Pheromone release by codling moth females and mating disruption dispensers

• Introduction
• Materials and methods
• Results and discussion
  • Female gland extracts
  • Female effluvia
  • Release from rubber septa
  • Pheromone concentration in field air
• References

Key words - sex pheromone, mating disruption, female effluvia, dispenser, release rate, air sampling, Cydia pomonella, Tortricidae, Lepidoptera

Introduction
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This study attempted a comparative analysis of the release rates of codling moth sex pheromone, (E,E)-8,10-dodecadien-1-ol (E8,E10-12OH; codlemone) (Roelofs et al. 1971), from calling females, trap lures, and mating disruption dispensers. The aerial concentration of codlemone in a mating disruption orchard was determined by air sampling, concurrently with field-EAG measurements (Koch et al. 1997).

Such data is needed for the further development of the mating disruption technique, including optimization of pheromone dosage and dispenser densities under different climatic and topographic conditions, and the interpretation of the induced behavioural modifications.

Materials and methods
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Effluvia from ten individual female codling moths were collected in an air-flow apparatus (Witzgall & Frérot 1989). The glands of these females were extracted after two hr of calling. Female glands were also extracted at various times before and after the onset of calling (N = 10). The release from ten-day-old red rubber septa (Thomas Scientific, Illinois), loaded with 100 µg of codlemone, was measured using the same apparatus as for the calling females (N = 5; Bäckman et al. 1997).

The release of codlemone was measured from field-aged Ecopom (Isagro Ricerca, Novara, Italy) and Isomate C+ (Shin-Etsu Chemical Co., Tokyo) mating disruption dispensers, containing initially 250 mg (Rama 1997) and 90 mg codlemone, respectively. The same dispensers (N = 3) were suspended in stoppered glass flasks for 0.5 to 2 hr, at 0, 8, 32 and 64 d after field exposure, and in the case of Isomate C+ also after 1 yr. The flasks were washed with solvent, which was analysed by gas chromatography (GC) (see Bengtsson et al. 1994). For measurements of isomeric composition, the extracts were concentrated until the E,Z; Z,E; and Z,Z isomers of codlemone were detectable.

The concentration of codlemone in orchard air (1000 Ecopom dispensers/ha) was determined by air sampling on Tenax filters (Bäckman et al. in prep.). The filters were carefully rinsed and sealed before use. Two weeks after dispenser application, 2.5 to 3.5 m3 of orchard air was drawn through a filter during 4 to 5 hr. Hexane filter extracts were evaporated under a stream of nitrogen and each extract was injected on two different GC columns. Tridecanol was used as internal standard.

Results and discussion
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Female gland extracts

The amount of codlemone in female glands increased after lights off, and peaked at 8.8 ± 2.7 ng after 2 hr of calling. This increase was accompanied by a decrease in gland proportion of dodecan-1-ol (12OH), from 56.0 ± 52.6 %, 1 hr before light off, to 10.7 ± 5.0 % after 2 hr of calling (Figure 1; Bäckman et al. 1997).

Dodecanol and tetradecanol (14OH) have been identified as minor pheromone components (Arn et al. 1985; Einhorn et al. 1986; Bartell et al. 1988; Causse et al. 1988). Isomate C+ dispensers contain these two compounds together with codlemone. The behavioural role of the saturated alcohols is still controversial (McDonough et al. 1993, 1994, 1995). The proportional decrease in female glands would argue against a behavioural role of 12OH, since behaviourally active minor components are expected to be produced in rather constant ratios. According to biosynthetic studies, 12OH stems from reduced codlemone precursors (Löfstedt & Bengtsson 1988), and the high proportion of 12OH before calling was probably due to an accumulation in the gland during an early phase of pheromone production.

Female effluvia
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The female release of codlemone was on average 6.7 ng/hr between 1 and 2 hr of calling. The individual variation was quite large (Figure 1), although all females were calling continuously for 2 hr without moving, with their glands close to the orifice of the glass capillary tube used for pheromone collection. The comparison of codlemone gland titer and release suggests a continuous production during calling, at a turnover rate of approximately 1 hr (Figure 1).

Figure 1 Average codlemone and 12OH gland titer (left side), at 2, 1 and 0 hr before onset of the scotophase, and at 0.1, 1, and 2 hr after onset of female calling (N = 10). Codlemone gland titer after 2 hr of calling, split into individual females (shaded, narrower bars; right side), and codlemone release by these females during 1 hr before gland extraction (empty bars) (data from Bäckman et al. 1997)

Release from rubber septa

Red rubber septa, loaded with 100 µg of codlemone and aged in the field for 10 days, released 60.6 ± 15.7 ng/hr (N = 5). Such septa, used for monitoring of codling moth males, released almost ten times more codlemone than a calling female.

Dispenser release
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The release rates of codlemone from fresh Ecopom and Isomate C+ dispensers were the same: this could be an artefact, due to rapid saturation of the glass walls with pheromone. The release rates then gradually decreased over 64 days; the decrease was slower in Ecopom than in Isomate C+ dispensers (Table). Ecopom dispensers are loaded with 250 mg of codlemone, the recommended density for codling moth control is 300 to 400 dispensers/ha; Isomate C+ dispensers are loaded with 90 mg/dispenser, the recommended density is 1000 dispensers/ha. All release rates were measured in static atmosphere, and are expected to be higher under air flow.

Compared to a calling female, fresh Ecopom mating disruption dispensers released approx. 3000 times, and a 64-day-old 600 times more codlemone. In one-year-old Isomate C+ dispensers, the release was still above 1 µg/hr (Table). It is important to note that dispensers left in orchards release enough codlemone to affect male behaviours during the following season, even if the orchard is treated with fresh dispensers.

The proportion of codlemone isomers (EZ, ZE, ZZ) was quite stable over 64 days; 1-yr-old Isomate C+ released 24.1 ± 2.8 % of the other isomers (Table). Both fresh and aged dispensers, including 1-yr-old Isomate C+, were attractive to codling moth males in the field (Bäckman et al. in prep.). The equilibrium blend of 61 % codlemone and 39 % of its geometric isomers has been reported to inhibit male attraction in the wind tunnel (McDonough et al. 1993).

Table Release of codlemone and its geometric isomers (EZ, ZE, ZZ) from Ecopom and Isomate C+ mating disruption dispensers (N = 3)

Dispenser age	0	8	32	64 days	1 year
Ecopom
codlemone (µg/hr)	21.9 ± 1.0	15.8 ± 0.6	11.0 ± 0.2	5.7 ± 0.1	-
other isomers (%)	6.4 ± 0.3	6.4 ± 0.2	6.9 ± 0.4	9.1 ± 0.2	-
Isomate C+
codlemone (µg/hr)	21.8 ± 1.6	7.9 ± 0.9	3.6 ± 0.1	2.9 ± 0.2	1.4 ± 0.1
other isomers (%)	7.6 ± 0.3	6.0 ± 1.1	6.4 ± 0.2	7.2 ± 0.1	24.1 ± 2.8

Pheromone concentration in field air

Orchard air contains a large number of volatile compounds, at high amounts (Figure 2). This made GC-analysis of codleomone collected from field air difficult, since some of these compounds elute close to codlemone. According to the successful measurements (codlemone peak visible on both polar and nonpolar capillary column in each sample), the aerial concentration of codlemone in an apple orchard with 1 000 Ecopom dispensers/ha, releasing ca. 12 mg/hr/ha (14 days old), was estimated to be in the order of 1 ng/m3 (1.1 ± 0.2; N=3). The air-sampling was done simultaneously with field-EAG measurements (Koch et al. 1997). The dispenser density at the site used for air sampling was 2.5 times higher than the recommended density for control of codling moth (Rama 1997).

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Figure 2 Gas chromatogram of 2.5 m3 orchard air sampled during 2.5 hr on a Tenax filter. First arrow: internal standard (10 ng 13:OH); second arrow: codlemone. Asterisks indicate a change in signal amplification (attenuation)

Figure 2 and Figure 4 in Koch et al. (1997) illustrate the selective perception of minute amounts of codlemone by the male antenna in orchard air, containing numerous other volatile compounds at much higher amounts. Nanogram concentrations of pheromone in this "odour environment" induce behavioural modifications leading to disruption of mating.

The aim of parallel air sampling and field-EAG measurements was to calibrate the antennal response measured in mV (Koch et al. 1997) with aerial pheromone concentrations. The field-EAG apparatus allows on-line measurements at a high temporal resolution - air sampling on filters over several hours produces only average measurements. It should be desirable to include a range of concentrations in the calibration measurements, but the field-EAG technique is not applicable at higher concentrations -- and air sampling for longer time intervals at lower pheromone concentrations is not possible either, due to the accumulation of plant volatiles on the filter. Simultaneous calibration measurements will therefore be pursued in clean air.

Acknowledgements

I thank Prof. U. T. Koch, Kaiserslautern for collaboration and stimulating discussions. This study was supported by the Swedish Council for Forestry and Agricultural Research (SJFR) and the Swedish Board of Agriculture (Jordbruksverket).

References
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