Chemical Senses vol. 30 no. 1 © Oxford University Press 2005; all rights reserved.
Volatile Organic Compounds as Signals in a Plant–Herbivore System: Electrophysiological Responses in Olfactory Sensilla of the Moth Cactoblastis cactorum
1 Max-Planck-Institut for Behavioral Physiology, Seewiesen, Germany, 3 Research School of Biological Sciences, Australian National University, Canberra ACT 0200, Australia and 4 Biosphere 2 Chemistry Unit, Columbia University, Oracle, AZ 85623, USA 2 Present address: Federal Office for Radiation Protection, D-85764 Oberschleissheim/Neuherberg, Germany 5 Present address: Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA
Correspondence to be sent to: PD Dr. Blanka Pophof, Bundesamt für Strahlenschutz, Ingolstädter Landstrasse 1, D-85764 Oberschleissheim/Neuherberg, Germany. E-mail: bpophof{at}bfs.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The morphological sensillum types on the antennae of male and female Cactoblastis cactorum were visualized by scanning electron microscopy. Electrophysiological recordings were performed for the first time on single olfactory sensilla of C. cactorum. The male sensilla trichodea house a receptor cell responding to the putative pheromone component (9Z,12E)-tetradecadienyl acetate. The sensilla trichodea of the females were much shorter than those of the males and contained specialized receptor cells responding to certain terpenoids, the most frequent being the nerolidol-sensitive cell. The sensilla auricillica and sensilla basiconica of both sexes contained cells responding less specifically to terpenoid compounds as well as to green leaf volatiles. Cells of the sensilla coeloconica responded to aliphatic aldehydes and acids. Eight volatile organic compounds emitted by Opuntia stricta, a host plant of C. cactorum, were identified using gas chromatography–mass spectrometry, ß-caryophyllene being the major compound. Five compounds identified by gas chromatography in the headspace of O. stricta elicited responses in olfactory receptor cells of C. cactorum, nonanal being the most active compound and therefore a candidate attractant of C. cactorum.
Key words: Cactoblastis cactorum, gas chromatography–mass spectrometry, Opuntia stricta, pheromones, single sensillum recordings, volatile organic compounds
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The interaction between phytophagous insects and their hosts is partially mediated by volatile organic compounds (VOCs) that are synthesized as products of plant metabolism and emitted into the environment. Within the context of moth olfactory orientation, VOCs often form sensory cues (e.g. Todd and Baker, 1993
; Anderson et al., 1995; Røstelien et al., 2000a,b; Stranden et al., 2002) that define host specificity and may provide information for behavioral choice of feeding and oviposition sites (Müller and Hilker, 2000, 2001; for a review see Honda, 1995). In addition, VOCs may act as cues for the selection of a particular individual of a given host species or as deterrents if they are specifically emitted by a damaged individual (De Morales et al., 2001). Volatiles induced by the attack of pest insects may even attract their natural enemies to the infested plants (e.g. Hilker et al., 2002; for reviews on induced volatiles, see Dicke and van Loon, 2000; Dicke and Hilker, 2003).
An examination of the role of VOCs in the interaction between the moth Cactoblastis cactorum and its cactus hosts of the genus Opuntia is interesting from several perspectives. First, C. cactorum has been a classic example of the successful biological control of a particularly catastrophic weed infestation: by 1925 an introduced plant, the prickly pear cactus Opuntia stricta had restricted human access to 25 million hectares of eastern Australia and was continuing its advance at 
100 hectares/h (Osmond and Monro, 1981). In 1925, after several unsuccessful attempts of chemical and biological control by other methods, C. cactorum, a native of Argentina and Paraguay, was introduced to Australia and proved to be a successful control agent by near-complete destruction of the cactus population. Eighty years later, the prickly pear and the moth continue to coexist, albeit at much lower and sustainable population densities.
Secondly and paradoxically, the biological control agent now shows potential as an insect pest to threaten a major native ecosystem in a different part of the world. Opuntia stricta cacti which became known as pest pears in Australia are native to Louisiana and its adjacent desert states in the USA. Until recently, the geographical separation between the original South American habitat of C. cactorum and the cactus habitats in North America was apparently sufficient to prevent infestation, but this has now been breached by human interference. In 1957, egg sticks were sent to Nevis in the Caribbean, in order to control local Opuntia spp. (Simmonds and Bennett, 1966). From Nevis, C. cactorum spread to other Caribbean islands, partially by deliberate introduction and partially by natural migration (Garcia-Tuduri et al., 1971; Bennett et al., 1985; Starmer et al., 1987). In 1989, the moth was discovered on the North American mainland in Florida. Cactoblastis cactorum is now established there, with the result that indigenous cacti such as the semaphore cactus, O. spinosissima, have become critically endangered (Zimmermann et al., 2001). A further spread of the moth towards the US desert states and Mexico is possible, adding relevance to research into mechanisms of host selection.
Finally, the Cactoblastis–Opuntia system is of particular theoretical interest because the host uses the crassulacean acid metabolism pathway (CAM, Osmond et al., 1979; Black and Osmond, 2003) for CO2 fixation. Although CAM includes all enzymes required for both C3 and C4 metabolism, resulting metabolic by-products may differ between CAM and C3 or C4 plants. CAM volatiles reported to date from non-cacti (e.g. in the tree Clusia rosea and pineapple; see Lerdau and Keller, 1997; Nogueira et al., 2001; Preston et al., 2003) do not notably differ from C3 or C4 plant VOCs. An objective of the present paper is the presentation of a survey of VOCs from the headspace of O. stricta.
From a chemoecological perspective, it is interesting that CAM metabolism allows the temporal separation between photosynthesis and CO2 fixation, meaning that the plants' stomata are often open in darkness, assimilating CO2 and releasing VOCs. Cactoblastis cactorum are nocturnally active moths. In previous work, it has been shown that the latter aspect is relevant for host finding by Cactoblastis: the CO2 gradient on the surface of assimilating cladodes of O. stricta forms a close-range sensory cue for oviposition (Stange et al., 1995), and artificially induced fluctuations of CO2 concentration deter oviposition (Stange, 1997). CO2, as a major atmospheric trace gas, is not a VOC in the strict sense and the constraints on its sensory perception are different from those applying to VOCs. In particular, the ubiquitous occurrence of CO2 sources or sinks makes it unlikely that CO2 gradients alone are sufficient sensory cues to exclusively define host specificity, and the mechanisms of atmospheric dispersal of CO2 gradients suggest that they are not important for long distance orientation. Therefore, it is desirable to supplement existing findings by addressing the role of more specific VOCs, and one objective of the present work is to obtain data on both the spectrum of VOCs emitted by the plant host and the spectrum of sensitivities found in the antennal sensory receptors of the moth.
Currently, no information exists about VOCs from O. stricta that may serve as olfactory cues for C. cactorum host plant finding. In fact, relatively little is known about cactus volatile chemistry. VOCs reported from cacti so far mostly include floral volatiles (Kaiser and Tollsten, 1995), such as the novel dehydrogeosmin and other terpenoids, benzyl derivatives, sulfur-containing compounds attractive to pollinating bats and lipoxogenase products, all from non-platyopuntias (Kaiser and Nussbaumer, 1990; Kaiser and Tollsten, 1995). In order to find chemical cues relevant for C. cactorum to locate its host plant, solid-phase microextraction (SPME) (Pawliszyn, 1999) was used to collect VOCs from O. stricta non-flowering cladodes followed by gas chromatography–mass spectrometry (GC-MS) to analyze these VOCs after desorption from the SPME fibers. As a prerequisite for electrophysiological recording, the morphology and distribution of the various sensillum types on the antennae of male and female C. cactorum was studied by scanning electron microscopy. Finally, the responses of these sensilla to identified and putative VOCs of O. stricta were characterized electrophysiologically by means of electroantennography and single sensillum recording techniques.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Animals
Cactoblastis-infested cladodes of O. stricta were collected in Araluen, New South Wales, Australia and kept in a greenhouse until pupation had occurred. For electrophysiological experiments, the pupae were shipped to Germany, sexed, and then kept at room temperature (20°C) and 16:8 light/dark regime until emergence. Adults were kept in Petri dishes at 7°C and high humidity (supplied by a water soaked cotton pad) until used for experiments. In total, 24 males (0–11 days after emergence) and 13 females (1–5 days after emergence) were used for electrophysiological recordings.
Morphology
Antennae of male and female C. cactorum were studied after standard preparation (air drying, gold sputtering; see Hallberg et al., 1994) in a Hitachi 4500 field emission scanning electron microscope.
VOC analysis by GC-MS
Solid phase microextraction fibers (Supelco, Bellefonte, PA; Pawliszyn, 1999) were used to collect VOCs from O. stricta followed by desorption into the GC for GC-MS analysis. Thirteen potted O. stricta plants were purchased from Hoak's Greenhouse & Nursery, Inc. Homestead, FL, and grown inside a greenhouse (25°C daytime temperature, natural light and day-length conditions near Oracle, Arizona, USA (32°35'N, 110°51'W, 1165 m elevation) for 10 weeks. One plant was removed from its 15 cm diam. pot and the soil was shaken from the roots and collected. The soil was returned to the same pot without the cactus plant to serve as a negative control for VOC analysis. Polyvinylacetate bags (Reynolds Inc., 1 l volume) (Raguso and Pellmyr, 1998) were placed around the soil-only pot, and around a single non-flowering O. stricta plant with a wire frame attached to the pot extending above the plant to prevent the bag from touching the plant. The soil-only pot was sampled after 17 h of enclosure in the bag (overnight) by piercing the bag with the SPME needle and extending a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber into the bag for 10 min to allow absorption of VOCs. The pot with O. stricta was sampled five times after 70 h (three overnights) in the same way with the SPME needle and PDMS/DVB fiber for 10 min. Fiber desorption was conducted for 5 min for each sample into the injection port of a Hewlett Packard 5890 GC using splitless injection and a Supelco Equity-1 column (100% PDMS; 30 m x 0.25 µm x 0.25 mm) and the following temperature program: 2 min at 45°C, increasing at 30°C/min up to 120°C; 5 min at 120°C, increasing at 30°C/min up to 220°C; finishing at 220°C for 10 min. VOCs eluting from the GC column were observed as a total ion current (TIC) chromatogram using Chemstation software and a Hewlett Packard 5971 mass selective detector (ionization energy 70 eV). Retention times of the VOCs found in O. stricta and seven commercially available standards (Sigma-Aldrich: 
-pinene, ß-pinene, nonanal, copaene, ß-caryophyllene, -caryophyllene; Treat USA, Lakeland, FL: germacrene D (40% minimum) from ylang-ylang oil) were compared.
Electrophysiological recordings
Electrophysiological recordings were performed using glass capillary Ag–AgCl electrodes. For electroantennographic (EAG) recordings, one antenna was cut off and slipped over the reference electrode; the recording electrode was inserted into the cut tip of the antenna. In this case, both electrodes had a tip diameter of 10 µm and were filled with hemolymph Ringer solution (Kaissling, 1995).
Single sensillum recordings were performed on living moths mounted in plastic pipette tips with antennae fixed by dental wax. The tip of one antenna was cut off and the reference electrode with a tip diameter of 10 µm, filled with hemolymph Ringer solution, was inserted into the open end. The recording electrodes were filled with sensillum–lymph Ringer solution (Kaissling, 1995) and positioned under a stereo microscope (Leitz, magnification x192). Recording electrodes with a tip diameter of 10 µm were slipped over the tips of single sensilla (s.) trichodea which had been cut using modified forceps (Kaissling, 1995). Electrodes with tip diameter of 1 µm were inserted at the base of s. auricillica or s. basiconica or into the cavities of s. coeloconica (Pophof, 1997).
The preparation was held in a continuous air stream (1 m/s) filtered through charcoal and humidified by percolation through distilled water. The odorants were loaded on pieces of filter paper (1 cm2) placed in glass cartridges (7 mm inner diameter). During stimulation the continuous air stream was passed for 500 ms through the cartridge.
Signals from olfactory sensilla were amplified using a custom-made amplifier with a low pass cut-off frequency of 2 kHz. Unfiltered data were sampled on-line using a Macintosh G3 computer and data acquisition program SuperScope II 2.31 (GW Instruments). Spikes of single receptor cells were discriminated visually according to their amplitude. The following parameters of responses to stimulation by volatiles were measured: amplitude of the electroannogram response, amplitude of the receptor potential recorded from single sensilla and peak spike frequency calculated from six consecutive spikes with the shortest interspike intervals.
Volatiles
In total, 14 pheromone candidates (14-carbon acetates, alcohols and aldehydes; see Table 2), chosen based on pheromone components known from other Phycitinae species (Arn et al., 1992), and 62 putative VOCs (Table 4) were tested. Pheromone components (from the stocks of the late E. Priesner, Seewiesen) were tested at a stimulus load of 10 µg (in 10 µl n-hexane) on filter paper. In a few cases, stock pheromone solutions of unknown concentrations had to be used (Table 2). The VOCs were purchased from Dragoco, Sigma, Aldrich and Fluka, the purity was in the range of 95–98%. Jasmine extract, synthetic jasmin oil (Dragoco) and orange oil (Treat) were mixtures of several terpenoids, the orange oil containing 40% germacrene D. The VOCs were tested at a dose of 1 µl loaded on filter paper in 10 µl solution (1 µl VOC added to 9 µl paraffin oil). Glass cartridges containing filter papers loaded with volatiles were used for several days. Between the experiments, they were enclosed in plastic vials and stored in a refrigerator at –20°C. For control, cartridges with clean filter paper without any odorant were used.
|
|
For volatiles which regularly excited the olfactory receptor cells [linalool, geraniol, nerolidol, limonene, citral, (E)-3-hexenol, (Z)-3-hexenal, heptanal, octanal, nonanal, nonanoic acid] dilution series were prepared. The maximum dose was either 1 or 5 µl, and dilution steps were a factor of 10. Pheromones were diluted in hexane (Roth), VOCs in paraffin oil (Roth). To register dose–response dependencies, stimuli of increasing intensity were given at 1 min intervals.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Morphology
The filiform antennae of C. cactorum consist of 100 segments, each carrying a complement of sensilla. As the antennal and sensillar morphology strongly resembles that of other pyralid species (Hallberg et al., 1994), the sensillum types were classified accordingly. In the male (Figure 1A), s. trichodea were the most conspicuous, occurring in three regular rows of 6–8 sensilla per segment. In addition, a field of distinct, much smaller, s. basiconica and auricillica was present on each segment, as well as 3–5 s. coeloconica. In the female (Figure 1B), s. auricillica and s. coeloconica were of identical appearance to those in the male but there were no regular rows of s. trichodea. The female s. trichodea were much shorter than in the male; they were straight, but still longer than the slightly bent s. basiconica.
|
GC-MS
Eight VOCs from O. stricta (24% of all VOCs by peak area) listed in Table 1 were identified with library search confidence levels >85% by matching mass spectra selected from individual total ion current peaks (Figure 2A) with spectra from the Wiley Registry of Mass Spectral Data, 6th Edition (containing 275,000 reference spectra). - and ß-caryophyllenes were detected with the highest confidence of all VOCs (98 and 99%, respectively). ß-Caryophyllene was clearly the major VOC detected by solid phase micro-extraction and GC-MS analysis. Meta-cymene (64% confidence level) was possibly an artifact of the SPME procedure (Zabaras and Wyllie, 2002). Retention time comparison between O. stricta VOCs and commercially available standards (see Materials and methods) confirmed the identities of the major VOCs shown in Table 1. Opuntia stricta VOC identity was also confirmed by comparing mass spectra to spectra obtained from commercially available standards. Numerous unidentified, branched hydrocarbons were also detected from O. stricta as other major and minor components (Figure 2A). No O. stricta VOCs were detected from the negative control sample (Figure 2B), which provided chromatogram peaks with mass spectra predominantly similar to industrial plastic components and stationary phase silica derivatives.
|
|
Physiology: electroantennograms
EAG recordings were performed on one male and three female antennae. In the male antenna, the putative pheromone component (9Z,12E)-14:Ac elicited a considerable response of 2.2 mV, followed in magnitude by nonanal which evoked a response of 0.9 mV. Other tested compounds—all pheromone candidates and some VOCs—elicited very weak or no responses (Table 2). In the female antenna, many of the VOCs elicited responses (Table 2). Responses with the largest amplitude were elicited by benzyl acetate (2.8 mV) and synthetic jasmine oil (2.6 mV). A weak response to (9Z,12E)-14:Ac was also observed.
Physiology: single sensillum recordings
Sensilla trichodea—males
In total, 28 male sensilla trichodea were used for recordings. According to the spike amplitude, usually two (in a few cases three) receptor cells could be distinguished. Seven sensilla were tested with VOCs: some terpenoid and cyclic compounds elicited weak or intermediate, predominantly excitatory responses (Table 3). Five sensilla were tested with a range of pheromone candidates. In all cases, the cell with the smaller spike amplitude responded to several 14-carbon acetates and to (9Z,12E)-tetradecadienylaldehyde, with the strongest responses being elicited by (9E,11E)-14:Ac. The cell with the larger spike amplitude gave equal responses of intermediate intensity to the three geometrical isomers of 10,12-tetradecadienol: Z,Z-, E,E- and E,Z- (Table 3).
|
A further 16 male s. trichodea, each containing a cell strongly and selectively responding to (9Z,12E)-14:Ac, were used to measure the dose-response dependence of the maximum receptor potential amplitude and the peak spike frequency (Figure 3A). Responses significantly higher than control (P = 0.0111 for receptor potential amplitude, P < 0.0001 for nerve impulse frequency, paired t-test) were elicited already by a 103-fold dilution of the original (9Z,12E)-14:Ac stock solution, the threshold being apparently close to a 104-fold dilution. The relatively high nerve-impulse responses to control stimuli (Figure 3A, lower graph) were probably due to contamination by pheromone from females which were used for experiments within the same setup.
|
Sensilla trichodea—females
Recordings were performed on 36 female s. trichodea (Table 4); these were about half as long as the corresponding male sensilla. Usually two receptor cells were present in each sensillum according to their different spike amplitudes.
Some of the tested terpenoid and aromatic compounds elicited responses in those cells. In three sensilla, quite unspecific inhibitory responses were observed. Excitatory responses were repeatedly elicited by nerolidol, geraniol, neryl acetate, synthetic jasmine oil, jasmine extract and orange oil. Most of the receptor cells were quite specific and responded to only one or few tested compounds. Most abundant was a cell (n = 6) present in 15% of the female s. trichodea and responding selectively and specifically to nerolidol, with a threshold stimulus dose at 10–3 µl (= ca. 1 µg) per filter paper (Figure 3B). In one case (Table 4, F4/t7), a nerolidol-sensitive cell responded with a similar intensity and even lower threshold to geraniol. The other five nerolidol-sensitive cells responded to geraniol only weakly or not at all (Table 4). The dose–response dependence of the maximum receptor potential amplitude and the peak spike frequency of the six nerolidol-sensitive cells is shown in Figure 3B. The relatively high nerve-impulse responses to control stimuli (Figure 3B, lower graph) result from previous activation of the cells with high doses of several of the tested compounds before the dose–response dependencies were measured.
The receptor cells of the female s. trichodea did not respond to saturated and unsaturated aliphatic alcohols, aldehydes and acids tested. A single weak response to the putative pheromone component (9Z,12E)-14:Ac was observed in a female s. trichodeum (Table 4, F12/t2).
Sensilla auricillica and basiconica
In total, 14 male and 8 female s. auricillica or s. basiconica were tested with a range of VOCs. No physiological or morphological differences between the sexes were observed. The sensilla contacted by the electrode were predominantly ear-shaped (s. auricillica) and were located at the border to the part of the antenna covered by scales, or even below the scales. A few very short hairs which seemed not to be flattened (s. basiconica) were also contacted, but the two morphological types were difficult to distinguish under the stereomicroscope. As physiological differences were not apparent either, they were all evaluated within one group (Table 5).
|
Usually up to three receptor cells were localized in these sensilla according to different spike amplitudes. They responded regularly with excitation or inhibition (Figure 4) to several terpenoid compounds (e.g. linalool, geraniol, limonene, citral, dimethyl-heptenol, jasmine extract and orange oil) as well as to aliphatic alcohols, aldehydes and acids (Table 5). Many of the cells were apparently generalists responding strongly to several related compounds. Strong responses to nonanal, octanal and heptanal (Figure 5) were recorded from
50% of sensilla tested with these compounds.
|
|
Sensilla coeloconica
Five s. coeloconica from two male moths could be contacted and were tested with several saturated and unsaturated aliphatic alcohols, aldehydes and acids. Usually at least three receptor cells were present according to the spike amplitudes. The receptor cells of the coeloconic sensilla did not respond to alcohols, while responses to aldehydes and acids were regularly obtained (Table 6 and Figure 6). Each cell responded best to a single compound, and much less or not at all to related compounds. In some cells, aldehydes elicited stronger responses than acids of the same chain length (Figure 6).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Putative pheromone components
In Phycitinae species closely related to C. cactorum the pheromone components known so far are predominantly 14-carbon acetates, alcohols and aldehydes with two double bonds at positions 9 and 12, with (9Z,12E)-14:Ac being one of the most common components within this group of moths (Arn et al., 1992; Witzgall et al., 2004). According to the responses obtained from receptor cells of male s. trichodea of C. cactorum, (9Z,12E)-14:Ac seems to be one of the pheromone components of C. cactorum, specifically exciting the pheromone receptor cell with a smaller spike amplitude. Although the absolute concentration of this compound was not known, the fact that this compound was effective even if diluted by a factor of 104 supports the idea that (9Z,12E)-14:Ac is indeed a pheromone component of C. cactorum. The cell with the larger spike amplitude responded weakly to (10Z,12Z)-tetradecadienol, (9E,12E)-tetradecadienol and (10E,12Z)-tetradecadienol. (9Z,12E)-Tetradecanol was not available for testing, but it may be another candidate pheromone component. Further quantitative studies are necessary.
The male pheromone-sensitive cells were excited additionally by several terpenoid compounds. This might be a general, receptor-independent effect similar to the inhibition of pheromone-sensitive cells of Antheraea pernyi by geraniol (Schneider et al., 1964) or the inhibition of pheromone sensitive cells of Bombyx mori by linalool (Kaissling et al., 1989; Pophof and van der Goes van Naters, 2002). A synergism between pheromones and plant odorants at the receptor level, as observed in Helicoverpa zea (Ochieng et al., 2002), was not studied in this paper, but it could occur as a consequence of consecutive stimulation with pheromones and other odorants.
The putative pheromone component (9Z,12E)-14:Ac elicited EAG responses also in female antennae (Table 2). These responses were weak, but comparable to EAGs elicited by nerolidol, which excited a considerable subset of receptor cells localized in female trichoid sensilla. Therefore, it seems probable that pheromone sensitive receptor cells occur on female antennae. Pheromone autodetection was observed in females of several moth species (Ljungberg et al., 1993; Todd and Baker, 1993; Schneider et al., 1998). For this reason, (9Z,12E)-14:Ac was tested also on female s. trichodea, but not in the other sensillum types, as in most moth species pheromone sensitive cells are localized exclusively in s. trichodea. Only one weak single-cell response to (9Z,12E)-14:Ac was observed in a single s. trichodeum (Table 4).
VOCs
Eight VOCs were identified in the headspace of the prickly pear O. stricta, a major host plant of C. cactorum, using GC-MS (Table 1). The present data do not show striking differences between VOCs of O. stricta and plants with C3 or C4 metabolism. The origin of plant constituents from CAM metabolism is usually proven by isotope ratio mass spectrometry (Preston et al., 2003). Several C6 and C9 aroma compounds were characterized by Weckerle et al. (2001) from extracts of Opuntia ficus indica cactus pear fruits using isotope ratio mass spectrometry and they showed that especially 1-hexanol, (E)-2-hexenol, (E)-2-nonenol and (2E,6Z)-nonandienol originate from the CAM metabolism. The only compound which has been found both in the headspace of O. stricta, and in extracts from fruits of the cactus pear, was nonanal. Nonanal has also been reported from non-platyopuntia cacti species in moderately low concentrations (Kaiser and Nussbaumer, 1990; Kaiser and Tollsten, 1995).
Six of the VOCs identified from O. stricta were tested electrophysiologically on the antennae and sensilla of both sexes of C. cactorum: the terpenoids -pinene, ß-pinene, -caryophyllene, ß-caryophyllene and germacrene D from orange oil elicited rather low responses in a few cells of female s. trichodea (Table 4) and male s. auricillica/basiconica (Table 5), as well as in female EAGs (Table 2). Nonanal seems to be a strong activator of a large proportion of receptor cells localized in male and female s. auricillica/basiconica and s. coeloconica (Tables 5 and 6). Therefore, nonanal, and possibly other related C9 compounds, might represent candidate attractants of C. cactorum and should be tested behaviorally.
Schneider et al. (1964) divided olfactory receptor cells into two groups: specialists, responding with high sensitivity to a narrow spectrum of compounds, which is identical for all cells of a certain cell type, and generalists, responding with lower sensitivity to different, but overlapping spectra of compounds. According to this classification, the s. trichodea of C. cactorum females house predominantly specialists (Table 4), as do trichoid sensilla of several other moth species, e.g. Trichoplusia ni (Todd and Baker, 1993) or Spodoptera littoralis (Anderson et al., 1995). The most abundant and best characterized receptor cell of C. cactorum specialized to a floral odor was the nerolidol-sensitive cell occurring in 15% of the female trichoid sensilla. Terpenes and terpene-alcohols, including nerolidol, are common odorants of moth pollinated flowers, including several cactus species (Kaiser and Tollsten, 1995). Accordingly, receptor cells specialized to such odorants occur on the antennae of several moth species. In three heliothine species, the most abundant specialist receptor responding to VOCs of plant origin was a cell specialized to germacrene D present in 50% of the female sensilla (Stranden et al., 2003). In Bombyx mori females, all trichoid sensilla are identical and contain two specialist receptor cells that respond best to 2,6-dimethyl-5-hepten-2-ol and benzoic acid (Priesner, 1979; Heinbockel and Kaissling, 1996).
Receptor cells of s. auricillica or s. basiconica contain specialists and generalists, responding to terpenes as well as to green leaf volatiles (Table 5). This is in accordance with recordings from the basiconic sensilla of Spodoptera littoralis (Noctuidae), which contain cell types specialized to certain terpenes (e.g. geraniol, -humulene and ß-caryophyllene) besides generalists responding to a broad range of green leaf volatiles (Anderson et al., 1995) and recordings from s. auricillica of Scoliopteryx libatrix (Noctuidae) which revealed receptor cells responding to 
-3-carene, (+/–)-linalool, -pinene and green leaf volatiles (Anderson et al., 2000).
Similarly to s. coeloconica of Bombyx mori (Pophof, 1997), the receptor cells of coeloconic sensilla of C. cactorum responded to aliphatic acids and aldehydes (Table 6). In contrast to B. mori, the cells of C. cactorum could distinguish between acids and aldehydes of the same chain length and responded stronger to aldehydes.
There was a certain overlap in the specificity of receptor cells housed in morphologically distinct sensillum types. Cells specialized to certain terpenoid compounds occurred in s. trichodea as well as in s. auricillica/basiconica, cells responding strongly to aldehydes occurred in s. auricillica/basiconica and s. coeloconica. However, no overlap between s. trichodea and s. coeloconica was found; cells from female s. trichodea never responded to aliphatic compounds tested in this study.
In C. cactorum, cells of many female sensilla did not respond to any of the tested compounds (Tables 3 and 4). Therefore, the odorants activating these cells remain undiscovered. Furthermore, only a small proportion of sensilla was contacted, and due to missing morphological data the total number of contacted neurons remains unknown. Weak EAG responses to compounds activating strongly specialized receptor cells (e.g. nerolidol, nonanal, Table 2) indicate that strong activation of a certain small subset of sensilla is not reflected by a large EAG- amplitude (see also Wibe, 2004). Therefore, in C. cactorum EAG seems not to be a suitable method to search for volatiles perceived by this moth species. On the other hand, strong activators of certain cell types might be overlooked by GC-MS analysis due to their low concentrations. Therefore, a search for behaviorally active VOCs could be continued more productively by GC linked to single sensillum recordings (GC-SCR), as used recently in several other moth species (e.g. Røstelien et al., 2000a,b; Stranden et al., 2002, 2003). Also, compounds present in trace amounts, which may be biologically active, may be pursued in future work using more sensitive mass spectrometric detection.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
We thank Koji Nakanishi for his support and expert advice in natural products chemistry, Roger Heady for performing the electron microscopy, Marit Stranden for providing orange oil for electrophysiological experiments and Barry Osmond and Karl-Ernst Kaissling for important, helpful discussions. LA was partially supported by NSF (CHE-0216226). We thank Office of the Executive Vice Provost, Columbia University (Michael Crow) and Barry Osmond for a program enhancement grant to GS. We also thank Edward P. Bass for supporting this work.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Anderson, P., Hansson, B.S. and Löfquist, J. (1995) Plant-odour-specific receptor neurones on the antennae of female and male Spodoptera littoralis. Physiol. Entomol., 20, 189–198.
Anderson, P., Hallberg, E. and Subchev, M. (2000) Morphology of antennal sensilla auricillica and their detection of plant volatiles in the Herald moth, Scoliopteryx libatrix L. (Lepidoptera: Noctuidae). Arthrop. Struct. Dev., 29, 33–41.
Arn, H., Tóth, M. and Priesner, E. (1992) List of Sex Pheromones of Lepidoptera and Related Attractants. International Organization for Biological Control, West Palearctic Regional Section, INRA, Montfavet.
Bennett, F.D., Cock, M.J.W., Hughes, I.W., Simmonds, F.J.S. and Yaseen, M. (1985) In: Cock, M.J.W. (ed.), A Review of Biological Control of Pests in the Commonwealth Caribbean and Bermuda up to 1982. Commonwealth Institute of Biological Controls, Technical Communication no. 9, 218.
Black, C.B. and Osmond, C.B. (2003) Crassulacean acid metabolism photosynthesis: ‘working the night shift’. Photosynth. Res., 76, 329–341.
De Morales, C.M., Mescher, M.C. and Tumlinson, J.H. (2001) Caterpillar-induced nocturnal plant volatiles repel conspecific females. Nature, 410, 577–580.[CrossRef][Medline]
Dicke, M. and Hilker, M. (2003) Induced plant defenses: from molecular biology to evolutionary ecology. Basic Appl. Ecol., 4, 3–14.[CrossRef]
Dicke, M. and van Loon, J.J.A. (2000) Multitrophic effects of herbivore-induced plant volatiles in an evolutionary context. Ent. Exp. Appl., 97, 237–249.[CrossRef]
Garcia-Tuduri, J.C., Martorell, L.F. and Medina Gaud, S. (1971) Geographical distribution and host plant list of the cactus moth Cactoblastis cactorum (Berg) in Puerto Rico and the United States Virgin Islands. J. Agric. Univ. Puerto Rico, 55, 130–134.
Hallberg, E., Hansson, B.S. and Steinbrecht, R.A. (1994) Morphological chracteristics of antennal sensilla in the European cornborer, Ostrinia nubilalis (Lepidoptera: Pyralidae). Tissue Cell, 26, 489–502.[Medline]
Heinbockel, T. and Kaissling, K.-E. (1996) Variability of olfactory receptor neuron responses of female silkmoths (Bombyx mori L.) to benzoic acid and (±)-linalool. J. Insect Physiol., 42, 565–578.[CrossRef]
Hilker, M., Kobs, C., Varama, M. and Schrank, K. (2002) Insect egg deposition induces Pinus sylvestris to attract egg parasitoids. J. Exp. Biol., 205, 455–461.
Honda, K. (1995) Chemical basis of differential oviposition by lepidopterous insects. Arch. Insect Biochem. Physiol., 30, 1–23.
Kaiser, R. and Nussbaumer, C. (1990) 13. 1,2,3,4,4a,5,8,8a-octahydro-4ß,8a -dimethylnapthalen-4ß-ol (=dehydrogeosmin), a novel compound occurring in the flower scent of various species of Cactacea. Helv. Chim. Acta, 73, 133–139.[CrossRef]
Kaiser, R. and Tollsten, L. (1995) An introduction to the scent of cacti. Flav. Fragr. J., 10, 153–164.
Kaissling, K.-E. (1995) Single unit and electroantennogram recordings in insect olfactory organs. In Spielman, A.I. and Brand, J.G. (eds), Experimental Cell Biology of Taste and Olfaction. Current Techniques and Protocols. CRC Press, Boca Raton, FL, pp. 361–377.
Kaissling, K.-E., Meng, L.Z. and Bestmann, H.J. (1989) Responses of bombykol receptor cells to (Z,E)-4,6-hexadecadiene and linalool. J. Comp. Physiol. A, 165, 147–154.[CrossRef]
Lerdau, M. and Keller, M. (1997) Controls on isoprene emission from trees in a subtropical dry forest. Plant Cell Environ., 20, 569–578.[CrossRef]
Ljungberg, H., Anderson, P. and Hansson, B.S. (1993) Physiology and morphology of pheromone-specific sensilla on the antennae of male and female Spodoptera littoralis (Lepidoptera: Noctuidae). J. Insect Physiol., 39, 253–260.[CrossRef]
Müller, C. and Hilker, M. (2000) The effect of a green leaf volatile on host plant finding by larvae of a herbivorous insect. Naturwissenschaften, 87, 216–219.[Medline]
Müller, C. and Hilker, M. (2001) Host finding and oviposition behavior in a chrysomelid specialist—the importance of host plant surface waxes. J. Chem. Ecol. 27, 985–994.[Medline]
Noguiera, P.C. de L., Bittrich, V., Shepard, G.J., Lopes, A.V. and Marsaioli, A.J. (2001) The ecological and taxonomic importance of flower volatiles of Clusia species (Guttiferae). Phytochem., 56, 443–452.[CrossRef]
Ochieng, S.A., Park, K.C . and Baker, T.C. (2002) Host plant volatiles synergize responses of sex pheromone-specific olfactory receptor neurons in male Helicoverpa zea. J. Comp. Phys. A, 188, 325–333.
Osmond, C.B. and Monro, J.B. (1981) Prickly pear. In Carr, D.J. and Carr, S.G.R. (eds), Plants and Man in Australia. Academic Press, Sydney, pp. 194–222.
Osmond, C.B., Nott, D.L. and Firth, P.M. (1979) Carbon assimilation patterns and growth of the introduced CAM plant Opuntia inermis in eastern Australia. Oecologia, 40, 331–350.[CrossRef][Web of Science]
Pawliszyn, J. (1999) Applications of Solid Phase Microextraction. In Smith, R.M. (ed.), RSC Chromatography Monographs, Vol. xviii. Royal Society of Chemistry, Cambridge, 655.
Pophof, B. (1997) Olfactory responses from sensilla coeloconica of the silkmoth Bombyx mori. Physiol. Entomol., 22, 239–248.
Pophof, B. and van der Goes van Naters, W. (2002) Activation and inhibition of the transduction process in silkmoth olfactory receptor neurons. Chem. Senses, 27, 435–443.
Preston, C., Richling, E., Elss, S., Appel, M., Heckel, F., Hartlieb, A. and Schreier, P. (2003) On-line gas chromatography combustion/pyrolysis isotope ratio mass spectrometry (HRGC-C/P-IRMS) of pineapple (Ananas comosus L. Merr.) volatiles. J. Agric. Food Chem., 51, 8027–8031.[Medline]
Priesner, E. (1979) Progress in the analysis of pheromone receptor systems. Ann. Zool. Ecol Anim., 11, 533–546.
Raguso, R.A. and Pellmyr, O. (1998) Dynamic headspace analysis of floral volatiles: a comparison of methods. Oikos, 81, 238–254.[CrossRef][Web of Science]
Røstelien, T., Borg-Karlson, A.K. and Mustaparta, H. (2000a) Selective receptor neurone responses to E-ß-ocimene, ß-myrcene, E,E-
-farnesene and homo-farnesene in the moth Heliothis virescens, identified by gas chromatography linked to electrophysiology. J. Comp. Physiol. A, 186, 833–847.[CrossRef][Medline]
Røstelien, T., Borg-Karlson, A.K., Faldt, J., Jacobsson, U. and Mustaparta, H. (2000b) The plant sesquiterpene germacrene D specifically activates a major type of antennal receptor neuron of the tobacco budworm moth Heliothis virescens. Chem. Senses, 25, 141–148.
Schneider, D., Lacher, V. and Kaissling, K.-E. (1964) Die Reaktionsweise und das Reaktionsspektrum von Riechzellen bei Antheraea pernyi (Lepidoptera, Saturniidae). Z. Vergl. Physiol., 48, 632–662.[CrossRef]
Schneider, D., Schulz, S., Priesner, E., Ziesmann, J. and Francke, W. (1998) Autodetection and chemistry of female and male pheromone in both sexes of the tiger moth Panaxia quadripunctata. J. Comp. Physiol. A, 182, 153–161.[CrossRef]
Simmonds, F.J. and Bennett, F.D. (1966) Biological control of Opuntia spp. by Cactoblastis cactorum in the Leeward Islands (West Indies). Entomophaga, 11, 183–189.
Stange, G. (1997) Effects of changes in atmospheric carbon dioxide on the location of hosts by the moth, Cactoblastis cactorum. Oecologia, 110, 539–555.[CrossRef][Web of Science]
Stange, G., Monro, J., Stowe, S. and Osmond, C.B. (1995) The CO2 sense of the moth Cactoblastis cactorum and its probable role in the biological control of the CAM plant Opuntia stricta. Oecologia, 102, 341–352.[CrossRef][Web of Science]
Starmer, W.T., Aberdeen, V. and Lachance, M.A. (1987) The yeast community associated with decaying Opuntia stricta (Haworth) in Florida with regard to the moth, Cactoblastis cactorum (Berg). Flav. Sci., 51, 7–11.
Stranden, M., Borg-Karlson, A.-K. and Mustaparta, H. (2002) Receptor neuron discrimination of the germacrene D enantiomers in the moth Helicoverpa armigera. Chem. Senses, 27, 143–152.
Stranden, M., Liblikas, I., König, W.A., Almaas, T.J., Borg-Karlsson, A.-K. and Mustaparta, H. (2003) (–)-Germacrene D receptor neurones in three species of heliothine moths: structure–activity relationships. J. Comp. Physiol. A, 189, 563–577.[CrossRef]
Todd, J.L. and Baker, T.C. (1993) Response of single antennal neurons of female cabbage loopers to behaviorally active attractants. Naturwissenschaften, 80, 183–186.[CrossRef]
Weckerle, B., Bastl-Borrmann, R., Richling, E., Hör, K., Ruff, C. and Schreier, P. (2001) Cactus pear (Opuntia ficus indica) flavor constituents—chiral evaluation (MDGC-MS) and isotope ratio (HRGC-IRMS) analysis. Flav. Frag. J., 16, 360–363.[CrossRef]
Wibe, A. (2004) How the choice of method influence on the results in electrophysiological studies of insect olfaction. J. Insect Physiol., 50, 497–503.[Medline]
Witzgall, P., Lindblom, T., Bengtsson, M. and Tóth, M (2004) The Pherolist. www-pherolist.slu.se
Zabaras, D. and Wyllie, S.G. (2002) Rearrangement of p-menthane terpenes by carboxen during HS-SPME. J. Sep. Sci., 25, 685–690.[CrossRef]
Zimmermann, H.G., Moran V.C. and Hoffmann J.H. (2001) The renowned cactus moth, Cactoblastis cactorum (Lepidoptera: Pyralidae): its natural history and threat to native opuntia floras in Mexico and the United States of America. Florida Entomol., 84, 543–551.
Accepted October 28, 2004
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||