Rhizophagus Grandis </Emphasis> (Coleoptera: Rhizophagidae)
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14 Chemoecology 3 (1992) 14-18 Orientation of Rhizophagus grandis (Coleoptera: Rhizophagidae) to oxygenated monoterpenes in a species-specific predator-prey relationship Jean-Claude Gr6goire 1, Daniel Couillien ~, Ralph Krebber 2, Winfried A. K6nig z, Holger Meyer 2, and Wittko Francke 2 1 Laboratoire de Biologie animale et cellulaire, CP 160, Section interfacultaire d'Agronomie, Universit6 Libre de Bruxelles, 50 av. F. D. Roosevelt, B-1050 Bruxelles, Belgium 2 Institut for Organische Chemie, Universit~it Hamburg, Martin-Luther-King-Platz 6, D-2000 Hamburg 13, Federal Republic of Germany Received July 14, 1991 / Revision accepted September 28, 1991 Summary some of the components (fenchol, terpinene-4-ol and bor- neol) did not influence the predators' response. However, The predator, Rhizophagus grandis, is linked the addition of (-)-a-terpineol increased the attractivity of to its specific prey, Dendroctonus micans, by semiochemical the synthetic blend to almost that of larval frass of the prey. signals regulating oviposition and long range orientation. A Ecological implications of the identified semiochemicals and mixture of simple oxygenated monoterpenes [(-)-fenchone, their use in pest management are discussed. (-)-pinocamphone, rac. camphor, terpinene-4-ol, borneol, fenchol and verbenone], identified from the frass of D: mi- cans has been found to be extremely active in a flight wind- Key words tunnel as a long-range attractant for the predator. The mix- ture elicited 84 07o of the response to larval frass of the prey. specificity, predation, long-range orientation, Excluding pinocamphone from the mixture did not influence windtunnel, Coleoptera, Rhizophagidae, Scolytidae, Rhizo- its activity. Also, changing the absolute configurations of phagus grandis, Dendroctonus micans Introduction comparative analysis of the larval frass of D. micans and of a North American species, D. valens (normally out of R. gran- Rhizophagus grandis Gyll. has been described dis's geographic rang e but eliciting high oviposition of R. since the beginning of this century as a specific predator, at- grandis in laboratory tests), resulted in the identification of 7 tacking exclusively the Eurasian greater spruce beetle, Den- simple oxygenated monoterpenes generated by the larvae of droctonus micans (Kug.) (Bergmiller t903; Francke-Gros- both bark beetle species: (-)-fenchone, pinocamphone, cam- mann 1954; Kobakhidze 1965; Gr6goire 1988). phor, terpinene-4-ol, borneol, fenchol and verbenone (Gr6- goire et al. 1991). Bioassays showed that a mixture of these One striking feature of this specific associa- compounds strongly induced oviposition. tion is the particularly high capacity of R. grandis to discover its prey. For instance, in Belgium, about 90°7o of the prey's We have now completed our investigations on brood chambers contain predators, even though D. micans is the enantiOmeric compositions of the natural compounds and quite sparse, with usually less than 4 brood chambers/hectare we present our results obtained by testing the same blend as a (Gr6goire 1984). This spectacular capacity for prey location long-range attractant for R. grandis. has been attributed to the predator's perception of, and re- sponse to still unknown volatile components of the larval Materials and methods frass of D. micans (Tondeur & Gr6goire 1980). To our knowl- Chemical analyses and synthetic compounds edge, long-range attractants for Rhizophagidae have only been described for R. depressus (F.) and R. ferrugineus Enantiomeric separations of oxygenated mo- (Payk.) which respond to a 10:1 mixture of ethanol and a- noterpenes were carried out by gas chromatography using cy- pinene (Schroeder & Linde16w 1989). clodextrins as stationary phases. Determination of enantiom- eric purity of commercially available (+)-terpinen-4-ol and An important aspect of the specific link be- (-)-ceterpineol was achieved by transformation to the tri- tween R. grandis and D. micans is oviposition in response to fluoroacetates and separation on a 30 m, 0.25 mm i.d. fused specific chemical signals issued by the prey larvae. Recently, a silica capillary coated with octakis-(3-0-butyryl-2,6-di-0-pen- tyl)-y-cyelodextrin (LipodexE) (KOnig et al. 1989) at 100°C. Enantiomers of all other terpenes were separated without der- © Georg Thieme Verlag Stuttgart. New York ivatization under isothermal conditions using a 30 m, 0.25 Orientation of R. grandis in a windtunnel Chemoecology 3 (1992) 15 Table 1 Source,chemical purity, enantiomeric excess (% ee) and rotation value of monoterpenesused in bioassays 3ompound Source Chemical Enantiomenc [a]d 2° Purity Excess (% ee) [-)-Fenchone Fluka 97% 80 -54.70 (c=2.51; EtOH) ac. Camphor Ndrich 99% [-)-Verbenone Bedoukian 98% 70 -172.8 ° (c=2.52; EtOH) ',-)-Isopinocamphone a) 98% 98 -12.5 ° (c=1.43; EtOH) [-)-Pinocamphone a) 98% 98 -23.8 ° (c=0.47; EtOH) ',+)-Fenchol Aldrich 89% 85 +9.7 o (c=2.01; EtOH) [-)-Fenchol b) 98% 96 -12.9 ° (c=1.30; EtOH) ù-)-Borneol Fluka 80% 85 -18.20 (c=2.43; EtOH) ',+)-Terpinene-4-ol Merck 95% 35 +10.2 o (c=0.99; EtOH) ù-)-~-Terpineol Merck 98% 91 -93.80 (c=1.36; EtOH) a) Zweifel & Brown (1964) b) Beckmann & Metzger (1956) Blend c. Identical to blend b, but racemic ter- pinene-4-ol, (-)-fenchol, racemic borneol, have been replaced ~o ~o .ao~O4~O by (+)-terpinene-4-ol, (+)-fenchol and (S)-(-)-borneol. Blend d. Identical to blend c plus an addition- 1 2 3 4 5 a], equal part of «-terpineol, a component also present in no- ticeable amounts in the natural frass of D. mieans (Grégoire et al. 1991). ~o~ ~où ~où ~où Bioassays The windtunnel used in our experiments is 6 7 8 9 2 m long, 1.2 m high and 0.6 m wide. The 'floor', 'ceiling' and lateral sides are made of 6 mm glass plates mounted on a Fig. 1 Structures of oxygenated monoterpenes identified from frass steel frame, with sliding plates on the lateral sides allowing of Dendroctonus micans larvae: 1 (-)-fenchone, 2 camphor, 3 verbe- easy access to every part of the arena. The two end panels none, 4 (-)-isopinocamphone, 5 (-)-pinocamphone, 6 (+)-fenchol, are made of Tergal gauze fixed on a removable frame. Air 7 (-)-borneol, 8 terpinen-4-ol, 9 G-terpineol is pushed into the windtunnel by an electric fan at a speed of 0.02-0.08 m/s as measured with a bot wire anemometer mm i.d. capillary with oktakis-(3-0-acetyl-2,6-di-0-pentyl)-7- (Testoven 4200). Before reaching the test arena, the airflow cyclodextrin as stationary phase: camphor, fenchone, isopino- passes through a 1.5 cm thick layer of activated carbon, then camphone and pinocamphone were separated at 100 °C, bor- through a 1.3 cm thick beehive structure (dimensions of the neol and fenchol at 110°C and verbenone at 140°C. Source, cells: 1.3 × 1.3 cm) Having passed this structure, the airflow is chemical purity, enantiomeric excess and rotation value of laminar, as checked during preliminary tests with ammonium compounds used in bioassays are shown in Table 1. The corre- chtoride 'smoke'. The windtunnel is lit from above by 5 fluo- sponding chemical structures are given in Figure 1. rescent tubes (Phillips, TLD 58W/33). Gross structures of naturally-occuring oxy- Experimental animals were produced in the genated monoterpenes have been assigned earlier (Grégoire et laboratory in "oviposition boxes" containing spruce bark al. 1991). Crude pentane extracts of larval frass of D. micans powder, fresh spruce bark, live D. micans larvae and pairs of feeding in the phloem of Picea abies, or of D. valens feeding adult predatôrs (Grégoire et al. 1984). As it was previously in Pinus lambertiana were used for analyses. Using synthetic established that flight ability in Rhizophagus grandis de- samples as references, absolute configurations of the natural creases with the time spent in cold storage at 4-5 °C (about products were determined under the conditions described 8 070 per month, D. Couillien, J.-C. Grégoire in prep.), only 1 above. to 3 months old insects were used. After sieving the insects under watet (to separate them from their bark powder storage Four blends of synthetic compounds were medium), they were sorted according to sex, and precondi- prepared for bioassays. tioned for 12 h in a Petri dish on moist paper at room temper- ature (20-23 °C) under the windtunnel light. 4 to 13 batches of Blend a. Equal mixtures of racemic terpinene- 50-80 insects were used for each test. Approximately equal 4-ol, (-)-c~-fenchol, (+)/(-)-borneol, (S)-(-)-verbenone, (-)- numbers of male and female batches were tun for each treat- fenchone, racemic camphor, (-)-pinocamphone and of 99 % ment. The same insects were allowed to fly only twice on the pure pentane (Merck). This blend was identical to the one same day in different tests. used in the oviposition tests (Grégoire et al. 1991). Bioassays were run according to the method Blend b. Identicai to blend a, minus (-)-pino- developed by T. Wyatt, A. Phillips, J.-C. Grégoire (in prep.). camphone. The stimuli were released from an artificial, black polyethy- 16 Chemoecology 3 (1992) Grégoire et al. lene "tree" 9 cm in diameter an 118 cm high, standing 10 cm Table 2 Absolute configuration and enantiomeric excess (% ee) of in front of the air inlet of the test arena, equidistant from oxygenated monoterpenes identified from frass of Dendroctonus mi- both side pannels. The stimuli were deposited on 4 × 6 × 6 cm cans and D. va&ns triangular pieces of blotting paper fixed onto two 50 ml Ep- O. mi¢ans D. va&ns pendorff pipette tips affixed to the "tree" at an angle of 45 °C Compound abs. conf. (% ee) abs. conf. (% ee) and at heights of respectively 20 and 100 cm from the floor. The number and height of the dispensers (pipette tips) was Fenchone (-) 98 (-} 98 previously established in preliminary tests with ammonium Camphor rsc.