands were extracted by rotating the mandible 180 ~ then gently pulling the mandible from the cranium with fine forceps. The resultant two sets of eight glands and mandibles of each species were placed in vials containing i ml dichloromethane and refrigerated at 5-10 ~ C. Similar extracts of Lestrimelitta limao and Trigona (Scaptotrigona)pectoralis were made from workers collected at their nests. The mandibular gland contents of L. lirnao consists of the two isomers of citral, geranial and neral, and is used during foraging raids on nests of other highly social bees (Blum et al., 1970; Sakagami and Laroca, 1963; Roubik, 1981). Bioassay of colony response to mandibular gland extracts was made by counting the number of bees leaving the nest a n d / o r biting the control stimulus, followed directly by another count after the presentation of one of the test extracts. The stimulus in both cases was a 7-ram filter paper disk held in place with an insect pin 2.5 cm over a dark blue nylon cloth ball, approximately 8 cm in diameter. Counts of bees leaving the nest were made for 1 rain when the stimulus was held 2-3 cm below the nest entrance; the second 1 min count was made after the filter paper was impregnated with 6 ~1 (approx. 0.1 female equivalents, FE) of a test extract. Bioassays using randomly chosen test substances were made at 15-rain intervals on each of 8 days. Tests on M. i. triptaridis were made between 0715 and 1000 hr local time, and those on M. fasciata between 1330 and 1645 hr. All tests were made in early April 1980. A second series of bioassays testing the ability of a single worker to elicit a defensive response from nestmates was made by holding a live worker 2 cm
1467
CONTENTS OF STINGLESS BEE GLANDS
below the nest entrance, not within visual range of the guard bees. Counts of exiting workers were made as in the above bioassay. The control treatment preceding the presentation of the live worker was presentation of the fingers in which the bee was later held. Chemical analysis was performed with mandibular glands extracted in 1.5 ml of hexane and stored in glass vials with Teflon-lined caps. After two weeks during shipping to the Uppsala University Ecological Station in Sweden, the samples were stored under nitrogen gas at 5 ~ C. Prior to analysis the samples were concentrated to 100 /~l by evaporation. Isolation and identification were performed with an LKB 2091 gas c h r o m a t o g r a p h - m a s s spectrometer fitted with a 25-m glass capillary column coated with WG-11 stationary phase. The temperature p r o g r a m was set from 50 to 200 ~ C / m i n after an initial isothermal time of 4 min.
RESULTS
Bioassay. Colony response of M. fasciata and M. i. triplaridis to test substances is shown in Figure 1. Workers of both species responded maximally to conspecific mandibular gland extracts ( P < 0.01, Wilcoxon signed ranks test). In addition, these species exhibited a biting response in the presence of their respective gland extracts; the latter was never or rarely observed with other extracts or controls. One to five M. i. triplaridis closed their mandibles on the nylon ball in 12 of 16 tests with their mandibular gland extract; the same numbers of M. fasciata showed this response to their glandular extract in all of eight tests. The response of M. fasciata to glandular extracts of L. limao, T. pectoral&, and M. i. triplaridis was similar. This response, while significantly less than that to'its own glandular extracts, was in all cases greater than the three controls; however, only the response to extracts of M. i. triplaridis was statistically higher ( P < 0.05). Biting behavior was elicited in one or two of eight trials with extracts of each of the other species tested. Thus M.fasciata displayed alarm and biting response to mandibular gland extracts of other species, while M. i. triplaridis did not. A single, live biting bee elicited alarm and biting responses from its nestmates for both Melipona species. Biting by exiting workers occurred only when a live bee was held under the nest entrance. The biting response was observed in seven of 13 trials with M.fasciata and in five of 10 trials with M. i. triplaridis. Furthermore, a significant alarm response was recorded in the number of bees leaving the nest after presentation of a live bee for both M. fasciata ( X control = 4.0 bees/rain; ~ with live bee = 10.1 bees/rain: P < 0.01, Wilcoxon test, N = 13) and M. i. triplaridis ( ~ control = 2.9 bees/min; .~ with live bee = 9.9 bees/min: P < 0.01, Wilcoxon test, N = 10).
M. i. t r i p l a r i d i s 10. D-control m-treatment
Z r > r .J Z
/i,
O-
ho
Mi
P
so L'I Treatment No.
T'p
fp
M. fasciata
15
O-control B-treatment
zl0 4" > .J Z
Mf
Mi
T~p
LI
Treatment
so
fp
ho
No.
FtG. 1. A l a r m response of Melipona species to the p r e s e n t a t i o n of test substances at the nest entrance. H o r i z o n t a l lines represent means, vertical lines the ranges, and boxes the s t a n d a r d deviations of the n u m b e r s of bees leaving the nest e n t r a n c e in l-rain periods after the presentations. O p e n boxes represent controls; closed boxes represent the response to the test substance. In M. i. triplaridis the only test substance which elicited a greater response t h a n the control was its own m a n d i b u l a r gland extract ( P < 0.01"), which also elicited a greater response t h a n any o t h e r test s u b s t a n c e ( P < 0.01 **). In M.faseiata only its o w n m a n d i b u l a r gland extract and t h a t of M. i. triplaridis elicited a higher response t h a n controls ( P < 0.01" and P < 0.05 *, respectively); the f o r m e r elicited a higher response t h a n any other test extract, and the latter elicited a higher response t h a n the fp, so, a n d ho tests ( P < 0.01"* and P ~ 0.05"*, respectively). Mi, m a n d i b u l a r gland extract of M. i. triplaridis; Mr, m a n d i b u l a r gland extract of M.fasciata; L1, m a n d i b u l a r gland extract of Lestrimelitta limao; Tp, m a n d i b u l a r gland extract of Trigonapectoralis; ho, honey f r o m a Melipona nest; so, solvent + filter paper; fp, filter paper; *Wilcoxon signed r a n k s test; **Kruskal-Wallis test.
1469
C O N T E N T S OF STINGLESS BEE G L A N D S
Chemical Analysis. Several major components were identified from the mandibular gland extracts. The component corresponding to peak 3 (Figure 2) has a mass spectrum, molecular ion, and retention time identical to that of 2-heptanol. This and other alcohols are common as constituents of the mandibular gland secretions of other species of stingless bees (Blum, 1979). Only M. i. triplaridis exhibited skatole (peak 13, Figure 2). The mass spectrum, molecular ion, and retention time of this component are identical to synthetic skatole. It is conceivable that skatole is present in M.fasciata, but in amounts which were undetectable. However, since three separate extracts of each species were analyzed, and the same amount was used in each case (approximately 2 FE), it is reasonable to conclude that skatole is present in much larger quantities in M. i. triplaridis. Qualitatively, the odor produced by a biting M. i. triplaridis corresponds to skatole, and that of M.fasciata is not distinguishable from 2-heptanol. Several hydrocarbons were present, most notable of which is undecane, which is present in substantial amounts in both species. This hydrocarbon has been reported as an alarm pheromone in several ant species (Parry and Morgan, 1979; Bergstrom and Lovquist, 1971). Nerol is present only in M. i. triplaridis and has previously been reported as a mandibular gland component of stingless bees (Johnson and Weimer, 1982). DISCUSSION
We have presented evidence that two species of Melipona respond to mandibular gland extracts with defensive behavior including alarm recruitment from the colony. Skatole, reported here for one species, has not been previously identified in any bee species. Kerr and Lello (1962), however, reported a foul-smelling odor released by workers of some stingless bee species. It is known to repel predators ofa triehopteran (Duffield et al., 1977), and the mandibular gland secretion of meliponine bees may function in this context as well. It is notable, in light of the species-specific response of M.fasciata, that the major volatile component, 2-heptanol, is the same for both species. This substance is the major component used in the odor trails used by some Trigona species in the recruitment of foragers to food resources (Blum, 1979). In the honeybee, Apis mellifera, a freshly excised sting apparatus will elicit a more pronounced response than isopentyl acetate alone, the major component ofa multicomponent alarm pheromone (Boch and Shearer, 1966). Grandperrin and Mauchamp (1982) show that a maximum alarm response in this species is elicited by mixtures containing both the major and trace constituents identified in sting extracts. The discrimination may then lie in the presence of quantitative variation in both the major and minor volatile
1470
SMITh AND ROUBIK
r i1
~vi'
r m
E
c~ =
isothermal-- I
( 13
I
12
'r.
11
260
isothermal
1(~0~
--~ r
10
200
o+
100~
FIG. 2. Capillary gas chromatograms of mandibular gland extracts of M. i. triplaridis (top) and M. fasciata (bottom). Identification of secondary alcohols was based on the molecular ion, m-18 ion, m / e 45 ion, and comparisons with the mass spectra and retention times of synthetic alcohols. Skatole and nerol were identified by comparison of mass spectra and retention times with those of synthetic skatole, nerol, geraniol, and linolool. Peak identification: 1, undecane; 2, undecene; 3, 2-heptanol; 4, 2-nonanol; 5, nerol; 6, hexadecane; 7, 2-undecanol; 8, heptadecene; 9, unidentified aromatic (m = 234); 10, contaminant; 11, unidentified aromatic (m =234); 12, ethyl hexadecanoate; 13, skatole.
CONTENTS OF STINGLESS BEE GLANDS
1471
constituents (Weaver et al., 1975). However, Johnson and Weimer (1982) show that in certain instances workers of T. fulviventris are not inclined toward leaving the nest upon presentation of the synthetic analog of the alarm pheromone. In the present investigation then, the observed decrease in response to the nonconspecific animal extracts may be explained either by the lack of trace constituents or by the alteration of the aggressive response by additional compounds. Work in progress with the synthetic compounds should indicate the relative importance of the various constituents, and how they vary within and between stingless bee species. For four species of Apis there are species-specific blends of acetates in the sting-associated glands (Koeniger et al., 1979); the most pronounced defensive response to an extract of these glands is elicited from the species from which the extract is made. The response to these volatiles greatly enhances nest defense (Boch et al., 1962). Explanation of the species-specific response in Melipona must take into consideration the behavior of the robber bee Lestrimelitta limao. This species raids the nests of some species of stingless bees (Sakagami and Laroca, 1963; Roubik, 1981). During such raids the robber bees release the secretion from the mandibular gland, and this eventually disrupts the ability of the raided bees to muster a defense. It has been reported that some stingless bee species are not normally susceptible to this form of attack (Michener, 1974). It may be that this lowered susceptibility to raids of robber bees is due to the evoluation of lowered or altered responses to citral, the major component of the mandibular gland secretion of L. limao, and further development of a specific pheromone blend. Such a mechanism would facilitate the discrimination between nestmates and non-nestmates, and the maintenance of clear communication channels, which are the preliminary requirements in the defense of the nest against this form of attack. Acknowledgments--We wish to thank the Smithsonian Tropical Research Institute for the use of the facilities by B.H.S. Support from the Smithsonian Institution Scholarly Studies grant 1234S002 to D.W.R. is gratefully acknowledged. Chemical analyses were facilitated by a fellowship from the American Scandinavian Foundation to B.H.S. All chemical analyses were done at the Uppsala University Ecological Station, Oland, Sweden. In addition we wish to t h a n k Ms. Boel Lanne, Ms. Ingela Johansson, J.H. Cane, and C.D. Michener for their assistance in various aspects of this work.
REFE RENCES
BARONI URBANI,C. 1979. Territoriality in social insects, pp. 91-120, in H.R. Herman (ed.). Social Insects. Academic Press, New York. BERGSTROM, G., and LOVQUIST, J. 1971. Camponotis ligniperda Latr. A model for composite volatile secretions of Dufour's gland in formicine ants, pp. 195-223, in A.S. Tahori (ed.). Chemical Releasers in Insects. Proc. 2nd Int. I U P A C Cong. Pest. Chem. in Tel Aviv. Vol. 3. Gorden & Breach, New York.
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BLUM, M.S. 1966 Chemical releasers of social behavior. VIII. Citral in the mandibular gland secretion of Lestrimelitta limao. Ann. Ent. Soc. Am. 59:962-964. BLUM, M.S. 1970. The chemical basis of insect sociality, pp. 61-94, in M. Beroza (ed.). Chemicals Controlling Insect Behavior. Academic Press, New York. BLUM, M.S. 1979. Hymenopterous pheromones: Optimizing the specificity and acuity Of the signal, pp. 201-211, in F.J. Ritter (ed.). Chemical Ecology: Odour Communication in Animals. Elsevier/North Holland. BLUM, M.S., and BRAND, J.M. 1972. Social insect pheromones: Their chemistry and function. Am. Zool. 12:553-576. BLUM, M.S., CREWE, R.M., KERR, W.E., KEITH, L.H., GARRISON, A.W., and WALKER,M.M. 1970. Citral in stingless bees: Isolation in trail laying and robbing. J. Insect PhysioL 16:1637-1648. BOCH, R., and SFIEARER~O.A. 1966. Iso-pentyl acetate in stings of honeybees of different ages. J. Apic. Res. 5:65-70. BocH, R., SHEARER,D.A., and STONE, B.C. 1962. Identification of iso-amyl acetate as an active component in the sting pheromone of the honeybee. Nature 195:1018-1020. DUEEIELD, R.M., BLUM,M.S., WALLACE,J.B., LLOYD,H.A., and REGNIER,F.E. 1977. Chemistry of the defensive secretion of the caddisfly Pycnopsyche scabripennis (Trichoptera: Limnephilidae). 9'. Chem. Ecol. 3:649-656. GARY, N.E. 1974. Pheromones that affect the behavior and physiology of honey bees, pp. 200-221, in M.C. Birch (ed.). Pheromones. American Elsevier, New York. GRANDPERRIN,D., and MAUCHAMP,B. 1982. The Koschewnikow gland as the principal organ secreting the sting alarm pheromone in the honeybee worker (Apis mellifera L.), p. 333, in M.D. Breed, C.D. Michener, and H.E. Evans (eds.). The Biology of Social Insects. Westview Press. JOHNSON, L.K. and WEIMER,D.F. 1982. Nerol: An alarm substance of the stingless bee Trigona fulviventris (Hymenoptera: Apidae). J. Chem. Ecol. 8:1167-1181. KEEPING, M.G., CREWE, R.M., and FIELD, B.I. 1982. Mandibular gland secretions of the old World stingless bee, Trigonagribodoi Magretti: Isolation, identification, and compositional changes with age. 3". Apic. Res. 21:65-73. KERR, W.E., and DE LELLO,E. 1962. Sting glands in stingless bees--a vestigal character. J. N. Y. Entomol. Soc. 70:190-214. KOENIGER, N., WEISS,J., MASCHEWITZ, U. 1979. Alarm pheromones of the sting in the genus Apis. J. Insect PhysioL 25(6):461-542. MICHENER, C.D. 1974. The Social Behavior of the Bees. Harvard University Press, Cambridge, Massachusetts. 404 pp. PARRY, K., and MORGAN, E.D., 1979. Pheromones of ants: A review. Physiol. Entomol. 4:161-189. ROUBIK, D.W. 1981. New species of Trigona and cleptobiotic Lestrimelitta from French Guiana (Hymenoptera: Apidae). Rev. Biol. Trop. 28:263-269. SAKAGAMI,S.F., and LAROCA,S. 1963. Additional observations on the habits of the cleptobiotic stingless bees, the genus Lestrimelina Friese. J. Fac. Sei. Hokkaido Univ. (VI, Zool.), 15:319-339. WEAVER,N., WEAVER,E.C., and CLARKE,E.T. 1975. Reactions of five species of stingless bees to some volatile chemicals and to other species of bees. J. Insect. PhysioL 21:87-94. WILSON, E.O. 1971. The Insect Societies. Harvard University Press, Cambridge, Massachusetts.
MANDIBULAR GLANDS OF STINGLESS BEES (HYMENOPTERA: APIDAE): Chemical Analysis of Their Contents and Biological Function in Two Species of Melipona
B.H. S M I T H 1 a n d D . W . R O U B I K 2 1Department o[" Entomology, University of Kansas Lawrence, Kansas 66045 Smtthsoman Tropical Research Institute P.O. Box 2072, Balboa, Republic of Panama 2
9
.
(Received November 24, 1981; revised January 14, 1983) Abstract Workers of Melipona fasciata and M. interrupta triplaridis respond to their respective mandibular gland extracts with alarm recruitment and defensive behavior. Workers rapidly exit from the nest entrance, land on an intruding object, and bite with the mandibles while vibrating the flight muscles. These behaviors are accompanied by the release of the contents of the mandibular glands. Colonies of both species exhibited greater response to their own mandibular gland extracts than to those of other stingless bee species. Chemical analysis identified 2-heptanol as the major component in hexane extracts of each species. Undecane was a constituent of both species; skatole and nerol were identified only in extracts of M. i. triplaridis. Key Words--Melipona, Hymenoptera, Apidae, mandibular glands, 2heptanol, skatole, nerol, undeeane, alarm response, stingless bees. INTRODUCTION A m a j o r fi~nction of a l a r m r e c r u i t m e n t p h e r o m o n e s of social insects is the release of d ef en s i v e b e h a v i o r in the p r o x i m i t y of the nest (Wilson, 1971; B l u m and Brand, 1972; P a r r y and M o r g a n , 1979). Volatile c o m p o u n d s of biting and stinging H y m e n o p t e r a are released f r o m glands associated with the sting a p p a r a t u s or m a n d i b l e s of w o r k e r s ( B a r o n i U r b a n i , 1979). G r o u p s of w o r k e r s , m a n y times those specialized f o r nest defense, r e s p o n d to the release o f a l a r m p h e r o m o n e s near the c o l o n y by l e av i n g the nest, after which a d d i t i o n a l stimuli m a y cause t h e m to a t t a c k an i n t r u d i n g object (Gary, 1974; 1465 0098-0331 / 83/1100-1465503.00/0 9 1983 Plenum Publishing Corporation
1466
SMITH AND ROUBIK
Blum, 1979); in some species, however, workers are less inclined toward leaving the nest (Johnson and Weimer, 1982). Among the highly social bees, i.e., the honeybees (Apinae) and the stingless honeybees (Meliponinae), alarm pheromones have been identified for all species of Apis and for several meliponine species of the tribe Trigonini (Blum, 1966, 1979; Keeping et al., 1982). Here we present the first description of possible alarm substances in the genus Melipona and of the inter- and intraspecific alarm recruitment responses to natural extracts. We also consider evolutionary reasons for species-specific responses to substances used in nest defense. METHODS AND MATERIALS
Studies with natural colonies of Melipona fasciata and M. interrupta triplaridis (Cockerell) near Gamboa, Panama, indicated pronounced response of workers to colony disturbance (Roubik, in preparation). One colony of each species was used as a source of mandibular glands and in testing colony response to the resultant extracts. For bioassay, eight workers of each Melipona species were taken from the nests; the glands were extracted by rotating the mandible 180 ~ then gently pulling the mandible from the cranium with fine forceps. The resultant two sets of eight glands and mandibles of each species were placed in vials containing i ml dichloromethane and refrigerated at 5-10 ~ C. Similar extracts of Lestrimelitta limao and Trigona (Scaptotrigona)pectoralis were made from workers collected at their nests. The mandibular gland contents of L. lirnao consists of the two isomers of citral, geranial and neral, and is used during foraging raids on nests of other highly social bees (Blum et al., 1970; Sakagami and Laroca, 1963; Roubik, 1981). Bioassay of colony response to mandibular gland extracts was made by counting the number of bees leaving the nest a n d / o r biting the control stimulus, followed directly by another count after the presentation of one of the test extracts. The stimulus in both cases was a 7-ram filter paper disk held in place with an insect pin 2.5 cm over a dark blue nylon cloth ball, approximately 8 cm in diameter. Counts of bees leaving the nest were made for 1 rain when the stimulus was held 2-3 cm below the nest entrance; the second 1 min count was made after the filter paper was impregnated with 6 ~1 (approx. 0.1 female equivalents, FE) of a test extract. Bioassays using randomly chosen test substances were made at 15-rain intervals on each of 8 days. Tests on M. i. triptaridis were made between 0715 and 1000 hr local time, and those on M. fasciata between 1330 and 1645 hr. All tests were made in early April 1980. A second series of bioassays testing the ability of a single worker to elicit a defensive response from nestmates was made by holding a live worker 2 cm
1467
CONTENTS OF STINGLESS BEE GLANDS
below the nest entrance, not within visual range of the guard bees. Counts of exiting workers were made as in the above bioassay. The control treatment preceding the presentation of the live worker was presentation of the fingers in which the bee was later held. Chemical analysis was performed with mandibular glands extracted in 1.5 ml of hexane and stored in glass vials with Teflon-lined caps. After two weeks during shipping to the Uppsala University Ecological Station in Sweden, the samples were stored under nitrogen gas at 5 ~ C. Prior to analysis the samples were concentrated to 100 /~l by evaporation. Isolation and identification were performed with an LKB 2091 gas c h r o m a t o g r a p h - m a s s spectrometer fitted with a 25-m glass capillary column coated with WG-11 stationary phase. The temperature p r o g r a m was set from 50 to 200 ~ C / m i n after an initial isothermal time of 4 min.
RESULTS
Bioassay. Colony response of M. fasciata and M. i. triplaridis to test substances is shown in Figure 1. Workers of both species responded maximally to conspecific mandibular gland extracts ( P < 0.01, Wilcoxon signed ranks test). In addition, these species exhibited a biting response in the presence of their respective gland extracts; the latter was never or rarely observed with other extracts or controls. One to five M. i. triplaridis closed their mandibles on the nylon ball in 12 of 16 tests with their mandibular gland extract; the same numbers of M. fasciata showed this response to their glandular extract in all of eight tests. The response of M. fasciata to glandular extracts of L. limao, T. pectoral&, and M. i. triplaridis was similar. This response, while significantly less than that to'its own glandular extracts, was in all cases greater than the three controls; however, only the response to extracts of M. i. triplaridis was statistically higher ( P < 0.05). Biting behavior was elicited in one or two of eight trials with extracts of each of the other species tested. Thus M.fasciata displayed alarm and biting response to mandibular gland extracts of other species, while M. i. triplaridis did not. A single, live biting bee elicited alarm and biting responses from its nestmates for both Melipona species. Biting by exiting workers occurred only when a live bee was held under the nest entrance. The biting response was observed in seven of 13 trials with M.fasciata and in five of 10 trials with M. i. triplaridis. Furthermore, a significant alarm response was recorded in the number of bees leaving the nest after presentation of a live bee for both M. fasciata ( X control = 4.0 bees/rain; ~ with live bee = 10.1 bees/rain: P < 0.01, Wilcoxon test, N = 13) and M. i. triplaridis ( ~ control = 2.9 bees/min; .~ with live bee = 9.9 bees/min: P < 0.01, Wilcoxon test, N = 10).
M. i. t r i p l a r i d i s 10. D-control m-treatment
Z r > r .J Z
/i,
O-
ho
Mi
P
so L'I Treatment No.
T'p
fp
M. fasciata
15
O-control B-treatment
zl0 4" > .J Z
Mf
Mi
T~p
LI
Treatment
so
fp
ho
No.
FtG. 1. A l a r m response of Melipona species to the p r e s e n t a t i o n of test substances at the nest entrance. H o r i z o n t a l lines represent means, vertical lines the ranges, and boxes the s t a n d a r d deviations of the n u m b e r s of bees leaving the nest e n t r a n c e in l-rain periods after the presentations. O p e n boxes represent controls; closed boxes represent the response to the test substance. In M. i. triplaridis the only test substance which elicited a greater response t h a n the control was its own m a n d i b u l a r gland extract ( P < 0.01"), which also elicited a greater response t h a n any o t h e r test s u b s t a n c e ( P < 0.01 **). In M.faseiata only its o w n m a n d i b u l a r gland extract and t h a t of M. i. triplaridis elicited a higher response t h a n controls ( P < 0.01" and P < 0.05 *, respectively); the f o r m e r elicited a higher response t h a n any other test extract, and the latter elicited a higher response t h a n the fp, so, a n d ho tests ( P < 0.01"* and P ~ 0.05"*, respectively). Mi, m a n d i b u l a r gland extract of M. i. triplaridis; Mr, m a n d i b u l a r gland extract of M.fasciata; L1, m a n d i b u l a r gland extract of Lestrimelitta limao; Tp, m a n d i b u l a r gland extract of Trigonapectoralis; ho, honey f r o m a Melipona nest; so, solvent + filter paper; fp, filter paper; *Wilcoxon signed r a n k s test; **Kruskal-Wallis test.
1469
C O N T E N T S OF STINGLESS BEE G L A N D S
Chemical Analysis. Several major components were identified from the mandibular gland extracts. The component corresponding to peak 3 (Figure 2) has a mass spectrum, molecular ion, and retention time identical to that of 2-heptanol. This and other alcohols are common as constituents of the mandibular gland secretions of other species of stingless bees (Blum, 1979). Only M. i. triplaridis exhibited skatole (peak 13, Figure 2). The mass spectrum, molecular ion, and retention time of this component are identical to synthetic skatole. It is conceivable that skatole is present in M.fasciata, but in amounts which were undetectable. However, since three separate extracts of each species were analyzed, and the same amount was used in each case (approximately 2 FE), it is reasonable to conclude that skatole is present in much larger quantities in M. i. triplaridis. Qualitatively, the odor produced by a biting M. i. triplaridis corresponds to skatole, and that of M.fasciata is not distinguishable from 2-heptanol. Several hydrocarbons were present, most notable of which is undecane, which is present in substantial amounts in both species. This hydrocarbon has been reported as an alarm pheromone in several ant species (Parry and Morgan, 1979; Bergstrom and Lovquist, 1971). Nerol is present only in M. i. triplaridis and has previously been reported as a mandibular gland component of stingless bees (Johnson and Weimer, 1982). DISCUSSION
We have presented evidence that two species of Melipona respond to mandibular gland extracts with defensive behavior including alarm recruitment from the colony. Skatole, reported here for one species, has not been previously identified in any bee species. Kerr and Lello (1962), however, reported a foul-smelling odor released by workers of some stingless bee species. It is known to repel predators ofa triehopteran (Duffield et al., 1977), and the mandibular gland secretion of meliponine bees may function in this context as well. It is notable, in light of the species-specific response of M.fasciata, that the major volatile component, 2-heptanol, is the same for both species. This substance is the major component used in the odor trails used by some Trigona species in the recruitment of foragers to food resources (Blum, 1979). In the honeybee, Apis mellifera, a freshly excised sting apparatus will elicit a more pronounced response than isopentyl acetate alone, the major component ofa multicomponent alarm pheromone (Boch and Shearer, 1966). Grandperrin and Mauchamp (1982) show that a maximum alarm response in this species is elicited by mixtures containing both the major and trace constituents identified in sting extracts. The discrimination may then lie in the presence of quantitative variation in both the major and minor volatile
1470
SMITh AND ROUBIK
r i1
~vi'
r m
E
c~ =
isothermal-- I
( 13
I
12
'r.
11
260
isothermal
1(~0~
--~ r
10
200
o+
100~
FIG. 2. Capillary gas chromatograms of mandibular gland extracts of M. i. triplaridis (top) and M. fasciata (bottom). Identification of secondary alcohols was based on the molecular ion, m-18 ion, m / e 45 ion, and comparisons with the mass spectra and retention times of synthetic alcohols. Skatole and nerol were identified by comparison of mass spectra and retention times with those of synthetic skatole, nerol, geraniol, and linolool. Peak identification: 1, undecane; 2, undecene; 3, 2-heptanol; 4, 2-nonanol; 5, nerol; 6, hexadecane; 7, 2-undecanol; 8, heptadecene; 9, unidentified aromatic (m = 234); 10, contaminant; 11, unidentified aromatic (m =234); 12, ethyl hexadecanoate; 13, skatole.
CONTENTS OF STINGLESS BEE GLANDS
1471
constituents (Weaver et al., 1975). However, Johnson and Weimer (1982) show that in certain instances workers of T. fulviventris are not inclined toward leaving the nest upon presentation of the synthetic analog of the alarm pheromone. In the present investigation then, the observed decrease in response to the nonconspecific animal extracts may be explained either by the lack of trace constituents or by the alteration of the aggressive response by additional compounds. Work in progress with the synthetic compounds should indicate the relative importance of the various constituents, and how they vary within and between stingless bee species. For four species of Apis there are species-specific blends of acetates in the sting-associated glands (Koeniger et al., 1979); the most pronounced defensive response to an extract of these glands is elicited from the species from which the extract is made. The response to these volatiles greatly enhances nest defense (Boch et al., 1962). Explanation of the species-specific response in Melipona must take into consideration the behavior of the robber bee Lestrimelitta limao. This species raids the nests of some species of stingless bees (Sakagami and Laroca, 1963; Roubik, 1981). During such raids the robber bees release the secretion from the mandibular gland, and this eventually disrupts the ability of the raided bees to muster a defense. It has been reported that some stingless bee species are not normally susceptible to this form of attack (Michener, 1974). It may be that this lowered susceptibility to raids of robber bees is due to the evoluation of lowered or altered responses to citral, the major component of the mandibular gland secretion of L. limao, and further development of a specific pheromone blend. Such a mechanism would facilitate the discrimination between nestmates and non-nestmates, and the maintenance of clear communication channels, which are the preliminary requirements in the defense of the nest against this form of attack. Acknowledgments--We wish to thank the Smithsonian Tropical Research Institute for the use of the facilities by B.H.S. Support from the Smithsonian Institution Scholarly Studies grant 1234S002 to D.W.R. is gratefully acknowledged. Chemical analyses were facilitated by a fellowship from the American Scandinavian Foundation to B.H.S. All chemical analyses were done at the Uppsala University Ecological Station, Oland, Sweden. In addition we wish to t h a n k Ms. Boel Lanne, Ms. Ingela Johansson, J.H. Cane, and C.D. Michener for their assistance in various aspects of this work.
REFE RENCES
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