Entomol. exp. appl. 53: 125-131, 1989. �9 1989 Kluwer Academic Publishers. Printed in Belgium. 125

Effect of substrate formulation on the efficacy of the pea moth, Cydia nigricana, sex pheromone lures

Antonin Horfik, 1 Ivan Hrd~, 2 Karel Kone6n~ 3 & Jan Vrko~ 3

Oseva, Research and Breeding Institute of Technical Crops and Legumes, 787 01 ~Sumperk - Temenice, Czechoslovakia," 2Insect Chemical Ecology Unit, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, U Salamounky 41, 158 00 Prague 5, Czechoslovakia; 3Institute o f

Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Flemingovo n6m. 2, 166 10 Prague 6, Czechoslovakia

Accepted: July 27, 1989

Key words: (E,E)-8,10-dodecadien-l-yl acetate, sex attractant, rubber dispensers, release rate, isomeri- zation, field experiments, Cydia nigricana, pea moth

Abstract

Field experiments have shown that the activity of (E,E)-8,10-dodecadien-1-yl acetate, the sex pheromone of the pea moth, Cydia nigricana (F.) (Lepidoptera, Tortricidae), is significantly affected by the type of rubber substrate used for the preparation of dispensers. Rubber substrates cured with elemental sulphur or accelerators based on organic sulphur compounds isomerized pea moth pheromone faster than rubber substrates cured by organic peroxides. The degree of isomerization increased during field use and/or storage. Average catches of males were further dependent on the release rate of pheromone from the dispenser. In a 2 x 3 factorial experiment arranged in 6 x 6 Latin square design a statistically highly significant dispenser x dose interaction was demonstrated (P < 0.01).

Introduction

The sex pheromone of the pea moth, Cydia nigri- cana (F.), is (E,E)-8,10-dodecadien-l-yl acetate (E8,E10-12:Ac) (Greenway & Wall, 1980; Greenway, 1984). Although E8,E10-12:Ac elic- ited high electoantennographic responses in the antennae of males (Wall etal., 1976), it was almost inactive in field trials (Horfik et aL, 1980; Horfik, 1985; Horfik & Hrd2~, 1988). The activity of E8,E10-12:Ac can decline after a day (Wall et al., 1976) or might persist up to three weeks on a rubber formulation without antioxidant (Greenway & Wall, 1981). Bournoville (1979;

1980) also noted the instability of E8,E10-12:Ac in this respect.

It has been generally considered that inactivity of E8,E10-12:Ac in field trials is caused by the inhibitory effect of its decomposition or isomeri- zation products (Greenway & Wall, 1982; Wall & Greenway, 1983), It was assumed that the undesirable effect was due to the presence of small amounts of other isomers of 8,10-12:Ac (Greenway & Wall, 1984; Davis et al., 1984).

Attempts at stabilization of E8,E10-12:Ac, with the aim of obtaining a good biological activity for C. nigricana, have not been satisfactory so far. Bournoville (1980) failed when using an anti-

126

oxidant or an antiultraviolet component, while Greenway & Wall (1982) stated that the addition of the antioxidant N-2-octyl-N'-phenyl-p-phenyl- enediamine (UOP 688) stabilised ES,E10-12:Ac for more than 60 days. According to Wall et al. (1987) freshly exposed lures from France (INRA) and the UK (Rothamsted) containing E8,E10-12:Ac attracted similar numbers of moths in both countries, but the INRA lures rapidly became less attractive because they did not contain antioxidant (UOP 688) to prevent the formation of a product repellent to male moths. The Rothamsted lure remained strongly active for more than four weeks.

In this paper we present the results of three field experiments on the isomerization and the release rates of the synthetic pheromone from different substrates.

Materials and methods

Pheromone dispensers: EE-RES caps, i.e. for- mulation E8,E10-12 :Ac with antioxidant UOP 688, used in field experiment li, were pre- pared in the Insecticides and Fungicides Depart- ment of Rothamsted Experimental Station, Harpenden, U.K. All other dispensers were pre- pared in the Institute of Organic Chemistry and Biochemistry of the Czechoslovak Academy of Sciences in Prague (I~IOCHB). Pheromone chemicals: (E,E)-8,10-dodecadien-l-yl acetate of 99~o purity with less than 0.1~o of the other iso- mers was prepared in I]OCHB. (E)-10-dode- cen-l-yl acetate of 98.5~o purity was purchased from the Wolfson Unit of Chemical Ecology, Department of Chemistry, University of Southampton, Southampton, U.K.

Formulation substrates: (A)Rubber septa CM-4408 ((~SN 620011) sulphur cured, thiuram accelerated blend of synthetic isoprene and styrene-butadiene rubber, diameter 18 mm, dis- penser weight 1.5 g (Gum~ne SNP n.p., Doln6 Vestenice, Czechoslovakia);(B) Natural rubber SMR 20 (82~/o wt), carbon black (16~o wt) cured with dicumyl peroxide (2~o wt), sheets 15 • 15 x 2 mm, dispenser weight 0.6 g (Institute

of Chemical Technology, Prague, Czechoslova- kia); (C) Natural rubber SMR 20, cured with 2~o dicumyl peroxide (2~o wt) sheets 15 x 15 x 2 mm, dispenser weight 0.55 g (Institute of Chemical Technology, Prague, Czechoslova- kia); (D) Natural rubber SMR 20 (90~o wt), zinc oxide (5~o wt), stearic acid (2~o wt), phenyl-3- napthyl amine (1~o wt), cured with tetramethyl- thiuram disulphide (2?/o wt); (E)Natural rubber SMR 20 (80~ wt), cured with dicumyl peroxide (4~o wt); (F) Natural rubber SMR 20 (82~o wt), carbon black HAF (16~o wt), cured with dicumyl peroxide (2~o wt). Shape, weight and producer of cups under (D), (E) and (F) are the same: slightly conical cups of 10 mm diameter, height 10 mm, wall thickness 1.7 ram, dispenser weight 0.6 g (JZD 'Kvrten', Nehvizdy, Czechoslovakia).

Impregnation of substrates and pheromone analysis: The tested substrates (n--3) were impregnated with pheromones, dissolved in toluene and isomerization analysis was done using the relative static method according to Baker e taL (1980). The methods used are de- scribed in detail in Vrko6 et al. (1988).

Pheromone traps: Cylindrical 'Etokap' traps (Chemika o.p., Bratislava, Czechoslovakia) were used. The traps are made of hard grey poly- styrene, 19.8 cm long and 9.0 cm diameter, pro- vided with detachable fronts and open at each end~ The removable foils (bottoms) were coated with adhesive - Chemstop 1 (1985) or Chemflor 1 (1986) (Chemika, Bratislava, Czechoslovakia). The dispensers were suspended in the traps at the middle of the longitudinal axis. In the field the traps were hung on rods so that the openings were at the height of the vegetation.

Arrangement of the field experiments: The traps were arranged according to the Latin square design 5 x 5 (Experiments li, lii) or 6 x 6 (Experi- ment 2), where the number of sites (columns) is equal to the number of occasions (rows) and simultaneously to the number of experimental treatments. After each evaluation of the catch the traps were relocated so that each trap was at each site once. This arrangement was proposed for pheromone trapping by Perry et al. (1980).

Experiments li and lii were done in a sowing of

peas (Pisum sativum L.) in the grounds of the Breeding Station Horni Mo~trnice (North Moravia) between 15th and 26th July, 1985. In experiment li five traps with dispensers 85, 50, 51, 52, EE-RES (see Table 1) were located randomly on an area of 12 ha. The minimum distance between the traps was 100 m. The wind condi- tions of the locality were undefined. The numbers of trapped males were checked daily in the morning. The experiment was terminated after five days and the traps left in the field; on day seven the dispenser EE-RES was replaced by the dispenser CN-85, dosed with E10-12:Ac. This experiment (lii) also lasted five days and was evaluated in the same way as experiment li.

Experiment2 lasted for 18 days, from June 16th to July 3rd, 1986. In the grounds of the Breeding Station Lu2any as P~e~tice (West Bohemia) six traps with dispensers 63, 64, 65, 66, 67 and 68 (Table 1) were located in an area of 21 ha of peas. The traps were arranged in a row and spaced 95 m apart. For technical reasons they had to be orientated along the wind, which blows predominantly in the NW or SW direction in this locality. The number of trapped males was recorded at three-day intervals, always in the morning.

Table 1. P h e r o m o n e d i s p e n s e r s u sed in field expe r imen t s for

mon i to r ing Cydia nigricana

Trea t - A t t r a c t a n t dose (mg) Dis - Field

m e n t s p e n s e r experi-

(dis- E 1 0 - 1 2 : A c E8 ,E10-12 :Ac sub- m e n t

p e n s e r s t ra te a

code)

CN-85 1.0 - A lii

84 - 1.0 A li lii

50 - 10.0 B li lii

51 - 2.0 C li lii

52 - 2.0 B li lii

E E - R E S - 0.1 ? li

63 - 0.2 D 2

64 - 2.0 D 2

65 - 0.2 E 2

66 - 2.0 E 2

67 - 0.2 F 2

68 - 2.0 F 2

a See Mate r ia l s and me thods .

127

A natural logarithmic transformation was used in all data analysis to equalize error variances and produce additivity effects. The transformed natural In(catch + 1) data were evaluated by ANOVA. The significance of the differences between the means of treatments was tested using the method of Tukey.

In experiment 2 the six treatments represent combinations of two factors (factorial experi- ment). The first factor is the dose of E8,E10- 12:Ac at two levels (0.2 and 2.0mg), the second factor is the substrate, and this had three levels (D, E and F - see Table 1). The effects of both factors were investigated and also the signifi- cance of some orthogonal comparisons.

Results

A comparison of the results of chemical analysis of the dispensers prepared from various sub- strates with the results obtained during the trapping of C. nigricana in experiments li and lii is summarized in Table 2. The results show that when a substrate was used in which a higher degree of isomerization takes place inactive dis- pensers were obtained.

In experiment li a total of 119 males of C. nigricana were caught. The highest catches were in traps baited with dispensers 52 (mean catch/day 10.2 males), 50 (6.6) and 51 (6.2), while in traps with dispensers 85 and EE-RES the catches were very low (0.6 and 0.2). The effects of the occasions (F = 1.28) and sites (F = 0.42) were not significant. The effect of treatments was highly significant (F = 21.01, P < 0.0005). An ANOVA showed that the low catches in treat- ments 85 and EE-RES [mean ln(N + 1): 0.35835 and 0.13863] in comparison with treatments 50, 51 and 52 (means: 1.94640, 1.92790 and 2.23182) are highly significant: DT(0 .o5 ) = 0.97317 DT(o.ol) = 1.26016. In this experiment we com- pared the 10.0 and 2.0 mg doses ofE8,E10-12: Ac on two different substrates cured by dicumyl peroxide, where the lower dose seems to be better.

Field experiment lii. The population density of

128

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pea moth was already low, in total only 67 males were caught. Higher catches were achieved in traps with dispensers CN-85 (mean catch/day 3.6 males), 52 (3.4), 51 (3.4) and 50 (2.8). In the trap with dispenser 85 mean catch/day only 0.2 was recorded. Under these conditions the effects of sites (F = 0.62), occasions (F -- 2.15) and treat- ments (F = 2.27) were not statistically significant. The low efficacy of the dispenser on sulphur cured substrate (85) is evident (mean 0.13863), and it differs strikingly in its effect from the dispenser with the same substrate CN-85 (mean 1.12336) but dosed with E10-12:Ac. So far E10-12:Ac is most frequently used as an attractant for monitor- ing of C. nigricana. All formulations of E8,E10- 12:Ac on dicumyl peroxide cured substrates were effective (means 50: 1.12696, 51: 1.21368, 52: 1.33941), and the catches did not differ signi- ficantly from those on CN-85. At the same time these E8,E10-12:Ac based dispensers were ex- posed to the effects of weather six days longer (in experiment li) than the E10-12:Ac based CN-85 dispenser.

In field experiment 2 further substrates were tested, when the low efficacy of E8,E10-12:Ac formulated on substrate D, expected on the basis of chemical analysis, was confirmed by low catches of males. The results of chemical analyses and the catches of the males in the traps are given in Table 3. In total 469 males were caught; mean catch/trap/3 days: 26.5 (65), 24.2 (67), 16.7 (68), 10.2 (66), 0.3 (63, 64).

The results of an ANOVA of transformed data accounted for only a small proportion of the total variability are shown in Table 4.

The effect of sites was highly significant (F = 6.57, P < 0.001). At the two end sites a total of 45.8~o of males was caught (upwind site I - 25.4Yo, downwind site VI - 20.4~o). This may have been due to trap interaction, but the dif- ference between sites could be significant for a number of other reasons.

Most significant was the effect of treatments (P < 0.005). Formulation substrate (first factor) has a much higher effect on the total variability of the values of catches than the pheromone dose (second factor). Substrates E and F do not differ,

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129

Table 4. A N O V A of t ransformed data accounted for only a small proport ion of the total variability in field experiment 2 (1986); 2 • 3 factorial exp. imposed on 6 x 6 Latin square; first f a c t o r - dose of E8,E 10-12:Ac (0.2 and 2.0 mg), second factor - d ispenser (D, E, F - see Materials and methods)

Source" d.f. Variance P < ratio F

Trea tments 5 143,33 0.0005 - doses 1 22.46 0.0005 - dispensers 2 340.67 0.0005

- substrate F/E 1 2.01 - substrate E-F /D 1 679.32 0.0005

- interaction 2 6.42 0.01 - substrate E/F/dose I 1.61 - substrate E-F /D/dose 1 11.23 0.005

Error 20

To taP 35

a Occasions and sites excluded

but these carriers of natural rubber cured with dicumyl peroxide exceed the effectiveness of the less suitable carrier D, i.e. rubber cured with tetra- methylthiuram disulphide (P < 0.0005). The larg- est catches were achieved at a lower dose (0.2 mg) of E8,E10-12 :Ac on carriers E and F, when com- pared with the higher dose (2.0 rag). This is con- firmed by the value F-- 11.23 (P < 0.005) for substrate E-F/D/dose interaction (Table4). These data are supported by the data on the rate of evaporation of the pheromone under labora- tory conditions. For dispensers 65 and 67 dosed with 0.2 mg of pheromone evaporation rate was 2.5 ng/h, and for dispensers 66 and 68 dosed with 2.0 mg of pheromone it was 20-30 ng/h.

Discussion

The results show that the main factor which affects the efficacy of E8,E10-12:Ac as an attractant for the males ofC. nigricana is the purity of the pheromone (E,E isomer), and that the next most important factor is the rate of release. The degree of isomerization of pheromone with a con- jugated system of double bonds on a rubber sub- strate is dependent on the curing agent used. The lowest isomerization was observed in substrates

130

cured with sulphur-free reagents (Brown & McDonough, 1986; Vrko6 et al., 1988). Although E8,E10-12:Ac is relatively least inclined to isomerization (with respect to the E,E-system of double bonds, see Vrko6 et al., 1988) a certain degree ofisomerization takes place in all instances immediately after application of the substrate. Generally, in a mixture of isomers the E,E isomer shows tendency to dominate (60-67~o of E,E is expected). If an unsuitable substrate is used for the formulation, isomerization proceeds to an equilibrium mixture of all our isomers. We con- sider that it was solely due to differences in the formulations, which had affected to various extents the degree of isomerization of the pea moth sex pheromone, that led to discrepancies in the results obtained by various authors (Wall etal., 1976; Bournoville, 1979; 1980; Hor~k et al., 1980; Greenway & Wall, 1981; Greenway et al., 1983; Hor/tk, 1985; Wall et aL 1987; Hor/tk & Hrd~,, 1988).

Hrd2~ et al. (1986) obtained similar results for Cydia pomonella, when demonstrating the rela- tionship between the biological activity of cod- lemone and the degradative effect of the carriers, and they stressed the importance of the uniform evaporation, without further admixture of iso- mers.

Reports on the positive activity of E8,E10-12: Ac in field trials, without details of the formu- lation substrates used and lacking data on the degree of isomerization and the gradient of the released substance, are interesting but not easy to reproduce (see Tshmir & Mittus, 1984; Kolesova & Tshmir, 1982).

Greenway & Wall (1981) tested E8,E10-12 : Ac down to 10 - 1 #g/fresh rubber lure. There was no significant effect of dose between 1 and 102/~g, but below this there was a decrease in activity. Wall et al. (1976) showed that traps containing 0.1 mg of E8,E 10-12: Ac were more effective than those containing 1.0 mg. In our earlier field experi- ment (Hor/tk & Hrd~,, 1988) E8,E10-12:Ac was not attractive in the range of doses 0.01-0.5 mg/rubber dispenser CM-4408. In field experiment 2 we have therefore chosen a rela- tively high range of doses (0.2 and 2.0 rag) to

demonstrate a good attractiveness of E8,E10-12: Ac on suitable dispensers for a sufficiently long period (18 days). The lowest tested dose (0.2 mg) was more effective than a higher dose (2.0 mg). It seems (see also Greenway & Wall, 1981; Wall & Perry, 1981 ; W.all et aL, 1987) that it would be useful to test still lower doses on suitable carriers.

Experiment 2 was based as a 2 x 3 factorial experiment in a 6 x 6 Latin square design. For experiments with semiochemicals this arrange- ment brings some advantage. Perry et al. (1980) recommended the Latin square design for experi- ment with one factor, and for a two-factor experi- ment a Graeco-Latin square design, under the assumption that there is no statistical interaction between the effects of these two sets of treat- ments. However, the results of the present study show that the statistical interaction between the type of carrier and the dose can be highly signifi- cant, and the arrangement according to the Graeco-Latin square design would be incorrect in this case. Nevertheless a knowledge of statistical interactions is very important, and helps to increase the efficacy of the development of phero- mone monitoring systems for insects by selection of suitable combinations of factors and their levels, e.g. dispenser, semiochemical, dose, glue, trap design.

Acknowledgements

We thank Dr M. Griga and Ing. J. Kreuzman of the Oseva, Research and Breeding Institute of Technical Crops and Legumes, Sumperk for their technical assistance.

R~sum~

Effet du substrat sur l'efficacit~ de leurres de phOro- mones sexuelles de Cydia nigricana

Dans la nature, l'action de (E,E)-8,10-dod6- cadien-1-yl-ac6tate, ph6romone sexuelle de C. ni- gricana F., est significativement modifi6e par le type de caoutchouc utilis6 po~r la r6alisation du

diffuseur. Le caoutchouc vulcanis6 avec du soufre ou avec des acc616rateurs ~t base de compos6s soufr6s isom6rise la ph6romone sexuelle de C. nigricana plus rapidement que le caoutchouc vulcanis6 avec des p6roxydes organiques. Le taux d'isom6risation augmente pendant l'utilisation dans la nature et/ou pendant le stockage. Les captures moyennes de mgles d6pendent avant tout de la lib6ration de la ph6romone par le dif- fuseur. Une 6tude de 2 x 3 facteurs organis6e en carr6 latin (6 x 6) a mis en 6vidence une inter- action hautement significative entre diffuseur et dose (P < 0,01).

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