Journal of Chemical Ecology, Vol. 20, No. 11, 1994
METHYL SALICYLATE AND (-)-(1R,5S)-MYRTENAL ARE PLANT-DERIVED REPELLENTS FOR BLACK BEAN APHID, Aphis Fabae SCOP. (HOMOPTERA: APHIDIDAE)
J I M H A R D I E , I R U F U S I S A A C S , 1'2 J O H N A . P I C K E T T , 2'* L E S T E R J. W A D H A M S , 2 and C H R I S T I N E M. W O O D C O C K 2 bAFRC Linked Research Group in Aphid Biology hnperial College at Silwood Park Ascot, Berks SL5 7PY, U.K. "-AFRC Institute of Arable Crops Research Rothamsted Experimental Station Harpenden, Herts AL5 2JQ, U.K. (Received October 4, 1993; accepted June 22, 1994) Abstract--Methyl salicylate and (-)-(IR,5S)-myrtenal stimulate specific olfactory cells in the primary rhinaria on the sixth and fifth antennal segments, respectively, of the black bean aphid, Aphis fabae. In behavioral studies employing a linear track olfactometer, both compounds were repellent to A. fabae and also inhibited attraction to volatiles from its host, broad bean (Ficia faba). Methyl salicylate is associated with secondary metabolite-based defense in plants, and the monoterpenoid ( -)-( 1R,5S I-myrtenal is metabolically related to (-)-(IS,5S)-t~-pinene, an abundant component of defensive resins produced by gymnosperms. It is argued that these two compounds are employed by A. fabae as indicators of nutritionally unsuitable or nonhost plants.
Key Words--Aphid, Aphis fabae, Homoptera, Aphididae, semiochemicals, chemical ecology, electrophysiology, salicylate, monoterpenoid.
INTRODUCTION E v i d e n c e is a c c u m u l a t i n g that alate a p h i d s e m p l o y volatile k a i r o m o n e s in host plant r e c o g n i t i o n p r i o r to l a n d i n g (Pickett et al., 1992). S t u d i e s o n a p h i d olfactory r e c e p t o r s , particularly u s i n g s i n g l e - c e l l r e c o r d i n g s ( S C R ) c o u p l e d with h i g h r e s o l u t i o n gas c h r o m a t o g r a p h y ( G C ) ( W a d h a m s , 1990), h a v e a l l o w e d identifi*To whom correspondence should be addressed. 2847 0098-033119411100-2847507.00/0 9 1994 Plenum PublishingCorporation
2848
HARDIEET AL.
cation of electrophysiologically active compounds, the behavioral roles of which are being examined (Campbell et al., 1993; Isaacs et al., 1993). The aphids Brevicoryne brassicae (L.) and Lipaphis erysimi (Kalt.), which specialize on plants in the Brassicaceae ( = Cruciferae), are attracted in a linear track olfactometer to 3-butenyl isothiocyanate (Nottingham et al., 1991), a glucosinolate catabolite typical of this family (Ju et al., 1982). Similarly, (-)-/5-caryophyllene, a ubiquitous plant component abundantly present in hops, Humulus lupulus L. (Cannabaceae), proved attractive to the damson-hop aphid, Phorodon humuli (Schrank), as was the fatty acid oxidation product (E)-2-hexenal (Campbell et al., 1993). The black bean aphid, Aphisfabae Scop., has also shown attraction to volatiles from its host, broad bean Viciafaba L. (Fabaceae = Leguminosae) (Nottingham et al., 1991). However, A. fabae and P. humuli also possess olfactory receptors for isothiocyanates (Nottingham et al., 1991; Pickett et al., 1992), although neither aphid colonizes brassicas, suggesting that these compounds could be employed as indicators of nonhost plants that may deter these aphids from landing. Indeed, olfactometer studies with alate A. fabae showed that 4-pentenyl and 3-butenyl isothiocyanates were repellent and also masked the attractancy of the host plant (Nottingham et al., 1991; Isaacs et al., 1993). For P. humuli, application of an isothiocyanate precursor decreased aphid numbers on hops in the field (C.A.M. Campbell, personal communication). With the cereal aphids Rhopalosiphum padi L. and Metopolophium dirhodum (Walk.), methyl salicylate, as a component of the rosaceous winter host, significantly reduced colonization by spring migrants on wheat and barley crops (Pettersson et al., 1994). Such masking of host attractancy by nonhost compounds has been documented in pioneering studies on the Colorado potato beetle, Leptinotarsa decemlineata Say (Thiery and Visser, 1987). The olfactory receptors of a number of aphid species have been investigated by GC-SCR, using extracts from a range of host and nonhost plants (Pickett et al., in preparation). Methyl salicylate and (-)-(1R,5S)-myrtenal, a component of the resins produced by gymnosperms, were identified as having significant electrophysiological activity in many species. It was considered possible that these two compounds could have host-masking effects with A. fabae, which does not naturally colonize rosaceous plants or gymnosperms. In the present study, electrophysiological threshold concentrations for methyl salicylate and (-)-(1R,5S)-myrtenal were established for olfactory receptors of A. fabae and behavioral activity was investigated. METHODS AND MATERIALS
Aphids. Alate virginoparous A. fabae were reared in crowded cultures on V. faba (cv. tick bean) at 15~ and 16 hr day length. For olfactometer studies, young adults were held over damp sand under glass tubes and starved for 24 hr prior to assays. Aphids were used only once.
APHID REPELLENTS
2849
Chemicals. Methyl salicylate and (-)-(IR,5S)-myrtenal were obtained from Aldrich Chemical Co. (purity 99% and 98%, respectively) and diluted in hexane for SCR studies. In olfactometer assays, compounds were placed in a 1- or 10#1 glass microcapillary (Drummond Sci. Co.) held at 45 ~ in one arm of the olfactometer and released into an airstream of 500 ml/min. Release rates from the capillaries were calculated by measuring the movement of the meniscus over time using an eyepiece graticule and microscope (Isaacs et al., 1993). Entrainment of Plant Volatiles. To ascertain whether methyl salicylate or ( - )-( lR,5S)-myrtenal was present in the volatiles produced by V. faba, seedlings (cv. Sutton Dwarf, ca. 20 cm tall) in potting compost were contained in a glass culture vessel (Quickfit FV range, 1 liter) and air, dried and purified by passage through activated 5.~ molecular sieve and charcoal, was drawn at 1 liter/rain over the plant material. Volatiles were entrained onto Porapak Q that had been purified by washing with ether (5 ml) and heating for 12 hr at 150~ under a stream of nitrogen. Collected volatiles were desorbed from the Porapak by elution with freshly distilled diethyl ether. The resulting extract was concentrated under a stream of nitrogen and stored in sealed glass ampoules at - 2 0 ~ Gas Chromatography-Mass Spectrometry. (GC-MS). GC employed an oncolumn injector and a 50-m x 0.32-mm-ID fused silica column with a crosslinked methylsilicone bonded phase (HP-1), 0.52-t~m film thickness, linked directly to a 70-250 mass spectrometer and integrated data system (VG Analytical). Tentative identifications by GC-MS were confirmed by comparison with authentic samples and then by peak enhancement on GC (Pickett, 1990). Fragmentograms were constructed using the data system to select intensity values of designated ion masses typical of the spectra from compounds under analysis. Conditions: GC, 30~ for 5 min, 5~ to 180~ isothermal; MS, electron impact at 70 eV, 230~ Electrophysiology. Recordings from cells associated with the olfactory receptors on the primary rhinaria of A. fabae alate virginoparae were made using tungsten microelectrodes. The indifferent electrode was placed in the first or second antennal segment, and the recording electrode was then brought into contact with the rhinarium until impulses were recorded. Permanent copies of the action potentials generated by the olfactory cells were obtained by standard methods (Wadhams et al., 1982). The stimulus was delivered into a purified airstream (1 liter/min) flowing continuously over the preparation. The delivery system, employing a filter paper in a disposable Pasteur pipet cartridge, has been described previously (Wadhams et al., 1982). The impulse frequency was determined as the number of impulses elicited during the first 1 sec after stimulus application. Olfactometry. Experiments were carried out in a linear track olfactometer (Sakuma and Fukami, 1985) made of transparent Perspex and brass rods and based on a design modified for use with aphids. Groups of ca. 25 young adult
2850
HARDIE ET AL.
alate A. fabae were placed in a pot with Fluon-lined walls in the bottom of the olfactometer. Only those aphids that climbed the vertical rod, in response to an overhead light, were scored for their initial turning choice at the T junction of the rods, either towards odor-laden or control (odor-free) airstreams. For control runs, aphids were assayed without chemical in either arm. In all bioassays, total numbers of aphids turning left or right during a 10-rain period were recorded, and the olfactometer was rotated 180 ~ between runs to remove any directional bias. Air drawn though the apparatus was exhausted from the room, and the olfactometer equipment was washed between runs with a 1% (v/v) solution of Teepol detergent and then a 75% (v/v) methanol solution. The mean numbers of aphids orienting towards different treatments were compared using two-tailed related sample t tests. Olfactometer assays to test the effect of nonhost chemicals on the orientation of A. fabae to V. faba (cv. Sutton Dwarf) odor employed 2 g of leaves freshly picked from greenhouse grown plants in one arm of the olfactometer, with damp filter paper (Whatman No. 1,4.25-cm diameter) at the other inlet to equalize humidity. White paper was placed either side of the T junction to prevent orientation to visual stimuli from the plant material, and synthetic compounds were released as before from the same arm as the plant material. Aphids were assayed for their response to host odor with and without ( - ) - ( 1 R , 5 S ) myrtenal and methyl salicylate. RESULTS AND DISCUSSION
In alate virginoparae of A. fabae, electrophysiological responses to methyl salicylate are confined to cells in the distal primary rhinarium (sixth antennal segment) (Figure 1), while cells in the proximal primary rhinarium (fifth antennal segment) respond to (-)-(1R,5S)-myrtenal (Figure 2). The threshold level obtained for these two compounds (10 -9 g) was two orders of magnitude lower than that observed previously for 4-pentenyl isothiocyanate (Nottingham et al., 1991). Responses for A. fabae in the linear track olfactometer to methyl salicylate and (-)-(1R,5S)-myrtenal are given in Tables 1 and 2, and show threshold repellency at release rates of 50 and 34 #g/hr, respectively, an order of magnitude lower than the behavioral threshold for the alkenyl isothiocyanates (Isaacs et al., 1993). V. faba leaf volatiles attracted A. fabae (Table 3) but, as shown previously with isothiocyanates, addition of threshold amounts of either methyl salicylate or (-)-(1R,5S)-myrtenal eliminated the response. To confirm that these two compounds are not produced by intact V. faba, entrained volatiles were analyzed in GC-MS by mass fragmentography at the abundant diagnostic ions m/z 92, 120, 152 and m/z 79, 107, respectively (Mass Spectrometry Data Centre, 1991). Neither methyl salicylate nor (-)-(1R,5S)-myrtenal could be detected in the sample.
APHID
REPELLENTS
2851
80
70
60
i so 40 "5 a. 30
_E
20 10 i
i
i
10-1o lO-e lO-e
i
10-7
Stimulus (log g)
FIG. 1. Dose-response curve of Aphisfabae olfactory cell (distal primary rhinarium) to methyl salicylate. Means of five preparations •
80 70 60 o
so 40
~. 30
s
20 10 I
i
i
10-1o 10-9 lO-a
i
lO-r
Stimulus (log g)
FIG. 2. Dose-response curve of Aphisfabae olfactory cell (proximal primary rhinarium) to (-)-(1R,5S)-myrtenal. Means of five preparations •
2852
HARDIE ET AL.
TABLE 1. RESPONSES OF ALATE Aphisfabae VIRGINOPARAE TO DIFFERENT RELEASE RATES OF METHYL SALICYLATE IN LINEAR TRACK OLFACTOMETER
Release rate (p.g/hr) 0 30 40 50 60 70 100
Aphids (mean 5: SE) moving towards Treatment 7.3 7.3 8.6 6.3 5.1 5.0 7.6
t
Significance (P)"
1.90 0.37 1.16 2.67 2.65 2.76 2.58
NS NS NS
METHYL SALICYLATE AND (-)-(1R,5S)-MYRTENAL ARE PLANT-DERIVED REPELLENTS FOR BLACK BEAN APHID, Aphis Fabae SCOP. (HOMOPTERA: APHIDIDAE)
J I M H A R D I E , I R U F U S I S A A C S , 1'2 J O H N A . P I C K E T T , 2'* L E S T E R J. W A D H A M S , 2 and C H R I S T I N E M. W O O D C O C K 2 bAFRC Linked Research Group in Aphid Biology hnperial College at Silwood Park Ascot, Berks SL5 7PY, U.K. "-AFRC Institute of Arable Crops Research Rothamsted Experimental Station Harpenden, Herts AL5 2JQ, U.K. (Received October 4, 1993; accepted June 22, 1994) Abstract--Methyl salicylate and (-)-(IR,5S)-myrtenal stimulate specific olfactory cells in the primary rhinaria on the sixth and fifth antennal segments, respectively, of the black bean aphid, Aphis fabae. In behavioral studies employing a linear track olfactometer, both compounds were repellent to A. fabae and also inhibited attraction to volatiles from its host, broad bean (Ficia faba). Methyl salicylate is associated with secondary metabolite-based defense in plants, and the monoterpenoid ( -)-( 1R,5S I-myrtenal is metabolically related to (-)-(IS,5S)-t~-pinene, an abundant component of defensive resins produced by gymnosperms. It is argued that these two compounds are employed by A. fabae as indicators of nutritionally unsuitable or nonhost plants.
Key Words--Aphid, Aphis fabae, Homoptera, Aphididae, semiochemicals, chemical ecology, electrophysiology, salicylate, monoterpenoid.
INTRODUCTION E v i d e n c e is a c c u m u l a t i n g that alate a p h i d s e m p l o y volatile k a i r o m o n e s in host plant r e c o g n i t i o n p r i o r to l a n d i n g (Pickett et al., 1992). S t u d i e s o n a p h i d olfactory r e c e p t o r s , particularly u s i n g s i n g l e - c e l l r e c o r d i n g s ( S C R ) c o u p l e d with h i g h r e s o l u t i o n gas c h r o m a t o g r a p h y ( G C ) ( W a d h a m s , 1990), h a v e a l l o w e d identifi*To whom correspondence should be addressed. 2847 0098-033119411100-2847507.00/0 9 1994 Plenum PublishingCorporation
2848
HARDIEET AL.
cation of electrophysiologically active compounds, the behavioral roles of which are being examined (Campbell et al., 1993; Isaacs et al., 1993). The aphids Brevicoryne brassicae (L.) and Lipaphis erysimi (Kalt.), which specialize on plants in the Brassicaceae ( = Cruciferae), are attracted in a linear track olfactometer to 3-butenyl isothiocyanate (Nottingham et al., 1991), a glucosinolate catabolite typical of this family (Ju et al., 1982). Similarly, (-)-/5-caryophyllene, a ubiquitous plant component abundantly present in hops, Humulus lupulus L. (Cannabaceae), proved attractive to the damson-hop aphid, Phorodon humuli (Schrank), as was the fatty acid oxidation product (E)-2-hexenal (Campbell et al., 1993). The black bean aphid, Aphisfabae Scop., has also shown attraction to volatiles from its host, broad bean Viciafaba L. (Fabaceae = Leguminosae) (Nottingham et al., 1991). However, A. fabae and P. humuli also possess olfactory receptors for isothiocyanates (Nottingham et al., 1991; Pickett et al., 1992), although neither aphid colonizes brassicas, suggesting that these compounds could be employed as indicators of nonhost plants that may deter these aphids from landing. Indeed, olfactometer studies with alate A. fabae showed that 4-pentenyl and 3-butenyl isothiocyanates were repellent and also masked the attractancy of the host plant (Nottingham et al., 1991; Isaacs et al., 1993). For P. humuli, application of an isothiocyanate precursor decreased aphid numbers on hops in the field (C.A.M. Campbell, personal communication). With the cereal aphids Rhopalosiphum padi L. and Metopolophium dirhodum (Walk.), methyl salicylate, as a component of the rosaceous winter host, significantly reduced colonization by spring migrants on wheat and barley crops (Pettersson et al., 1994). Such masking of host attractancy by nonhost compounds has been documented in pioneering studies on the Colorado potato beetle, Leptinotarsa decemlineata Say (Thiery and Visser, 1987). The olfactory receptors of a number of aphid species have been investigated by GC-SCR, using extracts from a range of host and nonhost plants (Pickett et al., in preparation). Methyl salicylate and (-)-(1R,5S)-myrtenal, a component of the resins produced by gymnosperms, were identified as having significant electrophysiological activity in many species. It was considered possible that these two compounds could have host-masking effects with A. fabae, which does not naturally colonize rosaceous plants or gymnosperms. In the present study, electrophysiological threshold concentrations for methyl salicylate and (-)-(1R,5S)-myrtenal were established for olfactory receptors of A. fabae and behavioral activity was investigated. METHODS AND MATERIALS
Aphids. Alate virginoparous A. fabae were reared in crowded cultures on V. faba (cv. tick bean) at 15~ and 16 hr day length. For olfactometer studies, young adults were held over damp sand under glass tubes and starved for 24 hr prior to assays. Aphids were used only once.
APHID REPELLENTS
2849
Chemicals. Methyl salicylate and (-)-(IR,5S)-myrtenal were obtained from Aldrich Chemical Co. (purity 99% and 98%, respectively) and diluted in hexane for SCR studies. In olfactometer assays, compounds were placed in a 1- or 10#1 glass microcapillary (Drummond Sci. Co.) held at 45 ~ in one arm of the olfactometer and released into an airstream of 500 ml/min. Release rates from the capillaries were calculated by measuring the movement of the meniscus over time using an eyepiece graticule and microscope (Isaacs et al., 1993). Entrainment of Plant Volatiles. To ascertain whether methyl salicylate or ( - )-( lR,5S)-myrtenal was present in the volatiles produced by V. faba, seedlings (cv. Sutton Dwarf, ca. 20 cm tall) in potting compost were contained in a glass culture vessel (Quickfit FV range, 1 liter) and air, dried and purified by passage through activated 5.~ molecular sieve and charcoal, was drawn at 1 liter/rain over the plant material. Volatiles were entrained onto Porapak Q that had been purified by washing with ether (5 ml) and heating for 12 hr at 150~ under a stream of nitrogen. Collected volatiles were desorbed from the Porapak by elution with freshly distilled diethyl ether. The resulting extract was concentrated under a stream of nitrogen and stored in sealed glass ampoules at - 2 0 ~ Gas Chromatography-Mass Spectrometry. (GC-MS). GC employed an oncolumn injector and a 50-m x 0.32-mm-ID fused silica column with a crosslinked methylsilicone bonded phase (HP-1), 0.52-t~m film thickness, linked directly to a 70-250 mass spectrometer and integrated data system (VG Analytical). Tentative identifications by GC-MS were confirmed by comparison with authentic samples and then by peak enhancement on GC (Pickett, 1990). Fragmentograms were constructed using the data system to select intensity values of designated ion masses typical of the spectra from compounds under analysis. Conditions: GC, 30~ for 5 min, 5~ to 180~ isothermal; MS, electron impact at 70 eV, 230~ Electrophysiology. Recordings from cells associated with the olfactory receptors on the primary rhinaria of A. fabae alate virginoparae were made using tungsten microelectrodes. The indifferent electrode was placed in the first or second antennal segment, and the recording electrode was then brought into contact with the rhinarium until impulses were recorded. Permanent copies of the action potentials generated by the olfactory cells were obtained by standard methods (Wadhams et al., 1982). The stimulus was delivered into a purified airstream (1 liter/min) flowing continuously over the preparation. The delivery system, employing a filter paper in a disposable Pasteur pipet cartridge, has been described previously (Wadhams et al., 1982). The impulse frequency was determined as the number of impulses elicited during the first 1 sec after stimulus application. Olfactometry. Experiments were carried out in a linear track olfactometer (Sakuma and Fukami, 1985) made of transparent Perspex and brass rods and based on a design modified for use with aphids. Groups of ca. 25 young adult
2850
HARDIE ET AL.
alate A. fabae were placed in a pot with Fluon-lined walls in the bottom of the olfactometer. Only those aphids that climbed the vertical rod, in response to an overhead light, were scored for their initial turning choice at the T junction of the rods, either towards odor-laden or control (odor-free) airstreams. For control runs, aphids were assayed without chemical in either arm. In all bioassays, total numbers of aphids turning left or right during a 10-rain period were recorded, and the olfactometer was rotated 180 ~ between runs to remove any directional bias. Air drawn though the apparatus was exhausted from the room, and the olfactometer equipment was washed between runs with a 1% (v/v) solution of Teepol detergent and then a 75% (v/v) methanol solution. The mean numbers of aphids orienting towards different treatments were compared using two-tailed related sample t tests. Olfactometer assays to test the effect of nonhost chemicals on the orientation of A. fabae to V. faba (cv. Sutton Dwarf) odor employed 2 g of leaves freshly picked from greenhouse grown plants in one arm of the olfactometer, with damp filter paper (Whatman No. 1,4.25-cm diameter) at the other inlet to equalize humidity. White paper was placed either side of the T junction to prevent orientation to visual stimuli from the plant material, and synthetic compounds were released as before from the same arm as the plant material. Aphids were assayed for their response to host odor with and without ( - ) - ( 1 R , 5 S ) myrtenal and methyl salicylate. RESULTS AND DISCUSSION
In alate virginoparae of A. fabae, electrophysiological responses to methyl salicylate are confined to cells in the distal primary rhinarium (sixth antennal segment) (Figure 1), while cells in the proximal primary rhinarium (fifth antennal segment) respond to (-)-(1R,5S)-myrtenal (Figure 2). The threshold level obtained for these two compounds (10 -9 g) was two orders of magnitude lower than that observed previously for 4-pentenyl isothiocyanate (Nottingham et al., 1991). Responses for A. fabae in the linear track olfactometer to methyl salicylate and (-)-(1R,5S)-myrtenal are given in Tables 1 and 2, and show threshold repellency at release rates of 50 and 34 #g/hr, respectively, an order of magnitude lower than the behavioral threshold for the alkenyl isothiocyanates (Isaacs et al., 1993). V. faba leaf volatiles attracted A. fabae (Table 3) but, as shown previously with isothiocyanates, addition of threshold amounts of either methyl salicylate or (-)-(1R,5S)-myrtenal eliminated the response. To confirm that these two compounds are not produced by intact V. faba, entrained volatiles were analyzed in GC-MS by mass fragmentography at the abundant diagnostic ions m/z 92, 120, 152 and m/z 79, 107, respectively (Mass Spectrometry Data Centre, 1991). Neither methyl salicylate nor (-)-(1R,5S)-myrtenal could be detected in the sample.
APHID
REPELLENTS
2851
80
70
60
i so 40 "5 a. 30
_E
20 10 i
i
i
10-1o lO-e lO-e
i
10-7
Stimulus (log g)
FIG. 1. Dose-response curve of Aphisfabae olfactory cell (distal primary rhinarium) to methyl salicylate. Means of five preparations •
80 70 60 o
so 40
~. 30
s
20 10 I
i
i
10-1o 10-9 lO-a
i
lO-r
Stimulus (log g)
FIG. 2. Dose-response curve of Aphisfabae olfactory cell (proximal primary rhinarium) to (-)-(1R,5S)-myrtenal. Means of five preparations •
2852
HARDIE ET AL.
TABLE 1. RESPONSES OF ALATE Aphisfabae VIRGINOPARAE TO DIFFERENT RELEASE RATES OF METHYL SALICYLATE IN LINEAR TRACK OLFACTOMETER
Release rate (p.g/hr) 0 30 40 50 60 70 100
Aphids (mean 5: SE) moving towards Treatment 7.3 7.3 8.6 6.3 5.1 5.0 7.6
t
Significance (P)"
1.90 0.37 1.16 2.67 2.65 2.76 2.58
NS NS NS
