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Volatile scent gland components of some tropical Hemiptera
J. Insect Physiol., 1975, Vol. 21, pp. 1219 to 1224. Pergamon Press. Printed in Great Britain.
VOLATILE SCENT GLAND COMPONENTS SOME TROPICAL HEMIPTERA
OF
J. K. MACLEOD,~I. HOWE,’ J. CABLE,~J. D. BLAKE,** J. T. BAEER,~and D. SMITHY 1 Research School of Chemistry, Australian National University, P.O. Box 4, Canberra, A.C.T. 2600; 2 Roche Research Institute of Marine Pharmacology, P.O. Box 255, Dee Why, N.S.W. 2099; and a Department
of Primary
Industries,
P.O. Box 83, Nambour,
Qld. 4560, Australia
(Received 12 November 1974) Abstract-The major volatile chemical constituents present in the scent glands of the bronze orange bug (Musgrawia sulciventris) and the larger horned citrus bug (Biprorulus bibax) have been identified by gas chromatography and mass spectrometry. The amount of trans-2-octenyl acetate present in M. sulciventris showed considerable seasonal variation but no evidence was found to support the postulated role of this compound as a sex pheromone in these insects.
INTRODUCTION THE PRESENCEof pheromones has been implied in a number of families of the Order Hemiptera (MACCONNELLand SILVERSTEIN,1973) but the only evidence in the family Pentatomoidae arises from the investigations by MITCHELL and MAU (1971) of Nezara viridula (L.). Using a simple Y-tube olfactometer these workers were able to demonstrate that the male produces a substance, the chemical nature of which was not elucidated, that is highly attractive to the females. GILBY and WATERHOUSE(1965) have identified 18 compounds which account for 99.9 per cent of the scent gland composition of this bug, but it is not known if any of these compounds are responsible for the pheromonal activity reported by MITCHELLand MAU. BUTENDANTand TAM (1957) identified truns-2hexenyl acetate as the major volatile component in the abdomina1 gland secretion obtained from the male giant belostomatid water bug, Lethocerus indicus (L. & S.) and proposed that since this compound was not found in the female it may play a r6le as a sex attractant. The identification of trans-2-octenyl acetate in the defensive secretion of the bronze orange bug, Musgraewia sulciventris, and trans-2-decenyl acetate in the larger horned citrus bug, Biprorulus bibax, led PARK and SUTHERLAND (1962) to speculate that these compounds may act as sex attractants by analogy with the proposed r61e of 2-hexenyl acetate in L. indicus. PATTENDENand STADDON(1970) subsequently showed that 2-hexenyl acetate is present in the glands of the female as well as the male giant water bug and have proposed that since the compound is not sex-specific it probably plays no r6le as a pheromone. Despite this finding, 2-octenyl acetate and l
Now at the Sugar Research Institute, P.O. Box 21,
Mackay, Qld. 4740, Australia.
2-decenyl acetate are still mentioned (MACCONNELL and SILVERSTEIN,1973) as possible pheromones in M. sulciventris and B. bibax respectively although there is no evidence to support this spectdation. In addition to expanding the earlier analyses of the chemical constituents of the defensive secretions of the bronze orange bug and the larger horned citrus bug using gas chromatographic-mass spectrometric techniques we were interested in investigating the r81e of 2-octenyl acetate in the bronze orange bug and in searching for any evidence of chemical communication between the male and female insects. The bronze orange bug and the larger horned citrus bug are pests of economic importance in the citrus orchards of N.S.W. (HELY, 1968) and Queensland (S~~IITH, 1973). In Queensland the present method of control involves spraying the trees with carbaryl, methidathion, or promecarb. MATERIALS
AND METHODS
Insects M. sulciventris and B. bibax specimens were collected during the summer months from citrus orchards near Nambour in Southern Queensland. The M. sulciventris adults employed in the behavioural studies were collected in early February from a field population which was mating actively. The insects were separated according to sex 1 week prior to conducting the ‘f-tube experiments. Collection of scent The scent gland constituents were collected either by causing the bug to eject the contents into a O-3 ml Reacti Vial (Pierce Chemical Co., Illinois) or by dissection of the gland and contents from specimens that had been anaesthetized with COB. Collections were made in Nambour and the Teflon-sealed vials
1219
J. K. MACLEOD,I. HOWE, J. CABLE,J. D. BLAKE,J. T. BAKER,AND D. SMITH
1220
maintained at dry-ice temperature during transit to Canberra. These samples showed no variation from those obtained from insects which were transported live to Canberra. Gas chromatography-mass
spectrometry
Gas chromatographic separation of the scent gland components was achieved using the following three columns. Column 1: 2 m x 3 mm Silanized stainless steel packed with 3% SE-30 on Varaport 30. Column 2: 2 m x 3 mm glass packed with 10% FFAP on Gas Crom Q. Column 3 : 2 m x 3 mm Silanized stainless steel packed with 3% OV-1 on Gas Chrom Q. The following procedure was employed in the GC analysis. An injection of O-1 ~1 of neat scent gland secretion was made with the column oven at 35°C. After a delay of 4 min the GC oven was programmed from 35 to 200°C at a rate of 4”C/min. The retention times and elution temperatures were noted as the compounds were detected and these results were found to be reproducible ( f 5 per cent) following the above procedure. On-line gas chromatography-mass spectrometry (GC-MS) was performed using the following instruments. (1) A Varian 1740 gas chromatograph interfaced to a GEC-AEI MS-902 mass spectrometer using a commercially available Varian V 5600 dual membrane separator. The details of the operation of this GC-MS combination have been published in an earlier report from this laboratory (BAKERet al., 1972). (2) AVarian 1400 gas chromatograph coupled to a Varian MAT CH-7 mass spectrometer via a line-of-sight Watson-Biemann separator. (3) A Varian 1440 gas chromatograph interfaced to a Varian MAT 111 mass spectrometer ia a variable slit separator. Behavioural
studies
Two glass Y-tube olfactometers were constructed with arms 70 cm long and 70 mm i.d. End cages were made by cutting a circular hole in the side of 0.5 gal ice-cream containers and lining the aperture with split Tygon tubing to provide a good seal between the container and the glass tubing. A small hole was cut in the lid of each container to allow air to be drawn through the system by attachment to a water pump. Alternatively, air was blown through the system using a compressor equipped with traps for oil and water vapour. RESULTS
AND DISCUSSION
Gas chromatography-mass
spectrometry
M. sulciventris. The scent gland contents form a biphasic system similar to that noted by GILBY and WATERHOUSE (1965) in the pentatomoid N. viridula. The upper clear phase consists primarily of hydrocarbons with tridecane accounting for at least 50
per cent of the phase. The yellow phase contains a large amount of tridecane but has a higher proportion of more polar compounds such as 4-0x0-2hexenal. In the adult where 20 to 40 ~1 of secretion could be obtained, the GC-MS investigation was carried out on the neat lower yellow phase. In the instars the quantity of secretion available ismuchless and carbon disulphide washings of the excised gland and contents were utilized for the study of the components of the scent gland. The instars and adults contained the same compounds in the scent gland secretion although some variation in concentrations was noted. Part of this variation is due no doubt to the differential solubility of the components in carbon disulphide as compared with the composition of the neat phases. The retention times and identity of 12 components which comprise at least 98 per cent of the scent gland secretion are given in Table 1. The compounds were identified by GC retention times and mass spectral comparison with authentic samples. The identification of GC peaks 1 to 8 confirmed the earlier analysis of the scent gland components by BAKER et aE. (1970) which was carried out using GC and chemical methods. 2-Octenyl acetate and dodecane have the same retention time on column 1 and since both compounds exhibit a molecular ion at m/e 170 the initial search for 2-octenyl acetate using this column for GC-MS presented problems. The mass spectrum of 2-octenyl acetate exhibits characteristic ions at m/e 128 (M+ - 42) and m/e 110 (Mf - 60), however, and using this information it was possible to determine from the mass spectra whether GC peak No. 6 was predominantly dodecane, 2-octenyl acetate, or a mixture. Quantitation of the 2-octenyl acetate was possible through the use of column 2 where the ester is well separated from dodecane. The scent gland secretion obtained from insects collected in early December contained approximately 3 per cent of 2-octenyl acetate. The secretions collected during late December and early January, however, contained about one-third of this quantity of 2-octenyl acetate. Only a trace amount of the ester could be detected in the material obtained in late January and early February. No significant or consistent variation was noted between the male and female insects collected at the same time during the summer or between the insects before and after mating. Gas chromatographic peaks 10 and 11 were identified as tridecanyl butyrate and tetradecanyl butyrate. In general, the simple esters obtained from other pentatomoids are unsaturated, e.g. 2-hexenyl acetate, 2-octenyl acetate, 2-decenyl acetate, and the longer-chain esters which have been identified as pheromones in other insects have remote sites of unsaturation, e.g. cis-9-tetradecenyl acetate (JACOBSON et al., 1970) and cis-lltetradecenyl acetate (ROELOFS and TETTE, 1970; KLUN and BRINDLEY, 1970). The long-chain esters
Volatile scent gland components Table
I. Retention
1 2 3 4 : 6 7 8 9 10 11
Compound
of the
Column 2 .___
1
Temperature (“C)
Trans-2-hexenal 4-Oxo-trans-2-hexenal Tram-2-octenal Undecane
55 72 90 100
4-0x0-trans-2-octenal Dodecane Tram-2-octenyl acetate Trans-2-decenal Tridecane Pentadecane Tridecanyl butyrate Tetradecanyl butyrate
102 115 115 120 129 141 196 205
found in the scent gland secretion from M. sulciventris have been identified by GC and MS comparison with authentic samples, but it is not known if these saturated esters have any pheromonal r81e in this insect. As mentioned earlier, the compounds present in the scent gland of the larval stages of the bronze orange bug are all found in the adult. No 2-octenyl acetate was detected in the larvae but in view of the wide seasonal variation of the abundance of this compound in the adult insect, and since large numbers of instars have not been examined, it would be unwise to place too much emphasis on this point. As a genera1 rule, the scent gland secretion from coreoids consists of saturated esters and aldehydes whereas that from pentatomoids is largely composed of o-/-3 unsaturated esters and aldehydes. While no difference in composition was noted between adults and larvae of the pentatomoid M. sulciventris, BAKER and JONES (1969) have reported that the Table 2.
1221
times (Rt) and temperatures of compounds identified by GC-MS scent gland secretion of M. sulciventris Column
Peak No.
of tropical hemiptera
Temperature Rt
(‘C)
min. sec. 8 40 13 10 17 20 19 30
68 124 101 54
23 19 23 25 27 30 43 45
30 55 30 05 20 05 10 20
__-
lit
min. sec. 11 35 26 20 20 30 7 30
72 116 128 90 112
12 24 27 17 23
adult coreoid Pternistria bispina (Stal) has a scent gland secretion composed of the expected saturated compounds whereas the larval secretion is composed of unsaturated aldehydes and esters. Thus while the generalization is very useful it probably should be restricted to adult insects. B. bibax. The scent gland contents form a taophase system similar to that encountered in M. sulciventris. The compounds identified in the yellow lower phase after examination by GC-MS are shown in Table 2. Apart from 2-decenol, which occurs to the extent of less than 1 per cent, the compounds listed have been identified as components of the scent gland secretions of other pentatomoids (CALAM and YOUDEOWI, 1968). The quantity of 2-decenyl acetate present is substantial and does not appear to be subject to the same seasonal variation in amount as was observed from 2-octenyl acetate in M. sulciventris. The small quantity of 2-decenol, identified by GC-MS
Retention times (Rt) and temperatures of compounds GC-MS of the scent gland secretion of B. bibax
identified by
Column 3 Peak SO.
1 2 3 4 5 6 7 8 9 10
Compound
Truns-2-hexenal 4-Oxo-tram-2-hexenal Trws-2-hexenyl acetate Undecane Dodecane Tranr-2-decenal 2-Decenol Tridecane Trons-2-decenyl acetate Pentadecane
25 10 30 ov 15
Percentage
10 10 10
Temperature
48 67 75 90 105 112 115 122 134 148
Rt min. sec. 6 15 11 00 12 50 16 40 20 25 22 00 22 40 24 20 27 20 30 10
1222
J. K. MACLEOD, I. HOWE,J. CABLE,J. D. BLAKE,J. T. BAKER,AND
D. SMITH
Table 3. Response of adult female bronze orange bugs to caged adult male bronze orange bugs in a Y-tube olfactometer No. of female bugs in Exp. No.
Females Caged released males
1
10
10
2*
10
10
Free carton
Target carton
Blank carton
Ytube
Time (hr)
8 2 2
0 0 0
0 5 1
2 3 7
t 10 2
* Citrus fruit placed in Y-tube. The results (see Table 3) demonstrate that the free females show no attractancy towards the caged males under the conditions of the experiment. In fact in experiment 1, half of the female population had moved into the blank carton after 10 hr, but none was found in the carton of caged males. It is possible that the females were repelled by the male defensive secretion (see other experiments below). In experiment 2, fresh citrus twigs, leaves, and fruits were placed in the Y-tube and after 2 hr, 7 out of 10 female bugs were found to be attached to the citrus material and preferred to remain there. (2) Male-to-female attractancy test: Caged female bronze orange bugs were placed in the target carton and free males released in another. The air current was maintained from the target and blank cartons towards the free carton (except where otherwise indicated). The results (see Table 4) demonstrate that the free males show no attractancy towards the caged females under the conditions of the experiment. Experiments 4 and 5 indicate that the bugs prefer to move towards the source of the air flow regardless of the contents of the carton towards which they are moving. Experiment 6 shows that the male bugs prefer to remain on the citrus fruit. (3) Female reaction to 2-octenyl acetate: A few drops of 2-octenyl acetate were placed in a O-3 ml Reacti Vial in the target carton and free females
comparison with an authentic sample, may be a consequence of the larger amount of 2-decenyl acetate present in these insects. The alcohol may play a part in the biosynthesis of the acetate or it may arise via a small amount of hydrolysis of the ester. Behavioural
studies
M. sulciventris. A Y-tube olfactometer was used to investigate possible attractancy of females towards males and vice versa, and to determine the behavioural response of the bugs to 2-octenyl acetate. The experiments were conducted at Nambour during February 1974, at a laboratory temperature of about 30°C. The bugs were in season to mate, although they were not necessarily virgin. Several mating pairs were found after a few hours when 10 males and 10 females were placed together in the same cage. (1) Female-to-mule attractancy test: Caged male bronze orange bugs were placed in the ‘target’ carton of the apparatus and free female bronze orange bugs were released in another carton (the ‘free’ carton). The air current (up to 10 l./min) was maintained from the target and blank cartons towards the free carton. The possibility of visual stimulus was excluded by partially masking the entrance to the target cage with filter paper.
Table 4. Response of adult male bronze orange bugs to caged adult female bronze orange bugs in a Y-tube olfactometer No. of male bugs in Exp. No.
Males released
Caged females
Free carton
Target carton
Blank carton
Ytube
Time (hr)
3
10
6
;
0 2
0 4
5 2
t 3
4*
10
6
0
9
0
1
1
5t
10
6
3
1
6
0
1
6f
10
6
1
0
0
9
2
* Air flow from target carton only. t Air flow from blank carton only. $ Citrus fruit placed in Y-tube.
Volatile scent gland components of tropical hemiptera were released in another. The air current was maintained from the target and blank cartons towards the free carton. The results (see Table 5) show that the females are not attracted by 2-octenyl acetate under the conditions of the experiment. In fact they appear to be repelled by the chemical, possibly associating the scent with an alarm secretion. In each experiment (7 and 8) 1 female ejected her defensive secretion in the Y-tube. It has been observed in the field that one ejection by a bronze bug tends to trigger off ejection by other bugs. The ejections observed here would support association of 2-octenyl acetate by the bugs with a defensive (or alarm) secretion. Table 5. Response of adult female bronze orange bugs to 2-octenyl acetate in a Y-tube olfactometer No. of bugs in Free carton
Exp. No.
Females released
7
10
3 3
8
10
4 3
Target carton
Blank carton
Ytube
Time (hr)
1 2
1 5
5 0
t
0 2
3 5
3 0
2 2
(4) Male reaction to 2-octenyl acetate: Males were released in one carton under the same conditions as the females in (3) above. The results (see Table 6) show that the males are not attracted by 2-octenyl acetate under the conditions of the experiment. In each experiment (9 and 10) one male ejection was seen to take place. Again this supports an association by the bugs of 2-octenyl acetate with a defensive secretion. Table 6. Response of adult male bronze orange bugs to 2-octenyl acetate in a Y-tube olfactometer No. of bugs in Males released
Free carton
9
10
7
10
10
3
Exp. No.
Blank carton
Ytube
Time (hr)
1
1
1
3
3
4
0
3
Target carton
B. bibax. Further Y-tube olfactometer tests will be undertaken when the insects become available since these insects have been examined only very cursorily from this viewpoint to date.
1223
have been reported in other pentatomoids. The remaining two compounds, tridecanyl butyrate and tetradecanyl butyrate, constitute the first reported examples of long-chain saturated esters occurring in the Pentatomoidae. Lower homologues of these esters have been noted in coreids, but the lower molecular weight esters reported in pentatomoids are usually unsaturated compounds, e.g. 2-hexenyl acetate, 2-octenyl acetate, and 2-decenyl acetate. The role of the two long-chain saturated esters in the secretion from M. sulciventris is not known. No evidence was found for a long-range sex attractant in M. sulciventris. The quantity of 2-octenyl acetate present displays considerable seasonal variation, but the observations made with a Y-tube olfactometer do not support the postulated role of this compound as a pheromone. It appears to function as an alarm substance as might be expected for a component of a defensive secretion. Careful observation of these insects indicates that a short-distance attraction mechanism between sexes may operate but it is not known whether this phenomenon involves chemical communication. In the orchard it is also not uncommon for M. sulciventris to aggregate in clumps of up to 20 individuals. The compounds identified in the scent gland secretion from B. bibax have been previously reported in the Pentatomoidae with the exception of 2-decenol. The corresponding unsaturated aldehyde and acetate are major components of the secretion and the small quantity of 2-decenol may be biosynthetically significant or it may arise from either of these compounds. The concentration of 2-decenyl acetate found in B. bibax is much larger than that of 2-octenyl acetate in M. sulciventris and does not apear to be subject to the seasonal variation. No behavioural studies have been carried out with B. bibax to investigate the possible role of 2-decenyl acetate as a pheromone but it is hoped to investigate this aspect when the insects become available in the summer. Acknowledgeraenrs-Financialsupport through a Rural Credits Development Fund Grant (R.C.D. F-4) is gratefully acknowledged. The assistance and co-operation of Mr. TONY WHILEY and the staff of the Queensland Department of Primary Industries’ Pineapple Research Laboratory proved invaluable during behavioural studies conducted at these premises in February 1974.
REFERENCES BAKERJ. T., BLAKE J. D., MACLEODJ. K., IRONSIDED. A., and JOHNSONI. C. (1972) The volatile constituents of the fruit spotting bug Amblypelta nitida. Aust. J.
Chem. 25, 393-400. CONCLUSION Ten of the 12 compounds indentified in the scent gland secretion obtained from M. sulciventris
BAKERJ. T. and JONES P. A. (1969) Volatile constituents of the scent gland reservoir of the nymph of the coreoid Pternistra bispina StHI. Aust. J. Chem. 22,
1793-1796.
1224
J. K. MACLEOD, I. HOWE, J. CABLE, J. D. BLAKE, J. T. BAKER, AND D. SMITH
BAKERJ. T., JON= P. A., MACLEOD J. K., and SMITH D. (1970) Scent gland constituents of tropical Hemiptera. ANZAAS Conference, X6-23 August, Port Moresby, TPNG. BUTENDANTA. and TAM N. (1957) Uber einen geschlechtsspezifischen Dufstoff der Wasserwanze Belostoma indica Vitalis (Letkocerus indicus Lep.). HoppeSeyler’s Z. pkysiol. Ckem. 308,277-283. CALAMD. H. and YOUDEOWIA. (1968) Identification and functions of secretion from the posterior scent gland of the fifth instar larva of the bug Dysderus intermedius. J. Insect Pkysiol. 14, 1147-1158. GILBY A. R. and WATERHOUSED. F. (1965) The composition of the scent of the green vegetable bug Nezara viridula. Proc. R. Sot. Lond. (B) 162, 105-120. HELY P. C. (1968) The entomology of citrus in New South Wales. Aust. ent. Sot.. Miscellaneous Publication No. 1. JACOBSON M., REDFERN R. E., JONES W. A., and ALDRIDCE M. H. (1970) Sex pheromones of the southern armyworm moth: isolation, identification and synthesis. Science, Wash. 170, 542-544. KLUN J. A. and BRINDLEY T. A. (1970) Cis-lltetradecenyl acetate, a sex stimulant of the European corn borer. J. econ. Ent. 63, 779-780.
MACCONNELLJ. G. and SILVER~TEINR. M. (1973) Recent results in insect pheromone chemistry, references to Cimicidae, Lygaeidae, Miridae, Pyrrhocoridae and Reduviidae. &geeu. Chem. int. Ed. i2, 644-654. MITCHELL W. C. and MAU W. L. F. (1971) Resoonse of the female southern green stink bug and its parasite, Trickopoda pennipes to male stink bug pheromones. J. econ. Ent. 64, 856-859. PARK R. J. and SUTHERLANDM. D. (1962) Volatile constituents of the bronze orange bug Rlrorrororis sulciventris. Awt. J. Ckem. 15, 172-174. PATTENDENG. and STADDONB. W. (1970) Observations on the metasternal scent glands of Letkocerus spp. (Heteroptera: Belostomatidae). Ann. efrt. Sor. Am. 63, 900-901. ROELOFSW. L. and TETTE P. L. (1970) Sex pheromone of the obliaue-banded leaf roller moth. Nature. Lond. 226, 1172. %ITH D. (1973) Unpublished report, Queensland Department of Primary Industries.
VOLATILE SCENT GLAND COMPONENTS SOME TROPICAL HEMIPTERA
OF
J. K. MACLEOD,~I. HOWE,’ J. CABLE,~J. D. BLAKE,** J. T. BAEER,~and D. SMITHY 1 Research School of Chemistry, Australian National University, P.O. Box 4, Canberra, A.C.T. 2600; 2 Roche Research Institute of Marine Pharmacology, P.O. Box 255, Dee Why, N.S.W. 2099; and a Department
of Primary
Industries,
P.O. Box 83, Nambour,
Qld. 4560, Australia
(Received 12 November 1974) Abstract-The major volatile chemical constituents present in the scent glands of the bronze orange bug (Musgrawia sulciventris) and the larger horned citrus bug (Biprorulus bibax) have been identified by gas chromatography and mass spectrometry. The amount of trans-2-octenyl acetate present in M. sulciventris showed considerable seasonal variation but no evidence was found to support the postulated role of this compound as a sex pheromone in these insects.
INTRODUCTION THE PRESENCEof pheromones has been implied in a number of families of the Order Hemiptera (MACCONNELLand SILVERSTEIN,1973) but the only evidence in the family Pentatomoidae arises from the investigations by MITCHELL and MAU (1971) of Nezara viridula (L.). Using a simple Y-tube olfactometer these workers were able to demonstrate that the male produces a substance, the chemical nature of which was not elucidated, that is highly attractive to the females. GILBY and WATERHOUSE(1965) have identified 18 compounds which account for 99.9 per cent of the scent gland composition of this bug, but it is not known if any of these compounds are responsible for the pheromonal activity reported by MITCHELLand MAU. BUTENDANTand TAM (1957) identified truns-2hexenyl acetate as the major volatile component in the abdomina1 gland secretion obtained from the male giant belostomatid water bug, Lethocerus indicus (L. & S.) and proposed that since this compound was not found in the female it may play a r6le as a sex attractant. The identification of trans-2-octenyl acetate in the defensive secretion of the bronze orange bug, Musgraewia sulciventris, and trans-2-decenyl acetate in the larger horned citrus bug, Biprorulus bibax, led PARK and SUTHERLAND (1962) to speculate that these compounds may act as sex attractants by analogy with the proposed r61e of 2-hexenyl acetate in L. indicus. PATTENDENand STADDON(1970) subsequently showed that 2-hexenyl acetate is present in the glands of the female as well as the male giant water bug and have proposed that since the compound is not sex-specific it probably plays no r6le as a pheromone. Despite this finding, 2-octenyl acetate and l
Now at the Sugar Research Institute, P.O. Box 21,
Mackay, Qld. 4740, Australia.
2-decenyl acetate are still mentioned (MACCONNELL and SILVERSTEIN,1973) as possible pheromones in M. sulciventris and B. bibax respectively although there is no evidence to support this spectdation. In addition to expanding the earlier analyses of the chemical constituents of the defensive secretions of the bronze orange bug and the larger horned citrus bug using gas chromatographic-mass spectrometric techniques we were interested in investigating the r81e of 2-octenyl acetate in the bronze orange bug and in searching for any evidence of chemical communication between the male and female insects. The bronze orange bug and the larger horned citrus bug are pests of economic importance in the citrus orchards of N.S.W. (HELY, 1968) and Queensland (S~~IITH, 1973). In Queensland the present method of control involves spraying the trees with carbaryl, methidathion, or promecarb. MATERIALS
AND METHODS
Insects M. sulciventris and B. bibax specimens were collected during the summer months from citrus orchards near Nambour in Southern Queensland. The M. sulciventris adults employed in the behavioural studies were collected in early February from a field population which was mating actively. The insects were separated according to sex 1 week prior to conducting the ‘f-tube experiments. Collection of scent The scent gland constituents were collected either by causing the bug to eject the contents into a O-3 ml Reacti Vial (Pierce Chemical Co., Illinois) or by dissection of the gland and contents from specimens that had been anaesthetized with COB. Collections were made in Nambour and the Teflon-sealed vials
1219
J. K. MACLEOD,I. HOWE, J. CABLE,J. D. BLAKE,J. T. BAKER,AND D. SMITH
1220
maintained at dry-ice temperature during transit to Canberra. These samples showed no variation from those obtained from insects which were transported live to Canberra. Gas chromatography-mass
spectrometry
Gas chromatographic separation of the scent gland components was achieved using the following three columns. Column 1: 2 m x 3 mm Silanized stainless steel packed with 3% SE-30 on Varaport 30. Column 2: 2 m x 3 mm glass packed with 10% FFAP on Gas Crom Q. Column 3 : 2 m x 3 mm Silanized stainless steel packed with 3% OV-1 on Gas Chrom Q. The following procedure was employed in the GC analysis. An injection of O-1 ~1 of neat scent gland secretion was made with the column oven at 35°C. After a delay of 4 min the GC oven was programmed from 35 to 200°C at a rate of 4”C/min. The retention times and elution temperatures were noted as the compounds were detected and these results were found to be reproducible ( f 5 per cent) following the above procedure. On-line gas chromatography-mass spectrometry (GC-MS) was performed using the following instruments. (1) A Varian 1740 gas chromatograph interfaced to a GEC-AEI MS-902 mass spectrometer using a commercially available Varian V 5600 dual membrane separator. The details of the operation of this GC-MS combination have been published in an earlier report from this laboratory (BAKERet al., 1972). (2) AVarian 1400 gas chromatograph coupled to a Varian MAT CH-7 mass spectrometer via a line-of-sight Watson-Biemann separator. (3) A Varian 1440 gas chromatograph interfaced to a Varian MAT 111 mass spectrometer ia a variable slit separator. Behavioural
studies
Two glass Y-tube olfactometers were constructed with arms 70 cm long and 70 mm i.d. End cages were made by cutting a circular hole in the side of 0.5 gal ice-cream containers and lining the aperture with split Tygon tubing to provide a good seal between the container and the glass tubing. A small hole was cut in the lid of each container to allow air to be drawn through the system by attachment to a water pump. Alternatively, air was blown through the system using a compressor equipped with traps for oil and water vapour. RESULTS
AND DISCUSSION
Gas chromatography-mass
spectrometry
M. sulciventris. The scent gland contents form a biphasic system similar to that noted by GILBY and WATERHOUSE (1965) in the pentatomoid N. viridula. The upper clear phase consists primarily of hydrocarbons with tridecane accounting for at least 50
per cent of the phase. The yellow phase contains a large amount of tridecane but has a higher proportion of more polar compounds such as 4-0x0-2hexenal. In the adult where 20 to 40 ~1 of secretion could be obtained, the GC-MS investigation was carried out on the neat lower yellow phase. In the instars the quantity of secretion available ismuchless and carbon disulphide washings of the excised gland and contents were utilized for the study of the components of the scent gland. The instars and adults contained the same compounds in the scent gland secretion although some variation in concentrations was noted. Part of this variation is due no doubt to the differential solubility of the components in carbon disulphide as compared with the composition of the neat phases. The retention times and identity of 12 components which comprise at least 98 per cent of the scent gland secretion are given in Table 1. The compounds were identified by GC retention times and mass spectral comparison with authentic samples. The identification of GC peaks 1 to 8 confirmed the earlier analysis of the scent gland components by BAKER et aE. (1970) which was carried out using GC and chemical methods. 2-Octenyl acetate and dodecane have the same retention time on column 1 and since both compounds exhibit a molecular ion at m/e 170 the initial search for 2-octenyl acetate using this column for GC-MS presented problems. The mass spectrum of 2-octenyl acetate exhibits characteristic ions at m/e 128 (M+ - 42) and m/e 110 (Mf - 60), however, and using this information it was possible to determine from the mass spectra whether GC peak No. 6 was predominantly dodecane, 2-octenyl acetate, or a mixture. Quantitation of the 2-octenyl acetate was possible through the use of column 2 where the ester is well separated from dodecane. The scent gland secretion obtained from insects collected in early December contained approximately 3 per cent of 2-octenyl acetate. The secretions collected during late December and early January, however, contained about one-third of this quantity of 2-octenyl acetate. Only a trace amount of the ester could be detected in the material obtained in late January and early February. No significant or consistent variation was noted between the male and female insects collected at the same time during the summer or between the insects before and after mating. Gas chromatographic peaks 10 and 11 were identified as tridecanyl butyrate and tetradecanyl butyrate. In general, the simple esters obtained from other pentatomoids are unsaturated, e.g. 2-hexenyl acetate, 2-octenyl acetate, 2-decenyl acetate, and the longer-chain esters which have been identified as pheromones in other insects have remote sites of unsaturation, e.g. cis-9-tetradecenyl acetate (JACOBSON et al., 1970) and cis-lltetradecenyl acetate (ROELOFS and TETTE, 1970; KLUN and BRINDLEY, 1970). The long-chain esters
Volatile scent gland components Table
I. Retention
1 2 3 4 : 6 7 8 9 10 11
Compound
of the
Column 2 .___
1
Temperature (“C)
Trans-2-hexenal 4-Oxo-trans-2-hexenal Tram-2-octenal Undecane
55 72 90 100
4-0x0-trans-2-octenal Dodecane Tram-2-octenyl acetate Trans-2-decenal Tridecane Pentadecane Tridecanyl butyrate Tetradecanyl butyrate
102 115 115 120 129 141 196 205
found in the scent gland secretion from M. sulciventris have been identified by GC and MS comparison with authentic samples, but it is not known if these saturated esters have any pheromonal r81e in this insect. As mentioned earlier, the compounds present in the scent gland of the larval stages of the bronze orange bug are all found in the adult. No 2-octenyl acetate was detected in the larvae but in view of the wide seasonal variation of the abundance of this compound in the adult insect, and since large numbers of instars have not been examined, it would be unwise to place too much emphasis on this point. As a genera1 rule, the scent gland secretion from coreoids consists of saturated esters and aldehydes whereas that from pentatomoids is largely composed of o-/-3 unsaturated esters and aldehydes. While no difference in composition was noted between adults and larvae of the pentatomoid M. sulciventris, BAKER and JONES (1969) have reported that the Table 2.
1221
times (Rt) and temperatures of compounds identified by GC-MS scent gland secretion of M. sulciventris Column
Peak No.
of tropical hemiptera
Temperature Rt
(‘C)
min. sec. 8 40 13 10 17 20 19 30
68 124 101 54
23 19 23 25 27 30 43 45
30 55 30 05 20 05 10 20
__-
lit
min. sec. 11 35 26 20 20 30 7 30
72 116 128 90 112
12 24 27 17 23
adult coreoid Pternistria bispina (Stal) has a scent gland secretion composed of the expected saturated compounds whereas the larval secretion is composed of unsaturated aldehydes and esters. Thus while the generalization is very useful it probably should be restricted to adult insects. B. bibax. The scent gland contents form a taophase system similar to that encountered in M. sulciventris. The compounds identified in the yellow lower phase after examination by GC-MS are shown in Table 2. Apart from 2-decenol, which occurs to the extent of less than 1 per cent, the compounds listed have been identified as components of the scent gland secretions of other pentatomoids (CALAM and YOUDEOWI, 1968). The quantity of 2-decenyl acetate present is substantial and does not appear to be subject to the same seasonal variation in amount as was observed from 2-octenyl acetate in M. sulciventris. The small quantity of 2-decenol, identified by GC-MS
Retention times (Rt) and temperatures of compounds GC-MS of the scent gland secretion of B. bibax
identified by
Column 3 Peak SO.
1 2 3 4 5 6 7 8 9 10
Compound
Truns-2-hexenal 4-Oxo-tram-2-hexenal Trws-2-hexenyl acetate Undecane Dodecane Tranr-2-decenal 2-Decenol Tridecane Trons-2-decenyl acetate Pentadecane
25 10 30 ov 15
Percentage
10 10 10
Temperature
48 67 75 90 105 112 115 122 134 148
Rt min. sec. 6 15 11 00 12 50 16 40 20 25 22 00 22 40 24 20 27 20 30 10
1222
J. K. MACLEOD, I. HOWE,J. CABLE,J. D. BLAKE,J. T. BAKER,AND
D. SMITH
Table 3. Response of adult female bronze orange bugs to caged adult male bronze orange bugs in a Y-tube olfactometer No. of female bugs in Exp. No.
Females Caged released males
1
10
10
2*
10
10
Free carton
Target carton
Blank carton
Ytube
Time (hr)
8 2 2
0 0 0
0 5 1
2 3 7
t 10 2
* Citrus fruit placed in Y-tube. The results (see Table 3) demonstrate that the free females show no attractancy towards the caged males under the conditions of the experiment. In fact in experiment 1, half of the female population had moved into the blank carton after 10 hr, but none was found in the carton of caged males. It is possible that the females were repelled by the male defensive secretion (see other experiments below). In experiment 2, fresh citrus twigs, leaves, and fruits were placed in the Y-tube and after 2 hr, 7 out of 10 female bugs were found to be attached to the citrus material and preferred to remain there. (2) Male-to-female attractancy test: Caged female bronze orange bugs were placed in the target carton and free males released in another. The air current was maintained from the target and blank cartons towards the free carton (except where otherwise indicated). The results (see Table 4) demonstrate that the free males show no attractancy towards the caged females under the conditions of the experiment. Experiments 4 and 5 indicate that the bugs prefer to move towards the source of the air flow regardless of the contents of the carton towards which they are moving. Experiment 6 shows that the male bugs prefer to remain on the citrus fruit. (3) Female reaction to 2-octenyl acetate: A few drops of 2-octenyl acetate were placed in a O-3 ml Reacti Vial in the target carton and free females
comparison with an authentic sample, may be a consequence of the larger amount of 2-decenyl acetate present in these insects. The alcohol may play a part in the biosynthesis of the acetate or it may arise via a small amount of hydrolysis of the ester. Behavioural
studies
M. sulciventris. A Y-tube olfactometer was used to investigate possible attractancy of females towards males and vice versa, and to determine the behavioural response of the bugs to 2-octenyl acetate. The experiments were conducted at Nambour during February 1974, at a laboratory temperature of about 30°C. The bugs were in season to mate, although they were not necessarily virgin. Several mating pairs were found after a few hours when 10 males and 10 females were placed together in the same cage. (1) Female-to-mule attractancy test: Caged male bronze orange bugs were placed in the ‘target’ carton of the apparatus and free female bronze orange bugs were released in another carton (the ‘free’ carton). The air current (up to 10 l./min) was maintained from the target and blank cartons towards the free carton. The possibility of visual stimulus was excluded by partially masking the entrance to the target cage with filter paper.
Table 4. Response of adult male bronze orange bugs to caged adult female bronze orange bugs in a Y-tube olfactometer No. of male bugs in Exp. No.
Males released
Caged females
Free carton
Target carton
Blank carton
Ytube
Time (hr)
3
10
6
;
0 2
0 4
5 2
t 3
4*
10
6
0
9
0
1
1
5t
10
6
3
1
6
0
1
6f
10
6
1
0
0
9
2
* Air flow from target carton only. t Air flow from blank carton only. $ Citrus fruit placed in Y-tube.
Volatile scent gland components of tropical hemiptera were released in another. The air current was maintained from the target and blank cartons towards the free carton. The results (see Table 5) show that the females are not attracted by 2-octenyl acetate under the conditions of the experiment. In fact they appear to be repelled by the chemical, possibly associating the scent with an alarm secretion. In each experiment (7 and 8) 1 female ejected her defensive secretion in the Y-tube. It has been observed in the field that one ejection by a bronze bug tends to trigger off ejection by other bugs. The ejections observed here would support association of 2-octenyl acetate by the bugs with a defensive (or alarm) secretion. Table 5. Response of adult female bronze orange bugs to 2-octenyl acetate in a Y-tube olfactometer No. of bugs in Free carton
Exp. No.
Females released
7
10
3 3
8
10
4 3
Target carton
Blank carton
Ytube
Time (hr)
1 2
1 5
5 0
t
0 2
3 5
3 0
2 2
(4) Male reaction to 2-octenyl acetate: Males were released in one carton under the same conditions as the females in (3) above. The results (see Table 6) show that the males are not attracted by 2-octenyl acetate under the conditions of the experiment. In each experiment (9 and 10) one male ejection was seen to take place. Again this supports an association by the bugs of 2-octenyl acetate with a defensive secretion. Table 6. Response of adult male bronze orange bugs to 2-octenyl acetate in a Y-tube olfactometer No. of bugs in Males released
Free carton
9
10
7
10
10
3
Exp. No.
Blank carton
Ytube
Time (hr)
1
1
1
3
3
4
0
3
Target carton
B. bibax. Further Y-tube olfactometer tests will be undertaken when the insects become available since these insects have been examined only very cursorily from this viewpoint to date.
1223
have been reported in other pentatomoids. The remaining two compounds, tridecanyl butyrate and tetradecanyl butyrate, constitute the first reported examples of long-chain saturated esters occurring in the Pentatomoidae. Lower homologues of these esters have been noted in coreids, but the lower molecular weight esters reported in pentatomoids are usually unsaturated compounds, e.g. 2-hexenyl acetate, 2-octenyl acetate, and 2-decenyl acetate. The role of the two long-chain saturated esters in the secretion from M. sulciventris is not known. No evidence was found for a long-range sex attractant in M. sulciventris. The quantity of 2-octenyl acetate present displays considerable seasonal variation, but the observations made with a Y-tube olfactometer do not support the postulated role of this compound as a pheromone. It appears to function as an alarm substance as might be expected for a component of a defensive secretion. Careful observation of these insects indicates that a short-distance attraction mechanism between sexes may operate but it is not known whether this phenomenon involves chemical communication. In the orchard it is also not uncommon for M. sulciventris to aggregate in clumps of up to 20 individuals. The compounds identified in the scent gland secretion from B. bibax have been previously reported in the Pentatomoidae with the exception of 2-decenol. The corresponding unsaturated aldehyde and acetate are major components of the secretion and the small quantity of 2-decenol may be biosynthetically significant or it may arise from either of these compounds. The concentration of 2-decenyl acetate found in B. bibax is much larger than that of 2-octenyl acetate in M. sulciventris and does not apear to be subject to the seasonal variation. No behavioural studies have been carried out with B. bibax to investigate the possible role of 2-decenyl acetate as a pheromone but it is hoped to investigate this aspect when the insects become available in the summer. Acknowledgeraenrs-Financialsupport through a Rural Credits Development Fund Grant (R.C.D. F-4) is gratefully acknowledged. The assistance and co-operation of Mr. TONY WHILEY and the staff of the Queensland Department of Primary Industries’ Pineapple Research Laboratory proved invaluable during behavioural studies conducted at these premises in February 1974.
REFERENCES BAKERJ. T., BLAKE J. D., MACLEODJ. K., IRONSIDED. A., and JOHNSONI. C. (1972) The volatile constituents of the fruit spotting bug Amblypelta nitida. Aust. J.
Chem. 25, 393-400. CONCLUSION Ten of the 12 compounds indentified in the scent gland secretion obtained from M. sulciventris
BAKERJ. T. and JONES P. A. (1969) Volatile constituents of the scent gland reservoir of the nymph of the coreoid Pternistra bispina StHI. Aust. J. Chem. 22,
1793-1796.
1224
J. K. MACLEOD, I. HOWE, J. CABLE, J. D. BLAKE, J. T. BAKER, AND D. SMITH
BAKERJ. T., JON= P. A., MACLEOD J. K., and SMITH D. (1970) Scent gland constituents of tropical Hemiptera. ANZAAS Conference, X6-23 August, Port Moresby, TPNG. BUTENDANTA. and TAM N. (1957) Uber einen geschlechtsspezifischen Dufstoff der Wasserwanze Belostoma indica Vitalis (Letkocerus indicus Lep.). HoppeSeyler’s Z. pkysiol. Ckem. 308,277-283. CALAMD. H. and YOUDEOWIA. (1968) Identification and functions of secretion from the posterior scent gland of the fifth instar larva of the bug Dysderus intermedius. J. Insect Pkysiol. 14, 1147-1158. GILBY A. R. and WATERHOUSED. F. (1965) The composition of the scent of the green vegetable bug Nezara viridula. Proc. R. Sot. Lond. (B) 162, 105-120. HELY P. C. (1968) The entomology of citrus in New South Wales. Aust. ent. Sot.. Miscellaneous Publication No. 1. JACOBSON M., REDFERN R. E., JONES W. A., and ALDRIDCE M. H. (1970) Sex pheromones of the southern armyworm moth: isolation, identification and synthesis. Science, Wash. 170, 542-544. KLUN J. A. and BRINDLEY T. A. (1970) Cis-lltetradecenyl acetate, a sex stimulant of the European corn borer. J. econ. Ent. 63, 779-780.
MACCONNELLJ. G. and SILVER~TEINR. M. (1973) Recent results in insect pheromone chemistry, references to Cimicidae, Lygaeidae, Miridae, Pyrrhocoridae and Reduviidae. &geeu. Chem. int. Ed. i2, 644-654. MITCHELL W. C. and MAU W. L. F. (1971) Resoonse of the female southern green stink bug and its parasite, Trickopoda pennipes to male stink bug pheromones. J. econ. Ent. 64, 856-859. PARK R. J. and SUTHERLANDM. D. (1962) Volatile constituents of the bronze orange bug Rlrorrororis sulciventris. Awt. J. Ckem. 15, 172-174. PATTENDENG. and STADDONB. W. (1970) Observations on the metasternal scent glands of Letkocerus spp. (Heteroptera: Belostomatidae). Ann. efrt. Sor. Am. 63, 900-901. ROELOFSW. L. and TETTE P. L. (1970) Sex pheromone of the obliaue-banded leaf roller moth. Nature. Lond. 226, 1172. %ITH D. (1973) Unpublished report, Queensland Department of Primary Industries.