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Volatile cephalic substances of the stingless bees, Trigona mexicana and Trigona pectoralis
J. Insect Physiol.,
1973,Vol.
19, pp.
1111to 1127. Pergamon
Press. Printed in Great Britain
VOLATILE CEPHALIC SUBSTANCES OF THE STINGLESS BEES, TRIGONA MEXICANA AND TRIGONA PECTORALIS JULIA M. LUBY,l FRED E. REGNIER,l ERIC T. CLARKE,2 ELIZABETH C. WEAVER,3 and NEVIN WEAVER3 l Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907; 2 Southwestern University, Georgetown, Texas 78626; and 3 Department of Biology, University of Massachusetts at Boston, Boston, Massachusetts 02116. (Received 26 October 1972)
Abstract-Extracts of the heads of the stingless bees, Trigona mexicana and T. pectoralis, contain mixtures of compounds that are identifiable by gas chromatography and mass spectrometry. These compounds form homologous series of aliphatic alcohols and ketones with an odd number of carbon atoms and functional groups at the 2-position. The alcohols and the ketones range from 7 to 17 carbon atoms. Benzaldehyde and a nitrogen containing compound are also present in the mixtures. The series of compounds from the two species are nearly identical qualitatively. They differ in the absence of 2-undecanol and 2-pentadecanol from the extracts of T. mexicana and T. pectoralis, respectively. The highest concentration of material is found in the 7-carbon fraction in T. mexicana and in the 13 to 15 carbon range in T. pectoralis. There is a major difference in the relative concentration of 2-heptanol and 2-heptanone in the two species with the concentration of the alcohol being one-fourth that of 2-heptanone in T. mexicana and ten times greater than the ketones in T. pectoralis. Both the alcohols and ketones are alarm pheromones. The alcohols are more active in inducing attack by the bees than are the ketones, but a mixture of the ketones and benzaldehyde was very active.
INTRODUCTION
SOCIALinsects live in colonies which, during much of the year, may contain food reserves and individuaIs of al1 castes in one or more stages of development. The colony must be protected from a wide variety of enemies (SCHWARZ,1948) and a system for quickly bringing a large number of adults to the defence of the colony may be necessary for colony survival. Many social insects employ alarm pheromones to recruit defenders (LAW and REGNIER, 1971; WILSON, 1971). The defence systems of the stingless bees (Tribe Meliponini) have not been extensively studied. LINDAUERand KERR (1960) and KERR et al. (1963) reported that several species of Meliponini have recruiting pheromones and described behaviour that must have resulted from alarm pheromones. Recently BLUM (1966) and BLUM et al. (1970) reported that geranial and neral occur in the mandibular glands of both Lestrimelitta limao, Fr. Smith and Trigona subterranea Friese, and that in the latter species it is both a recruiting pheromone and an alarm pheromone. In a recent 1111
1112
J. M. LUBY, F. E. RIXGNIER,E. T. CLARKE,E. C. WEAVER,ANDN. WEAVER
review of the chemical basis for insect sociality, BLUM (1971) refers to an unpublished study on T. postica, T. xanthotricha, and T. tubiba. The mandibular gland of T. postica reportedly contained benzaldehyde, undecane, tridecane, and odd carbon number 2-alkanones ranging from 7 to 25 carbon atoms while those of T. xanthotricha contain benzaldehyde, aliphatic hydrocarbons, and 2-heptanone. The mandibular glands of T. tubiba secrete benzaldehyde, undecane, tridecane, 2-heptanone, and 2-nonanone. Benzaldehyde was highly attractive to workers of T. tubiba and did not elicit aggressive behaviour as did 2-heptanone and 2-nonanone. The potency of benzaldehyde as an attractant to T. post& workers was reported to be enhanced by the presence of 2-undecanone and 2-tridecanone. Unfortunately, the experimental details of these studies are still not available. Other than the data cited above, little is known of the volatile cephalic material used by the many Trigona species in the tropics for chemical communication. This study of two species of stingless bees from Mexico, T. mexicana and T. pectoralis, was undertaken to attempt to clarify the chemical nature and significance of these volatile compounds in a natural communication system.
MATERIALS
AND METHODS
Biological material Four colonies of Trigona (Scaptotrigona) mexicana Guerin (Hymenoptera : Apidae: Meliponini) were found nesting in the archaeological ruins of El Tajin near Papa&a, Veracruz, Mexico; three of the nest entrances led through cracks in perpendicular walls to the interior of the mounds; the other nest was at least partially exposed deep in a niche that was an architectural feature of the building, Several colonies of T. (Scuptotrigona) pectoralis Dalla Torre were found in the hollows of trees in Yaxcaba, Yucatan, Mexico. Bees were collected by placing a net bag over the entrance to the nest; they were anaesthetized with chloroform and decapitated or alternately, the mandibular glands were removed. The heads of T. mexicana were extracted with chloroform ; heads and mandibular glands of T. pectoralis were extracted with hexane. The extracts were reduced to a volume of 0.25 ml under a stream of nitrogen and transferred to Kontes microflex vessels. The extract from T. mexicana was designated as ‘sample A’, and that from the heads of T. pectoralis as ‘sample B’. Reagents. Benzaldehyde and the 2-alkanones were obtained from Aldrich Wisconsin, while the sodium borohydride Chemical Company, Inc., Milwaukee, was supplied by Ventron of Beverly, Mass., and Apiezon-L by Anspec Company, Ann Arbor, Michigan. Diethylether, chloroform, and hexane were obtained from the J. T. Baker Chemical Co., Phillipsburg, N.J. P and 3% Dexsil 300 on 100/120 Gas chromatography packings. Gas-Chrom mesh Gas-Chrom Q were supplied by Applied Science Laboratories, Inc., State College, Pa. Apparatus. Gas chromatographic analyses were carried out on a Varian Aerograph Model 1200 instrument fitted with a hydrogen flame detector and
VOLATILE
CEPHALIC
SUBSTANCES
OF THE
STINGLESS
BEES
1113
6 ft x & in. stainless steel column packed with 3% Dexsil 300 on lOO/lZO mesh Gas-Chrom Q. The stainless steel injection port inserts supplied with the instruInjection port and detector temperament were used as the precolumn reactors. tures were 150 and 277°C respectively, while the column oven was programmed from 68 to 250°C at 4”C/ min. A 100 ml/min flow of nitrogen through the column was used. Combination gas chromatographic-mass spectral analyses. Combination gas analyses were performed on an LKBchromatographic-mass spectral (GC/MS) 9000 instrument fitted with an 8 ft x & in. stainless steel column packed with 3% conditions Dexsil 300 on 100/120 mesh Gas-Chrom Q. The chromatographic described previously were used in these analyses. The mass spectrometer was operated at an electron energy of 70 eV, with an accelerator voltage of 3.5 kV and ion source temperature of 250°C. Reaction gas chromatography. Reaction gas chromatographic (RGC) supports were prepared as described previously (REGNIER and HUANG, 1970). were prepared by sodium boroPreparation of 2-alkanols. The 2-alkanols hydride reduction of the corresponding 2-alkanones (DAUBEN, 1955). of components in Chromatographic quantitation. The absolute concentration the bee extracts and the relative reactivity of these components in flow reactors was determined using n-pentadecane as an internal standard. Relative peak areas were calculated by weighing. Field experiments Whole bees or parts of bees were mashed between the fingers and presented near the entrance of the nest, and the behaviour of the bees was compared to the behaviour when a clean hand was presented. Known chemicals were tested by dipping wooden rods (toothpicks) into vials of chemicals and presenting them about 1 to 2 cm in front of, and just below the level of the lower edge of the entrance tube. A subjective scale was used to judge the severity of attacks on the rod and the investigator. Some of the chemicals were diluted 1 : 9 (v/v) with diethyl ether and tested on small glass rods. In conducting the biological assays the experimenter did not know the identity of the chemical he was testing, and he handled nothing except the clean ends of rods.
RESULTS Analytical gas chromatography on a 3% Dexsil 300 column resolves the volatile components from the heads of the stingless bees, T. mexicana (sample A) and T. pectoralis (sample B) as shown in the chromatograms (Figs. 1, 2). The symbols used to identify the peaks in these chromatograms will be used later when referring to the mass spectra of these compounds. Slashes on the chromatograms indicate points at which mass spectra were obtained. The concentrations of components in these samples are listed in Table 1. Relative retention times are related to an internal standard, n-pentadecane, shown in the chromatograms.
1114 J. M. LUBY, F. E. REGNIER,E. T. CLARKE,E. C. WEAVER,AND N. WEAVER
SSUOdSaJ
aSUOdSaJ
Ja,lJOC’a~
JSpJODi%i
VOLATILE CEPHALIC SUBSTANCES OF THE STINGLESS BEES TABLE
~-THE
1115
CONCENTRATION OF VOLATILECEPHALICSUBSTANCES IN THE HEADSOF T. mexicana AND T. pectoralis T. pectoralis
T. mexicana Peak* number
Relative+ concentration
Peaks number
Absolute concentration per worker kg)
A, A* A, A4f A, A, A,
23 100 40 6 10 2
B, B, B, B* B, B, B,
4.4 0.4 5.8 1.8 0.9 0.4 -
A, A, A,, A,, A,,
14 12 3
B, B, B 10 B 11 B 12
2.2 44 9.7 6.2 -
B 13
4
* Peak number refers to peak indicated on the gas chromatograph in Fig. 1. t_ Relative concentration is expressed relative to the most abundant component. 1 Position of compound is indicated by a dash on the chromatogram in Fig. 1. 4 Peak number refers to peak indicated on the gas chromatogram in Fig. 2.
Reaction gas chromatography
Further analysis by reaction gas chromatography yielded information on functional group composition of components. By this method, compounds containing a specific functional group (such as a carbonyl or hydroxyl group) may be subtracted from the carrier gas as the mixture passes through a flow reactor packed with a specific reagent (REGNIER and HUANG, 1970). Reactive species are converted into non-volatile derivatives and are identified by their absence in the chromatogram. Flow reactors containing O-5 and 5% boric acid coates supports were used to identify alcohols while sodium borohydride (NaBH,) reactors were used to identify aldehydes and ketones. The boric acid flow reactor was operated at both 123 and 150°C. When the flow reactor was maintained at 123”C, compound B, was completely subtracted while at 150°C its subtraction was incomplete. It is probable that at 150°C the low molecular weight compounds are almost exclusively in the vapour phase and the residence time in the flow reactor is not sufficient for derivatization. By lowering the reactor temperature highly volatile compounds are in contact with the boric acid for a sufficient time to be subtracted. Compounds A,, B,, and B, showed 100 per cent removal by the boric acid reactor and little reactivity toward sodium borohydride. This behaviour suggests that these compounds are alcohols (REGNIER and HUANG, 1970).
1116
J. M. LUBY,F. E. REGNIER, E. T. CLARKE, E. C.
WEAVER, AND
N. WEAVER
Compounds As and B, exhibited high reactivities toward sodium borohydride (92 and 59 per cent removal respectively) and are probably aldehydes (REGNIERand HUANG, 1970). Other compounds, such as A,, A,, A,, A,,. A1s, B,, B,, B,, B,, B rO, and Bra, which show low reactivity (1040 per cent subtraction) with NaBH, are suspected of being ketones. Mass spectrometry. Mixtures A and B from the heads of T. mexicana and T. pectoralis, respectively, were analysed by gas chromatography-mass spectrometry to determine molecular size and the type and position of functional groups present in the molecules. Discussion of mass spectra will be limited to those fragment ions which are characteristic of functional groups and to those which help in locating functional groups in a molecule. In the presentation of mass spectral data, components of the two samples will be grouped on the basis of probable functional group, apparent molecular weight, and relative retention time. Ketones will be discussed first in the order of increasing molecular weight. Examination of the spectra of A,, A,, A,, B,, A,, B,, Arc,, Bra, Ars, and B,, indicated that all of these compounds produce fragment ions at m/e 43, 58, M-43, M-18, M-15, and M where the symbol M indicates the molecular ion. The molecular weights of these compounds compose a family 28 atomic mass units (AMU) apart ranging from 142 to 254. The fragment ion at m/e 58 is unusual in that it represents a rearranged product. It has been demonstrated in many cases that the m/e 58 fragment ion is characteristic of 2-alkanones (BUDZIKIEWIEZet al., 1966). The major fragment ions in these compounds are produced by two fragmentation mechanisms, one cleaving the a bond on either side of the carbonyl group and the second cleaving the bond in the position /3 to the carbonyl group. Additional fragment ions result from the loss of water (M-18) and a methyl group (M-15) from the parent ion. The spectra indicate that these substances are saturated 2-alkanones with an odd number of carbon atoms ranging from 7 to 17. Comparison of the characteristic fragment ions from the ketones in samples A and B and standard ketones can be seen in Table 2. The spectral data for compounds A,, As, B,, Alo, and B,, closely match that of standard compounds. Compounds A,, A,, Blo, and A,, show fragment ion intensities that are either higher at m/e 43 or lower at m/e 58 than the corresponding standards. We attribute this to contamination of these components with other compounds. Since the Dexsil 300 column used in these analyses is not capable of complete resolution of a 2-alkanone and the corresponding 2-alkanol, contamination of the ketones with alcohols was a problem. The mass spectra of compounds A,, A,, Blo, and A,, all had enhanced ions at m/e 43 and 45. As can be seen in the spectrum of 2-heptanol (Fig. 6), the fragment ions at m/e 43 and 45 are intense in 2-alkanols. If an enhanced m/e 43 peak is the base peak in the spectrum, it will cause all other peaks including m/e 58 to be of lower intensity than standards. Compounds A, and B, were suspected of being aldehydes on the basis of their reactivity with NaBH, in the reaction gas chromatographic analyses cited above. Fragment ions at m/e 77 and 51 in these compounds strongly suggest the presence of an aromatic ring as is seen in the spectrum of B, (Fig. 3). By the addition of
VOLATILECEPHALICSUBSTANCES OF THE STINGLESS BEES TABLE 2-A
III7
TABULATION OF THE CHARACTERISTIC IONSIN THE VOLATILEKETONESPECTRA OF T. mexicana, T. pectoralis, ANDSOMESTANDARD 2-ALKANONES Relative intensity of various fragment ions
Compound
43
58
M-43
M-18
M-IS
A, 2-Heptanone A, 2-Nonanone A, 2-Undecanone A* Bg 2-Tridecanone Ai, B Z%entadecanone Ai, B 13
100 100 100 100 100 100 83 93 92 81 100 90 100 97
49 56 87 88 85 100 100 100 100 100 87 100 80 100
12 14 2 2 2 2 2 2 2 I 2 2 1 I
2 1 I I I I 2 1 1 1 2 2 I I
2 3 3 3 2 3 3 4 3 2 2 3 2 2
M* 5 5 7 8 4 6 7 9 8 7 6 7 4 4
(114) (114) (142) (142) (170) (170) (198) (198) (198) (226) (226) (226) (254) (254)
* The mass of the molecular ion is indicated in parentheses.
106
m/e FIG. 3. Mass spectrum of compound Bs. Instrumentation, sample introduction, and operating conditions are described in the experimental section.
1118
J. M. LUBY,F. E. REGNIER, E. T. CLARKE, E. C. WEAVER,ANDN. WEAVER
29 AMU to this aromatic ring, one obtains the molecular ion (m/e 106). Several fragment ions could exhibit this characteristic such as [-CH,CH,]+ or [-CHO]+. The presence of an intense fragment ion at M - 1 (m/e 105) strongly favours the aldehyde (MCCOLLUMand MEYERSON,1963). A comparison of the mass spectra of compounds A,, B, (Fig. 3), and benzaldehyde (Fig. 4) showed that these compounds were identical. 06(M)
’k 60T) a5 P e
40-
E 0 z 20
-
‘**
%
100
50
I, 1.
m/e
FIG. 4. Mass spectrum of benzaldehyde. Sample introduction was through the gas chromatograph.
Other conditions were the same as those in Fig. 3.
All of the compounds tentatively identified as alcohols by reaction gas chromatography (A,, B,, A,, and B,) have intense fragment ions at m/e 45. This fragment ion suggests that these alcohols are 2-alkanols (BUDZIKIEWICZ et al., 1968) and thus biosynthetically related to the 2-alkanones present in these samples. It appears from the spectrum of A, (Fig. 5) that the molecular weight is 115. This could only be true if the molecule contained nitrogen. Since no nitrogen containing fragment ions are observed in the spectrum, it may be concluded that this compound does not contain nitrogen and that m/e 115 is not the molecular ion. The molecular weight must be greater than 115. Previous studies have shown that 2-alkanols characteristically lose a proton to give fragment ions at M-l. On this basis, the molecular weight of A, would be 116 and fragment ions at m/e 101 and 98 are the result of the loss of a methyl group (M-15) an d water (M-18) from the parent ion. Comparison of the spectra of A, and 2-heptanol (Fig. 6) shows that they are identical. In addition to A,, B,, A,, and B,, the intense fragment ion at m/e 45 was found in the spectra of B,, A,, AlI, and B,, whose concentration was too low for RGC
VOLATILE
CEPHALIC
SUBSTANCES
OF THE STlNGLESS
1119
BEES
5
FIG. 5. Mass spectrum of compound A,. Information on instrumentation analytical procedures are available in the experimental section.
and
100
I-
-I m/e
FIG. 6. Mass spectrum of 2-heptanol. Instrumentation, operating conditions, and sample introduction are the same as those used in Fig. 4.
1120
J. M. LUBY, F. E. REGNIER,E. T. CLARKE,E. C. WEAVER,ANDN. WEAVER
characterization. A comparison of the characteristic ions in the spectra of all unknown alcohols and standard Z-alkanols can be seen in Table 3. TABLE 3-A TABULATION OF THE CHARACTERISTIC IONSIN THE VOLATILEALCOHOL SPECTRAOF T. ?TWXiCana, T. peCtO?diS, ANDSOMESTANDARD 2-ALKANOLS Relative intensity of various fragment ions Compound
Al Bl
2-Heptanol A4 R, 2-Nonanol R, 2-Undecanol A, 2-Pentadecanol A,1
45
M-18
M-15
100 100 100 100 100 100 100 100 100 100 100
10 9 9 5 8 5 9 7 9 7 3
8 7 8 3 5 3 3
3 3 1 1
M-l 1 1 1 1 1 1 1 1 1 1 1
M* 0 0 0 0 0 0 0
0 0 0 0
(116) (116) (116) (144) (144) (144)
(172) (172) (228) (228) (256)
* The mass of the molecular ion is indicated in parentheses.
Both compounds A, and B, have an apparent molecular ion at m/e 168. The 18 AMU difference between this ion and an m/e 150 ion in the mass spectrum suggest that these molecules contain oxygen. Additional even mass ions at m/e 110, 68, and 54 further suggest that these molecules also contain nitrogen. Compound A,, could not be identified from the mass spectrum. It appears that the eventual identification of these compounds will necessitate additional chemical analyses. Although the mass spectra of all the alcohols, aldehydes, and ketones of samples A and B were virtually identical to the various standard compounds, it is conceivable that compounds with slight differences in carbon skeleton could give similar mass spectra. Gas chromatographic retention times. Final identification of the components in these mixtures was achieved by comparing their gas chromatographic retention times with those of the standards (Table 4). Retention times for 2-heptadecanol and 2-heptadecanone were determined from a plot of relative retention times v. carbon number for other members of homologous series (JAMES, 1959). Since the retention times of branched chain compounds are decreased on Dexsil300 columns (REGNIER et al., 1972), non-identity of carbon skeletons would be easily discernible. Field experiments
Mashed whole bees or heads elicited attacks from both species of bees. T. also made less severe attacks on the hand that had mashed the thorax and abdomen of a bee; since the bees that were decapitated were always rather severely
pectoralis
VOLATILE
CEPHALIC
SUBSTANCES
OF THE STINGLESS
alarmed when they were captured, the legs and may have become contaminated with secretion colony was already alarmed, the bees circling piece of wood two or more metres from the nest TABLE
~-RELATIVE
RETENTION
Compound
Al Bl
2-Heptanol
A, R, 2-Heptanone -4, B, Benzaldehyde A, R, 2-Nonanol ALI B, 2-Nonanone‘ B, 2-Undecanol
TIMES
Tr* 0.23 0.23 0.25 0.26 0.26 0.26 0.34 0.35 0.36 0.51 0.49 0.50 0.53 0.52 0.53 0.78 0.77
OF
Trigona
1121
BBF.8
possibly other parts of the body from the mandibular gland. If a the investigator would attack a if a mashed bee was rubbed on it.
CEPHALIC
SUBSTANCES
Compound
A, B*
2-Undecanone
A* B, 2-Tridecanone 2-Pentadecanol AI0 B 2-‘ientadecanone A,, B 2-&eptadecanol AI, B 2%eptadecanone
AND
STANDARDS
T,
041 0.80 0.80 I.06 1.07 1.05 1.25 I.28 1.29 1.27 1.49 1.46 1.521_ 1.51 1 a49 1.56t
* The symbol T, designates relative retention time. t Relative retention times for 2-heptadecanol and 2-heptadecanone were determined from a plot of relative retention times vs. carbon number for other members of homologous slices.
Table 5 shows the visible reactions of T. mexicana and T. pectoralis to authentic samples of some of the compounds that have been identified from their heads. In order to make tests as comparable as possible, all of the tests in a series (one column in the table) were completed within half a day using a single colony. In the case of the purified compounds, a series included compounds that were offered both neat and diluted. Increasing numbers of + ‘s in the table indicate increasing severity of attacks on an odour source and on the experimenter. Five ‘f’s are used to indicate an attack of maximum severity. Other types of behaviour recorded in Table 5 were probably mild alarm reactions that did not result in attack. With the behaviour indicated as ‘investigate’, the bees typically clung with the hind legs to the inside of the entrance tube at a point near the source of the chemical and stretched toward it. The ‘roar’ was caused by the beating of wings inside the nest, and in T. mexicana and some other species studied, it might either precede an attack or occur when there was no attack. It is probable that the nests of T. pectoralis were so deep inside the tree that the noise from within was not often
*2
+ 0 +
*2
+ + + + *3 + 0 +4
*2 Roar; *3 Excite;
0 +
*1
++
++
+++ +I +++ + ? + 0 + *1
** Repeal.
o’? + O? + *1
+ *1
*1
*2
0 0 0 0 0 0 0 0 *1
*1
0
Neat
0
Neat
Neat
+ + + *3 0 *3 -
++
*l
Neat
SUBSTANCES
+ 0 0 0 0 0 -t-? -
*1
0
0
Neat
TESTED)
Dilute
Neat
4'S UP TO A MAXIMUM
CEPHALIC
AT THE
0 0 *2 0 0 0 0 0 0 -
-
0 ++ + 0 0 + 0 + 0 -
+
Dilute
0 0 -
-
0 +
-
ENTRANCE
O? ++ + + +? + 0 + 0 -
+++
Neat
*1
-
-
:? 0 O? + + + *1 *3
Neat
*1
0
0 ++ + + 0
+++
+t **
Dilute
NEST
-
-t-
+ 0 + + 0 -I-t0 +
Dilute -
TO THE
OF 5 +'S;O:NODISCERNABLEREPONSE;
PRESENTED
Purified chemicals
NOT
OF
Commercial chemicals
-:
NUMBERS
TO VOLATILE
T. pectoralis
Neat
*3
T. pectoralis BY INCREASING
T. mexicana
++++ ++ *3
0
AND
INDICATED
T. mexicana
+ *2
Neat
*i Investigate;
2-Heptanol 2-Heptanone 2-Nonanol 2-Nonanone 2-Undecanol 2-Undecanone 2-Tridecanol 2-Tridecanone 2-Pentadecanol 2-Pentadecanone Benzaldehyde
Chemical
OF
SEVERE ATTACKS
~-F&ACTIONS
(INCREASINGLY
TABLE
VOLATILE CEPHALIC SUBSTANCES OF THE STINGLESS BEES
1123
audible above the normal environmental noises. The behaviour designated ‘excite’ consisted of various types of rather quick and nervous-appearing movements. When the bees were ‘repelled’ the guards that normally lined the mouth of the . entrance tube retreated far back into it, sometimes out of sight. As can be seen in Table 5, both species were more likely to attack in response to 2-alkanols than 2-alkanones with the same carbon skeleton. Within the alcohol series, they attacked most readily in response to the lower molecular weight compounds. 2-Heptanol occurred in fairly high concentration in both species, and significant amounts of 2-nonanol occurred in T. pectoralis, but otherwise there was no apparent relationship between the concentration of the chemicals in the bees and the reactions of the bees to individual chemicals. In both species, for instance, the ketones occur in much higher concentrations than do the alcohols, but the alcohols were more excitatory. Benzaldehyde also occurs in significant concentrations in both species, and although the bees always responded to it in some way, it caused a very mild attack on only one occasion. When a mixture of 10 mg of 2-heptanone, 1 mg each of 2-nonanone, 2-undecanone, and 2-tridecanone, and 5 mg of benzaldehyde in 1 ml of hexane was presented on a wooden rod at the entrance of 3 colonies of T. peetorah, an immediate and vigorous attack occurred every time. It will be noted that the results with the diluted and undiluted compounds were about the same. This is probably because the techniques used in presenting compounds to the bees did not result in large concentration differences in the air at the nest. The bees also appeared to react in about the same way to the commercial grade and the purified compounds, though the tests on the two grades were not conducted on the same day and therefore were not strictly comparable. The bees varied from day to day in their reactions to test compounds, and on some days the attacks were so severe that it was impossible to complete a meaningful series of tests. After the bees attacked they were left alone for several minutes and were not tested again until they did not react to a blank. When the bees were especially fractious, hexane or nonane was used as a control. Both colonies of T. pectoralis used to assay chemicals, and some other colonies of that species which were used for occasional tests, varied greatly in their readiness to attack on different days. Sometimes the extreme fractiousness of a colony persisted for several days. For instance, while trying to complete a series of tests on one of the colonies it was necessary to leave the area of the nest after each test. When the experimenter returned after 1 or 2 hr, the bees often attacked him. At the end of 3 days only 7 compounds had been tested. In these tests both Z-tridecanol and Z-pentadecanol led to fairly strong attack reactions (+ + + and + +, respectively) following a negative nonane control. Unfortunately there was no opportunity to test the ketones during this series. Most of the compounds that have been identified as alarm pheromones in insects (LAW and REGNIER, 1971) were tested on these bees, and most of the chemicals did not cause any response. However, 2-octanol was only slightly less active than 2-heptanol for both species. 2-Methyl-2 heptanol caused attacks by 35
1124
J. M. LUBY, F. E. REGNIER, E. T. CLARKE,E. C. WEAVER,AND N. WEAVER
T. mexicana on both tests, and it excited T. pectoralis. Several secondary and tertiary alcohols caused attack or strong excitement on one occasion and will have to be studied further. Among normal primary alcohols with from 8 to 16 carbons, only 1-decanol elicited mild attack from T. pectoralis on one occasion. Heptadecane almost always caused mild attack. The bees usually investigated hexadecane and octadecane; while the other members of the series were without visible effect. DISCUSSION
From the examination of mass spectral and gas chromatographic data of the cephalic compounds of T. mexicana and T. pectoralis, conclusions may be drawn as to the identities of many of these unknowns. Because of equivalent gas chromatographic and mass spectral characteristics, the following structures are assigned to the unknowns: A, and B, are 2-heptanol; A, is 2-heptanone; A, and B, are benzaldehyde; A, and B, are 2-nonanol; A, is 2-nonanone; B, is 2-undecanone; As and B, are 2-tridecanone; Ag is 2-pentadecanol; and A,, is 2-pentadecanone. The identifications of A,, A,, As, and A,, are further substantiated by gas chromatographic data in which the co-injection of 2-heptanone, 2-nonanone, and 2-tridecanone, and 2-pentadecanone with sample A resulted in enhancement of the peaks of the sample compounds with no loss of peak symmetry. Unequivocal identification of compounds A,, A, Arr, Ala, B,, B,, B,, BIO, B12, and B,, is not possible from our data. B, and 2-heptanone have identical relative retention times, but no mass spectrum was obtained for B, because no peak was seen in the gas chromatogram of the GCMS analysis. During simple GC analysis of this mixture, the resolution of the column was better and a distinct B, peak was observed that could be quantitated. From the m/e 58 fragment ion in the mass spectra of B,, A,, A12, and Ars, it is apparent that these compounds are 2-alkanones. As noted in the Results, the primary problem in the identification of these substances is contamination of the ketones with the alcohols. The mass spectra of A,, Br,,, and A,, have enhanced ions at m/e 43 and 45 and as shown in the spectrum of 2-heptanol (Fig. 6), the fragment ions at m/e 43 and 45 are intense in 2-alkanols. Therefore, it is probable that A, is 2-undecanone, B,, is 2-pentadecanone, and A,, The identification of A, as 2-undecanone is further subis 2-heptadecanone. stantiated by gas chromatographic data in which the co-injection of 2-undecanone with sample A resulted in enhancement of peak A, with no loss of symmetry. Due to the fact that synthetic 2-heptadecanone was not available for analysis, the identification of A,, and B,, is based on the similarity of their spectra to synthetic An unequivocal identification of B, is not possible because of 2-pentadecanone. the unexplainable enhancement of the ion at m/e 58. The prominent ion at m/e 4.5 in the mass spectra of B,, Arr, and B,, indicates that these compounds are 2-alkanols. It has been pointed out in the Results that compound B, and 2-undecanol have many common mass spectral fragmentation characteristics, but that the spectra of B, and 2-undecanol were not identical. Additional fragment ions at m/e 50, 97, 105, and 152 suggest that the compound
VOLATILE
CEPHALIC
SUBSTANCES
OF THE STINGLESS
BEES
1125
contains impurities or has a carbon skeleton different from 2-undecanol. From the gas chromatographic retention times (Table 4), we may conclude that B, is not a branched chain compound since its retention time is not shorter than that of standard 2-undecanone. It may be concluded that the compound is contaminated and that the additional fragment ions in the mass spectrum of B, are due to a second compound which co-eluted from the gas chromatographic column with B,. Since 2-heptadecanol was not available for analysis, the identification of compounds A,, and B,, must be equivocal. An inspection of the mass spectra of compounds A,, and B,, showed that the mass spectrum of A,, is very similar to that of 2-pentadecanol. It is therefore probable that compound A,, is 2-heptadecanol. The mass spectrum of B12, however, has additional ions at m/e 43 and 55. Since we do not have synthetic 2-heptadecanol for comparison, it is difficult to determine whether this compound has a different carbon skeleton or whether it is contaminated. On the basis of prior experiences with this mixture, it is probable that B,, is also contaminated. Positive identification of A,, B,, and B,, is not possible from the data available. From an intense search of the literature, it appears that A,, B,, and B,, have not been identified previously. It is therefore unlikely that the structures of these new compounds may be determined by mass spectrometry. Additional instrumental and chemical analyses will be necessary for their identification. T. mexicana and T. pectoralis are classified in the subgenus Scaptotrigona on the basis of their anatomical characteristics. The similarity of the volatile substances from their heads is further evidence of the close kinship of these two species. The major difference which we found is in the quantitative composition of the chemical mixtures and particularly in 2-heptanone and 2-tridecanone. We have examined the volatile chemicals and studied the responses to a wide variety of chemicals of three species of Trigona in other subgenera and have found them quite different from these two species. Benzaldehyde or the individual ketones usually did not cause attack by the bees, but a mixture of these substances caused strong attack. It could well be to the advantage of the colony to react strongly to mixtures that closely resemble the mixture secreted by an alarmed bee of the same species. Otherwise the colony might be subject to frequent false alarms. Ketones are fairly common alarm substances of a number of ant species (LAW and REGNIER, 1971) and ants are common near the nests of these bees. For instance, a small ant was often found nesting near the colonies of T. pectoralis; when 2-heptanone was tested on the bees the nearby ants of this species exhibited typical alarm behaviour ; when the ants were crushed, they gave off a strong odour of 2-heptanone. Although our technique of assay was suitable to demonstrate alarm reactions under conditions that often prevail when the colony is endangered, it was not sufficiently refined to study threshold concentrations, the precise ranking of activity of different compounds, nor the precise synergistic effect of mixtures of compounds. Such studies are planned for the future if a suitable adaptation of the technique of WILSON et al. (1969) can be developed. The presentation of chemicals
1126
J. M. LUBY, F. E. REGNIER, E. T.
CLARKE, E. C. WEAVER, ANDN. WEAVER
in the open air where there was often considerable wind was particularly unsuitable for studying the alarm pheromone activity of the higher molecular weight compounds. Since 2-tridecanone, 2-pentadecanone, and 2-pentadecanol were solids at the temperature in the field, it was surprising when they elicited an attack response under the conditions of our tests. They might, however, have strong alarm activity in more confined quarters such as when deposited on an intruder that has penetrated the nest. It is also possible that compounds of low volatility might exert their influence on a blend of volatile compounds by modifying the vapour pressure of individual components and thus changing the composition of the blend in the vapour phase. It is quite possible, of course, that the less volatile substances have some role independent of alarm. The higher molecular weight compounds might not normally reach a vapour concentration high enough to cause alarm but could still act to mark a food source or a possible new next site. Indeed, BLUM et al. (1970) and BLUM (1971) claim that high concentrations of neral geranial, and 2-alkanones act as alarm pheromones, but that low concentrations act as trail markers to recruit other bees to a source of food. This is an attractive idea, but these authors present no data to support their claim. It is also possible that the same substances act as alarm pheromones and as defence chemicals. Benzaldehyde is known to be a space repellent for the honeybee (TOWNSEND,1963), and it might serve a similar role against some of the species that attack the nests of stingless bees. CONCLUSIONS From the reaction gas chromatographic analyses, mass spectral data, and retention indices it may be concluded that both T. mexicana and T. pectoralis produced homologous series of normal aliphatic alcohols and ketones with functional groups at the 2-position of the carbon skeleton. The compounds all contain odd numbers of carbon atoms ranging from C, to C,,. In addition, both species produce benzaldehyde and several unidentified compounds. The primary difference between the volatile mixtures produced by these two species is in the quantitative composition of the blends. Both the alcohols and ketones are alarm pheromones. The alcohols are more active in inducing attack by the bees than are the ketones, but a mixture of the ketones and benzaldehyde was very active. Acknowledgements-This work was supported in part by National Science Foundation Grant No. G&30179X. The authors wish to thank Dr. A. WILLE of the University of Costa Rica for identifying the bees. This is Journal Paper No. 4903 of the Purdue Agricultural Experiment Station. REFERENCES
BLUMM. S. (1966) Chemical releasers of social behaviour-VIII. gland secretion of Lestrimelitta Sot. Am. 59, 962-964.
limao (Hymenoptera:
Apidae:
Citral in the mandibular Melitidae). Ann. mt.
VOLATILECEPHALICSUBSTANCES OF THE STINGLESS BEES
1127
BLUM M. S. (1971) Dimensions of chemical sociality. Chemical Releasers in Insects 3, 147162. BLUM M. S., CREWER. M., KEER W. E., KEITH L. H., GARRISON A. W., and WALKERM. M. (1970) Citral in stingless bees: isolation and functions in trail-laying and robbing. J. Insect Physiol. 16, 1637-1648. BUDZIKIEWICZ H., DJERA~SIC., and WILLIAMS D. H. (1968) Interpretation of Mass Spectra of Organic Compounds, p. 38. Holden-Day, San Francisco. BUDZIKIEWICZH., PENSELAWC., and DJERASSIC. (1966) Mass spectrometry in structural and stereochemical problems. Tetrahedron 22, 1391-1398. DAUBENW. G., HANCEP. D., and HAYES W. K. (1955) Acid catalyzed transformations of X-santonin. r. Am. them. Sot. 77,4609-4612. JAMESA. T. (1959) Determination of the degree of unsaturation of long chain fatty acids by gas-liquid chromatography. r. Chromatog. 2, 552-561. KERR W. E., FERREIRAA., and SIMOESDE MATTOSN. (1963) Communication among stingless bees-additional data (Hymenoptera: Apidae). r. N. Y. ent. Sot. 71, 80-90. LAW J. H. and RECNIER F. E. (1971) Pheromones. A. Rew. Biochem. 40, 533-548. LINDAUERM. and KERR W. E. (1960) Communication between the workers of stingless bees. Bee World 41, 29-41, 65-71. MCCOLLUMJ. D. and MEYERSONS. (1963) Organic ions in the gas phase-X. Decomposition of benzaldehyde under electron impact. J. Am. them. Sot. 85, 1739-1741. REGNIER F. E. and HUANG J. (1970) The determination of oxygen containing functional groups by reaction gas chromatography. J. Chrom. Sci. 8, 267-271. REGNIERF. E., NIEH M., and HOLLDOBLERB. (1972) Dufours gland components of Pogonomyrmex badins and Pogonomyrmex rugosus. J. Insect Physiol. In press. SCHWA= F. (1948) Stingless bees (Meliponidae) of the western hemisphere. Bull. Am. Mus. nut. Hist. 90. TOWNSENDF. (1963) Benzaldehyde: a new repellent for driving bees. Bee World 44, 146149. WILSON E. 0. (1971) The Insect Societies. p. 272. Cambridge, Mass.: Harvard University Press. WILSON E. O., BOSSERTW. H., and REGNIERF. E. (1969) A general method of estimating threshold concentrations of odorent molecules. J. Insect Physiol. 15, 597-610.
36
1973,Vol.
19, pp.
1111to 1127. Pergamon
Press. Printed in Great Britain
VOLATILE CEPHALIC SUBSTANCES OF THE STINGLESS BEES, TRIGONA MEXICANA AND TRIGONA PECTORALIS JULIA M. LUBY,l FRED E. REGNIER,l ERIC T. CLARKE,2 ELIZABETH C. WEAVER,3 and NEVIN WEAVER3 l Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907; 2 Southwestern University, Georgetown, Texas 78626; and 3 Department of Biology, University of Massachusetts at Boston, Boston, Massachusetts 02116. (Received 26 October 1972)
Abstract-Extracts of the heads of the stingless bees, Trigona mexicana and T. pectoralis, contain mixtures of compounds that are identifiable by gas chromatography and mass spectrometry. These compounds form homologous series of aliphatic alcohols and ketones with an odd number of carbon atoms and functional groups at the 2-position. The alcohols and the ketones range from 7 to 17 carbon atoms. Benzaldehyde and a nitrogen containing compound are also present in the mixtures. The series of compounds from the two species are nearly identical qualitatively. They differ in the absence of 2-undecanol and 2-pentadecanol from the extracts of T. mexicana and T. pectoralis, respectively. The highest concentration of material is found in the 7-carbon fraction in T. mexicana and in the 13 to 15 carbon range in T. pectoralis. There is a major difference in the relative concentration of 2-heptanol and 2-heptanone in the two species with the concentration of the alcohol being one-fourth that of 2-heptanone in T. mexicana and ten times greater than the ketones in T. pectoralis. Both the alcohols and ketones are alarm pheromones. The alcohols are more active in inducing attack by the bees than are the ketones, but a mixture of the ketones and benzaldehyde was very active.
INTRODUCTION
SOCIALinsects live in colonies which, during much of the year, may contain food reserves and individuaIs of al1 castes in one or more stages of development. The colony must be protected from a wide variety of enemies (SCHWARZ,1948) and a system for quickly bringing a large number of adults to the defence of the colony may be necessary for colony survival. Many social insects employ alarm pheromones to recruit defenders (LAW and REGNIER, 1971; WILSON, 1971). The defence systems of the stingless bees (Tribe Meliponini) have not been extensively studied. LINDAUERand KERR (1960) and KERR et al. (1963) reported that several species of Meliponini have recruiting pheromones and described behaviour that must have resulted from alarm pheromones. Recently BLUM (1966) and BLUM et al. (1970) reported that geranial and neral occur in the mandibular glands of both Lestrimelitta limao, Fr. Smith and Trigona subterranea Friese, and that in the latter species it is both a recruiting pheromone and an alarm pheromone. In a recent 1111
1112
J. M. LUBY, F. E. RIXGNIER,E. T. CLARKE,E. C. WEAVER,ANDN. WEAVER
review of the chemical basis for insect sociality, BLUM (1971) refers to an unpublished study on T. postica, T. xanthotricha, and T. tubiba. The mandibular gland of T. postica reportedly contained benzaldehyde, undecane, tridecane, and odd carbon number 2-alkanones ranging from 7 to 25 carbon atoms while those of T. xanthotricha contain benzaldehyde, aliphatic hydrocarbons, and 2-heptanone. The mandibular glands of T. tubiba secrete benzaldehyde, undecane, tridecane, 2-heptanone, and 2-nonanone. Benzaldehyde was highly attractive to workers of T. tubiba and did not elicit aggressive behaviour as did 2-heptanone and 2-nonanone. The potency of benzaldehyde as an attractant to T. post& workers was reported to be enhanced by the presence of 2-undecanone and 2-tridecanone. Unfortunately, the experimental details of these studies are still not available. Other than the data cited above, little is known of the volatile cephalic material used by the many Trigona species in the tropics for chemical communication. This study of two species of stingless bees from Mexico, T. mexicana and T. pectoralis, was undertaken to attempt to clarify the chemical nature and significance of these volatile compounds in a natural communication system.
MATERIALS
AND METHODS
Biological material Four colonies of Trigona (Scaptotrigona) mexicana Guerin (Hymenoptera : Apidae: Meliponini) were found nesting in the archaeological ruins of El Tajin near Papa&a, Veracruz, Mexico; three of the nest entrances led through cracks in perpendicular walls to the interior of the mounds; the other nest was at least partially exposed deep in a niche that was an architectural feature of the building, Several colonies of T. (Scuptotrigona) pectoralis Dalla Torre were found in the hollows of trees in Yaxcaba, Yucatan, Mexico. Bees were collected by placing a net bag over the entrance to the nest; they were anaesthetized with chloroform and decapitated or alternately, the mandibular glands were removed. The heads of T. mexicana were extracted with chloroform ; heads and mandibular glands of T. pectoralis were extracted with hexane. The extracts were reduced to a volume of 0.25 ml under a stream of nitrogen and transferred to Kontes microflex vessels. The extract from T. mexicana was designated as ‘sample A’, and that from the heads of T. pectoralis as ‘sample B’. Reagents. Benzaldehyde and the 2-alkanones were obtained from Aldrich Wisconsin, while the sodium borohydride Chemical Company, Inc., Milwaukee, was supplied by Ventron of Beverly, Mass., and Apiezon-L by Anspec Company, Ann Arbor, Michigan. Diethylether, chloroform, and hexane were obtained from the J. T. Baker Chemical Co., Phillipsburg, N.J. P and 3% Dexsil 300 on 100/120 Gas chromatography packings. Gas-Chrom mesh Gas-Chrom Q were supplied by Applied Science Laboratories, Inc., State College, Pa. Apparatus. Gas chromatographic analyses were carried out on a Varian Aerograph Model 1200 instrument fitted with a hydrogen flame detector and
VOLATILE
CEPHALIC
SUBSTANCES
OF THE
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1113
6 ft x & in. stainless steel column packed with 3% Dexsil 300 on lOO/lZO mesh Gas-Chrom Q. The stainless steel injection port inserts supplied with the instruInjection port and detector temperament were used as the precolumn reactors. tures were 150 and 277°C respectively, while the column oven was programmed from 68 to 250°C at 4”C/ min. A 100 ml/min flow of nitrogen through the column was used. Combination gas chromatographic-mass spectral analyses. Combination gas analyses were performed on an LKBchromatographic-mass spectral (GC/MS) 9000 instrument fitted with an 8 ft x & in. stainless steel column packed with 3% conditions Dexsil 300 on 100/120 mesh Gas-Chrom Q. The chromatographic described previously were used in these analyses. The mass spectrometer was operated at an electron energy of 70 eV, with an accelerator voltage of 3.5 kV and ion source temperature of 250°C. Reaction gas chromatography. Reaction gas chromatographic (RGC) supports were prepared as described previously (REGNIER and HUANG, 1970). were prepared by sodium boroPreparation of 2-alkanols. The 2-alkanols hydride reduction of the corresponding 2-alkanones (DAUBEN, 1955). of components in Chromatographic quantitation. The absolute concentration the bee extracts and the relative reactivity of these components in flow reactors was determined using n-pentadecane as an internal standard. Relative peak areas were calculated by weighing. Field experiments Whole bees or parts of bees were mashed between the fingers and presented near the entrance of the nest, and the behaviour of the bees was compared to the behaviour when a clean hand was presented. Known chemicals were tested by dipping wooden rods (toothpicks) into vials of chemicals and presenting them about 1 to 2 cm in front of, and just below the level of the lower edge of the entrance tube. A subjective scale was used to judge the severity of attacks on the rod and the investigator. Some of the chemicals were diluted 1 : 9 (v/v) with diethyl ether and tested on small glass rods. In conducting the biological assays the experimenter did not know the identity of the chemical he was testing, and he handled nothing except the clean ends of rods.
RESULTS Analytical gas chromatography on a 3% Dexsil 300 column resolves the volatile components from the heads of the stingless bees, T. mexicana (sample A) and T. pectoralis (sample B) as shown in the chromatograms (Figs. 1, 2). The symbols used to identify the peaks in these chromatograms will be used later when referring to the mass spectra of these compounds. Slashes on the chromatograms indicate points at which mass spectra were obtained. The concentrations of components in these samples are listed in Table 1. Relative retention times are related to an internal standard, n-pentadecane, shown in the chromatograms.
1114 J. M. LUBY, F. E. REGNIER,E. T. CLARKE,E. C. WEAVER,AND N. WEAVER
SSUOdSaJ
aSUOdSaJ
Ja,lJOC’a~
JSpJODi%i
VOLATILE CEPHALIC SUBSTANCES OF THE STINGLESS BEES TABLE
~-THE
1115
CONCENTRATION OF VOLATILECEPHALICSUBSTANCES IN THE HEADSOF T. mexicana AND T. pectoralis T. pectoralis
T. mexicana Peak* number
Relative+ concentration
Peaks number
Absolute concentration per worker kg)
A, A* A, A4f A, A, A,
23 100 40 6 10 2
B, B, B, B* B, B, B,
4.4 0.4 5.8 1.8 0.9 0.4 -
A, A, A,, A,, A,,
14 12 3
B, B, B 10 B 11 B 12
2.2 44 9.7 6.2 -
B 13
4
* Peak number refers to peak indicated on the gas chromatograph in Fig. 1. t_ Relative concentration is expressed relative to the most abundant component. 1 Position of compound is indicated by a dash on the chromatogram in Fig. 1. 4 Peak number refers to peak indicated on the gas chromatogram in Fig. 2.
Reaction gas chromatography
Further analysis by reaction gas chromatography yielded information on functional group composition of components. By this method, compounds containing a specific functional group (such as a carbonyl or hydroxyl group) may be subtracted from the carrier gas as the mixture passes through a flow reactor packed with a specific reagent (REGNIER and HUANG, 1970). Reactive species are converted into non-volatile derivatives and are identified by their absence in the chromatogram. Flow reactors containing O-5 and 5% boric acid coates supports were used to identify alcohols while sodium borohydride (NaBH,) reactors were used to identify aldehydes and ketones. The boric acid flow reactor was operated at both 123 and 150°C. When the flow reactor was maintained at 123”C, compound B, was completely subtracted while at 150°C its subtraction was incomplete. It is probable that at 150°C the low molecular weight compounds are almost exclusively in the vapour phase and the residence time in the flow reactor is not sufficient for derivatization. By lowering the reactor temperature highly volatile compounds are in contact with the boric acid for a sufficient time to be subtracted. Compounds A,, B,, and B, showed 100 per cent removal by the boric acid reactor and little reactivity toward sodium borohydride. This behaviour suggests that these compounds are alcohols (REGNIER and HUANG, 1970).
1116
J. M. LUBY,F. E. REGNIER, E. T. CLARKE, E. C.
WEAVER, AND
N. WEAVER
Compounds As and B, exhibited high reactivities toward sodium borohydride (92 and 59 per cent removal respectively) and are probably aldehydes (REGNIERand HUANG, 1970). Other compounds, such as A,, A,, A,, A,,. A1s, B,, B,, B,, B,, B rO, and Bra, which show low reactivity (1040 per cent subtraction) with NaBH, are suspected of being ketones. Mass spectrometry. Mixtures A and B from the heads of T. mexicana and T. pectoralis, respectively, were analysed by gas chromatography-mass spectrometry to determine molecular size and the type and position of functional groups present in the molecules. Discussion of mass spectra will be limited to those fragment ions which are characteristic of functional groups and to those which help in locating functional groups in a molecule. In the presentation of mass spectral data, components of the two samples will be grouped on the basis of probable functional group, apparent molecular weight, and relative retention time. Ketones will be discussed first in the order of increasing molecular weight. Examination of the spectra of A,, A,, A,, B,, A,, B,, Arc,, Bra, Ars, and B,, indicated that all of these compounds produce fragment ions at m/e 43, 58, M-43, M-18, M-15, and M where the symbol M indicates the molecular ion. The molecular weights of these compounds compose a family 28 atomic mass units (AMU) apart ranging from 142 to 254. The fragment ion at m/e 58 is unusual in that it represents a rearranged product. It has been demonstrated in many cases that the m/e 58 fragment ion is characteristic of 2-alkanones (BUDZIKIEWIEZet al., 1966). The major fragment ions in these compounds are produced by two fragmentation mechanisms, one cleaving the a bond on either side of the carbonyl group and the second cleaving the bond in the position /3 to the carbonyl group. Additional fragment ions result from the loss of water (M-18) and a methyl group (M-15) from the parent ion. The spectra indicate that these substances are saturated 2-alkanones with an odd number of carbon atoms ranging from 7 to 17. Comparison of the characteristic fragment ions from the ketones in samples A and B and standard ketones can be seen in Table 2. The spectral data for compounds A,, As, B,, Alo, and B,, closely match that of standard compounds. Compounds A,, A,, Blo, and A,, show fragment ion intensities that are either higher at m/e 43 or lower at m/e 58 than the corresponding standards. We attribute this to contamination of these components with other compounds. Since the Dexsil 300 column used in these analyses is not capable of complete resolution of a 2-alkanone and the corresponding 2-alkanol, contamination of the ketones with alcohols was a problem. The mass spectra of compounds A,, A,, Blo, and A,, all had enhanced ions at m/e 43 and 45. As can be seen in the spectrum of 2-heptanol (Fig. 6), the fragment ions at m/e 43 and 45 are intense in 2-alkanols. If an enhanced m/e 43 peak is the base peak in the spectrum, it will cause all other peaks including m/e 58 to be of lower intensity than standards. Compounds A, and B, were suspected of being aldehydes on the basis of their reactivity with NaBH, in the reaction gas chromatographic analyses cited above. Fragment ions at m/e 77 and 51 in these compounds strongly suggest the presence of an aromatic ring as is seen in the spectrum of B, (Fig. 3). By the addition of
VOLATILECEPHALICSUBSTANCES OF THE STINGLESS BEES TABLE 2-A
III7
TABULATION OF THE CHARACTERISTIC IONSIN THE VOLATILEKETONESPECTRA OF T. mexicana, T. pectoralis, ANDSOMESTANDARD 2-ALKANONES Relative intensity of various fragment ions
Compound
43
58
M-43
M-18
M-IS
A, 2-Heptanone A, 2-Nonanone A, 2-Undecanone A* Bg 2-Tridecanone Ai, B Z%entadecanone Ai, B 13
100 100 100 100 100 100 83 93 92 81 100 90 100 97
49 56 87 88 85 100 100 100 100 100 87 100 80 100
12 14 2 2 2 2 2 2 2 I 2 2 1 I
2 1 I I I I 2 1 1 1 2 2 I I
2 3 3 3 2 3 3 4 3 2 2 3 2 2
M* 5 5 7 8 4 6 7 9 8 7 6 7 4 4
(114) (114) (142) (142) (170) (170) (198) (198) (198) (226) (226) (226) (254) (254)
* The mass of the molecular ion is indicated in parentheses.
106
m/e FIG. 3. Mass spectrum of compound Bs. Instrumentation, sample introduction, and operating conditions are described in the experimental section.
1118
J. M. LUBY,F. E. REGNIER, E. T. CLARKE, E. C. WEAVER,ANDN. WEAVER
29 AMU to this aromatic ring, one obtains the molecular ion (m/e 106). Several fragment ions could exhibit this characteristic such as [-CH,CH,]+ or [-CHO]+. The presence of an intense fragment ion at M - 1 (m/e 105) strongly favours the aldehyde (MCCOLLUMand MEYERSON,1963). A comparison of the mass spectra of compounds A,, B, (Fig. 3), and benzaldehyde (Fig. 4) showed that these compounds were identical. 06(M)
’k 60T) a5 P e
40-
E 0 z 20
-
‘**
%
100
50
I, 1.
m/e
FIG. 4. Mass spectrum of benzaldehyde. Sample introduction was through the gas chromatograph.
Other conditions were the same as those in Fig. 3.
All of the compounds tentatively identified as alcohols by reaction gas chromatography (A,, B,, A,, and B,) have intense fragment ions at m/e 45. This fragment ion suggests that these alcohols are 2-alkanols (BUDZIKIEWICZ et al., 1968) and thus biosynthetically related to the 2-alkanones present in these samples. It appears from the spectrum of A, (Fig. 5) that the molecular weight is 115. This could only be true if the molecule contained nitrogen. Since no nitrogen containing fragment ions are observed in the spectrum, it may be concluded that this compound does not contain nitrogen and that m/e 115 is not the molecular ion. The molecular weight must be greater than 115. Previous studies have shown that 2-alkanols characteristically lose a proton to give fragment ions at M-l. On this basis, the molecular weight of A, would be 116 and fragment ions at m/e 101 and 98 are the result of the loss of a methyl group (M-15) an d water (M-18) from the parent ion. Comparison of the spectra of A, and 2-heptanol (Fig. 6) shows that they are identical. In addition to A,, B,, A,, and B,, the intense fragment ion at m/e 45 was found in the spectra of B,, A,, AlI, and B,, whose concentration was too low for RGC
VOLATILE
CEPHALIC
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OF THE STlNGLESS
1119
BEES
5
FIG. 5. Mass spectrum of compound A,. Information on instrumentation analytical procedures are available in the experimental section.
and
100
I-
-I m/e
FIG. 6. Mass spectrum of 2-heptanol. Instrumentation, operating conditions, and sample introduction are the same as those used in Fig. 4.
1120
J. M. LUBY, F. E. REGNIER,E. T. CLARKE,E. C. WEAVER,ANDN. WEAVER
characterization. A comparison of the characteristic ions in the spectra of all unknown alcohols and standard Z-alkanols can be seen in Table 3. TABLE 3-A TABULATION OF THE CHARACTERISTIC IONSIN THE VOLATILEALCOHOL SPECTRAOF T. ?TWXiCana, T. peCtO?diS, ANDSOMESTANDARD 2-ALKANOLS Relative intensity of various fragment ions Compound
Al Bl
2-Heptanol A4 R, 2-Nonanol R, 2-Undecanol A, 2-Pentadecanol A,1
45
M-18
M-15
100 100 100 100 100 100 100 100 100 100 100
10 9 9 5 8 5 9 7 9 7 3
8 7 8 3 5 3 3
3 3 1 1
M-l 1 1 1 1 1 1 1 1 1 1 1
M* 0 0 0 0 0 0 0
0 0 0 0
(116) (116) (116) (144) (144) (144)
(172) (172) (228) (228) (256)
* The mass of the molecular ion is indicated in parentheses.
Both compounds A, and B, have an apparent molecular ion at m/e 168. The 18 AMU difference between this ion and an m/e 150 ion in the mass spectrum suggest that these molecules contain oxygen. Additional even mass ions at m/e 110, 68, and 54 further suggest that these molecules also contain nitrogen. Compound A,, could not be identified from the mass spectrum. It appears that the eventual identification of these compounds will necessitate additional chemical analyses. Although the mass spectra of all the alcohols, aldehydes, and ketones of samples A and B were virtually identical to the various standard compounds, it is conceivable that compounds with slight differences in carbon skeleton could give similar mass spectra. Gas chromatographic retention times. Final identification of the components in these mixtures was achieved by comparing their gas chromatographic retention times with those of the standards (Table 4). Retention times for 2-heptadecanol and 2-heptadecanone were determined from a plot of relative retention times v. carbon number for other members of homologous series (JAMES, 1959). Since the retention times of branched chain compounds are decreased on Dexsil300 columns (REGNIER et al., 1972), non-identity of carbon skeletons would be easily discernible. Field experiments
Mashed whole bees or heads elicited attacks from both species of bees. T. also made less severe attacks on the hand that had mashed the thorax and abdomen of a bee; since the bees that were decapitated were always rather severely
pectoralis
VOLATILE
CEPHALIC
SUBSTANCES
OF THE STINGLESS
alarmed when they were captured, the legs and may have become contaminated with secretion colony was already alarmed, the bees circling piece of wood two or more metres from the nest TABLE
~-RELATIVE
RETENTION
Compound
Al Bl
2-Heptanol
A, R, 2-Heptanone -4, B, Benzaldehyde A, R, 2-Nonanol ALI B, 2-Nonanone‘ B, 2-Undecanol
TIMES
Tr* 0.23 0.23 0.25 0.26 0.26 0.26 0.34 0.35 0.36 0.51 0.49 0.50 0.53 0.52 0.53 0.78 0.77
OF
Trigona
1121
BBF.8
possibly other parts of the body from the mandibular gland. If a the investigator would attack a if a mashed bee was rubbed on it.
CEPHALIC
SUBSTANCES
Compound
A, B*
2-Undecanone
A* B, 2-Tridecanone 2-Pentadecanol AI0 B 2-‘ientadecanone A,, B 2-&eptadecanol AI, B 2%eptadecanone
AND
STANDARDS
T,
041 0.80 0.80 I.06 1.07 1.05 1.25 I.28 1.29 1.27 1.49 1.46 1.521_ 1.51 1 a49 1.56t
* The symbol T, designates relative retention time. t Relative retention times for 2-heptadecanol and 2-heptadecanone were determined from a plot of relative retention times vs. carbon number for other members of homologous slices.
Table 5 shows the visible reactions of T. mexicana and T. pectoralis to authentic samples of some of the compounds that have been identified from their heads. In order to make tests as comparable as possible, all of the tests in a series (one column in the table) were completed within half a day using a single colony. In the case of the purified compounds, a series included compounds that were offered both neat and diluted. Increasing numbers of + ‘s in the table indicate increasing severity of attacks on an odour source and on the experimenter. Five ‘f’s are used to indicate an attack of maximum severity. Other types of behaviour recorded in Table 5 were probably mild alarm reactions that did not result in attack. With the behaviour indicated as ‘investigate’, the bees typically clung with the hind legs to the inside of the entrance tube at a point near the source of the chemical and stretched toward it. The ‘roar’ was caused by the beating of wings inside the nest, and in T. mexicana and some other species studied, it might either precede an attack or occur when there was no attack. It is probable that the nests of T. pectoralis were so deep inside the tree that the noise from within was not often
*2
+ 0 +
*2
+ + + + *3 + 0 +4
*2 Roar; *3 Excite;
0 +
*1
++
++
+++ +I +++ + ? + 0 + *1
** Repeal.
o’? + O? + *1
+ *1
*1
*2
0 0 0 0 0 0 0 0 *1
*1
0
Neat
0
Neat
Neat
+ + + *3 0 *3 -
++
*l
Neat
SUBSTANCES
+ 0 0 0 0 0 -t-? -
*1
0
0
Neat
TESTED)
Dilute
Neat
4'S UP TO A MAXIMUM
CEPHALIC
AT THE
0 0 *2 0 0 0 0 0 0 -
-
0 ++ + 0 0 + 0 + 0 -
+
Dilute
0 0 -
-
0 +
-
ENTRANCE
O? ++ + + +? + 0 + 0 -
+++
Neat
*1
-
-
:? 0 O? + + + *1 *3
Neat
*1
0
0 ++ + + 0
+++
+t **
Dilute
NEST
-
-t-
+ 0 + + 0 -I-t0 +
Dilute -
TO THE
OF 5 +'S;O:NODISCERNABLEREPONSE;
PRESENTED
Purified chemicals
NOT
OF
Commercial chemicals
-:
NUMBERS
TO VOLATILE
T. pectoralis
Neat
*3
T. pectoralis BY INCREASING
T. mexicana
++++ ++ *3
0
AND
INDICATED
T. mexicana
+ *2
Neat
*i Investigate;
2-Heptanol 2-Heptanone 2-Nonanol 2-Nonanone 2-Undecanol 2-Undecanone 2-Tridecanol 2-Tridecanone 2-Pentadecanol 2-Pentadecanone Benzaldehyde
Chemical
OF
SEVERE ATTACKS
~-F&ACTIONS
(INCREASINGLY
TABLE
VOLATILE CEPHALIC SUBSTANCES OF THE STINGLESS BEES
1123
audible above the normal environmental noises. The behaviour designated ‘excite’ consisted of various types of rather quick and nervous-appearing movements. When the bees were ‘repelled’ the guards that normally lined the mouth of the . entrance tube retreated far back into it, sometimes out of sight. As can be seen in Table 5, both species were more likely to attack in response to 2-alkanols than 2-alkanones with the same carbon skeleton. Within the alcohol series, they attacked most readily in response to the lower molecular weight compounds. 2-Heptanol occurred in fairly high concentration in both species, and significant amounts of 2-nonanol occurred in T. pectoralis, but otherwise there was no apparent relationship between the concentration of the chemicals in the bees and the reactions of the bees to individual chemicals. In both species, for instance, the ketones occur in much higher concentrations than do the alcohols, but the alcohols were more excitatory. Benzaldehyde also occurs in significant concentrations in both species, and although the bees always responded to it in some way, it caused a very mild attack on only one occasion. When a mixture of 10 mg of 2-heptanone, 1 mg each of 2-nonanone, 2-undecanone, and 2-tridecanone, and 5 mg of benzaldehyde in 1 ml of hexane was presented on a wooden rod at the entrance of 3 colonies of T. peetorah, an immediate and vigorous attack occurred every time. It will be noted that the results with the diluted and undiluted compounds were about the same. This is probably because the techniques used in presenting compounds to the bees did not result in large concentration differences in the air at the nest. The bees also appeared to react in about the same way to the commercial grade and the purified compounds, though the tests on the two grades were not conducted on the same day and therefore were not strictly comparable. The bees varied from day to day in their reactions to test compounds, and on some days the attacks were so severe that it was impossible to complete a meaningful series of tests. After the bees attacked they were left alone for several minutes and were not tested again until they did not react to a blank. When the bees were especially fractious, hexane or nonane was used as a control. Both colonies of T. pectoralis used to assay chemicals, and some other colonies of that species which were used for occasional tests, varied greatly in their readiness to attack on different days. Sometimes the extreme fractiousness of a colony persisted for several days. For instance, while trying to complete a series of tests on one of the colonies it was necessary to leave the area of the nest after each test. When the experimenter returned after 1 or 2 hr, the bees often attacked him. At the end of 3 days only 7 compounds had been tested. In these tests both Z-tridecanol and Z-pentadecanol led to fairly strong attack reactions (+ + + and + +, respectively) following a negative nonane control. Unfortunately there was no opportunity to test the ketones during this series. Most of the compounds that have been identified as alarm pheromones in insects (LAW and REGNIER, 1971) were tested on these bees, and most of the chemicals did not cause any response. However, 2-octanol was only slightly less active than 2-heptanol for both species. 2-Methyl-2 heptanol caused attacks by 35
1124
J. M. LUBY, F. E. REGNIER, E. T. CLARKE,E. C. WEAVER,AND N. WEAVER
T. mexicana on both tests, and it excited T. pectoralis. Several secondary and tertiary alcohols caused attack or strong excitement on one occasion and will have to be studied further. Among normal primary alcohols with from 8 to 16 carbons, only 1-decanol elicited mild attack from T. pectoralis on one occasion. Heptadecane almost always caused mild attack. The bees usually investigated hexadecane and octadecane; while the other members of the series were without visible effect. DISCUSSION
From the examination of mass spectral and gas chromatographic data of the cephalic compounds of T. mexicana and T. pectoralis, conclusions may be drawn as to the identities of many of these unknowns. Because of equivalent gas chromatographic and mass spectral characteristics, the following structures are assigned to the unknowns: A, and B, are 2-heptanol; A, is 2-heptanone; A, and B, are benzaldehyde; A, and B, are 2-nonanol; A, is 2-nonanone; B, is 2-undecanone; As and B, are 2-tridecanone; Ag is 2-pentadecanol; and A,, is 2-pentadecanone. The identifications of A,, A,, As, and A,, are further substantiated by gas chromatographic data in which the co-injection of 2-heptanone, 2-nonanone, and 2-tridecanone, and 2-pentadecanone with sample A resulted in enhancement of the peaks of the sample compounds with no loss of peak symmetry. Unequivocal identification of compounds A,, A, Arr, Ala, B,, B,, B,, BIO, B12, and B,, is not possible from our data. B, and 2-heptanone have identical relative retention times, but no mass spectrum was obtained for B, because no peak was seen in the gas chromatogram of the GCMS analysis. During simple GC analysis of this mixture, the resolution of the column was better and a distinct B, peak was observed that could be quantitated. From the m/e 58 fragment ion in the mass spectra of B,, A,, A12, and Ars, it is apparent that these compounds are 2-alkanones. As noted in the Results, the primary problem in the identification of these substances is contamination of the ketones with the alcohols. The mass spectra of A,, Br,,, and A,, have enhanced ions at m/e 43 and 45 and as shown in the spectrum of 2-heptanol (Fig. 6), the fragment ions at m/e 43 and 45 are intense in 2-alkanols. Therefore, it is probable that A, is 2-undecanone, B,, is 2-pentadecanone, and A,, The identification of A, as 2-undecanone is further subis 2-heptadecanone. stantiated by gas chromatographic data in which the co-injection of 2-undecanone with sample A resulted in enhancement of peak A, with no loss of symmetry. Due to the fact that synthetic 2-heptadecanone was not available for analysis, the identification of A,, and B,, is based on the similarity of their spectra to synthetic An unequivocal identification of B, is not possible because of 2-pentadecanone. the unexplainable enhancement of the ion at m/e 58. The prominent ion at m/e 4.5 in the mass spectra of B,, Arr, and B,, indicates that these compounds are 2-alkanols. It has been pointed out in the Results that compound B, and 2-undecanol have many common mass spectral fragmentation characteristics, but that the spectra of B, and 2-undecanol were not identical. Additional fragment ions at m/e 50, 97, 105, and 152 suggest that the compound
VOLATILE
CEPHALIC
SUBSTANCES
OF THE STINGLESS
BEES
1125
contains impurities or has a carbon skeleton different from 2-undecanol. From the gas chromatographic retention times (Table 4), we may conclude that B, is not a branched chain compound since its retention time is not shorter than that of standard 2-undecanone. It may be concluded that the compound is contaminated and that the additional fragment ions in the mass spectrum of B, are due to a second compound which co-eluted from the gas chromatographic column with B,. Since 2-heptadecanol was not available for analysis, the identification of compounds A,, and B,, must be equivocal. An inspection of the mass spectra of compounds A,, and B,, showed that the mass spectrum of A,, is very similar to that of 2-pentadecanol. It is therefore probable that compound A,, is 2-heptadecanol. The mass spectrum of B12, however, has additional ions at m/e 43 and 55. Since we do not have synthetic 2-heptadecanol for comparison, it is difficult to determine whether this compound has a different carbon skeleton or whether it is contaminated. On the basis of prior experiences with this mixture, it is probable that B,, is also contaminated. Positive identification of A,, B,, and B,, is not possible from the data available. From an intense search of the literature, it appears that A,, B,, and B,, have not been identified previously. It is therefore unlikely that the structures of these new compounds may be determined by mass spectrometry. Additional instrumental and chemical analyses will be necessary for their identification. T. mexicana and T. pectoralis are classified in the subgenus Scaptotrigona on the basis of their anatomical characteristics. The similarity of the volatile substances from their heads is further evidence of the close kinship of these two species. The major difference which we found is in the quantitative composition of the chemical mixtures and particularly in 2-heptanone and 2-tridecanone. We have examined the volatile chemicals and studied the responses to a wide variety of chemicals of three species of Trigona in other subgenera and have found them quite different from these two species. Benzaldehyde or the individual ketones usually did not cause attack by the bees, but a mixture of these substances caused strong attack. It could well be to the advantage of the colony to react strongly to mixtures that closely resemble the mixture secreted by an alarmed bee of the same species. Otherwise the colony might be subject to frequent false alarms. Ketones are fairly common alarm substances of a number of ant species (LAW and REGNIER, 1971) and ants are common near the nests of these bees. For instance, a small ant was often found nesting near the colonies of T. pectoralis; when 2-heptanone was tested on the bees the nearby ants of this species exhibited typical alarm behaviour ; when the ants were crushed, they gave off a strong odour of 2-heptanone. Although our technique of assay was suitable to demonstrate alarm reactions under conditions that often prevail when the colony is endangered, it was not sufficiently refined to study threshold concentrations, the precise ranking of activity of different compounds, nor the precise synergistic effect of mixtures of compounds. Such studies are planned for the future if a suitable adaptation of the technique of WILSON et al. (1969) can be developed. The presentation of chemicals
1126
J. M. LUBY, F. E. REGNIER, E. T.
CLARKE, E. C. WEAVER, ANDN. WEAVER
in the open air where there was often considerable wind was particularly unsuitable for studying the alarm pheromone activity of the higher molecular weight compounds. Since 2-tridecanone, 2-pentadecanone, and 2-pentadecanol were solids at the temperature in the field, it was surprising when they elicited an attack response under the conditions of our tests. They might, however, have strong alarm activity in more confined quarters such as when deposited on an intruder that has penetrated the nest. It is also possible that compounds of low volatility might exert their influence on a blend of volatile compounds by modifying the vapour pressure of individual components and thus changing the composition of the blend in the vapour phase. It is quite possible, of course, that the less volatile substances have some role independent of alarm. The higher molecular weight compounds might not normally reach a vapour concentration high enough to cause alarm but could still act to mark a food source or a possible new next site. Indeed, BLUM et al. (1970) and BLUM (1971) claim that high concentrations of neral geranial, and 2-alkanones act as alarm pheromones, but that low concentrations act as trail markers to recruit other bees to a source of food. This is an attractive idea, but these authors present no data to support their claim. It is also possible that the same substances act as alarm pheromones and as defence chemicals. Benzaldehyde is known to be a space repellent for the honeybee (TOWNSEND,1963), and it might serve a similar role against some of the species that attack the nests of stingless bees. CONCLUSIONS From the reaction gas chromatographic analyses, mass spectral data, and retention indices it may be concluded that both T. mexicana and T. pectoralis produced homologous series of normal aliphatic alcohols and ketones with functional groups at the 2-position of the carbon skeleton. The compounds all contain odd numbers of carbon atoms ranging from C, to C,,. In addition, both species produce benzaldehyde and several unidentified compounds. The primary difference between the volatile mixtures produced by these two species is in the quantitative composition of the blends. Both the alcohols and ketones are alarm pheromones. The alcohols are more active in inducing attack by the bees than are the ketones, but a mixture of the ketones and benzaldehyde was very active. Acknowledgements-This work was supported in part by National Science Foundation Grant No. G&30179X. The authors wish to thank Dr. A. WILLE of the University of Costa Rica for identifying the bees. This is Journal Paper No. 4903 of the Purdue Agricultural Experiment Station. REFERENCES
BLUMM. S. (1966) Chemical releasers of social behaviour-VIII. gland secretion of Lestrimelitta Sot. Am. 59, 962-964.
limao (Hymenoptera:
Apidae:
Citral in the mandibular Melitidae). Ann. mt.
VOLATILECEPHALICSUBSTANCES OF THE STINGLESS BEES
1127
BLUM M. S. (1971) Dimensions of chemical sociality. Chemical Releasers in Insects 3, 147162. BLUM M. S., CREWER. M., KEER W. E., KEITH L. H., GARRISON A. W., and WALKERM. M. (1970) Citral in stingless bees: isolation and functions in trail-laying and robbing. J. Insect Physiol. 16, 1637-1648. BUDZIKIEWICZ H., DJERA~SIC., and WILLIAMS D. H. (1968) Interpretation of Mass Spectra of Organic Compounds, p. 38. Holden-Day, San Francisco. BUDZIKIEWICZH., PENSELAWC., and DJERASSIC. (1966) Mass spectrometry in structural and stereochemical problems. Tetrahedron 22, 1391-1398. DAUBENW. G., HANCEP. D., and HAYES W. K. (1955) Acid catalyzed transformations of X-santonin. r. Am. them. Sot. 77,4609-4612. JAMESA. T. (1959) Determination of the degree of unsaturation of long chain fatty acids by gas-liquid chromatography. r. Chromatog. 2, 552-561. KERR W. E., FERREIRAA., and SIMOESDE MATTOSN. (1963) Communication among stingless bees-additional data (Hymenoptera: Apidae). r. N. Y. ent. Sot. 71, 80-90. LAW J. H. and RECNIER F. E. (1971) Pheromones. A. Rew. Biochem. 40, 533-548. LINDAUERM. and KERR W. E. (1960) Communication between the workers of stingless bees. Bee World 41, 29-41, 65-71. MCCOLLUMJ. D. and MEYERSONS. (1963) Organic ions in the gas phase-X. Decomposition of benzaldehyde under electron impact. J. Am. them. Sot. 85, 1739-1741. REGNIER F. E. and HUANG J. (1970) The determination of oxygen containing functional groups by reaction gas chromatography. J. Chrom. Sci. 8, 267-271. REGNIERF. E., NIEH M., and HOLLDOBLERB. (1972) Dufours gland components of Pogonomyrmex badins and Pogonomyrmex rugosus. J. Insect Physiol. In press. SCHWA= F. (1948) Stingless bees (Meliponidae) of the western hemisphere. Bull. Am. Mus. nut. Hist. 90. TOWNSENDF. (1963) Benzaldehyde: a new repellent for driving bees. Bee World 44, 146149. WILSON E. 0. (1971) The Insect Societies. p. 272. Cambridge, Mass.: Harvard University Press. WILSON E. O., BOSSERTW. H., and REGNIERF. E. (1969) A general method of estimating threshold concentrations of odorent molecules. J. Insect Physiol. 15, 597-610.
36