Electrophysiological responses of Thaumetopoea pityocampa females to host volatiles: implications for host selection of active and inactive terpenes

Q.-H. Zhang et al.: Electrophysiological responses of Thaumetopoea pityocampa females to host volatiles 103 Anz. Schädlingskunde J. Pest Science 76, 103 107 Ó 2003, Blackwell Verlag, Berlin ISSN 1436-5693 1 Chemical Ecology, Department of Crop Science, Swedish University of Agricultural Sciences, P. O. Box 44, SE-230 53 Alnarp, Sweden; 2 DAAPV - Entomologia, Università di Padova, Via Romea 16a, Agripolis 35020 Legnaro PD, Italy; 3 Chemical Ecology, Department of Botany, Göteborg University, Box 461, SE-405 30 Göteborg, Sweden; 4 Present address: USDA-ARS Chemicals Affecting Insect Behavior Laboratory, BARC-West, B-007, 10300 Baltimore Avenue, Beltsville, MD 20705, USA Electrophysiological responses of Thaumetopoea pityocampa females to host volatiles: implications for host selection of active and inactive terpenes By Q.-H. Zhang 1,4,F.Schlyter 1,A.Battisti 2,G.Birgersson 3 and P. Anderson 1 Abstract Volatiles from newly cut branches with needles of Pinus sylvestris L. were collected with headspace sampling technique, and then identified and quantified by combined gas chromatographic-mass spectrometry (GC-MS). The response of antennae of the female pine processionary moth, Thaumetopoea pityocampa, to these volatiles was recorded by coupled gas chromatographic-electroantennographic detection (GC-). Surprisingly, the most common and major monoterpene hydrocarbons (MT), a-pinene, 3-carene, and b-pinene were antennally inactive. Female antennae responded strongly only to four minor MT components, myrcene, b-phellandrene, trans-b-ocimene, and terpinolene. Weaker, but repeatable responses were also found to limonene, cis-b-ocimene, and c-terpinene. Further recordings with two synthetic MT mixtures supported our findings from the natural material. When separating the two enantiomers of limonene by running different synthetic MT mixtures, the response was found only to the ())-enantiomer, but not to the opposite (+)-enantiomer. -responses were also found to some less volatile compounds, such as sesquiterpenes (SqT), active at ng-levels. The sensitivity and specificity of the antenna to a select number of active host MTs and SqTs suggest that these play a role in the host selection process of T. pityocampa females. 1 Introduction The pine processionary moth, Thaumetopoea pityocampa Schiff.; is one of the most destructive pine defoliators in the Mediterranean region (Schmidt, 1989; Zhang and Paiva, 1998). It not only causes significant economic damage by severe defoliation (Devkota and Schmidt, 1990), but also serious allergic reactions to humans and other mammals by the urticating hairs of the late instar larvae (Lamy, 1990). Many indigenous and exotic Pinus species are host plants of T. pityocampa in Southern Europe, North Africa, and the Near East (Avtzis, 1986, Schopf and Avtzis, 1987; Battisti, 1988; Devkota and Schmidt, 1990; Zhang and Paiva, 2003). In addition to tree shape, host volatiles (kairomones or allomones) are suggested to play a role in the habitat selection (Mendel, 1988; Tiberi et al., 1999). A study in Portugal on the needle monoterpene compositions from 12 pine species showed a statistical correspondence between the intensity of the attack by T. pityocampa and amounts and ratios of two major monoterpene hydrocarbons (Mateus et al., 1998). The presence of a-pinene and b-pinene, and their specific ratios, has been reported as stimuli for the oviposition preference of Choristoneura fumiferana (Städler, 1974) and Panolis flammea (Leather, 1987). We chose the gas chromatographic-electroantennographic detection (GC-) technique to investigate the olfactory host selection mechanisms of T. pityocampa females and to determine the potential kairomone chemicals involved. The pine odours are a complex mixture (Da Silva et al., 2001), and the chemical identification of all components or, alternatively, the fractionation of such a blend for subsequent behavioural testing would be a demanding task. The GC- method will pinpoint the physiologically active components of a mixture (Arn et al. 1975), but it will not reveal their behavioural function (attractive, repellent or other). The antennally active components will, however, define a biologically relevant group of compounds for subsequent behavioural testing. In a first step of the elucidation of the kairomonal signal for the pine processionary moth, we report here the results of GC- tests of females on volatiles from P. sylvestris. 2 Materials and methods 2.1 Insects The pupae of T. pityocampa were collected in June 1999 in a mixed stand of Pinus in NE Italy (Colli Euganei, Padova 45 16 N, 11 44 E, 100 130 m.a.s.l.) and were put individually into plastic containers, and kept at 25 C, 70 % R. H. and 16:8 (L:D) photoperiod. The emerged adults were used for electrophysiological recordings on the day of emergence or the following day, in Alnarp during July and August of the same year. 2.2 Volatile collection Headspace collection was made in a P. sylvestris L. stand, 30 km east of Lund, Sweden, in 2 May 1998. Volatiles from three newly cut branches of P. sylvestris with needles (each ca. 35 cm long, with total fresh weight of 118 g) were collected by enclosing them in a polyester bag (MenyÒ ToppitsÒ, 35 43 cm, England) through which activated charcoal filtered air was sucked at 300 ml/min by a mini-pump. The effluent volatiles were absorbed on 30 mg of Porapak QÒ (mesh 50 80, Supelco, USA) in a 3-mm ID TeflonÒ tube for 1.5 h. Air temperature inside the sampling bag was recorded during the U.S. Copyright Clearance Center Code Statement: 1436 5693/2003/7604 0103 $15.00/0

104 Q.-H. Zhang et al.: Electrophysiological responses of Thaumetopoea pityocampa females to host volatiles aeration with a digital Min-Max thermometer, as 21 22.5 C. The Porapak Q filter was rinsed with 300 ll diethyl ether (Fluka puriss p.a.), and the aeration extract was stored at )20 C until GC- and GC-MS analyses. 2.3 GC- and GC-MS analyses About 1 ll of the pine needle aeration samples were injected splitless into a HP 5890 GC equipped with a HP-INNOWAX column (30 m 0.25 mm 0.25 lm) and a 1:1 effluent splitter that allowed simultaneous flame ionisation detection () and electroantennographic detection () of the separated volatile compounds (see Zhang et al., 1999 for details). GC- recordings were done using excised antennae of T. pityocampa females suspended between two saline-filled wells (Zhang et al., 1997). Antennal signals were amplified (JoAC, Lund, Sweden) before they were stored on a PC equipped with an IDAC-card and the program ver. 2.3 (Syntech, Hilversum, The Netherlands). Using the same technique, we injected two synthetic monoterpene reference mixtures, with different enantiomers if available, (1 ll/injection) to confirm compound identity and study enantioselective responses. The reference mixtures contained well-known Pinus major monoterpenes and those compounds that were candidates for the active peaks, based on retention times and GC-MS identifications from the natural extract. For b-phellandrene, no reference was available. All samples were run at least three times. The criterion for activity was a response clearly different from noise, observed at least two times for the natural sample and three times for the synthetic mixes. Chemical analyses were made on a HP 5890 series II gas chromatograph coupled with a HP 5972 mass selective detector (GC-MSD, as described by Zhang et al., 1999). Volatiles were identified by comparison of retention times and mass spectra with those of synthetic compounds for monoterpenes, with computer data in the NBS75K library, and with our own KEM-EKOL library for sesquiterpenes or other less volatile compounds. 3 Results GC- analysis of the headspace sample of P. sylvestris needles showed that T. pityocampa female antennae responded strongly to 4 minor monoterpene hydrocarbons (MT) components; myrcene, b-phellandrene, trans-b-ocimene and terpinolene (fig. 1A). Weaker, but repeatable responses were also found to 3 other components: limonene, cis-b-ocimene, and c-terpinene. However, the two major MTs, a-pinene and D3-carene, and one of the most common MTs of Pinus spp.; b-pinene, elicited no antennal responses even at the large amounts present in the natural extract (fig. 1A). Further recordings with the two reference MT mixtures fully supported our findings from the natural materials, with the strong, repeatable antennal responses again to the same 4 components: myrcene, cis-b-ocimene, transb-ocimene, and terpinolene (figs. 1B and 1C). When presenting the two enantiomers of limonene in two different reference MT mixtures to the antenna, an response could not be found to the (+)-enantiomer (fig. 1B) but only to the opposite enantiomer, ())- limonene (fig. 1C). One of the strongly active MTs from the pine-needle aeration sample, b-phellandrene, was not available as enantiomer for inclusion in the synthetic mixtures. In addition to the compounds in the highly volatile MT fraction, the T. pityocampa female antennae also showed GC--responses to as many as 15 compounds with lower volatility occurring later in the chromatogram (fig. 2). Clear responses were found, in spite of the low amounts emitted from the pine needles and presented to the antennae. Two aromatic compounds, 2-ethyl-1-hexanol, and several sesquiterpenes (SqT) were clearly active at the ng-level (fig. 2). The most dominant SqT in the headspace sample, active peak No 15 (fig. 2), was only 1/60 of that of the major MT, D3-carene. 4 Discussion Our data are the first electrophysiological evidence that T. pityocampa female antennae respond to the host terpenes. The responses show that T. pityocampa female antennae are able to detect several minor monoterpene and sesquiterpene components emitted from the pine needles. Surprisingly, the most dominant host volatiles in the pine needle emission, a-pinene, b-pinene, and D3-carene elicited no responses by the female antennae at the doses tested. These 3 monoterpenes are released from most Pinus species at high levels (Mateus et al., 1998; Da Silva et al., 2001). In contrast, the minor MT components and less volatile SqTs to which the antennae were sensitive may show more qualitative and quantitative variation among Pinus host trees than so far investigated. The phytophagous insects might effectively exploit such variation in minor constituents for their host finding. A similar pattern of low, but in this case still existing, response to major MTs was found in the pine shoot beetles, Tomicus piniperda and T. minor (Schlyter et al. 2000). Some of the -active MTs found in this study are chiral monoterpenes, e.g. limonene and b-phellandrene. The enantiomeric-specific response was found only to the ())-limonene, even though both enantiomers occur in needles of P. sylvestris and other pines with variable ratios (Sjödin et al., 1996; Da Silva et al., 2001). Separation of antennal activity to the enantiomers of b-phellandrene has not been done yet. Further GC- analyses with chiral columns and standards seems to be necessary. A previous study in Portugal examined in detail the host chemistry, including enantiomer composition, of needle MTs in 10 pine species (Da Silva et al., 2001). A correlation of the intensity of the attack by T. pityocampa (mean number of larval winter nests/tree) and the host monoterpenes was found (Paiva et al., 1998; Mateus et al., 1998). The 3 major MTs (a-pinene, b-pinene, and limonene) as well as their ratios showed statistically significant correlations. These correlations might be non-causal, or, alternatively, the correlations of chemistry host choice might be caused by collinearity among monoterpene components due to their shared basic biosynthetic pathways. In the light of our GC- findings, exclusion of the -inactive MTs from their principle component analyses might reveal the biologically significant correlations between the attack levels and antennally active minor MTs.

Q.-H. Zhang et al.: Electrophysiological responses of Thaumetopoea pityocampa females to host volatiles 105 α-pinene (600) camphene (10) β-pinene (60) sabinene (130) 3-carene (1200) myrcene (160) limonene (30) α-terpinene (15) β-phellandrene (165) cis-β-ocimene (1 ) γ-terpinene (35) trans-β-ocimene (5) p-cymene (10) terpinolene (230) A α-pinene(50) β-pinene (55) 3-carene (55) myrcene (10) α-terpinene(55) (+)-limonene (60) cis-β-ocimene (5) trans-β-ocimene (20) terpinolene (60) B α-pinene (50) β-pinene (55) 3-carene (55) myrcene (10) α-terpinene (55) ( )-limonene (60) cis-β-ocimene (5) trans-β-ocimene (20) terpinolene (60) C 4.0 5.0 6.0 7.0 8.0 9.0 Retention time (min) Fig. 1. GC- responses of Thaumetopoea pityocampa female antennae to monoterpenes from headspace samples of Pinus sylvestris needles (A) and from two synthetic MT-mixtures (B and C). Mixtures in B) and C) differ only in the enantiomers of limonene. The amount of each compound directed toward the antenna was estimated and presented as ng (in parenthesis). Compound names in bold are those compounds giving a repeatable antennal response.

106 Q.-H. Zhang et al.: Electrophysiological responses of Thaumetopoea pityocampa females to host volatiles 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10.0 11.0 12.0 13.0 14.0 15.0 Retention time (min) Fig. 2. GC- responses of Thaumetopoea pityocampa female antennae to less volatile compounds, e.g. sesquiterpenes (SqT), from headspace sample of Pinus sylvestris needles. The active GC- peaks (numbered) were tentatively identified (based on retention time index and mass spectra) and quantified (ng) as follows: (1): 1-Ethyl-4-methoxy-benzene (1); (2): 1-methyl, ethoxybenzene (1); (3): oxygenated MT (3); (4): 2-ethyl-1-hexanol (1); (5): un-identified (1); (6): un-identified (1); (7): un-identified SqT (1); (8): SqT (c-muurolene, 3); (9): SqT (b-caryophyllene, 11); (10): SqT (c-gurijunene, 1); (11): SqT (selinene, 1); (12): a-humulene, 2); (13): SqT (germacrene D, 7); (14): SqT (germacrene B, 5); (15): SqT (d-cadinene, 20). GC- analysis is an efficient approach to find antennally active compounds from complex odour blends (Arn et al., 1975), but -active volatiles may or may not be behaviourally active. Therefore, behavioural studies with these -active MTs or SqTs are needed to confirm any potential kairomonal or deterrent activity, and to elucidate their roles in the host selection process of T. pityocampa. The antennally active less volatile compounds, including SqTs, are particularly interesting as they may, due to their lower volatility but high activity, give females important information both before and after landing. Recently, limonene (both enantiomers) has been found deterrent to egg laying of T. pityocampa females by Tiberi et al. (1999). It should be pointed out that the headspace sample was taken from the needles of P. sylvestris in southern Sweden, a nondistribution area of T. pityocampa. There are significant geographical variations in the MT composition within this pine species (Fäldt, 2000). Further GC- analysis of other pine species and moth populations from the distribution area, combined with behavioural tests, will provide more ecologically relevant information on the host selection mechanisms. Guided by the present results, such studies are now underway, in the multi-national EU project PROMOTH. Acknowledgements We are grateful to Dr. A.-C. Bäckman (Alnarp, Sweden) for help with the recordings, especially the portable antennae holder. We also thank Prof. M. R. Paiva and Dr Y. Hillbur for valuable comments. The EU project PROMOTH ÔÔGlobal change and pine processionary moth: a new challenge for integrated pest managementõõ (QLRT-2001-00852) provided support for the computer evaluation of the electrophysiological data and for the compilation of this paper. References Arn, H.; Städler, E.; Rauscher, S. (1975): The electroantennographic detector - a selective and sensitive tool in the gas chromatographic analysis of insect pheromones. Z. Naturforsch. 30c, 722 725. Avtzis, N. (1986): Development of Thaumetopoea pityocampa (Den. & Schiff.) in relation to food consumption. For. Ecol. & Mngmnt 15, 65 68. Battisti, A. (1988): Host-plant relationship and population dynamics of the pine processionary caterpillar, Thaumetopoea pityocampa (Den. & Schiff.). J. Appl. Entomol. 105, 393 402. Devkota, B.; Schmidt, G.H. (1990): Larval development of Thaumetopoea pityocampa (Den. & Schiff.) from Greece as influenced by different host plants under laboratory conditions. J. Appl. Entomol. 109, 321 330. Fäldt, J. (2000): Volatile constituents in conifers and coniferrelated wood-decaying fungi: Biotic influences on the monoterpene compositions in pines. Ph.D. thesis. Royal Inst. of Techn., Stockholm. Da Silva, M.D.R.G.; Mateus, E.P.; Munha, J.; Drazyk, A.; Farall, M.H.; Paiva, M.P.; Das Neves, H.J.C.; Mosandl, A. (2001): Differentiation of ten pine species from central Portugal by monoterpene enantiomer-selective composition analysis using multidimensional gas chromatography. Chromatographia (Suppl. Part 2) 53, S412 S416.

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