PROBLEMS AND OPPORTUNITIES WITH PHEROMONES

Raymond Baker Department of Chemistry, The University, Southampton, S09 5NH, UK

Abstract - The main areas for development in the use of pheromones in insect control are reviewed and a number of examples given which indicate the nature of the prob­lems which require investigation. Requirements for purity, isomeric and otherwise, vary according to insect species and the method of application. This is illustrated by discussion of a synthesis of the pheromone of Japanese beetle, Popillia ¿aponioa, and also faranal, a trail pheromone for the ant Monomovium pharaonis. Two projects are described which exemplify current procedures beginning with identification of pheromones to their use as control agents as mating disruption agents. Three novel spiroacetals have been identified as pheromones of the olive fly, Daous oleae, and the stereochemistry of 3- and 4-hydroxy-l,7-dioxaspiro[5.5]undecane, which comprise two of these components, has been assigned. Details of the use of the major compon­ent, 1,7-dioxaspiro[5.5]undecane, for mating disruption are reviewed. The pheromone of the forest pest, Panolis flammea, has been shown to consist of three monounsat-urated C14- and C^-acetates and the detailed behavioural response of the male moth to each component described. Large scale field trials for mating disruption of two forestry pests, Panolis flammea and Rhyacionia buoliana are described. For the latter, the population has been demonstrated to fall by ca . 70% as a result of one year's pheromone treatment.

INTRODUCTION

The last two decades have seen extensive studies leading to the structural elucidation of insect pheromones and to evaluation of the behavioural responses which they elicit. It has become increasingly clear that pheromone systems are multicomponent and that individual components are, in general, responsible for different aspects of behaviour. As a result of the complexity of insect behaviour, the assignment of chemical structures has proceeded significantly more quickly than the accumulation of biological data required for a thorough understanding of chemical communication.

This observation explains, in part, the apparent slowness of the advent of the application of pheromones in insect control. Other factors such as questionable patent protection, doubts regarding satisfactory commercial returns and the selectivity of pheromones which, in some cases, can be positively disadvantageous, have also been widely debated. The problems regarding pheromones are mostly associated with application and formulation. Field assess­ment is both time consuming and labour intensive and any venture associated with pheromones must, therefore, be carefully considered.

The main emphasis for development would appear to be the following:-

1. Development of new synthetic routes which might be adapted to larger scale.

2. Assessment of the level of purity required for specific applications.

3. Improvement of control programmes by the use of pheromone monitoring systems with partic­ular emphasis on relationships between trap catch and insect population levels. For example, pheromone traps have been used for the detection and estimation of the spiny bollworm, Earias insulana, adult populations in cotton fields (1). Eggs of Earias insulana are particularly hard to detect and larval penetration of the cotton bolls occurs soon after hatching. A pheromone system is, therefore, of particular value for detection of potential larval infestations. Although reliable estimates of adult popu­lations have been made on the basis of trap catch the relationship between male catch and larval density was not assessed. Nevertheless, more data along these lines must be obtained. It should be anticipated that the use of commercial monitoring systems such as those used for early warning of egg hatching in pea moth, Cydia nigvioana, (2) and popu­lation monitoring of codling moth, Laspeyvesia pomonella, (3) will grow steadily.

67

68 R. Baker

4. Mass trapping programs in situations in which certain requirements are met. The most interesting mass trapping program reported is that concerned with the campaign against the bark beetle, Ips typographus, which is the major pest of Norway spruce, Picea abies (4). Beginning in 1979, some 600,000 traps containing the three component aggregation pheromone mixture, 2-methyl-3-butene, ¿r£s-verbenol and ipsdienol, were used throughout Norway. The average number of beetles caught per trap was 4,850 and about three million trees were killed by beetle attack. This program was continued into 1982 when the number of trees killed was reported to be 300,000. Factors other than the trapping program have almost certainly contributed to the decrease in tree death but it is clear that the use of pheromones in this case must be regarded as successful.

Few successful mass trapping programs utilising sex pheromones have been reported but Sternlicht et al. (5) have established procedures for control of the citrous moth, Prays citri. About 140 traps per hectare are used containing the pheromone. Sticky traps cost ca_. $2 and the cost of the pheromone is less than $1 per trap per year. The initial evaluation of the merits of the system involved a comparison of the efficiacy of the trapping system against an area treated with insecticides. Samples of flowers were taken and the number of eggs and their viability was assessed. The pheromone system is rein­forced by biological control and is now highly regarded by farmers; almost two-thirds of the total lemon growing area in Israel is now protected in this manner. One of the reasons for the apparent success of this approach is the nature of citrous crop in Israel; relatively small areas of lemon trees are often surrounded by other crops and, presumably, migration of mated female P. oitri is minimised. Thus, in spite of the fact that the pheromone is specific for catching males, this particular control program appears to have real merit. The use of aggregation pheromones or the development of female attractants which could be used in conjunction with sex pheromones are potential areas for development.

5. Control programs involving mating inhibition. Particular emphasis here must be placed on improvements in formulation techniques for which reliable release characteristics can be obtained. Levels of pheromone release must be assessed for effective behaviour modification and these studies must be linked with those for data collection which gives estimates of the change of insect population and crop damage assessment. Fundamental studies on the effectiveness of the number and strength of point sources of the pheromone formulation and analysis of the effectiveness of single component or multicomponent systems are also required.

It is now appropriate to turn to the contribution which our group in Southampton is making in some of these areas and to place them in context with overall developments.

SYNTHETIC STUDIES

New syntheses are being developed for pheromones which appear to have field application. Additionally, the stereospecificity requirements of these syntheses is also of substantial interest. It would appear to be inappropriate to prepare material >99% isomerically pure if the requirement in the field is less critical. Future studies will examine relationships between biological activity and chemical composition.

Japanese beetle, Popillia japónica This insect is a damaging pest of a variety of ornamental trees and cultivated crops in the U.S.A. Its pheromone was isolated from virgin females and the structure assigned as (R,Z)-5-dec-l-enyloxacyclopentan-2-one (5). The unique feature of the compound is that even minute amounts of the (S,Z)-enantiomer decreases the response of the male to the pheromone under field conditions (6). Contamination of the (R,Z) with 2% of its enantiomer results in a three-fold decrease in activity compared to the optically pure pheromone and no activity is found with material of 94% enantiomeric excess (e.e.). The requirement for any commercial synthesis is, therefore, to produce material with >96% e.e. at reasonable cost. Previous syntheses of this pheromone have been either from optically active starting material (6) or by long synthetic routes which included resolution (7).

We have now shown that material of high optical purity can be obtained from a procedure which involves asymmetric reduction followed by enhancement of optical purity by a simple resolu­tion. Our synthetic strategy involved the choice of methyl-4-oxotetradec-5-ynoate (1) as a suitable starting material. This was prepared in a single step in 68% yield by inverse addition of dec-1-ynylmagnesium bromide in tetrahydrofuran to a solution of 3-methoxycarbonyl propionyl chloride in the same solvent at -65°C. This acetylenic ketone (1) was reduced with 9-pinan-3-yl-9-borabicyclo[3.3.l]nonane (2) (3 mol. equiv.) when stirred at room temperature for three days. A 70% yield of (3) was obtained as a 87:13 mixture of the (R) and (S)-enan-tiomers. The alcohol (3) was stirred with sodium hydride in benzene and then distilled; reduction with Lindlar catalyst gave 5-decen-l-yloxacyclopentan-2-one (4) with an optical purity of 73%.

Problems and Opportunities with Pheromones 69

To obtain optically pure (£) a resolution procedure was used to enhance the percentage e.e. of (3). This was converted to its phthalic half ester (6) and resolved with (+)-a-methyl-benzylamine. The white crystalline salt was recrystallised from diethyl ether-light petrol­eum and the optically pure half-ester (6) liberated from the salt by acidification with dilute hydrochloric acid. Hydrolysis with lN-NaOH gave the corresponding y-hydroxyacid which on slow distillation yielded pure R-(-)-(<£). The acetylenic lactone was converted into the optically pure natural pheromone (R,Z)-(5), [a]D = -69.7°.

This method of enrichment of the desired enantiomer does not appear to be totally satis­factory for larger scale syntheses but other procedures could be followed. Midland and Nauyen (8) have reported that the cyclohexylamine salt of the hydroxy acid obtained from (3) can be recrystallised from acetonitrile. Two recrystallisations were reported to yield material which eventually gave pheromone of high optical purity.

Pharaoh's ant, Monomovium pharaonis This ant is now well established as a major pest in hospitals, bakeries and food stores in the United Kingdom (8). The most active component of the trail pheromone, given the name faranal, has recently been shown to be (6E,10Z)-3,4,7,ll-tetramethyltrideca-6,10-dienal (9) and its absolute configuration has been demonstrated to be (3S,4R) (9a). Although the naturally occuring isomer is the most biologically active, a mixture of (3S,4R)- and (3R,4S)-faranal has been shown to possess considerable activity (10).

We have now developed a convergent, highly stereospecific, synthesis of racemic (3S,4R/3R,4S) faranal in which the stereospecific formation of a trisubstituted double bond and a substi­tuted vinyl iodide is controlled by addition of alkylcopper complexes to terminal acetylenic intermediates and the relative configuration of the methyl groups at C-3 and C-4 were established by the use of <r£s-4,5-dimethylcyclohexene (Scheme 1).

The trail following activity of the natural pheromone is extremely high and faranal is active at 0.005ng/cm of trail. It is already clear that material containing other isomers of faranal would still retain substantial activity. We have, therefore, developed a much shorter synthesis of faranal which yields a mixture of two diastereomers. The electro­chemical dimerisation of ethyl crotonate was examined as a possible route to diethyl-3,4-dimethyl-l,6-hexanedioate (10). Using the conditions used by Baizer (11) a 60% yield of (9) was obtained. The electrochemical dimerisation of ethyl crotonate can give rise to three isomeric diesters, the meso compound (10a) and the (3S,4S) (10c) and (3R,4R) (lOd) isomers. The racemic component (10c+10d) and the meso derivative (l£a) proved inseparable by g.c. 13C studies, however, indicated that the mixture contained 33% (K).a) and 67% (10c+10d). Hydrolysis of this mixture of diesters to a mixture of diacids, followed by pyrolysis to a mixture of 3,4-disubstituted cyclopentanones and oxidation of this mixture of cyclopentanones to a mixture of lactones by the methods described earlier gave (lla-d) in 84% yield. G.l.c. analysis showed that the mixture consisted of 25% (lla+llb) and 75% (llc+lld). These lactones were converted into 5-iodo-3,4-dimethyl-(tetrahydropyran-2-yloxy)pentanes by the method described earlier and were employed in the synthesis of a 3:1 diastereomeric mixture in which the minor isomer was racemic faranal. Nevertheless, the mixture has considerable biological activity and could have application in control methods for Monomorium pharaonis.

70 R. Baker

*enantiomer also formed Scheme 1

Reagents: i) EtCu(Me2S)MgBrI; ii) ; iii) p-TolS02Cl,py; iv) = -Li,DMSO,NH2CH2CH2NH2;

v) MeCu(Me2S).MgBrI; vi) I2; vii) t-BuLi, ether; viii) LiAlH4; ix) MeSO Cl,py;

x) NaH-LiAlH4(l:l); xi) Cr03; xii) Ba(OH)2 xiii) MCPBA; xiv) HBr,EtOH;

xv) NaI,CH3GOCH_; xvi) p-TsOH,MeOH; xvii) PCC,alumina.

Problems and Opportunities with Pheromones 71

Olive fly, Dacus oleae

This fly, which is widely distributed throughout the Mediterranean basin is a major pest of olives. We had previously shown that the major sex pheromone component of D. oleae is 1,7-dioxaspiro[5.5]undecane (12), which was prepared conveniently and efficiently by reaction of ô-valerolactone with sodium ethoxide followed by treatment with acid (12). Two novel hydroxyspiroacetals, 3- and 4-hydroxy-l,7-dioxaspiro[5.5]undecane (15 and 14) have now been isolated, as minor components, from the rectal gland of the female olive fly (13).

The structures of these two compounds were assigned on the basis of fragmentation patterns obtained from high resolution mass spectroscopy coupled with solid sample gas chromatography. These structures were then confirmed by synthesis and by detailed n.m.r. studies.

4-Hydroxy-l,7-dioxaspiro[5.5]undecane (14) was prepared by a route which involved reaction of the acetylenic derivative (15) with 6-valerolactone. Catalytic hydrogenation of (16) in anhydrous methanol over 5% Pd/BaS04 poisoned with quinoline afforded the corresponding mono-ene which gave (14) on reaction in cone, hydrochloric acid/water/THF (1:5:20) for 24 h at room temperature in 60% yield (Scheme 2). Analysis by GC and GC-MS showed that the reaction was very stereoselective, producing the diastereomers (14a) and (14b) in a ratio of 20:1, and these were readily separated by flash chromatography (ether/petroleum ether, 1:1); *H n.m.r. (C6D6): (14a) 6 4.1 (IH, t of t, Ja,d = Ja,e = HHz, Ja>b = Ja,c = 5.5Hz), 3.5-3.6 (4H,m), 2.0 (IH, d of d of d, Jb,d = 13Hz, Ja,b = 5.5Hz, Jb,G = 2Hz), 1.1-1.9 (10H,m), 1.1-1.9 (10H, m); addition of a lanthanide shift reagent Eu(fod)3 to (2b); 6 4.7 (Hb, d of d of d, Jb c =

Jb e = 12Hz, Jb,d = 3Hz). From the *H n.m.r. data of (14a) it was observed that the signal

for Ha was significantly deshielded by a 1,3-diaxial interaction with a ring oxygen (ô 4.1) and appeared as seven evenly spaced lines, intensity ratio 1:2:3:4:3:2:1). The remaining four protons on carbons adjacent to oxygen had similar chemical shifts to each other, as two were equatorial (deshielded by hyperconjugation) and two were axial and had a 1,3-diaxial interaction with an oxygen. Thus, the *H n.m.r. data indicated that (14a) was exclusively in the anomerically stabilised conformation, as shown. From the *H n.m.r. of (14b), after addition of D2O, it was apparent that Ha and Hb had similar chemical shifts (<5 3.8-4.2,2H,m), Hb having two 1,3-diaxial interactions with oxygens. Data from lanthanide shift experiments showed that Ha and Hb in (14b) were shifted downfield of Hc-He. The signal for Ha in (14b) was an unresolved multiplet, narrow in comparison to that of Ha in (14a). The coupling constants observed for Hb were also consistent with the structure shown. The stereoselec­tivity of the reaction could be accounted for by epimerisation at the spirocarbon (C^) favouring the sterically less hindered diastereoisomer (14a). The mass spectra and gas chromatographic properties of (14a) were found to be identical to those of the natural product.

72 R. Baker

Scheme 3

The second component (13) was prepared by hydroboration of l,7-dioxaspiro[5.5]undec-2-ene (17) (BH3.THF, then alkaline hydrogen peroxide) (Scheme 3). The two diastereoisomers (13a) and (13b) were purified by preparative HPLC (Zorbax Sil, ether/heptane, 1:1), and analysed by GC, GC^MS and XH n.m.r. (C6D6): 03a) ô 3.3-3.8 (5H,m) 1.1-2.2 (llH,m); (13b) 6 3.3-3.7 (5H,m), 1.1-2.2 (HH,m). This data does not in itself allow assignment of the specific conformations formed in the reaction. Data from lanthanide shift experiments, however, were fully consis­tent with formation of the anomerically stabilised conformations (13a) and (13b) ; -H n.m.r. (C6D6): (13a) + Eu(fod)3 6 5.6 (Ha,bm), 5.2 (Hb,Hc,m), 3.9 (Hd,He,m); (13b) + Eu(fod)3 6 5.0 (Ha,m), 4.6 (Hb,d of m, Jb>c - 12Hz), 4.2 (Hc, d of d, Jh>ç = 12Hz, JajC = 2Hz), 3.9 (Hd,m), 3.7 (He,m). The protons on methylenes adjacent to oxygen'in (13a) were shifted in pairs (Hb/Hc and H¿/He), whereas the corresponding protons Ha-He in (13b) were shifted to differing extents (Hd/He overlap slightly). This suggested that the hydroxyl group was equatorial in (13a) and axial in (13b). The signal for Ha in (13a) was broad compared to that for Ha in (13b). Coupling constants for (13a) were not readily seen owing to overlap of signals, but the coupling of Hb with Hc in (13b) was observed (JbjC

= 12Hz). A small observed coupling constant (Ja c = 2Hz) is consistent with the fact that Ha and H¿ each have a trans-coplanar C-0 bond. Tne coupling of Ha to Hb would also be reduced by a C-0 bond trans-copianar to the C-Ha bond, but was confused by a further weak coupling to Hf.

Field studies have been conducted to assess the potential of the major component of the phero-mone (12) for use in a monitoring trap for the olive fly. A suitable formulation for release of the~i>heromone at a predictable rate was first established and it was found that polyethyl­ene vials gave best results, compared with rubber septa or micro-fibres. High loadings (20-25mg) of the spiroacetal were required to obtain adequate response levels. Several trap designs were tested and the results are summarised (Table 1).

TABLE 1. Capture of Daous oleae in various trap designs.

Trap Design Number of D. oleae caught

Males Females

White triangular trap 613a 2c

Yellow triangular trap 779a 25c

Horizontal brown sticky board 534a 25c

*Vertical yellow trap - no lure 685a 122d

*Vertical yellow trap - with lure 2592b 61e

These traps were coated with sticky material on both sides. All traps unless otherwise stated were baited with 25mg spiroacetal in poly­ethylene vials. Data were transformed using /x + 1, and analysed using an analysis of variance followed by a multiple range test. Comparing the data for each sex separately, totals followed by the same letter are not significantly different at P = 0.05.

Trap Design

White triangular trap

Yellow triangular trap

Horizontal brown sticky

*Vertical yellow trap -

Vertical yellow trap -

- board

no lure

with lure

Number

Males

613a

779a

534a

685a

2592b

of D. oleae caught

Females

2c

25c

25c

122d

61e

Problems and Opportunities with Pheromones 73

Triangular traps, both white and yellow, gave catches which were roughly equal to a vertical yellow trap with no lure but when the yellow trap was tested with the lure the catch of males was four times higher. The pheromone had no influence on the capture of females but the col­our component of the yellow traps did increase their capture. The height of the traps on the olive tree was found not to influence the catch but that there were significant differences in the catch on the two faces of the vertical yellow trap when they were placed on the out­side of the canopy - the innermost face caught higher numbers. Previously recorded compon­ents of the sex pheromone, (E)-6-nonen-l-ol, (Z)-6-nonen-l-ol and p-cymene (14) did not attract the flies in the field and showed no synergistic effect when combined with the spiro-acetal. Combined monitoring of Prays oleae and Dacus oleae using their respective pheromones in the same trap appears to be feasible in the case of the triangular trap.

The major component of the sex pheromone of Dacus oleae was also formulated by Celamerck GmbH in plastic sachets to give a slow release formulation suitable for mating disruption. A 1 hectare site was selected for treatment consisting of 88 trees at 12 metres apart at a site in Granada. Two pheromone sachets were placed on opposite sides of each tree within this area which were designed to give a release rate which remained more or less constant over 30 days. A control area was also studied which was situated about 100 metres away from the test site. To monitor Dacus oleae mating behaviour nine sticky traps baited with the pheromone were placed 25m apart centrally within both test and control plots. Both triangular and vertical yellow traps were used. Control traps with no lure were also used to give an indication of the colour component in the attraction of the flies to the traps. The capture of D. oleae males in all the traps was recorded five days prior to placing the sachets in the treatment plot. All the traps were then monitored every five days over the subsequent two months. Capture of D. oleae in both treated and control plots is summarised (Table 2).

The disruption treatment had no effect on female catches in triangular traps but in the case of the vertical yellow traps the number caught in the treated area was significantly less than control catches. In the case of male catches, the total numbers caught on vertical yellow traps and in triangular traps was significantly lower in the treated area compared with the control area, with a percentage reduction of 80.6% in vertical yellow and 94% in delta traps immediately post treatment. The percentage reduction over the whole period was 76.7% in vertical yellow traps and 77% in triangular traps. This straight percentage reduc­tion in catch is not, however, corrected for the colour component in the attraction of the flies to the traps, and when the control catch is first subtracted from the total catches in both treated and control areas, the resulting corrected percentage reduction is greater than 99% immediately post-treatment and is still greater than 99% in yellow traps but only 91.5% in triangular traps over the whole post-treatment period.

TABLE 2. Catches of Dacus oleae within and outside the mating disruption area.

Total number of D. oleae males caught.

Traps No. Pre-Treatment Immediately Total 30.9.81 - post-treatment post-treatment 5.10.81 6.10.81 - 0 0 6.10.81 - 0 0

18.10.81 °R °RL 15.12.81 °K °RL

<D :

? * Vertical yellow 3 559 125 80.6 >99 406 76.7 >99.9

H < Triangular S 101 16 94 >99 223 77.1 91.5

2 ^ Vertical yellow .? 437 643 1748

Q £ Triangular 6 195 266 974

o w Vertical yellow 1 ^ gQ 14?

ti B no Iure C TO

u H Triangular I S 9 26

%R = Percent Reduction %RC = Percent Reduction corrected for control catches

The depression of trap catch, brought about by a pheromone 'blanket' is, therefore, not as straightforward in this fly species as it is with a lepidopteran one. The colour component of the trap still operates despite the total negation of the effects of the pheromone and must, therefore, be taken into account when monitoring mating disruption experiments for flies using trap-catch-depression as a measure of efficiency. No measure of fruit infesta­tion in the two plots was possible during this experiment because of the confusing effect of earlier generations having infested the same fruit, but since significant trap-catch-depress­ion was obtained in this preliminary trial more detailed trials along the same lines but with

T3

4-> TO TO <D <D

H <

O u +-> oj C CD O U U <

ntr

ol

aps

o ^

Traps No.

Vertical yellow

Triangular 6

Vertical yellow

Triangular 6

Vertical yellow no lure

Triangular 1

3

,?

1

Pre-Treatment 30.9.81 -5.10.81

559

101

437

195

24

5

Immediately post-treatment 6.10.81 • 18.10.81

125

16

643

266

90

9

%R

80.6

94

%RC

>99

>99

Total post-treatment 6.10.81 15.12.81

406

223

1748

974

147

26

" %R

76.7

77.1

%RC

>99.9

91.5

74 R- Baker

infestation studies will now follow.

Pine shoot moth, Rhyacionia buoliana The pine shoot moth, Rhyacionia buoliana, is a common and important pest of young plantations in Southern Britain. The larvae bore into the buds of trees, causing bud death, deformation by destruction of the terminal bud and 'post-horn' deformities. The adults are on the wing from late June until mid-August; first instar larvae hatch 2-3 weeks after egg laying and feed at the base of needles. By September the larvae attack side-buds, in which they over­winter. In the Spring the overwintering larvae move to a fresh bud, which is hollowed out to build a larger 'resin tent' of silk and resin. In early June the larvae pupate for emergence from late June onwards. The pheromone of R. buoliana was shown by Smith et al. (15) to be (E)-9-dodecenyl acetate.

At Kilvey Hill in South Wales, an area of lodgepole pine, Pinus contorta, was seriously affected by -R. buoliana. The estimated adult population in this area for 1979 was 1.06 x 10 adults ha-1. The possibility of using (E)-9-dodecenyl acetate for mating inhibition was investigated initially in 1979 and in 1980 a full-scale disruption program was initiated on ca. 30ha of lodgepole pine. The whole of the treated site was saturated with pheromone in e¥rly June 1980 and June 1981. Pheromone was formulated in Conrel fibres and applied manually. Application was 2g ha-1 and the formulation was designed to release pheromone over the entire adult flight period (<90 days). The fibres, attached to double sided tape, were placed around 1cm diameter branches approximately lm above the ground.

Population assessments commenced in June 1979 and the numbers of larvae and/or pupae counted in the resin tents in selected trees. Parasitism were assessed from numbers of parasite cocoons present by dissection or by breeding to allow emergence of the parasites. Assess­ments made in 1980 and 1981 were carried out in late May before pheromone treatment and the commencement of adult flight. The results are summarised (Table 3).

TABLE 3. Pine Shoot Moth - Insect Population following Pheromone Treatments

Insects per % Para. ( . No. adults % tree (mean) (mean) o) ha-1 (est.) Change

Area to be treated 57.1 25.8 42.5 106,250

Control 50.3 22.3 39.1 97,750

Area to be treated 48.8 21.3 38.4 96,000 -9.6

Control 54.6 20.5 43.4 108,500 +10.0

Treated Area 29.7 45.8 13.61 34,007 -67.4 ino1

1981 Control 50.4 26.9 36.8 92,000 -15.2

Treated area 22ha (15ha control) - 2500 trees/ha.

The overall insect population (less parasitism) was approximately 1 x 10 larvae/pupae ha~ in 1979 and 1980 in both the designated treatment area and control site. Application of pheromone took place after population assessment in 1980 and the net change in population resulting from this is shown by the 1981 insect count. Insect density in the control area remained largely unchanged but the fall in population in the area applied with pheromone fell by ca.. 70%. This substantial fall in population is apparently due to two main factors. Pheromone treatment appears to have yielded a smaller insect population but the net fall in density is also due to the percentage rise in parasitism of the pupae. This result is encouraging in that the success of a control method utilising pheromones is clearly enhanced by the operation of biological control.

Pine beauty moth, Panolis flammea This species has been associated with Scots pine, Pinus sylvestris, and there have been severe outbreaks on this host in Central Europe. However, in the U.K., outbreak populations in large areas of forest have been restricted to Lodgepole pine, Pinus contorta, in Scotland over the last five years. Control of these outbreaks is normally accomplished by aerial spraying with fenitrothion. Areas to be treated are delineated by extensive pupal surveys, backed by selective egg counts in the following Spring, immediately prior to the spray operation. Pupal surveys are arduous to carry out, as the pines are normally at the thicket stage and in accessible areas. A monitoring system based on pheromone-baited traps could be deployed more easily than a pupal survey, although the results might not be as easy to interpret in terms of population levels than the more conventional sampling methods.

Area to be treated

Control

Area to be treated

Control

Treated Area

Control

Insects per tree (mean)

57.1

50.3

48.8

54.6

29.7

50.4

% Para, (mean)

25.8

22.3

21.3

20.5

45.8

26.9

(A-B%)

42.5

39.1

38.4

43.4

13.61

36.8

No. adults ha"1 (est.)

106,250

97,750

96,000

108,500

34,007

92,000

% Change

-9.6

+ 10.0

-67.4

-15.2

1979

1980

1981

Problems and Opportunities with Pheromones 75

We have now determined that the major components of the sex pheromone system of P. flammea are (Z)-9-tetradecenyl acetate (Z:9:14Ac) (18), (Z)-11-hexadecenyl acetate (Z-ll:14Ac) (19) and (Z)-ll-tetradecenyl acetate (Z-ll:16Ac) (20) in the ratio 100:1:5 (16).

In field experiments using sticky traps baited with synthetic samples, Z-9:14Ac on its own caught males, but neither of the Z-ll compounds, nor their binary mixture, were attractive. Both of the binary mixtures which included Z-9:14Ac were more effective than Z-9:14Ac alone, but the three-component mixture was no more attractive than either. Thus the "primary com­ponents" of the sex pheromone of P. flammea are apparently Z-9:14Ac, plus either one of the two Z-ll compounds.

Combinations of these components have been studied in a laboratory windtunnel. Video record­ings were made of the behaviour of male moths involved in upwind flight towards the pheromone source, landings on the platform on which the lure was placed and attempted copulation with the source of chemicals. The component Z-ll:14Ac was shown to be essential for landing but all three components were essential to elicit the full sequence of flight-landing and copula­tion. A more detailed examination has lead to the proposal that there is a heirarchy of stimuli which release various stages in the behaviour of a male pine beauty moth approaching a female. Long range attraction is mediated by Z-9:14Ac in combination with one or both of Z-ll:14Ac and Z-ll:16Ac. As the concentration of Z-ll:14Ac increases near to its source, it reaches a level which initiates a search for a landing site, guided by the plume of Z-9:14Ac, and, presumably, visual and tactile stimuli. After landing, the male approaches the female, guided by Z-9:14Ac and probably visual stimuli, and copulation is initiated by a high concentration of Z-ll:16Ac and Z-9:14Ac. There are almost certainly other stages not included in this scheme, including changes of velocity and flight pattern in the last few metres of approach. Although the precise roles of the two Z-ll components in long range orientation have not been investigated in these experiments, the scheme suggests that the moth retains one compound for orientation throughout, while relying on the other two to "switch" the sequential stages of close-range behaviour.

Only two of the components were found to be necessary in the monitoring system for Panolis flammea. A 4:1 mixture of (Z)-9- and (Z)-11-tetradecenyl acetates has been found to be efficient for good trap catches and factors such as trap design, capacity, height, alignment, position and amount of pheromone have also been examined. Correlations between trap catch and population densities require further study, but we already have positive indications that trap catch numbers are valuable in assessing insect population.

Preliminary investigations have been made into mating disruption of P. flammea. Small forest plots were treated with pheromone dispensed from Conrel fibres. Effectiveness of applications were assessed on the basis of trap catches placed in the treatment area and control plots. In a number of experiments it was demonstrated that Z-9:14Ac alone was equally, or more, effective in depressing trap catch compared to binary mixtures of Z-9:14Ac and the other two components. An application rate of 5g ha""1 and release rate of 2mg h-1 ha~l was shown to be effective.

A larger scale trail was conducted in ca. 40ha of Caithness forest in Spring 1982. The pheromone, (Z)-9:Ci40Ac was microencapsulated and sprayed aerially in March. An equivalent area of forest was utilised as a control site and mating disruption was assessed by trap catch measurements in both areas. The formulation appeared to be totally effective over a period of four weeks but over the next two weeks, towards the end of April, trap catches in both areas were very similar. They were, however, extremely low and it is anticipated that the formulation remained active over the major flight period. Further assessment of the effect of pheromone permeation in the treated area will be made.

76 R. Baker

Acknowledgement - I am privileged in this review to present a summary of the research efforts of a group of particularly talented chemists and biologists. The results obtained for the range of programs have only been achieved by their close collaborations and innova­tion. I would mention, in particular, Drs D. Billington, J.W.S. Bradshaw, C.E. Broomfield, R.H. Herbert, P.E. Howse, O.T. Jones, V. Bhaskar Rao and W.S. Speed but we have benefited from the dedicated efforts of all the members of the Chemical Entomology Unit in Southampton. The invaluable support of Dr J. Stoakley and Mr D. Bevan of the Forestry Commission in the United Kingdom is gratefully acknowledged. Thanks are also due to Dr P. Ramos of the Estación Experimental del Zaiden, Granada, Spain and to the staff of the Institute of Sub­tropical Plants and Olives, Chania, Crete, for their co-operation and collaborations. Financial support from S.E.R.C, is also acknowleged.

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