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Characteristic odour components of the scent of stink bugs
J. Ins. Physiot., 1961, Vol. 6, pp. 113to 121. Pergamon Press Ltd., London. Printed in Great
CHARACTERISTIC ODOUR COMPONENTS SCENT OF STINK BUGS D. F. ~~rATER~O~S~,* * Division of Entomology,
C.S.I.R.O.,
Britain
OF THE
D. A. FORSS,I_ and R. I-i. ~AC~~~AN* Canberra,
and t Dairy Research
Section,
C.S.I.R.O.,
Melbourne, Australia (Receitzd 3 October 1960)
Abstract-The characteristic stink of three pentatomid bugs was shown to be largely due to a mixture of hex-2-enal, a dicarbonyl compound, and either act-2-enal or dec-2-enal. In two coreid bugs n-hexanal was a major odour component. These or similar compounds occur in plants on which the bugs feed, but it is possible that the bugs may also produce them metabolically. The odours almost certainly have a defensive role.
INTRODUCTION CONSIDERABLE interest has developed in recent years in the nature of the chemical compounds used by insects for offence, defence, as sex attractants, and as poisons. Although it is commonplace knowledge that many species belonging to the hemipterous superfamily Pentatomoidea have detestable odours, the identity of even the major constituents responsible does not appear to have been determined. CARIUS (1860) reported a characteristic, mildly rancid, fatty acid, cimicic acid, C,, H, 0,and another unpleasant smelling material, probably an aldehyde, in the bug lihuphs’gaster punctipmnis Illig. However, even this amount of characterization is exceptional, and, where any further information is available, it is of a much more general nature. For example, the coreid bug Anasa t&is (DeG.) produces a Iight yellow, volatile oil with a sharp odour reminiscent of amyl acetate, an acid reaction to litmus, and an acrid taste. The adults and larvae produced a similar, but not identical, odour (MOODY,1930). The secretion of Nezara oiridzda (L.) is irritant to the lips, has a strong aromatic odour, and is alkaline to litmus (MALOUF, 1933). The adult pentatomoid odours are due to light yellow oily materials produced by paired tubular glands and accessory glands. Their secretions are accumulated in a single reservoir (occ~ionally in paired reservoirs) situated ventrally in the metathorax. This reservoir opens to the exterior at each side between the middle and hind coxae. In the larvae, stink glands are present in some of the dorsal segments of the abdomen. On moulting to the adult the dorsal abdominal glands are repiaced by those in the ventral region of the metathorax, apparently without significant change in function. In adult Oncopeltus fusciatw (Dallas) the secretion from the tubular glands is not unpleasant, whereas the stink is either the product of the accessory giands or it may result from a reaction between these two secretions. Even in the larvae 113
114
D. F. WA~HOUSE, D. A. FORSS,ANDR. H. HACKMAN
which carry their unpaired dorsal scent glands on the fourth and fifth abdominal segments, the secretions of each differ. The anterior gland is responsible for the typical unpleasant odour, whereas the posterior gland secretes an inoffensive material (JOHANSSON,1957). EXPERIMENTAL 1. P~epara~~o~ of Samples
Field collected specimens of five species of bug (Table 1) were decapitated after cooling to about 3°C in order to inactivate the bugs and to reduce loss of the TABLEI-COMPONENTSOFTHEODORIFEROUS OIL OFFIVEPENTATOMOID BUGS Compounds identified
Insect
Pentatomidae Nezara v&&da var. smarag&la (F.)
~~oecoco~s s~lck~nt~&(&al)
Poe~~Lorn~~~s strikatus Westw. Coreidae Mictis profana F. .4morbus rubiginosus Guer.
Common name
Green vegetable bug Bronze orange bug -
-t + +
-I-
Crusader bug Eucalyptus bug
odoriferous oil while the glands, together with a tiny piece of adhering cuticular tissue, were rapidly dissected out. Glands from each species were then subjected to steam distillation and the distillates examined as follows: (a) (all five species) The steam distillate was treated with excess 2,4-dinitrosoluble in light phenylhydr~ine in 2N I-&SO,. The 2,4-dinitrophenylhydr~ones petroleum (boiling point below 4O”C, free of aromatic and carbonyl compounds) were fractionated on Celite-nitromethane liquid partition columns according to the method of DAY et al. (1960), whereas those insoluble in light petroleum, but soluble in benzene, were purified on alumina liquid adsorption chromatographic columns. (b) (only Nexava and Rhoecocuris) The steam distillate was extracted with light petroleum, the extract concentrated and then fractionated on a silicone-oil gas
115
CHARACTERISTIC ODOUR COMPONENTS OF THE SCENT OF STINK BUGS
from 50 to 132°C. chromatographic column (FORSS et al., 1960a) programmed The fractions thus obtained were treated with 2,4_dinitrophenylhydrazine as described in (a). 2. Characterization of Odour Constituents When the steam distillates from the five bugs were treated with 2,4-dinitrophenylhydrazine the stink from the three pentatomid bugs disappeared, indicating that the major odour components were carbonyl compounds. However, there remained a slight ester smell from the two coreid bugs, indicating that noncarbonyl compounds (possibly esters) contribute to the odour of these species. A. The green vegetable bug, Nezara
viridula
The five principal constituents (shown in Table 2) accounted for most of the odoriferous material. Each was treated with 2,4_dinitrophenylhydrazine and precipitates were obtained for fractions 1, 2, and 4. TABLE ~-GAS
CHROMATOGBAPHIC FRACTIONS FROM
-
-
Nezara
viridula
Fraction
No.
Time [min)
Identified as
Relative amount
_-
Odour
.-
19f
+++
Hex-2-enal
Strongest and most important component, but needs to be combined with fraction 4 to approach Nezara stink
28
Probably a dicarbonyl
Minor
34
+++ +++ +
84*
+++
Dee-2-enal
+++++ +++i-+ +++++
Tridecane
108
3
* Retention
odour
component,
relatively
pleasant
sweet odour Minor odour component, ester odour, orangepeel odour The most unpleasant component and next in importance after fraction 1 Weak odour
-
times of these fractions were identical with those
of authentic
samples
of the
respective aldehydes. t Did not react with 2,4-dinitrophenylhydrazine.
(a) Fraction 1 : hex-2-enal. The 2,4-dinitrophenyihydrazone was soluble in light petroleum, gave h maxEton 372 rnp and x maxELon_Kaon 455 mp, identical with that of the 2,4_dinitrophenylhydrazone of authentic hex-2-enal, and had a melting point of 145-146°C identical with, and undepressed when mixed with, the 2,4-dinitrophenylhydrazone of authentic hex-2-enal. The 2,4-dinitrophenylhydrazones of fraction 1 and of synthetic hex-2-enal were compared on paper chromatograms by the method of LYNN et al. (1956) and both moved in an identical fashion. (b) Fraction 2 : probably a dicarbonyl compound. The 2,4_dinitrophenylhydrazone was an orange precipitate insoluble in light petroleum, but soluble in
116
D. F. WATERHOUSE, D. A. FORSS,AND R. H. HACKMAN
benzene, suggesting a dicarbonyl. It had a melting point of 268°C and gave X maxcncl, 410 rnp and h maxEton_&oH 561 mp. From an infra-red examination of Nujol mulls of the 2,4_dinitrophenylhydrazone which had been purified on an alumina chromatographic column, the following was deduced: a band at 1692 cm-l was assigned to an (Y,p-unsaturated acid. Unsaturation was also indicated by a band at 983 cm-l, the frequency showing the influence of conjugation on the C-H deformation of a trans double bond. A band at 1746 cm-l was assigned to a keto ester (other than ,Gketo). Since this band was not present (see later) in the otherwise identical spectra of Rhoecocoris or Poecilometis, it is inferred that a compound of structure 0
H
III R-C-C=C--C
/”
i
H
\
OH
together with an ester of this acid is probably present. Some caution, however, must be exercised in accepting the validity of the absorption bands other than that at 983 cm-l, since it has recently been found (HORWOOD, unpublished) that chromatography on Celite-nitromethane columns modifies the infra-red spectra of some 2,4_dinitrophenylhydrazones and introduces foreign bands in the carbonyl region. A similar, but much weaker, modification was observed for the 2,4-dinitrophenylhydrazone of n-pentanal after passage through an alumina chromatographic column. (c) Fraction 3 : unidentzj?ed. This fraction did not react with 2,4_dinitrophenylhydrazine and, since it was present in small amount and did not contribute materially to the characteristic odour of Nexara, it was not investigated further. (d) Fraction 4 : dec-2-enal. The 2,4_dinitrophenylhydrazone was soluble in light petroleum, gave h maxEtOH 372 rnp and h maxEtOH_NaOH455 rnp, identical with that of the 2,4_dinitrophenylhydrazone of authentic dec-2-enal, had a melting point of 127-128°C undepressed when mixed with the authentic dec-2-enal derivative of melting point 124125°C. The 2,4-dinitrophenylhydrazones of fraction 4 and of synthetic dec-2-enal moved in an identical fashion when compared on paper chromatograms by the method of LYNN et al. (1956). (e) Fraction 5 : tridecane. This was the most abundant fraction obtained from the steam distillate but made no important contribution to the characteristic stink bug odour. It was identical in retention time with an authentic sample of tridecane and this identity was confirmed by means of mass spectrographic examination. B. The bronze orange bug, Rhoecocoris
sulciventris
Five main fractions were also obtained by gas chromatography of the bronze orange bug odoriferous material (Table 3). (a) Fraction 1 : hex-Zenal. Identical with fraction 1 from Nexara, but present in much lower proportion in the steam distillate and hence an unimportant constituent of the odour.
CHARACTFZISTIC
117
ODOUR COMPONENTS OF THE SCENT OF STINK BUGS
(b) Fraction 2 : probably a dicarbonyl compound. The orange 2,4_dinitrophenylhydrazone was insoluble in light petroleum, soluble in benzene, gave h max&.,, 561 mp, and had a melting point of 262°C. Its infra-red 41° mtLYh m=EtOH-NsOH TABLE ~--GAS
CHROMATOGRAPHICFRACTIONS FROM Rhoecocoris sulciventris
Fraction
No.
Relative amount
Time (min)
Identified
as
Odour
20*
c
Hex-2-enal
28
+++++
40*
+++++ +++++ +++++ ++ ++++++ ++++++ ++++++ ++++++ ++++++ ++++++ ++++++ ++++++
Same as fraction 2 of Nezuru. Probably a dicarbonyl Ott-2-enal
67 108
Similar to Nezuru but quantity small to be important Minor odour component
Strongest and component
important
Minor odour component Minor odour contributor, occurs in largest amount
?t
Tridecane Identical fraction from Nezura
most
5
* Retention times of these fractions were idehtical with those of authentic respective aldehydes. t Did not react with 2,4-dinitrophenylhydrazine.
too
but
samples of the
spectrum was identical with that from fraction 2 of Nezara, except that there was no band at 1746 cm-i, so that only 0
H
II I R-C-C=C-C
//O
I
H
\
OH
and not its ester is present in this bug. (c) Fraction 3 : act-Zenal. The 2,4_dinitrophenylhydrazone was soluble in light petroleum, gave A maxxton 372 rnp and X maxEtoH_N&H454 mp, identical with that of the 2,4-dinitrophenylhydrazone of authentic act-Zenal, and had a melting point of 127-128°C identical with, and undepressed when mixed with, the 2,4-dinitrophenylhydrazone of authentic act-2-enal. The 2,4_dinitrophenylhydrazones of fraction 3 and of synthetic act-Zenal moved in an identical fashion when compared on paper chromatograms by the method of LYNN et aZ. (1956). 8
118
D. F. WATERHOUSE, D. A. Fo~ss, ANDR. H. HACKMAN
(d) Fraction 4 : unidentified. This was present in small amount, was a minor component of the odour, and did not react with 2,4*dinitrophenylhydr~ine. (e) Fraction 5 : tridecune. This was identical with fraction 5 from iVezara and was the most abundant fraction obtained. It was a minor odour component. C. Poecilometis strigatus The odoriferous secretion from this bug, which could be distinguished by smell from that of both Nexara and Rhoecocoris, was examined in less detail. However, the presence was detected of hex-Z-enal, act-Z-enal, and a dicarbonyl identical with fraction 2 from ~~oecoco~~. D. Mictis profana and Amorbus rubiginosus The preliminary examination of odoriferous material from Mictis and Amorbus gave essentially similar results. The yellow 2,4_dinitrophenylhydrazones were soluble in light petroleum and were fractionated on a Celite-nitromethane column. Both species yielded a single constituent which was identified as the n-hexanal derivative with the following properties: h maxEtoH 358 rnp, h maxEtonNILoW 432 mp, with a smaller peak at 518 rnp, which had disappeared after 2 hr, indicating a saturated aldehyde and not a saturated ketone, identical in behaviour with the 2,4_dinitrophenylhydrazone of authentic n-hexanal; melting point 107-108°C identical with, and undepressed when mixed with, the 2,4-dinitrophenylhydrazone of synthetic n-hexanal; these natural and synthetic derivatives moved in an identical fashion on paper chromatograms (LYNN et al., 1956). Synthetic n-hexanal, which was purified on a silicone-oil gas chromatographic column, was appraised as contributing importantly to the odours of both Mictis and Amorbus, although other materials were also present in the odoriferous secretions to give each bug a characteristically different odour. DISCUSSION To the human nose the odours produced by all five bugs are distinct, although there is a far greater basic similarity within the families Pentatomidae and Coreidae than between these families. It is interesting, therefore, to note that a six-carbon aldehyde contributed to the odour of all five bugs. In two of the bugs act-Z-enal was an important constituent and in one bug dec-Zenal. The compounds reported in Table 1 are probably the major contributors to the odours of the Pentatomidae investigated. However, in addition to n-hexanal there are certainly other important, unidentified odour constituents in the two coreid bugs. The sharp amyl alcohollike odour reported by MOODY(1930) for the coreid A. tristis suggests that it may be basically similar to that of the coreid bugs we have examined. All of the compounds identified in this study have been isolated from skim milk, which has acquired an oxidized (cardboard) flavour (FORSSet al., 1955). In addition some of the above constituents are also known to occur naturally, although mainly in plants (GUENTHER,1949). Thus n-hexanal is found in various Eu~~l~~tus oils, where it contributes to the disagreeable properties of certain of these oils. In view of this it is relevant to note that Amorbus lives on eucalypts.
CHARACTERISTIC
ODOUR COMPONENTSOF THE SCENTOF STINKBUGS
119
Much more widespread in its distribution in plants is hex-Z-enal, which has been reported in the oils derived from the green leaves of numerous plant species (e.g. tea, mulberry, acacia) (GUNTHER, 1949). Because of its strong and pungent odour of green leaves, hex-2enal is sometimes used in perfumes and flavours. It is said that it forms part of the flavour of green tea and that the aroma of tea is primarily due to this compound (see ROTH et al., 1956). Hex-2-enal and hex-3-enol are together responsible for the attraction of silkworm larvae to leaves (WATANABE, 1959). Hex-Zenal was found to be one of the carbonyl compounds responsible for the odour of whale oil (see ROTH et al., 1956), although it may well have been formed in the oil after extraction, for it has also been isolated from a variety of oxidized fats and oils. Thus FORSSet al. (1955, 1960a) have identified it in oxidized skim milk and butterfat. Hex-Zenal and other unsaturated aldehydes are thought to be derived from unsaturated fatty acids through hydroperoxide formation and cleavage at the a-methylene group (BADINGS,1959; SWIFT et al., 1949). The only fully substantiated record of the natural occurrence of hex-Z-enal in animals, therefore, appears to be the careful work of ROTH et al. (1956) from both adult males and females of the cockroach EurycotisJIoridana (Walker). Pure tram hex-Zenal was the only material present in the yellow liquid produced by a gland opening on the ventral surface of the abdomen. Interestingly, in an old report, HEBARD(1917) states that the odour of this cockroach strongly suggests that of the pentatomid Brochymena annulata (E.). A related compound, tram hex-2-enol-l-acetate, occurs in abdominal glands of males of the waterbug, Be~sto~ indica Vitalis, where it is thought perhaps to function as a sex odour (BUTENANDTand TAM, 1957). This material is employed in south-east Asia as a seasoning for fatty foods. An unidentified aliphatic LY,p-unsaturated ketone has been isolated from the defensive glands of the water beetle Dytiwus marginalis (SCHILDKNECHTand HOLOUBEK,1959). Naturally occurring act-Zenal does not appear to have been reported, although it is formed during oxidation of skim milk, contributes to cardboard flavours ( FORSS et al., 1955), and has been isolated from oxidized cottonseed oil (SWIFT et al., 1949). n-Octanal is an important constituent of orange oil (GUENTHER, 1949),which isof interest in view of the great importance of act-Zenal in the odour of the bronze orange bug which lives on citrus. Non-2-enal has been isolated from cucumbers, oxidized skim milk, and oxidized butterfat (FORSS et al., 1955, 1960a, b) although it has not been detected in the five bug odours examined. Dee-2-enal has been found in the essential oii of several plants, including the orange (GUENTHER, 1949),but does not appear to have been reported previously from animals. It does, however, occur in oxidized skim milk (FORSSet al., 1955). The mechanism of production of the odoriferous materials by the bugs is unknown. In view of the occurrence in plants of many of the materials responsible it is possible that some constituents are simply con~en~ated from the very large volume of sap passed through the alimentary canal and that the amount of odoriferous material accumulated by the bug varies with the amount available from
120
D. F.
WATERHOUSE, D. A. Foass,
AND
R. H.
HACKMAN
the host plant. This might apply more particularly to species with a restricted host plant range. However, this is unlikely to be the only method of production. Thus the odour of the green vegetable bug (Nezura) is quite characteristic of this species although it may feed on any of a very wide range of plants. These plants presumably would provide unsaturated aldehydes in very different amounts and, unless Nexara possessed a special mechanism for taking up the wanted constituents only in certain definite proportions and rejecting closely related compounds, the components of the sap of the host plant would tend to influence greatly the nature of the odour. We have not determined whether there are quantitative differences between the odour components of samples of Nezuru collected at different times, although both qualitatively and to the nose they are similar. The larval odours are also similar to the nose. It is probable, therefore, that the odour components are produced, at least in part, metabolically and that a more or less constant composition is maintained. No information is available on their possible precursors, but this topic is worthy of experimental investigation. It is generally agreed that these odours are probably of repugnatorial value to the bugs which produce them (MOODY,1930; WEBER,1930), although HEIKERTINGER (1922) dissents from this opinion. There is no indication that they regularly play any part simply as excretory products, as sex attractants, or to assist in species recognition, for the adults are seldom gregarious, although young larvae of some species (e.g. of Nezuru viridulu) may be. Some interesting experiments have been carried out by EISNERet al. (1959) on the cockroach E. floridunu, which can spray its hex-2-enal as far as 3 ft when disturbed and can aim with some degree of-accuracy in the general direction of the stimulus. Ants (Pogonomyrmex bud&s (Latr.) and Cumponotus pennsylvunicus (Degeer) ) were repelled, although an unidentified tarantula which routinely feeds on Eurycotis in the laboratory was not affected (EISNER, personal communication). When a bug is picked up the characteristic disagreeable odour is usually evident immediately. In many instances the oily liquid is merely forced out on to the surface of the cuticIe where rapid evaporation takes place. Some bugs, however, are able to emit a spray, as for example Anusu t&is which, when strongly irritated, may discharge several drops of liquid as far as 5 in. in a latero-posterior direction from the body (MOODY, 1930). The pentatomid Tesserutomu pupillosu Thunb. is reported to be able to eject its liquid to a distance of 6-10 in. (MUIR, 1907). Under some circumstances the secretions may be toxic even to the bugs for, when a large number of Rhoecocoris were placed in a lightly closed container on a warm day, many were dead within an hour. It is evident that, for a particular compound, attraction, repellency, or toxic action may be simply a function of concentration. Experiments carried out with an unidentified pentatomid and several coreids demonstrated a strong deterrent effect to attack by predators, including ants, amphibia, birds, and mice. With birds there was obvious rejection only when the secretion appeared to have hit the eyes. With some species the secretion is ejected in response to movement or vibration nearby and no direct contact was necessary. Thus a caged bird (Cyunocittu stellerz] was repeatedly sprayed when merely landing
CHARACTERISTIC ODOURCOMPONENTS OF THE SCENTOF STINKBUGS
121
near a coreid. Moreover, this bug aimed its spray quite accurately by adjusting body orientation and stance to enable it to fire a broadside and then discharged its gland from the stimulated side of the body only (EISNFR, personal communication). Ac~~~~~t~We wish to acknowledge the help of W. STARKand J. F. Ho~woon of the Dairy Research Section, C.S.I.R.O., the former for assistance with gas chromatography and the latter for infra-red analyses. REFERENCES BADINGSH. T. (1959) Isolation and identification of csrbonyl compounds formed by autooxidation of ammonium linoleate. J. Amer. OiZ Chem. Sot. 36, 448-650. BUTENANDTA. and TAM N. (1957) Ub er einen g~~hle~h~spezi~s~hen DuftstofI der Wasserwanze 3e~ost~ indica Vitalis (Lethocew indicus Lep.). Hoppe-Sag. Z. 308, 277-283. CARIUSL. (1860) Ueber eine neue Slure der Reihe Cn Hrn_a 0,. Ann. Chem. Phwm. 114, 147-156. DAY E. A., B~ETTE R., and KEENEYM. (1960) Identification of volatile carbonyl compounds from cheddar cheese. J. Dairy Sci. 43,463-474. EISNERT., MCKITTRICKF.,‘and PAYNER. (1959) Defense sprays of roaches. Pest Control 27 (6), 9, 11-12, 44-45. FORSSD. A., DUNSTONEE. A., and STARKW. (1960a) Fishy Aavour in dairy products-II. The volatile compounds associated with fishy flavour in butterfat. J. Dairy Res. 27, 211-219. Fonts D. A., DUNSTONEE. A., and STARKW. (1960b) Aldehydes of cucumber. Unpublished work. Fonts D. A., PONT E. G., and STARKW. (1955) The volatile compounds associated with oxidized flavour in skim milk. y, Dairy Res. 22,91-102. GUENTHERE. (1949) The Essentaial Oils, Vol. 2. Van Nostrand, New York. HEBARDM. (1917) The Blattidae of North America north of the Mexican boundary. Mem. Amer. ent. Sac. 2, l-284. HEIKXRTINGERF. (1922) Sind die Wanzen (Hemipte~-Heteropte~) durch Ekelgeruch geschiitz 7 Biol. 261. 42, 441-464. JOHANSSON A. S. (1957) The functional anatomy of the metathoracic scent glands of the milkweed bug, Oncopeltusfasciatus (Dallas) (Heteroptera: Lygaeidae). Nor& mt. Tidsskr. 10, 95-109. LYNN W. S., STEELEL. A., and STAPLEE. (1956) Separation of 2,4-dinitrophenylhydraxones of aldehydes and ketones by paper chromatography. AnaZyt. Chem. 28,132-133. MALOUF N. S. R. (1933) Studies on the internal anatomy of the “stink bug” Nezuru viriduln L. Br&. Sot. est. .&ypte 17, 96-119. MCJ~DYD. L. (1930) The morphology of the repugnatory glands of Anasa tristis De Geer. Ann. ent. Sot. Amer. 23,81-104. MUIR F. (1907) Notes on the stridulating organ and stink glands of Tessaratomu papillosa, Thunb. Trans. ent. Sot. Land. (1907), 256-258. ROTH L. M., NIECISCHW. D., and STAHL W. H. (1956) Occurrence of 2-hexenal in the cockroach Eurycotis flwidana. Science 123, 670-671. SCHILDKNECHT H. and HOLOUBBKK. (1959) Uber einen Inhaltsstoff der Wehrdriisen des Gelbrandkltiers-III. Mitteilung ilber Insektenabwehrstoffe. Astgew. Chem. 71,524-525. Sw~pr C. E., O'CONNOR R. T., BROWSEL. E., and DOLLEARF. G. (1949) The aldehydes produced during the autoxidation of cottonseed oil. J. Ames. Oil Chem. Sot. 26,297-300. WATANABET. (1959) Studies on the volatile components of mulberry leaves-V. Attraction of p-y-hexenol and u-/3-hexenal to silkworm larvae. J. se&. Sci., Tokyo 28,23-26. WEERR H. (1930) Biologic der Hemipteren. Springer, Berlin.
CHARACTERISTIC ODOUR COMPONENTS SCENT OF STINK BUGS D. F. ~~rATER~O~S~,* * Division of Entomology,
C.S.I.R.O.,
Britain
OF THE
D. A. FORSS,I_ and R. I-i. ~AC~~~AN* Canberra,
and t Dairy Research
Section,
C.S.I.R.O.,
Melbourne, Australia (Receitzd 3 October 1960)
Abstract-The characteristic stink of three pentatomid bugs was shown to be largely due to a mixture of hex-2-enal, a dicarbonyl compound, and either act-2-enal or dec-2-enal. In two coreid bugs n-hexanal was a major odour component. These or similar compounds occur in plants on which the bugs feed, but it is possible that the bugs may also produce them metabolically. The odours almost certainly have a defensive role.
INTRODUCTION CONSIDERABLE interest has developed in recent years in the nature of the chemical compounds used by insects for offence, defence, as sex attractants, and as poisons. Although it is commonplace knowledge that many species belonging to the hemipterous superfamily Pentatomoidea have detestable odours, the identity of even the major constituents responsible does not appear to have been determined. CARIUS (1860) reported a characteristic, mildly rancid, fatty acid, cimicic acid, C,, H, 0,and another unpleasant smelling material, probably an aldehyde, in the bug lihuphs’gaster punctipmnis Illig. However, even this amount of characterization is exceptional, and, where any further information is available, it is of a much more general nature. For example, the coreid bug Anasa t&is (DeG.) produces a Iight yellow, volatile oil with a sharp odour reminiscent of amyl acetate, an acid reaction to litmus, and an acrid taste. The adults and larvae produced a similar, but not identical, odour (MOODY,1930). The secretion of Nezara oiridzda (L.) is irritant to the lips, has a strong aromatic odour, and is alkaline to litmus (MALOUF, 1933). The adult pentatomoid odours are due to light yellow oily materials produced by paired tubular glands and accessory glands. Their secretions are accumulated in a single reservoir (occ~ionally in paired reservoirs) situated ventrally in the metathorax. This reservoir opens to the exterior at each side between the middle and hind coxae. In the larvae, stink glands are present in some of the dorsal segments of the abdomen. On moulting to the adult the dorsal abdominal glands are repiaced by those in the ventral region of the metathorax, apparently without significant change in function. In adult Oncopeltus fusciatw (Dallas) the secretion from the tubular glands is not unpleasant, whereas the stink is either the product of the accessory giands or it may result from a reaction between these two secretions. Even in the larvae 113
114
D. F. WA~HOUSE, D. A. FORSS,ANDR. H. HACKMAN
which carry their unpaired dorsal scent glands on the fourth and fifth abdominal segments, the secretions of each differ. The anterior gland is responsible for the typical unpleasant odour, whereas the posterior gland secretes an inoffensive material (JOHANSSON,1957). EXPERIMENTAL 1. P~epara~~o~ of Samples
Field collected specimens of five species of bug (Table 1) were decapitated after cooling to about 3°C in order to inactivate the bugs and to reduce loss of the TABLEI-COMPONENTSOFTHEODORIFEROUS OIL OFFIVEPENTATOMOID BUGS Compounds identified
Insect
Pentatomidae Nezara v&&da var. smarag&la (F.)
~~oecoco~s s~lck~nt~&(&al)
Poe~~Lorn~~~s strikatus Westw. Coreidae Mictis profana F. .4morbus rubiginosus Guer.
Common name
Green vegetable bug Bronze orange bug -
-t + +
-I-
Crusader bug Eucalyptus bug
odoriferous oil while the glands, together with a tiny piece of adhering cuticular tissue, were rapidly dissected out. Glands from each species were then subjected to steam distillation and the distillates examined as follows: (a) (all five species) The steam distillate was treated with excess 2,4-dinitrosoluble in light phenylhydr~ine in 2N I-&SO,. The 2,4-dinitrophenylhydr~ones petroleum (boiling point below 4O”C, free of aromatic and carbonyl compounds) were fractionated on Celite-nitromethane liquid partition columns according to the method of DAY et al. (1960), whereas those insoluble in light petroleum, but soluble in benzene, were purified on alumina liquid adsorption chromatographic columns. (b) (only Nexava and Rhoecocuris) The steam distillate was extracted with light petroleum, the extract concentrated and then fractionated on a silicone-oil gas
115
CHARACTERISTIC ODOUR COMPONENTS OF THE SCENT OF STINK BUGS
from 50 to 132°C. chromatographic column (FORSS et al., 1960a) programmed The fractions thus obtained were treated with 2,4_dinitrophenylhydrazine as described in (a). 2. Characterization of Odour Constituents When the steam distillates from the five bugs were treated with 2,4-dinitrophenylhydrazine the stink from the three pentatomid bugs disappeared, indicating that the major odour components were carbonyl compounds. However, there remained a slight ester smell from the two coreid bugs, indicating that noncarbonyl compounds (possibly esters) contribute to the odour of these species. A. The green vegetable bug, Nezara
viridula
The five principal constituents (shown in Table 2) accounted for most of the odoriferous material. Each was treated with 2,4_dinitrophenylhydrazine and precipitates were obtained for fractions 1, 2, and 4. TABLE ~-GAS
CHROMATOGBAPHIC FRACTIONS FROM
-
-
Nezara
viridula
Fraction
No.
Time [min)
Identified as
Relative amount
_-
Odour
.-
19f
+++
Hex-2-enal
Strongest and most important component, but needs to be combined with fraction 4 to approach Nezara stink
28
Probably a dicarbonyl
Minor
34
+++ +++ +
84*
+++
Dee-2-enal
+++++ +++i-+ +++++
Tridecane
108
3
* Retention
odour
component,
relatively
pleasant
sweet odour Minor odour component, ester odour, orangepeel odour The most unpleasant component and next in importance after fraction 1 Weak odour
-
times of these fractions were identical with those
of authentic
samples
of the
respective aldehydes. t Did not react with 2,4-dinitrophenylhydrazine.
(a) Fraction 1 : hex-2-enal. The 2,4-dinitrophenyihydrazone was soluble in light petroleum, gave h maxEton 372 rnp and x maxELon_Kaon 455 mp, identical with that of the 2,4_dinitrophenylhydrazone of authentic hex-2-enal, and had a melting point of 145-146°C identical with, and undepressed when mixed with, the 2,4-dinitrophenylhydrazone of authentic hex-2-enal. The 2,4-dinitrophenylhydrazones of fraction 1 and of synthetic hex-2-enal were compared on paper chromatograms by the method of LYNN et al. (1956) and both moved in an identical fashion. (b) Fraction 2 : probably a dicarbonyl compound. The 2,4_dinitrophenylhydrazone was an orange precipitate insoluble in light petroleum, but soluble in
116
D. F. WATERHOUSE, D. A. FORSS,AND R. H. HACKMAN
benzene, suggesting a dicarbonyl. It had a melting point of 268°C and gave X maxcncl, 410 rnp and h maxEton_&oH 561 mp. From an infra-red examination of Nujol mulls of the 2,4_dinitrophenylhydrazone which had been purified on an alumina chromatographic column, the following was deduced: a band at 1692 cm-l was assigned to an (Y,p-unsaturated acid. Unsaturation was also indicated by a band at 983 cm-l, the frequency showing the influence of conjugation on the C-H deformation of a trans double bond. A band at 1746 cm-l was assigned to a keto ester (other than ,Gketo). Since this band was not present (see later) in the otherwise identical spectra of Rhoecocoris or Poecilometis, it is inferred that a compound of structure 0
H
III R-C-C=C--C
/”
i
H
\
OH
together with an ester of this acid is probably present. Some caution, however, must be exercised in accepting the validity of the absorption bands other than that at 983 cm-l, since it has recently been found (HORWOOD, unpublished) that chromatography on Celite-nitromethane columns modifies the infra-red spectra of some 2,4_dinitrophenylhydrazones and introduces foreign bands in the carbonyl region. A similar, but much weaker, modification was observed for the 2,4-dinitrophenylhydrazone of n-pentanal after passage through an alumina chromatographic column. (c) Fraction 3 : unidentzj?ed. This fraction did not react with 2,4_dinitrophenylhydrazine and, since it was present in small amount and did not contribute materially to the characteristic odour of Nexara, it was not investigated further. (d) Fraction 4 : dec-2-enal. The 2,4_dinitrophenylhydrazone was soluble in light petroleum, gave h maxEtOH 372 rnp and h maxEtOH_NaOH455 rnp, identical with that of the 2,4_dinitrophenylhydrazone of authentic dec-2-enal, had a melting point of 127-128°C undepressed when mixed with the authentic dec-2-enal derivative of melting point 124125°C. The 2,4-dinitrophenylhydrazones of fraction 4 and of synthetic dec-2-enal moved in an identical fashion when compared on paper chromatograms by the method of LYNN et al. (1956). (e) Fraction 5 : tridecane. This was the most abundant fraction obtained from the steam distillate but made no important contribution to the characteristic stink bug odour. It was identical in retention time with an authentic sample of tridecane and this identity was confirmed by means of mass spectrographic examination. B. The bronze orange bug, Rhoecocoris
sulciventris
Five main fractions were also obtained by gas chromatography of the bronze orange bug odoriferous material (Table 3). (a) Fraction 1 : hex-Zenal. Identical with fraction 1 from Nexara, but present in much lower proportion in the steam distillate and hence an unimportant constituent of the odour.
CHARACTFZISTIC
117
ODOUR COMPONENTS OF THE SCENT OF STINK BUGS
(b) Fraction 2 : probably a dicarbonyl compound. The orange 2,4_dinitrophenylhydrazone was insoluble in light petroleum, soluble in benzene, gave h max&.,, 561 mp, and had a melting point of 262°C. Its infra-red 41° mtLYh m=EtOH-NsOH TABLE ~--GAS
CHROMATOGRAPHICFRACTIONS FROM Rhoecocoris sulciventris
Fraction
No.
Relative amount
Time (min)
Identified
as
Odour
20*
c
Hex-2-enal
28
+++++
40*
+++++ +++++ +++++ ++ ++++++ ++++++ ++++++ ++++++ ++++++ ++++++ ++++++ ++++++
Same as fraction 2 of Nezuru. Probably a dicarbonyl Ott-2-enal
67 108
Similar to Nezuru but quantity small to be important Minor odour component
Strongest and component
important
Minor odour component Minor odour contributor, occurs in largest amount
?t
Tridecane Identical fraction from Nezura
most
5
* Retention times of these fractions were idehtical with those of authentic respective aldehydes. t Did not react with 2,4-dinitrophenylhydrazine.
too
but
samples of the
spectrum was identical with that from fraction 2 of Nezara, except that there was no band at 1746 cm-i, so that only 0
H
II I R-C-C=C-C
//O
I
H
\
OH
and not its ester is present in this bug. (c) Fraction 3 : act-Zenal. The 2,4_dinitrophenylhydrazone was soluble in light petroleum, gave A maxxton 372 rnp and X maxEtoH_N&H454 mp, identical with that of the 2,4-dinitrophenylhydrazone of authentic act-Zenal, and had a melting point of 127-128°C identical with, and undepressed when mixed with, the 2,4-dinitrophenylhydrazone of authentic act-2-enal. The 2,4_dinitrophenylhydrazones of fraction 3 and of synthetic act-Zenal moved in an identical fashion when compared on paper chromatograms by the method of LYNN et aZ. (1956). 8
118
D. F. WATERHOUSE, D. A. Fo~ss, ANDR. H. HACKMAN
(d) Fraction 4 : unidentified. This was present in small amount, was a minor component of the odour, and did not react with 2,4*dinitrophenylhydr~ine. (e) Fraction 5 : tridecune. This was identical with fraction 5 from iVezara and was the most abundant fraction obtained. It was a minor odour component. C. Poecilometis strigatus The odoriferous secretion from this bug, which could be distinguished by smell from that of both Nexara and Rhoecocoris, was examined in less detail. However, the presence was detected of hex-Z-enal, act-Z-enal, and a dicarbonyl identical with fraction 2 from ~~oecoco~~. D. Mictis profana and Amorbus rubiginosus The preliminary examination of odoriferous material from Mictis and Amorbus gave essentially similar results. The yellow 2,4_dinitrophenylhydrazones were soluble in light petroleum and were fractionated on a Celite-nitromethane column. Both species yielded a single constituent which was identified as the n-hexanal derivative with the following properties: h maxEtoH 358 rnp, h maxEtonNILoW 432 mp, with a smaller peak at 518 rnp, which had disappeared after 2 hr, indicating a saturated aldehyde and not a saturated ketone, identical in behaviour with the 2,4_dinitrophenylhydrazone of authentic n-hexanal; melting point 107-108°C identical with, and undepressed when mixed with, the 2,4-dinitrophenylhydrazone of synthetic n-hexanal; these natural and synthetic derivatives moved in an identical fashion on paper chromatograms (LYNN et al., 1956). Synthetic n-hexanal, which was purified on a silicone-oil gas chromatographic column, was appraised as contributing importantly to the odours of both Mictis and Amorbus, although other materials were also present in the odoriferous secretions to give each bug a characteristically different odour. DISCUSSION To the human nose the odours produced by all five bugs are distinct, although there is a far greater basic similarity within the families Pentatomidae and Coreidae than between these families. It is interesting, therefore, to note that a six-carbon aldehyde contributed to the odour of all five bugs. In two of the bugs act-Z-enal was an important constituent and in one bug dec-Zenal. The compounds reported in Table 1 are probably the major contributors to the odours of the Pentatomidae investigated. However, in addition to n-hexanal there are certainly other important, unidentified odour constituents in the two coreid bugs. The sharp amyl alcohollike odour reported by MOODY(1930) for the coreid A. tristis suggests that it may be basically similar to that of the coreid bugs we have examined. All of the compounds identified in this study have been isolated from skim milk, which has acquired an oxidized (cardboard) flavour (FORSSet al., 1955). In addition some of the above constituents are also known to occur naturally, although mainly in plants (GUENTHER,1949). Thus n-hexanal is found in various Eu~~l~~tus oils, where it contributes to the disagreeable properties of certain of these oils. In view of this it is relevant to note that Amorbus lives on eucalypts.
CHARACTERISTIC
ODOUR COMPONENTSOF THE SCENTOF STINKBUGS
119
Much more widespread in its distribution in plants is hex-Z-enal, which has been reported in the oils derived from the green leaves of numerous plant species (e.g. tea, mulberry, acacia) (GUNTHER, 1949). Because of its strong and pungent odour of green leaves, hex-2enal is sometimes used in perfumes and flavours. It is said that it forms part of the flavour of green tea and that the aroma of tea is primarily due to this compound (see ROTH et al., 1956). Hex-2-enal and hex-3-enol are together responsible for the attraction of silkworm larvae to leaves (WATANABE, 1959). Hex-Zenal was found to be one of the carbonyl compounds responsible for the odour of whale oil (see ROTH et al., 1956), although it may well have been formed in the oil after extraction, for it has also been isolated from a variety of oxidized fats and oils. Thus FORSSet al. (1955, 1960a) have identified it in oxidized skim milk and butterfat. Hex-Zenal and other unsaturated aldehydes are thought to be derived from unsaturated fatty acids through hydroperoxide formation and cleavage at the a-methylene group (BADINGS,1959; SWIFT et al., 1949). The only fully substantiated record of the natural occurrence of hex-Z-enal in animals, therefore, appears to be the careful work of ROTH et al. (1956) from both adult males and females of the cockroach EurycotisJIoridana (Walker). Pure tram hex-Zenal was the only material present in the yellow liquid produced by a gland opening on the ventral surface of the abdomen. Interestingly, in an old report, HEBARD(1917) states that the odour of this cockroach strongly suggests that of the pentatomid Brochymena annulata (E.). A related compound, tram hex-2-enol-l-acetate, occurs in abdominal glands of males of the waterbug, Be~sto~ indica Vitalis, where it is thought perhaps to function as a sex odour (BUTENANDTand TAM, 1957). This material is employed in south-east Asia as a seasoning for fatty foods. An unidentified aliphatic LY,p-unsaturated ketone has been isolated from the defensive glands of the water beetle Dytiwus marginalis (SCHILDKNECHTand HOLOUBEK,1959). Naturally occurring act-Zenal does not appear to have been reported, although it is formed during oxidation of skim milk, contributes to cardboard flavours ( FORSS et al., 1955), and has been isolated from oxidized cottonseed oil (SWIFT et al., 1949). n-Octanal is an important constituent of orange oil (GUENTHER, 1949),which isof interest in view of the great importance of act-Zenal in the odour of the bronze orange bug which lives on citrus. Non-2-enal has been isolated from cucumbers, oxidized skim milk, and oxidized butterfat (FORSS et al., 1955, 1960a, b) although it has not been detected in the five bug odours examined. Dee-2-enal has been found in the essential oii of several plants, including the orange (GUENTHER, 1949),but does not appear to have been reported previously from animals. It does, however, occur in oxidized skim milk (FORSSet al., 1955). The mechanism of production of the odoriferous materials by the bugs is unknown. In view of the occurrence in plants of many of the materials responsible it is possible that some constituents are simply con~en~ated from the very large volume of sap passed through the alimentary canal and that the amount of odoriferous material accumulated by the bug varies with the amount available from
120
D. F.
WATERHOUSE, D. A. Foass,
AND
R. H.
HACKMAN
the host plant. This might apply more particularly to species with a restricted host plant range. However, this is unlikely to be the only method of production. Thus the odour of the green vegetable bug (Nezura) is quite characteristic of this species although it may feed on any of a very wide range of plants. These plants presumably would provide unsaturated aldehydes in very different amounts and, unless Nexara possessed a special mechanism for taking up the wanted constituents only in certain definite proportions and rejecting closely related compounds, the components of the sap of the host plant would tend to influence greatly the nature of the odour. We have not determined whether there are quantitative differences between the odour components of samples of Nezuru collected at different times, although both qualitatively and to the nose they are similar. The larval odours are also similar to the nose. It is probable, therefore, that the odour components are produced, at least in part, metabolically and that a more or less constant composition is maintained. No information is available on their possible precursors, but this topic is worthy of experimental investigation. It is generally agreed that these odours are probably of repugnatorial value to the bugs which produce them (MOODY,1930; WEBER,1930), although HEIKERTINGER (1922) dissents from this opinion. There is no indication that they regularly play any part simply as excretory products, as sex attractants, or to assist in species recognition, for the adults are seldom gregarious, although young larvae of some species (e.g. of Nezuru viridulu) may be. Some interesting experiments have been carried out by EISNERet al. (1959) on the cockroach E. floridunu, which can spray its hex-2-enal as far as 3 ft when disturbed and can aim with some degree of-accuracy in the general direction of the stimulus. Ants (Pogonomyrmex bud&s (Latr.) and Cumponotus pennsylvunicus (Degeer) ) were repelled, although an unidentified tarantula which routinely feeds on Eurycotis in the laboratory was not affected (EISNER, personal communication). When a bug is picked up the characteristic disagreeable odour is usually evident immediately. In many instances the oily liquid is merely forced out on to the surface of the cuticIe where rapid evaporation takes place. Some bugs, however, are able to emit a spray, as for example Anusu t&is which, when strongly irritated, may discharge several drops of liquid as far as 5 in. in a latero-posterior direction from the body (MOODY, 1930). The pentatomid Tesserutomu pupillosu Thunb. is reported to be able to eject its liquid to a distance of 6-10 in. (MUIR, 1907). Under some circumstances the secretions may be toxic even to the bugs for, when a large number of Rhoecocoris were placed in a lightly closed container on a warm day, many were dead within an hour. It is evident that, for a particular compound, attraction, repellency, or toxic action may be simply a function of concentration. Experiments carried out with an unidentified pentatomid and several coreids demonstrated a strong deterrent effect to attack by predators, including ants, amphibia, birds, and mice. With birds there was obvious rejection only when the secretion appeared to have hit the eyes. With some species the secretion is ejected in response to movement or vibration nearby and no direct contact was necessary. Thus a caged bird (Cyunocittu stellerz] was repeatedly sprayed when merely landing
CHARACTERISTIC ODOURCOMPONENTS OF THE SCENTOF STINKBUGS
121
near a coreid. Moreover, this bug aimed its spray quite accurately by adjusting body orientation and stance to enable it to fire a broadside and then discharged its gland from the stimulated side of the body only (EISNFR, personal communication). Ac~~~~~t~We wish to acknowledge the help of W. STARKand J. F. Ho~woon of the Dairy Research Section, C.S.I.R.O., the former for assistance with gas chromatography and the latter for infra-red analyses. REFERENCES BADINGSH. T. (1959) Isolation and identification of csrbonyl compounds formed by autooxidation of ammonium linoleate. J. Amer. OiZ Chem. Sot. 36, 448-650. BUTENANDTA. and TAM N. (1957) Ub er einen g~~hle~h~spezi~s~hen DuftstofI der Wasserwanze 3e~ost~ indica Vitalis (Lethocew indicus Lep.). Hoppe-Sag. Z. 308, 277-283. CARIUSL. (1860) Ueber eine neue Slure der Reihe Cn Hrn_a 0,. Ann. Chem. Phwm. 114, 147-156. DAY E. A., B~ETTE R., and KEENEYM. (1960) Identification of volatile carbonyl compounds from cheddar cheese. J. Dairy Sci. 43,463-474. EISNERT., MCKITTRICKF.,‘and PAYNER. (1959) Defense sprays of roaches. Pest Control 27 (6), 9, 11-12, 44-45. FORSSD. A., DUNSTONEE. A., and STARKW. (1960a) Fishy Aavour in dairy products-II. The volatile compounds associated with fishy flavour in butterfat. J. Dairy Res. 27, 211-219. Fonts D. A., DUNSTONEE. A., and STARKW. (1960b) Aldehydes of cucumber. Unpublished work. Fonts D. A., PONT E. G., and STARKW. (1955) The volatile compounds associated with oxidized flavour in skim milk. y, Dairy Res. 22,91-102. GUENTHERE. (1949) The Essentaial Oils, Vol. 2. Van Nostrand, New York. HEBARDM. (1917) The Blattidae of North America north of the Mexican boundary. Mem. Amer. ent. Sac. 2, l-284. HEIKXRTINGERF. (1922) Sind die Wanzen (Hemipte~-Heteropte~) durch Ekelgeruch geschiitz 7 Biol. 261. 42, 441-464. JOHANSSON A. S. (1957) The functional anatomy of the metathoracic scent glands of the milkweed bug, Oncopeltusfasciatus (Dallas) (Heteroptera: Lygaeidae). Nor& mt. Tidsskr. 10, 95-109. LYNN W. S., STEELEL. A., and STAPLEE. (1956) Separation of 2,4-dinitrophenylhydraxones of aldehydes and ketones by paper chromatography. AnaZyt. Chem. 28,132-133. MALOUF N. S. R. (1933) Studies on the internal anatomy of the “stink bug” Nezuru viriduln L. Br&. Sot. est. .&ypte 17, 96-119. MCJ~DYD. L. (1930) The morphology of the repugnatory glands of Anasa tristis De Geer. Ann. ent. Sot. Amer. 23,81-104. MUIR F. (1907) Notes on the stridulating organ and stink glands of Tessaratomu papillosa, Thunb. Trans. ent. Sot. Land. (1907), 256-258. ROTH L. M., NIECISCHW. D., and STAHL W. H. (1956) Occurrence of 2-hexenal in the cockroach Eurycotis flwidana. Science 123, 670-671. SCHILDKNECHT H. and HOLOUBBKK. (1959) Uber einen Inhaltsstoff der Wehrdriisen des Gelbrandkltiers-III. Mitteilung ilber Insektenabwehrstoffe. Astgew. Chem. 71,524-525. Sw~pr C. E., O'CONNOR R. T., BROWSEL. E., and DOLLEARF. G. (1949) The aldehydes produced during the autoxidation of cottonseed oil. J. Ames. Oil Chem. Sot. 26,297-300. WATANABET. (1959) Studies on the volatile components of mulberry leaves-V. Attraction of p-y-hexenol and u-/3-hexenal to silkworm larvae. J. se&. Sci., Tokyo 28,23-26. WEERR H. (1930) Biologic der Hemipteren. Springer, Berlin.