Appendix for Defra project HH3103 TTF: Biocontrol approaches to aphid control; chemical ecology and natural enemies

Dr Jean Fitzgerald

East Malling Research

Dr Guy Poppy

University of Southampton

Dr Lester Wadhams

Rothamsted Research


Techniques used- Objective 2:

Gas chromatography (GC) and coupled gas chromatography-mass spectrometry (GC-MS) at Rothamsted Research and University of Southampton

Rothamsted Research

Analysis of solvent samples. Solvent samples were analyzed by gas chromatography on both polar (HP-wax, 30 m x 0.23 mm ID x 0.5 mm film thickness) and non-polar (HP-1, 50 m x 0.32 mm ID x 0.52 mm film thickness) capillary columns, using a HP6890 GC (Agilent Technologies, UK) fitted with a cool on-column injector and a flame ionisation detector (FID). The oven was kept at 30°C for 1 min., heated at 5°C min-1 to 150°C and then 10°C min-1 to 250°C (220°C for the wax column), where it was maintained for 20 min. The carrier gas was hydrogen. Kovats Retention Index (KI) was determined for the key semiochemicals.

Analysis by thermal desorption. The Tenax TA tubes were placed into a programmed temperature vaporisation (PTV) unit for analysis. The samples were analysed on a Hewlett Packard 6890 GC, fitted with a non-polar HP-1 cross-linked methyl silicone capillary column (50 m, 0.32 mm ID, 0.82 mm film thickness) and an FID. Desorption inside the PTV unit was performed using a rapid temperature ramp starting at 30°C (temperature of the PTV injector during introduction of the sample) and then programmed at 16°C s-1 to 220°C. The GC oven temperature was maintained at 30°C for 30 s and then programmed at 5oC min-1 to 120°C, then 10°C min-1 to 240°C. The carrier gas was hydrogen. KIs were worked out for peaks of interest.

Coupled gas chromatography-mass spectrometry (GC-MS). A capillary GC column (50 m x 0.32 mm i.d. HP-1) fitted with an on-column injector was directly coupled to a mass spectrometer (VG Autospec, Fisons Instruments). Ionization was by electron impact at 70 eV, 250oC. The oven temperature was maintained at 30oC for 5 min and then programmed at 5°C min-1 to 250°C. Tentative identification by GC-MS was confirmed by peak enhancement with authentic samples on both polar and non-polar GC columns (18).

Quantification of the components of the sex pheromone. A multiple point external standard method was used to quantify the amount of each component present in the air entrainment samples. Standards of nepetalactol (I), nepetalactone (III) (Figure 2.1) and dolichodial were made at various concentrations (5 ng/ml, 10 ng/ml, 25 ng/ml, 50 ng/ml, 75 ng/ml and 100 ng/ml). Three 1 ml injections of each sample were analyzed by GC on a non-polar HP-1 capillary column. A regression analysis was performed on the corresponding area of chemical and amount injected (Table 2.1). Using the regression equation, the ratios and total amount of nepetalactol (I), nepetalactone (III) and dolichodial produced by D. plantaginea oviparae was calculated.

GC and GC-MS at the University of Southampton

Analysis of P. lanceolata volatiles. Samples captured onto Tenax TA were desorbed using an Optic 2 programmable injector (Anatune, Cambridge, UK) fitted to an Agilent 6890N GC with flame ionisation detector. Injector conditions were equilibrated (1 min) then ramped from 40°C to 220°C at 16°C /sec whilst continuously operated in splitless mode. The non-polar fused silica capillary column (50 m x 0.32 mm i.d.) was coated with HP-1 (0.32 μm film). The carrier gas was helium (constant pressure 18 psi) and oven temperature was held at 40°C for 5 min then programmed at 5°C/min to 150°C, then at 10°C/min to 250°C and held for 13 min; detector temperature was 300°C. Data were captured and analysed using ChemStation Plus (Rev. A09.03). Samples on Poropak Q were eluted with redistilled diethyl ether and concentrated under nitrogen. Samples were initially analysed on a Hewlett-Packard 5890 Series II linked to a Hewlett-Packard 5971 Mass Selective Detector. The column used was a non-polar fused capillary column (30m x 0.25mm i.d.) coated with HP-1MS (0.25 μm film). The carrier gas was helium (constant 9 psi) and oven temperature was held at 40°C for 2 min then programmed at 5°C/min to 150°C then 10°C /sec to 250°C and held for 16 min. Injector temperature was 220°C and detector temperature was 260°C. Samples were injected (1 μl) in splitless mode (1 min) and data were captured and analysed by Enhanced ChemStation software (Vers A.03.00). Spectra searches were done on internal, Wiley275 and NIST98 databases. The same samples in solution were then injected (1 μl) onto the Tenax TA packed in liners and these were analysed after thermal desorption on the GC as above. Tentative identifications were based on good matches with spectra library searches and available Kovats Indices. Wherever possible, identities were confirmed with authenticated standards which were run on both the GC after thermal desorption and on the GC-MS.

Analysis of D. plantaginea sex pheromone components. Solvent samples were analysed on a Hewlett-Packard 5890 Series II Gas Chromatograph (GC) using flame ionisation detection. The column used was a non-polar fused capillary column (30 m x 0.32 mm i.d.) coated with SOLGEL-1 (0.25 μm film; HP1 equivalent SGE, Australia). The carrier gas was helium (constant 35 cm/s) and oven temperature was held at 40°C for 1 min then programmed at 10°C/min to 220°C and held for 2 min. Injector temperature was 260°C. Samples were injected (2 μl) in splitless mode and data was captured using a 35900 HPIB interface and analysed using HP 3365 series 2 ChemStation (ver. A.03.01). When peaks were very small or not detected, samples were further concentrated before reinjection. Identities of the two known aphid sex pheromone components were confirmed by coinjection and peak enhancement with authenticated samples of nepetalactol (I) and nepetalactone (III) on both non-polar (SOLGEL-1) and polar (CARBOWAX) columns. Sample identities were also checked by mass spectrometry on a HP5890 GC linked to a HP5971 MS. To confirm that the GC instrument was behaving consistently throughout the analysis, a selection of samples representing a range of different ratios were re-injected at the end of the analysis.

Identification of nepetalactol and nepetalactone enantiomers.

Separation of enantiomers of nepetalactol and nepetalactone via chiral GC. The enantiomers of nepetalactol (I and II) and nepetalactone (III and IV) (Figure 2.1) were analyzed by GC on a b-cyclodextrin chiral capillary column (30 m x 0.25 mm ID x 0.25 mm film thickness) using a HP5890 GC (Agilent Technologies, UK) fitted with a cool on-column injector and an FID. The GC oven temperature was maintained at 30°C for 1 min after sample injection and then raised by 5°C min-1 to 80°C, where it was maintained for 20 min, then 5°C min-1 to 180°C where it was maintained for 20 min. The carrier gas was hydrogen. Although the enantiomers of nepetalactol were unresolvable by chiral GC (b-cyclodextrin column), full structure confirmation was achieved by microscale NMR.

Derivatisation of volatile sample from oviparae. Air entrainment samples of D. plantaginea oviparae, containing approximately 84 ml of nepetalactol, were concentrated under a stream of nitrogen and dissolved in dichloromethane (0.5 ml) under nitrogen. A solution of (S)-(+)-a-methoxy-a-(trifluoromethyl)phenylacetyl chloride (40mg, 0.16mmol) and pyridine (25 ml) in dichloromethane (0.5 ml), prepared under nitrogen, was added, together with a few crystals of dimethylaminopyridine, and the reaction stirred overnight. The solvent was then removed under a stream of nitrogen and the residue partially redisolved in 10% diethyl ether in petroleum ether (40-60°C boiling fraction). The insoluble material was discarded by decanting off the soluble portion. The solvent was then removed under a stream of nitrogen and the residue redissolved in deuteriochloroform for NMR. The NMR was compared to diastereoisomeric derivatives from the two enantiomers of synthetic nepetalactol (I and IVII) (21).

NMR Analysis: 1H, 13C and 19F NMR spectroscopy was performed using a Bruker 500 Avance NMR spectrometer with 1H referenced to CDCl3 (7.25 ppm), 13C to CDCl3 (77.0 ppm) and 19F to CFCl3 (0 ppm). Quantitative 1H NMR spectroscopy was performed using a pulse angle of 30°, an acquisition time 5T1 (with T1 measured to be 2.5 sec) and a delay of 5 sec.

Synthesis of chemicals used

4,8-Dimethyl-(E)-nona-1,3,7-triene (DMNT) and 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene (TMTT). The synthesis of TMTT was achieved starting from the commercially available (E,E)-farnesol. The latter was catalytically oxidized using a perruthenate derivative to give the corresponding aldehyde. The Wittig reaction, reaction between a phosphonium ylide and an aldehyde, allowed the formation of the desired product with high yield and, more importantly, high chemical and stereochemical purity (>98% purity by GC). In the same way, the synthesis of DMNT (>99% purity by GC) was achieved starting from the commercially available geraniol.

·  (E)-Ocimene. The synthesis of (E)-ocimene was achieved by alkylation of a protected isoprene unit and its subsequent deprotection. Isoprene was reacted with sulfur dioxide under high pressure for 6 days to give 2,5-dihydro-methyl-3-methylthiophene-1,1-dioxide. The latter was subjected to facile deprotonation using a sodium amide base followed by alkylation with prenyl bromide to give a prenylated methyl sulpholene. The sulpholene derivative was refluxed in diethyl ether in presence of LiAlH4 to eliminate sulfur dioxide and give (E)-ocimene. Gas chromatography showed that the purity of the product was greater than 99%.

·  Dolichodial. The aerial parts of cat-thyme, Teucrium marum (102.58 g), were extracted in chloroform (2 x 800 mL) for 24 hr at ambient temperature. The solvent was removed under reduced pressure to yield a golden-brown gum (2.97 g). The extract was subjected to liquid chromatography over Florisil using hexane/diethyl ether (1:1), to yield a pale oil (912 mg). Bulb-to-bulb distillation using a Kugelrohr apparatus (90oC, 2mmHg) yielded 4 fractions, one of which was shown by comparison of 1H and 13C NMR data with literature values to contain dolichodial and epidolichodial in a 9:1 ratio.

·  Germacrene D. Samples of (−)-germacrene D were obtained by incubation of farnesyl pyrophosphate with purified, expressed (+) or (−)- germacrene D synthase, and subsequent hexane extraction and purification through a short column of silica gel (BDH, 40–63 µm)/magnesium sulfate (10:1).

·  (1R,4aS,7S,7aR)-Nepetalactol (I) and (1S,4aR,7R,7aS)-nepetalactol (II). The nepetalactol used was synthesised as stated in (22).

·  (4aS,7S,7aR)-Nepetalactone (III) and (4aR,7R,7aS)-nepetalactone (IV). The enantiomers of nepetalactone (III and IV) were synthesised from the corresponding enantiomers of nepetalactol (I and II).

Pyridium chlorochromate (320 mg, 1.5 mmol) was suspended in dichloromethane (5 ml) and the respective nepetalactol (50 mg, 0.29 mmol) was added. The reaction was stirred for 4 hr. The solvent was then removed under a stream of nitrogen and the residue partially redisolved in 30% diethyl ether in petroleum ether (40-60°C boiling fraction). The residue was purified on a florisil column eluted with 30% diethyl ether in petroleum ether (40-60°C boiling fraction). Fractions containing nepetalactone were combined. All other compounds were obtained from a commercial source with purity greater than 95%

Insect bioassays

Aphid bioassays – Pettersson four-way olfactometer (RR). A Perspex 4-way olfactometer was used to investigate the responses of aphids to the various stimuli (Table 2.2). The olfactometer had a diameter of 110 mm and comprised 3 layers of 6 mm perspex held together with plastic nuts and bolts. The bottom of the apparatus was lined with filter paper. In order to eliminate any visual stimuli, the olfactometer was placed in the centre of a black-walled box with an observation opening at the front. Air was drawn from the centre of the olfactometer by a vacuum pump, buffered by a 1l jar and adjusted with a flow meter to 350 ml min-1. Air was thus pulled equally through each of the four side arms (verified using airflow meters). Teflon tubing was used to attach a glass vessel (100 ml) and a flow meter to each of the four side arms. PTFE tape was used to ensure airtight seals between the olfactometer and the Teflon tubing. All five holes were covered with a layer of muslin to prevent access by aphids during the bioassays.

Aphid bioassays – ‘Pettersson-style’ four-way olfactometer (EMR). This was similar to that described in (24, 25) and was used to investigate the responses of aphids and parasitoids to the various stimuli (Table 2.3). The olfactometer had a diameter of 115 mm and consisted of 3 layers of 6 mm ground glass. The bottom two layers of glass were bonded together and formed the arena. The third layer of glass completed the top of the arena and was held in place with four perspex clips. A small circular glass lid was fitted in the centre of the top layer of glass, which allowed easy access for the introduction of the insects. A hole drilled centrally through the lid, was fitted with a spigot opening. Air was drawn through the central opening by a small pump at 400 ml min-1 so that air was pulled through each of the four side arms at 100 ml min-1 (verified by airflow meters). A plastic inlet port was spigotted into each of the four side arms, to allow odour delivery. PTFE tape was used to ensure airtight seals between the glass and the plastic inlet ports. Finally, the inlet ports were held firmly in place by tensioning a rubber band around the outside of the olfactometer. Four glass tubes (25 mm diameter), and tapered at one end were pressed firmly onto the inlet ports. The stimulus was introduced into the glass tubes. All five ports were covered with a layer of muslin to prevent access during the bioassays. To eliminate visual stimuli during bioassays with parasitoids the chamber was surrounded by a cylinder of cardboard, with holes cut to allow it to fit over the glass arms.


References

1. SIMON J-C., BAUMANN S., SUNNUCKS S.P., HERBERT P.D.N., PIERRE J-S., GALLIC J-F. and DEDRYVER C-A. (1999). Reproductive mode and population genetic structure of the cereal aphid Sitobion avenae studied using phenotypic and microsatellite markers. Molecular Ecology 8 531-545.

2. HARVEY N. , FITZGERALD J. and SOLOMON M. (2001). Multiple molecular markers applied to Dysaphis aphid populations. Proceedings Aphid Symposium, Rennes, France. 114.

3. HARVEY, N.G., FITZGERALD, J.D., JAMES, C.J. & SOLOMON, M.G. (2003). Isolation of microsatellite markers from the rosy apple aphid Dysaphis plantaginea. Molecular Ecology Notes 3, 111-112.