Uwe T. Koch, Wolfgang Lüder, Stephan Clemenz and Liliana I. Cichon1
Department of Biology, University of Kaiserslautern, 67653 Kaiserslautern, Germany
Abstract - Pheromone measurements using a field EAG system were made in apple orchards treated for mating disruption of codling moth, Cydia pomonella. We present the first example of simultaneous pheromone concentration measurements, using a field EAG system and gas chromatographic analysis of air filters. This allows an absolute calibration of the antennal response to pheromone dilutions in oil. Original recordings are shown illustrating the interaction of pheromone and non-pheromone stimuli in the EAG signal, and the instantaneous reaction to the transition from pheromone-free to a pheromone-loaded air current.
Key words - sex pheromone, mating disruption, field EAG, aerial pheromone concentrations, pome fruit orchard, Cydia pomonella, Tortricidae, Lepidoptera
Measurements of aerial pheromone concentrations can be an important contribution to the further development of the mating disruption technique. Investigations of wind effects, plant-surface interactions and concentration profiles in vertical and horizontal dimensions all require the acquisition of many measurements in time intervals which can be considered short in comparison to the time course of changes in temperature or wind situation.
Methods using air sampling and subsequent analysis by gas chromatography (e.g., Flint et al. 1993) offer absolute average concentration values, but they are not fast enough to yield a sufficient number of measurements in the required time. Field electroantennogram (EAG) measurements of pheromone concentrations can offer reproducible concentration values on a relative scale. The field EAG measurement system developed in Kaiserslautern has been used to measure pheromone concentrations in vineyards (Milli 1990; Sauer 1991; Färbert 1992, 1995; Karg 1992; Koch et al. 1992; Termer 1992; Karg & Sauer 1995), in cotton fields (Cardé et al. 1993; Färbert & Koch 1993; Färbert 1995; Färbert et al. 1996), in pea fields (Bengtsson et al. 1994) and in apple orchards (Milli 1993; Karg et al. 1994; Suckling et al. 1994; Milli et al. 1996). The newer versions of our system make use of a signal superposition technique to suppress the influence of plant odors and other non-pheromone airborne stimuli on the pheromone concentration measurements.
Up to now, field EAG measurement results had only been given in relative units, defined by the properties of the calibration sources in our system. In collaboration with the Chemical Ecology Group at Alnarp (see Bäckman 1997) we have simultaneously measured pheromone concentration with the field EAG and the classical filter adsorption technique. This permits to transfer the relative concentration units to absolute concentration values. In addition, we present measurement examples with highly dynamic changes in airborne pheromone concentration values demonstrating the linear superposition of pheromone- and non-pheromone-generated EAG signals.
Field EAG System
The field EAG system used in these experiments has been described in detail (Färbert et al. 1996a,b). It consists of an antenna holder, which is attached to the bottom of a vertical tube in which a steady current of air (14 ml/s) is maintained using a suction pump. A charcoal filter placed at the tube upper entrance removes all stimulating odor components from the incoming air. Three calibration sources, consisting of glass syringes, containing a vial with a pheromone/oil mixture (Sauer 1989), are connected to the tube in such a way that activation of the syringe piston generates an air puff (0.25 ml, 0.6 s duration) with defined pheromone content which is injected into the main airstream. An excised antenna of codling moth, Cydia pomonella, placed into the antennal holder, was used for measurements of airborne codlemone, (E,E)-8,10-dodecadien-1-ol.
The antennal responses to puffs from the calibration syringes with pheromone concentrations in three decadic steps are used to construct a dose response curve characterizing the properties of the antenna. When the charcoal filter is removed from the tube, outside air reaches the antenna and produces a rise in the EAG signal similar to a step function. The height of this step is caused by background odors as well as pheromones, and cannot be used as a measure for pheromone concentration. While the filter remains open, additional calibration pulses are released. The additional response of the EAG signal to the superimposed calibration puffs is used to calculate the airborne pheromone concentration: a small or disappearing additional response indicates high pheromone concentration, whereas an EAG response to the superimposed stimulus which is almost as strong as in the calibration mode indicates a very low level of ambient pheromone concentration, close to the reaction threshold of the antenna.
The relative pheromone concentration units used up to now in all our experiments are defined as follows: a concentration of 10-6 relative units is the concentration present in the headspace of a calibration syringe containing a vial with 106 parts of paraffin oil (Merck No.7161) and one part of pheromone.
The EAG measurement system including pumps, calibration syringes and associated step motor drives is mounted on a compact probe which is fully remote controlled and can be operated on a pole up to 5.5 m high. Wind velocity and direction are recorded in 40-ms intervals by two sensitive vector anemometers, one mounted on the EAG probe, the other at 5.7 m height.
Parallel air adsorption measurements
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These experiments were done in an experimental apple orchard at Alnarp (Sweden). The tree height was 2 m, tree spacing was 2.5 m, and the rows were 2 m apart. A surface of 0.5 ha had been treated with Ecopom dispensers (Isagro, Novara) loaded with 250 mg of codlemone and 250 mg of codlemone acetate, (E,E)-8,10-dodecadien-1-yl acetate, placed at a density of 1000 dispenser/ha.
The first air sampling experiment was made on July 8, 1996 (Bäckman 1997). The air filter was positioned in the center of the orchard, at equal distance from the closest dispensers, at 2.0 m height. The field EAG probe was positioned at the same height. The EAG probe and the air filter were positioned on a line perpendicular to the wind direction, 0.5 m apart. While the orchard air was drawn through the filter, measurements were done continuously with the field EAG. The individual readings of the EAG system were corrected for temperature dependence of the pheromone vapor pressure in the calibration syringes and then averaged to yield a representative overall pheromone concentration reading representing the time span during which the air sampling had taken place.
Interaction of pheromone and background
These recordings were made in an orchard at Solnäs, 10 km north of Lund (Sweden). Trees (2.20 m high) were spaced 1.6 m apart, at a row distance of 3.3 m. A total of approximately 14 ha had been treated with Ecopom dispensers (Isagro; 250 mg codlemone/dispenser) on May 27 at a density of 400 dispensers/ha. The field EAG recordings were taken on June 6, 21 hrs, on a transect parallel to the rows leading to the southern edge of the orchard.
Simultaneous EAG and absorption measurements
Figure 1 shows the results of the field EAG measurements made on July 8, concurrently with a filter adsorption measurement. The pheromone concentration readings were averaged in four sections. During each section the temperature was sufficiently constant. The four averages were then individually corrected for the temperature dependence of the pheromone vapor pressure in the calibration syringes. Finally, the four averages were again averaged to yield one overall average concentration: 6.8 ± 0.5 * 10-7 relative units. The result of the adsorption measurement was 1.1 ng/m3 (Bäckman 1997): 1.0 * 10-6 relative units of codlemone are accordingly equivalent to 1.6 ± 0.3 ng/m3.
The repetition of this measurement (12:30 to 17:30 hrs) took place on July 12 at the same location (Figure 2). Our overall averaged relative concentration reading was
Figure 1 Pheromone concentration measured in relative units (for definition see text) plotted versus time. Between 16:10 and 19:57, the orchard air was pumped through a pheromone-adsorbing filter (Bäckman 1997). Thin line error bars indicate the confidence interval of an individual concentration reading. Thick line error bars (without delineator) indicate standard error generated by averaging individual pheromone concentration readings. Open circles indicate the sensitivity threshold of the antenna in use. Large filled squares are averages taken over four time sections during which temperature was sufficiently constant, with temperature correction applied. The large filled circle indicates overall average of pheromone concentration in relative units. The right vertical axis was adjusted in such a way that the absolute concentration measured by adsorption technique reaches the same position as the overall average of the field EAG
6.1 ± 0.5 * 10-6 relative units. The result of the adsorption measurement was 1.3 ng/m3 (Bäckman 1997): 1.0 * 10-6 relative units of codlemone are accordingly equivalent to 2.1 ± 0.4 ng/m3. The measurements of July 8 and 12 are in agreement within the range of the errors.
Pheromone-background interaction
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During most of these measurements, the wind came from southern directions, thus the southern edge of the orchard received pheromone-free air coming from the neighbouring sugar beet field. Figure 3 shows three superimposed EAG traces recorded 4 m upwind from the orchard and at the orchard edge. As there is no pheromone in the air, the plateau response in Figure 3 is caused only by background odors. Note that although the position was not changed, the plateau height changed significantly. This must have been caused at least in part by a change of background odor concentration. In contrast, the additional response to the superimposed stimulus remains the same, indicating the unchanged pheromone concentration near the threshold of the system.
Figure 2 Repetition of the measurement of Figure 1 at the same location, 4 days later. The equivalence between relative and absolute concentration units found in Figure 1 is confirmed within the confidence intervals of both experiments
Figure 4 shows three superimposed EAG traces taken at different positions along the transect, oriented in north-south direction. At 4 m upwind from the orchard edge, the calibration pulses superimposed on the plateau (generated by opening the charcoal filter) were almost the same height as during the calibration. At the edge and inside the orchard 30 m downwind from the edge, we observed strong fluctuations of the EAG signal. Whenever the superimposed pulses occured during a strong upward excursion of the EAG signal, the net response to the pheromone stimulus was strongly reduced - but the absolute height of the peak of the response to the superimposed stimuli remained the same (see arrows in Figure 4). This antennal behavior is consistent with the concept of linear superposition of the EAG voltages generated by backgoung stimuli and pheromone stimuli.
Figure 3 EAG signals recorded at the upwind edge of an apple orchard treated for mating disruption. Solid and dashed traces: EAG voltage; dotted trace: activation of calibration syringes (columns of different height indicate activation of syringe with different pheromone content and opening of charcoal filter; plateau signal). The left part of the figure shows the calibration phase, in which the syringes are activated while the charcoal filter is in place. The right part shows the reactions to ambient air and superimposed calibration stimuli. Since the air in this measurement situation is pheromone-free, the plateau appearing on the opening of the charcoal filter is generated by background odors. Note that although the plateau height increases for the thick solid trace, the net reaction to the syringe stimulus stays constant.
Another measurement is shown in Figure 5. Here, the probe was positioned 6 m upwind from the orchard edge. Initially, only background signals were recorded, until the wind direction shifted by about 120° (as shown by the traces of the vector anemometer). After the wind was coming from the treated orchard, the air was loaded with pheromone, leading to an EAG trace with a very high plateau, in which both low dose syringe pulses disappeared completely. The evaluation of the pheromone concentration readings yields 5.5 * 10-8 or 0.10 ng/m3 and 1.8 * 10-6 or 3.3 ng/m3 for the two traces in Figure 5. This is a 33-fold increase in concentration without a change of the position of the probe, at a time lag of only 50 s between the two measurements.
Simultaneous measurements
The calibration of the field EAG system via sampling of airborne pheromone on filters represents a substantial improvement. Up to now, attempts were only made to determine the absolute pheromone concentration in the headspace of our calibration syringes (Sauer 1991; de Kramer pers. comm.; Koch & Cardé in prep.). Even if a precise knowledge of the syringe headspace concentration was available, a calculation of
Figure 4 EAG signals recorded at different positions in the orchard. The response peaks to the calibration stimuli remain at the same absolute height; but the traces between these stimuli show strong variations, resulting from pheromone-loaded air packets arriving at the probe
the overall sensitivity of our system would require data on the mixing ratio between the syringe puffs and the ambient air entering the measurement system.
The values determined here represent the performance of the field EAG system as a whole, and therefore offer a much higher reliability. The feasibility of aerial pheromone measurements by filter adsorption offers the possibility to experimentally test the sensitivity of the field EAG to different types and levels of background signals.
Figure 5 Comparison of responses to pheromone-free (dotted line) and pheromone-loaded (solid line) air occuring within 50 s due to a sudden shift in wind direction. Top traces: EAG voltage; bottom traces: vector anemometer x and y components. The y-component (thick line) of the anemometer output changes sign in the second interval, indicating a shift in wind direction by approximately 120°. This causes air from the treated orchard instead of clean air to reach the probe. Note that due to the high pheromone concentration, the responses to the low and medium dose syringes disappear in the pheromone plateau
Pheromone-background interactions
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The signal superposition technique used in our field EAG measurements rests on the assumption that the EAG signal voltages generated by pheromone add linearly to the EAG signal voltages generated by background odors. Recently, Rumbo et al. (1995) presented recordings in which the validity of this assumption was questioned. The circumstances of the recordings of Rumbo et al. were not documented to full extent; it is possible that the level of pheromone stimulation was so high, that saturation of the EAG response occurred. In addition, the air speed was extremely high, and they did not use an isolated antenna, but a whole-animal preparation. Furthermore, we have also found antennae which had a moderate pheromone response but an extremely strong response to background stimuli. In these cases, a linear superposition cannot be achieved, these antennae were discarded. The small amount of interference documented in Figures 3 and 4 results in a slightly higher pheromone concentration reading. But since the readings resulting from records as in Figure 3 are close to the threshold of the antenna, they do not enhance the uncertainty of measurements in this concentration range.
The results presented in Figure 5 clearly show examples of the highly dynamic effects occuring in the field EAG signal and underline the importance of concomitant recordings of wind speed and wind direction at a high time resolution.
Acknowledgements
We gratefully acknowledge the hospitality and support we experienced during our stay at the SLU, Alnarp, and the collaboration with Prof. Jan Löfqvist, Peter Witzgall, Anna-Carin Bäckman and Marie Bengtsson. We are also indebted to Christoph Klos, Georg Haas and Christian Trautwein who took part in our venture and contributed their work force and ideas. This work was made possible by grants from the Stiftung Rheinland-Pfalz für Innovation and the Swedish Council for Forestry and Agricultural Research (SJFR). Special help was also provided by the University of Kaiserslautern central electronics and central mechanics workshops. Finally, we gratefully acknowledge the support given by Prof. Dr. U. Bässler (Kaiserslautern).
Bengtsson M, Karg G, Kirsch PA, Löfqvist J, Sauer A, Witzgall P (1994) Mating disruption of pea moth Cydia nigricana F. (Lepidoptera: Tortricidae) by a repellent blend of sex pheromone and attraction inhibitors. J chem Ecol 20, 871-887
Bäckman A-C (1997) Pheromone release by codling moth females and mating disruption dispensers (This volume)
Cardé RT, Mafra-Neto A, Staten RT, Koch UT, Färbert P (1993) Evaluation of communication disruption in the pink bollworm in field wind tunnels. IOBC wprs Bulletin 16 (10), 23-28
Färbert P (1992) Aufbau und Anwendung eines Systems zur Simultanmessung von Windvektor und Pheromonverteilung zur Unterstützung der Paarungsstörmethode bei Lobesia botrana. Diplomarbeit Universität Kaiserslautern
Färbert P (1995) Pheromondichte-Messungen bei Freiland-Experimenten zur Paarungsstörung bei Pectinophora gossypiella und Lobesia botrana. Ph.D. Dissertation, Universität Kaiserslautern
Färbert P, Koch UT (1993) Measurements of the pheromone density in cotton fields and vineyards by EAG-method. IOBC wprs Bulletin 16 (10), 301
Färbert P, Koch UT, Färbert A, Staten RT, Cardé RT (1996a) Measuring pheromone concentrations in cotton fields with the EAG in Cardé RT, Minks AK (eds.) Pheromone Research: New Directions. Chapman & Hall, New York in press
Färbert P, Koch UT, Färbert A, Staten RT, Cardé RT (1996b) Pheromone concentration measured with EAG in cotton fields treated for mating disruption of Pectinophora gossypiella (Lepidoptera: Gelechiidae). Environ Entomol in press
Flint HM, Yamamoto AK, Parks NJ, Nyomura K (1993) Aerial concentrations of gossyplure, the sex pheromone of the pink bollworm (Lepidoptera, Gelechiidae), within and above cotton fields treated with long-lasting dispensers. Environ Entomol 22, 43-48
Karg G (1992) Untersuchungen zur Ausbreitung von Pheromonen in zur Paarungsstörung des Traubenwicklers behandelten Weinbergen. Ph.D. Dissertation, Universität Kaiserslautern
Karg G, Sauer AE (1995) Spatial distribution of pheromone in vineyards treated for mating disruption of the grape vine moth Lobesia botrana measured with electroantennograms. J chem Ecol 21, 1299-1314
Karg G, Suckling DM, Bradley SJ (1994) Absorption and release of pheromone of Epiphyas postvittana (Lepidoptera: Tortricidae) by apple leaves. J chem Ecol 20, 1825-1841
Koch UT, Färbert P, Termer A, Sauer AE, Milli R, Karg G, de Kramer JJ (1992) Pheromone density measurements by EAG method in vineyards, p. 145 in Arn H, Ioriatti C (eds.) Use of pheromones and other semiochemicals in integrated control. IOBC wprs Bulletin 15(5)
Milli R (1990) Messung der Pheromonverteilung im Freiland zur Unterstützung der Paarungsstörmethode bei Lobesia botrana Schiff. Diplomarbeit Universität Kaiserslautern.
Milli R (1993) Messung der räumlichen und zeitlichen Verteilung von synthetischem Apfelwickler-Pheromon in Apfelanlagen mit einem EAG-System. PhD Thesis, Universität Kaiserslautern
Milli R, Koch UT, de Kramer JJ (1996) Measurement of pheromone distribution in apple orchards treated for mating disruption of Cydia pomonella. Entomol exp appl in press
Rumbo ER, Suckling DM, Karg G (1995) Measurement of airborne pheromone concentrations using electroantennograms: interactions between environmental volatiles and pheromone. J Ins Physiol 41, 465-471
Sauer AE (1989) Ein System zur Messung von Pheromonkonzentrationen im Freiland mit Elektroantennogrammen. Diplomarbeit, Universität Kaiserslautern
Sauer AE (1991) Bestimmung von Pheromonkonzentrationen im Freiland mit Elektroantennogrammen zur Unterstützung der Paarungsstörmethode bei den Traubenwicklerarten Lobesia botrana und Eupoecilia ambiguella. Ph.D. Dissertation Universität, Kaiserslautern
Suckling DM, Karg G, Bradley SJ, Howard CR (1994) Field-electroantennogram and behavioral responses of Epiphyas postvittana (Lepidoptera: Tortricidae) under low pheromone and inhibitor concentrations. J econ Entomol 87, 1477-1487
Termer A (1992) Aufbau und Anwendung eines Systems zur Messung der Pheromonverteilung bei der Bekämpfung von Lobesia botrana durch Paarungsstörung. Diplomarbeit, Universität Kaiserslautern
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