J Comp Physiol A (1999) 185: 419±425
Ó Springer-Verlag 1999
ORIGINAL PAPER
R. P. J. Potting á P. M. LoÈsel á J. Scherkenbeck
Spatial discrimination of pheromones and behavioural antagonists by the tortricid moths Cydia pomonella and Adoxophyes orana
Accepted: 18 July 1999
Abstract Male moths responding to their species-speci®c sex pheromone, may cease their upwind ¯ight when pheromone components of sympatric species are added to the mixture. The interspeci®c interaction between the pheromone response of the tortricid moths Cydia pomonella and Adoxophyes orana was investigated in ®eld-trapping and wind-tunnel studies. Addition of the A. orana pheromone [(Z9)-tetradecenylacetate and (Z11)-tetradecenylacetate] to a source containing the C. pomonella pheromone [(E8, E10)-dodecadienol] resulted in a signi®cant inhibition of attraction by male C. pomonella. It is demonstrated that this behavioural antagonist for C. pomonella must be emitted from the same point source to induce this inhibitory eect. A spatial separation of the two interspeci®c pheromones (at 14 cm, 5 cm and 0.5 cm crosswind) restored the attraction of the conspeci®c pheromone for male C. pomonella. In contrast to C. pomonella, male A. orana were not inhibited by point sources releasing both the C. pomonella and A. orana pheromone. We suggest that the discrepancy in the interspeci®c pheromone interaction between these two tortricids can be explained if we consider the evolutionary ecology of interspeci®c pheromone communication in C. pomonella and A. orana. Key words Cydia pomonella á Adoxophyes orana á Sex pheromone á Interspeci®c interruption á Evolution Abbreviations Z9-14:Ac (Z9)-tetradecenylacetate Z11-14:Ac (Z11)-tetradecenylacetate á E8, E10-12:OH (E8, E10)-dodecadienol R.P.J. Potting (&)1 á P.M. LoÈsel á J. Scherkenbeck BAYER AG, Central Research Division, Agricultural Research Centre Monheim, Building 6220, D-40789, Monheim, Germany Present address: Entomology and Nematology Department, IACR-Rothamsted, Harpenden, Herts., AL5 2JQ, UK, e-mail:
[email protected] Fax: +44-1582-760981
1
Introduction The evolution of speci®c pheromone communication systems has played an important role in reproductive isolation in moths. One of the most important premating isolating mechanism for moths is the speci®city of the sex attractant signals, which are emitted by the females and perceived by the males. The precise blend production by calling females is usually matched with a corresponding speci®city in perception by conspeci®c male moths. The attraction response of male moths to their species-speci®c pheromone can be modulated by certain compounds that either synergize or inhibit the response of males. For several moth species compounds that inhibit the attraction to the sex pheromone of the species have been identi®ed. Many of these compounds are geometrical isomers of the pheromone, as for example in Cydia pomonella (Roelofs et al. 1972; Arn et al. 1974) and Adoxophyes orana (Minks and Voerman 1973; Guerin et al. 1986) and are often pheromone components in closely related species. Recent wind-tunnel experiments with inhibitors has revealed important knowledge on the physical structure of odour plumes. The study of Baker et al. (1998) combined with the elegant experiments of Vickers and Baker (1994) and Mafra-Neto and Carde (1994) demonstrated that an odour plume consists of strands of odour ®laments interspersed with pockets of clean air. Vickers and Baker (1994) demonstrated that a ¯ying male moth orienting in an odour plume, responds to the onset and disappearance of each odour strand by ¯ying straight upwind when it encounters a pheromone ®lament and by casting ¯ight when it is not reinforced by a subsequent pheromone ®lament. Vickers and Baker (1997) demonstrated that a male H. virescens ceases to ¯y upwind when it encounters a pheromone ®lament that is contaminated with an inhibitor. The ability to respond to intact pheromone ®laments enables an orienting male moth to orient towards a conspeci®c pheromone signal, without being disturbed
420
by background chemical noise that may originate from pheromone plumes of related sympatric species. For several species of moth it has been shown that the male antennae have speci®c receptors tuned to inhibitory pheromone components of closely related species (e.g. O'Connell et al. 1983; Renou and Lucas 1994). We investigated the nature of the interspeci®c response towards pure and contaminated pheromone sources for two tortricid moth species: the codling moth, C. pomonella L. (Lepidoptera: Tortricidae: Grapholitinae), a pest of world-wide importance in pome fruit crops, and the summerfruit tortrix moth A. orana (F.v.R.) (Lepidoptera: Tortricidae: Tortricinae), an important secondary pest in pome fruits in Europe. The main sex pheromone component of C. pomonella attracting males in the ®eld was identi®ed by Roelofs et al. (1972) as (E8, E10)-dodecadienol (E8, E10-12:OH; codlemone). Although several secondary pheromone components released by female C. pomonella have been identi®ed, they at most only play a marginal role in long range attraction of males (see Ebbinghaus et al. 1998 for recent discussion and references). The main sex pheromone components attracting male A. orana were identi®ed as a 9:1 mixture of (Z9)tetradecenylacetate (Z9-14:Ac) and (Z11)-tetradecenylacetate (Z11-14:Ac) (Meijer et al. 1972). Additional pheromone components for A. orana have been identi®ed by Guerin et al. (1986), but these compounds do not play a signi®cant role in long range attraction. Minks et al. (1973) demonstrated that the exact ratio of Z914:Ac and Z11-14:Ac is very important in determining the attractiveness of pheromone sources for A. orana and other sympatric tortricids using combinations of these two 14:Ac isomers. In European pomefruit-growing areas A. orana and C. pomonella are sympatric. A. orana and C. pomonella are phylogenetically related in the sense that they both belong to the family Tortricidae; however, within the Tortricidae they are taxonomically distinct in that A. orana belongs to the sub-family Tortricinae, which comprise mainly larval leaf feeders, and C. pomonella belongs to the subfamily Olethreutinae, which comprise mainly larval fruit or stem borers (Alford 1984). Both sub-families not only have a distinct ecological niche, they also have evolved a distinct pheromone communication system. The Tortricinae mainly use 14-carbon compounds as pheromone components (e.g. isomers of 14:Ac in A. orana), which are widespread in several lepidopteran families, whereas the Olethreutinae mainly use 12-carbon compounds as their sex pheromone, of which 12:OH pheromone components (e.g. 12:OH and E8, E10-12:OH in C. pomonella) are quite uncommon and seem to have evolved quite recently (Roelofs and Brown 1982; LoÈfstedt and Bengtsson 1988). We studied the interspeci®c interaction of the pheromones of A. orana and C. pomonella. An a priori hypothesis would be that A. orana is not disturbed by the relatively `novel' pheromone of C. pomonella, whereas C. pomonella may
behaviourally respond to the A. orana pheromone by detecting the ancestral 14:Ac components.
Materials and methods Odour sources The pheromone components used in the odour sources consisted of the main pheromone component of C. pomonella, E8, E10-12:OH (Dr. Baan, Hungarian Academy of Science, Budapest, Hungary; 97% purity) and the main pheromone components of A. orana, a 9:1 mixture of Z9-14:Ac and Z11-14:Ac (Aldrich Chemical; 98% purity). The compounds were applied as hexane solutions on rubber dispensers (IPO, Wageningen) or contained in a castor-oil based paste (LoÈsel et al., unpublished manuscript). The concentration of pheromone in the paste was 0.1% E8, E10-12:OH for C. pomonella and a 1% of a 9:1 mixture of Z9-14:Ac and Z11-14:Ac for A. orana. As a standard odour source 100-ll droplets of paste were used. In the experiments where dispensers were used as the odour source, the pheromone load per dispenser was 1 mg. Field trapping The experimental site was a 0.5-ha plot in an apple orchard at Laacherhof near Monheim, Germany. The height of the trees was up to 3 m, the width between the rows 3 m and the distance between trees 1.5 m. Trapping experiments were carried out in 1995 and 1996. Traps were of the covered funnel type (Fruit Consult International) and contained plastic blocks releasing dichloorphos to kill insects which entered the trap. The traps were hung in the trees at a height of 1.70 m, on average 10 m apart. The number of moths caught in the traps were recorded at least once a week from May 23 until August 28 in 1995 and from May 29 until September 16 in 1996. The odour sources were replaced after 4 weeks. Treatments were tested in randomised block design. In 1995, three types of odour sources were tested in two blocks: (1) a dispenser containing the A. orana pheromone (ADOX), (2) a dispenser containing the C. pomonella pheromone (CYDIA), and (3) two dispensers, one containing the A. orana pheromone and the other one containing the C. pomonella pheromone placed on top of each other (ADOX + CYDIA-separated). In 1996, three types of odour sources were tested in three blocks: (1) a plastic bottle lid with 100 ll of paste containing the A. orana pheromone (ADOX), (2) a bottle lid with 100 ll of paste containing the C. pomonella pheromone (CYDIA), and (3) a bottle lid with 100 ll of paste containing the A. orana pheromone and the C. pomonella pheromone (ADOX + CYDIA-combined). Insects Pupae of C. pomonella and A. orana were obtained from a laboratory culture (Andermatt Biocontrol, Switzerland), were separated by sex and were held in Petri dishes at 23 1 °C, 70 10% RH, 10:14 h dark:light regime. Moths used in the experiments were 2±3 days old unmated males. Wind tunnel studies Observations on the close-range behaviour of male moths approaching an odour source were done in a small wind tunnel (1.5 m ´ 0.4 m ´ 0.4 m). Air was blown through the tunnel at a windspeed of 0.20 m s)1. The tunnel was illuminated from above by two red-coloured photographic safelights (light intensity 5 lx). Temperature was maintained at 22 1 °C. Experiments on the spatial discrimination of odour sources were conducted in a large wind tunnel (3 m ´ 1 m ´ 1.5 m), as described by Ebbinghaus et al. (1998).
421 Close range behaviour
Combined odour source (®eld experiments)
The close-range ¯ight behaviour of individual male C. pomonella approaching a pheromone source (codlemone) with or without the A. orana pheromone was investigated in a small wind tunnel. Experiments were carried out 1 h after onset of the scotophase. One odour source consisted of a plastic bottle lid containing 0.1 g of paste with 0.1% codlemone + 0.1 g `empty' paste. The combined odour source consisted of 0.1 g paste with 0.1% codlemone + 0.1 g paste with 1% A. orana pheromone was placed on a 10 cm ´ 10 cm cardboard platform, at a height of 15 cm at the upwind end of the wind tunnel. Male C. pomonella (2±3 days old) were individually con®ned in a small Petri dish. The Petri dish was opened after it had been placed at the bottom of the downwind end of the wind tunnel. The behaviour of individual males was observed for 5 min. Parameters recorded were: Take o = leaving Petri dish; Land = land on platform; Contact = contact odour source.
The results of the ®eld-trapping experiment of 1996 are summarised in Fig. 1C, D. In this experiment we compared the attractiveness of the species-speci®c pheromone in pure form or in combination with the foreign pheromone. The dierence from the 1995 experiment was that in the combined odour source, both the A. orana and C. pomonella pheromone were now released from the same point source (plastic lid with oily paste containing both pheromones). There was no signi®cant dierence in the catch rate of male A. orana in the traps containing the combined or pure A. orana pheromone bait (Fig. 1C, t-test, T = 0.05, df = 32, P = 0.96). However, the addition of the A. orana pheromone almost completely inhibited the attractive eect of codlemone on male C. pomonella. There was a signi®cant reduction in the catch rate of male C. pomonella in the traps containing the combined pheromone bait compared to traps baited with the pure codlemone bait (Fig. 1D, t-test, T = )2.55, df = 32, P = 0.015).
Spatial discrimination The eect of spatially separating the A. orana pheromone and C. pomonella pheromone on the behaviour of male C. pomonella was examined in a series of experiments in the large wind tunnel. Odour sources consisted of plastic lids with 100 ll of paste containing the pheromone of C. pomonella or A. orana. In the experiments male C. pomonella were oered a choice between two odour sources. The odour sources were placed 90 cm apart on a metal frame at a height of 30 cm and 15 cm from the upwind diusing screen of the tunnel. Responding males were trapped by placing a transparent sheet (30 cm ´ 10 cm), coated with an insect-trapping compound (Tanglefoot, USA), below each odour source. For each test replicate 15± 20 male C. pomonella were released from a Petri dish at the downwind end of the tunnel (1 h after onset of the scotophase) and the number of males caught on the two traps was recorded 24 h after release. Each treatment was repeated four to six times (i.e. 70±120 males per treatment). To avoid any asymmetrical bias in the set up, the position of the odour sources was exchanged after each experiment. The following treatments were tested: (1) combined odour source, with both the A. orana and C. pomonella pheromone versus a pure C. pomonella pheromone source, and (2, 3, 4) a codlemone odour source ¯anked by two odour sources containing the A. orana pheromone at a distance of 14 cm, 5 cm and 0.5 cm versus a codlemone odour source ¯anked by two control odour sources containing paste with no pheromone at a distance of 14 cm, 5 cm and 0.5 cm.
Results Separated odour sources (®eld experiments) The ®eld-trapping data of 1995, where we compared the attractiveness of the species-speci®c pheromone in the presence or absence of a dispenser containing the other species' pheromone, are summarised in Fig. 1. The addition of a dispenser containing the pheromone of C. pomonella to a bait with a dispenser containing the A. orana pheromone did not signi®cantly aect the attraction of male A. orana (Fig. 1A, t-test, T = )0.82, df = 22, P = 0.42). Also for C. pomonella we did not ®nd a signi®cant inhibitory eect of the addition of a dispenser containing the A. orana pheromone to a bait containing a dispenser with the C. pomonella pheromone. There was no signi®cant dierence in the capture rates of male C. pomonella in traps baited with the C. pomonella pheromone or traps baited with both the A. orana and the C. pomonella pheromone (Fig. 1B, t-test, T = )0.72, df = 22, P = 0.48).
Close-range behaviour (wind tunnel experiments) The results of the observations of the close-range behaviour of male C. pomonella responding to a pure or combined odour are presented in Fig. 2. There was no signi®cant dierence in the initial response (i.e. take-o from dish) towards both odour sources (G-test, v2 = 0, P = 1). However, there was a signi®cant reduction in the number of landings on the platform (G-test, v2 = 4.41, P = 0.04) and subsequent number of contacts (G-test, v2 = 12.45, P = 0.0004) with the odour source for males responding to the combined odour source compared to males responding to the pure pheromone source. Thus, the attractive eect of codlemone seems to be inhibited by the A. orana pheromone at a close distance (20±30 cm) from the odour source. Spatial discrimination (wind tunnel experiments) The discrepancy in the ®eld-trapping results of 1995 (no inhibitory eect of A. orana pheromone) and 1996 (strong inhibitory eect of A. orana pheromone) suggested that the spatial separation of the pheromones may be responsible for the loss of the inhibitory eect of the A. orana pheromone for C. pomonella males. This was investigated in more detail in the large wind tunnel. The results are summarised in Fig. 3. When male C. pomonella were oered a choice between a source containing the pure pheromone and a combined point source containing both the A. orana and C. pomonella pheromone almost all males landed on the trap containing the pure pheromone source (binomial probability function, n = 45 trapped males, p = 0.5, P < 0.00001). When the C. pomonella pheromone and
422 Fig. 1A±D Comparison of attractiveness of single versus multi-species pheromone source. A, B Field trapping experiments (1995) with as bait a single or double dispenser per trap. Black bars (A) indicate mean trap catches for A. orana (n = 778) and white bars (B) indicate mean trap catches for C. pomonella (n = 230). Traps were baited with dispensers loaded with 1 mg of C. pomonella pheromone (Cydia); 1 mg of A. orana pheromone (Adox); two separate dispensers as bait: one with C. pomonella pheromone, the other one with A. orana pheromone (Separate). C, D Fieldtrapping experiments (1996) with oil paste as bait containing a single species or multi-species pheromone. Black bars (C) indicate mean trap catches for A. orana (n = 1638) and white bars (D) indicate mean trap catches for C. pomonella (n = 37). Traps were baited with 100 ll of paste with: 0.1% C. pomonella pheromone (Cydia); 1% A. orana pheromone (Adox); 0.1% C. pomonella pheromone + 1% A. orana pheromone (Combined). Asterisks indicates signi®cant dierence in trap catch, n.s. indicates non-signi®cant dierence in trap catch (t-test, P < 0.05)
A. orana pheromone were spatially separated at a distance of 14 cm, 5 cm and 0.5 cm, there were no signi®cant dierences in the numbers of male C. pomonella caught on traps with both pheromones and the trap containing the pure pheromone source [binomial probability functions: (1) 14 cm: n = 40 trapped males, p = 0.5, P = 0.437; (2) 5 cm: n = 33 trapped males, p =0.5, P = 0.500; (3) 0.5 cm: n = 28 trapped males, p = 0.5, P = 0.425].
and wind-tunnel experiments, show that the A. orana pheromone interrupts the pheromone-mediated orientation of male C. pomonella, resulting in a signi®cant reduction of trap catches compared to traps baited with the pheromone alone. In contrast, the C. pomonella pheromone does not interrupt the pheromone-mediated attraction of male A. orana.
Discussion
The inter-speci®c pheromone interaction between C. pomonella and A. orana is only in one direction: male C. pomonella are strongly inhibited by the pheromone of A. orana, but male A. orana are not inhibited by the pheromone of C. pomonella. A possible explanation for
The behavioural response of male C. pomonella and A. orana to their conspeci®c pheromone in the presence and absence of an interspeci®c pheromone, in both ®eld
Evolution of communication
423 Fig. 2 Close-range behaviour of male C. pomonella responding to its own pure pheromone (black bars) or to its own pheromone in combination with the A. orana pheromone (white bars) as measured in direct observations in the small wind tunnel. Pure 1 mg of paste with 0.1% (E8, E10)-dodecadienol (E8, E10-12:OH). Combined 1 mg of paste with 0.1% E8, E10-12:OH + 1% of a 9:1 mixture of (Z9)-tetradecenylacetate + (Z11)-tetradecenylacetate. Recorded behaviours: Take o ¯ying upwind; Land landing on platform; Contact contacting odour source. Asterisks indicate signi®cant dierence in response to odour sources (G-test, P < 0.05)
this phenomenon can be found if we consider the evolution of pheromone communication in tortricids. In their review on pheromone evolution in the Tortricidae, Roelofs and Brown (1982) show that there was a general trend for species in the subfamily Tortricinae to use 14-carbonchain sex pheromones, whereas species in the subfamily Olethreutinae utilise 12-carbon-chain sex pheromones. Phylogenetically the Olethreutinae are supposed to have derived from the more primitive Tortricinae. Since the Olethreutinae (to which C. pomonella belongs) are supposed to have evolved from the ancestral Tortricinae, we hypothesise that at one time the ancestor of C. pomonella used 14:Ac structures as its sex pheromone components and thus had speci®c receptors tuned to 14:Ac components. It is possible that since the evolution of 12-carbon aldehydes communication in the Olethreutinae, these receptors on male antennae tuned to 14:Ac compounds were retained and later in evolutionary time used to
recognise inter-speci®c females. In contrast, the Tortricinae (to which A. orana belongs) did not follow the evolutionary pathway leading to communication with 12carbon structures. Thus, A. orana may not have receptors tuned to 12:OH structures, which may explain why the pheromone response of male A. orana is not inhibited by E8, E10-12:OH, the sex pheromone of C. pomonella. Fig. 3 Eects of the spatial separation of two pheromone sources on response of male C. pomonella. Indicated are the choices of the responding (i.e. trapped) males in large wind tunnel. White bars indicate moths attracted towards combined pheromone source, where C. pomonella pheromone and A. orana pheromone were released from the same paste droplet (combined) or where a droplet containing C. pomonella pheromone was ¯anked by two droplets containing A. orana pheromone at 14 cm, 5 cm and 0.5 cm distance. Black bars indicate moths attracted to droplet with C. pomonella pheromone ¯anked by empty control paste droplets. Asterisks indicates signi®cant dierence in response to odour sources (binomial probability function, P < 0.0001)
424
From studies correlating the electrophysiological response of lepidopteran sensilla with their morphology, it is known that primary pheromone components involved in upwind orientation are detected by receptor neurones housed in Sensilla trichodea (e.g. Preiss and Priesner 1978; Mayer and Mankin 1985; Akers and O'Conell 1988; Almaas and Mustaparta 1990). For several moth species it has been demonstrated that male antennae have speci®c receptors for inhibitory pheromone components of closely related species (e.g. Renou and Lucas 1994 and references therein). These neurons tuned to antagonistic compounds are very often co-compartmentalised within the same sensillum hair as the neurons tuned to known agonists, which optimises the detection of non-coincidental arrival of odour strands to which each neuron is tuned (Baker et al. 1998). Since stimulation of isolated antennae of male C. pomonella with both Z9-14:Ac and Z11-14:Ac elicited signi®cant EAG responses (D. Ebbinghaus, unpublished observations), it is tempting to speculate that the response originates at least in part, in as yet unidenti®ed neurones tuned speci®cally to these molecules. In single-cell recordings from codlemone-sensitive S. trichodea Ebbinghaus et al. (1998) found three dierent types of cells, of which two responded to the main component E8, E10-12:OH, but the third one did not respond to any of the C. pomonella pheromone components. It is tempting to suggest that this third cell may be tuned to inhibitory compounds. Filamentous nature of odour plume Male C. pomonella do not orient upwind when the pheromone source contains the pheromone of A. orana. However, C. pomonella males successfully orientated to a source of codlemone with two sources of the inhibitory A. orana pheromone placed only 0.5 cm away from it. This corroborates the wind-tunnel studies of Witzgall and Priesner (1991), Liu and Haynes (1992), Rumbo et al. (1993) and Baker et al. (1998) and gives further evidence for the ®lamentous structure of an odour plume, by demonstrating that the odour ®laments emanating from the odour sources are suciently separated to be distinguished by ¯ying moths and that the inhibitor must be detected simultaneously with the pheromone to in¯uence the behaviour of the moth. The fact that an odour plume consists of separate pheromone ®laments is also evidenced by the fact that separating the main pheromone components of species which require a multi-component pheromone for upwind ¯ight disrupts the orientation of males, as has been demonstrated for the cabbage looper moth Trichoplusia ni (Linn and Gaston 1981) and the spotted stemborer Chilo partellus (Lux et al. 1994). In wind-tunnel studies we demonstrated that if the two pheromone components responsible for long-range attraction (Z9-14:Ac and Z11-14:Ac) in male A. orana are spatially separated, the attraction rate decreased dramatically (R.P.J. Potting et al., unpublished data).
Acknowledgements We thank Tanja Nepute for technical assistance and Dr. Dirk Ebbinghaus for critically reading a previous version of the manuscript.
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