Electrophysiological responses of eucalyptus brown looper Thyrinteina arnobia to essential oils of seven Eucalyptus species

Eucalyptus is frequently attacked by the Brazilian eucalyptus brown looper, Thyrinteina arnobia. This caterpillar is regarded as the main lepidopterous pest of Eucalyptus and yet no practical and environmentally acceptable method of control currently exists. Electroantennographic techniques (EAG) have never before been used to detect semiochemicals that affect the behavior of T. arnobia. Thus, in this work, the ability of T. arnobia males and females to detect volatile essential oils of seven Eucalyptus species was investigated by EAG. We demonstrated that T. arnobia antennal olfactory system clearly showed differential sensitivity to several compounds, by coupled gas chromatography-electroantennographic detection (GC-EAD). Twenty-eight compounds were identified that elicited responses in T. arnobia, indicating that GC-EAD analysis may well be a useful means of screening active plant extracts for compounds that contribute to the observed behavior of this defoliator. The results also suggest that this species uses several volatile cues to find its host.


Introduction
The genus Eucalyptus, native to Australia, is widely cultivated in Brazil, covering an area of more than three million hectares. This country is the world's leading producer of eucalyptus-based fiber and of the 6.3 million tons of cellulose produced here annually, the majority is extracted from this exotic species. Eucalyptus plantations also supply wood to the furniture industry and help to reduce the pressure on native forests. 1 The Brazilian eucalyptus brown looper, Thyrinteina arnobia (Stoll, 1782) (Lepidoptera: Geometridae), is the most harmful of the Eucalyptus pests in Brazil, causing severe losses in wood production through defoliation. This moth attacks several genera of the Myrtaceae family, including six Psidium species, eight Campomanesia species, nine Eugenia species and twenty Eucalyptus species. Biological data on T. arnobia, reared on leaves of Eucalyptus spp, have been recorded by a number of groups. [2][3][4][5][6] Several strategies have been tried and considerable effort spent on the development of methods to control this pest, yet no practical and environmentally acceptable one currently exists. Control is made very difficult by the huge area of plantations and by the height of the trees. 4 Herbivorous insects often interact with plants by means of volatile semiochemicals, particularly to locate hosts upon which to feed or oviposit. [7][8][9] Essential oil is typically a blend of compounds, principally terpenoids, which represent the dominant class in Eucalyptus leaves. Because the essential oils of different species often share the same monoterpenes, 10 a particular species' odor may have components typical of one or more species. Thus, knowledge of these volatiles could be used as the basis for a control strategy.
Some species of the genus Eucalyptus exhibit high essential oil contents in their leaves and others have medium or low contents. Some Eucalyptus species are characterized by their sclerophyllous foliage, 11 i.e., high specific leaf weight or leaf toughness, 12  To understand the interactions mediated by semiochemicals, it is necessary to study the factors involved in the olfactory perception of these compounds in order to identify those that could attract T. arnobia. Up to now, neither potentially attractive semiochemicals, nor electroantennographic techniques have been applied to T. arnobia. Thus, we compared the detection sensitivity of T. arnobia males and females to volatile components of essential oils of seven Eucalyptus species, using the electroantennographic technique (EAG). Moreover, gaschromatography linked on-line to electroantennography (GC-EAD) study was performed with volatiles sampled from E. grandis essential oil.

Experimental
Mass Spectra were recorded on a Shimadzu QP5000 (EIMS) at 70 eV. Gas chromatography was performed in a Shimadzu GC-17A with H 2 as carrier and using a DB-5 (30 m x 0.25 μm ID, 0.25 mm film thickness) capillary column with helium as carrier gas at a flow-rate of 1.6 mL min -1 . The temperature was held initially at 60 °C for 2 min and then increased at a rate of 3 °C min -1 to 240 °C. The injector was in the split mode at 225 °C. The interface temperature was 250 °C.

Eucalyptus essential oil extraction and analysis
Fresh leaves of E. grandis Hill ex Maiden, E. citriodora Hook, E. camaldulensis Dehnh., E. saligna Sm., E. urophylla S. T. Blake, E. cloesiana were collected in the Universidade Estadual Paulista (UNESP), Botucatu, SP, Brazil and E. maculate was collected in Universidade Federal de Viçosa, MG, Brazil. The collects were done in forest species arboretum, located in the experimental plantation of these universities and identified by the Dr. C. F. Wilcken. The leaves were randomly collected from approximately six-years-old Eucalyptus trees.
The fresh leaves (400 g) of each species were submitted to steam distillation for 4 h, using a Clevenger apparatus. The essential oils in the distillate were dried over anhydrous Na 2 SO 4 and kept in the freezer. The essential oil that provoked the highest depolarization in T. arnobia female antennae was analyzed by GC-MS. Components were identified by determination of their retention indices relative to those of a homologous series of n-alkanes 13 by co-injection with authentic samples, and by comparing fragmentation patterns in mass spectra with those stored on the spectrometer database and bibliography. 14 Insects T. arnobia specimens were obtained from a laboratory where they were grown on artificial diets, 4 established in the Insect Bioassay Laboratory of the Universidade Federal de São Carlos, SP, Brazil. Pupae were sexed and placed individually in plastic vials (6 cm x 6 cm ID) for emergence of adults. Male and female pupae were maintained in a growth chamber at 25 ± 1 °C and 60 ± 5 % r.h., in a 12:12 L:D photoperiod.

Electroantennography and coupled Gas Chromatography-Electroantennographic Detection (GC-EAD)
The electroantennographic and the coupling of Gas Chromatography-Electroantennographic Detection (GC-EAD) were done as described in the literature. 7,[13][14][15] Antennae of 1-2 day old male and female moths were used for electroantennographic experiments (EAG) and electroantennographic detection (EAD). 16 Each antenna was pulled from the head with forceps and a few segments were cut off at the base and the tip. 17 The antenna was 557 Electrophysiological Responses of Eucalyptus Brown Vol. 17, No. 3, 2006 then fixed between two stainless steel electrodes by pushing the base and tip into droplets of an electrically conductive gel (Spectra 360 ® electrode gel) applied to the metal electrodes.
EAG experiments were performed in order to elucidate the sensitivity and selectivity of the antennal receptors of T. arnobia. The EAG response was evaluated as follows: the volatile compounds or control were released from Pasteur pipettes containing a piece of filter paper (ca. 0.8 cm 2 ) previously impregnated with 10 μL of a freshly prepared solution of each test essential oil in hexane, after the solvent had evaporated. The puff containing the test essential oil (from seven Eucalyptus species) was delivered into a continuously humidified and purified air stream (1.2 L min -1 ) passing for 0.3 seconds through the impregnated filter paper in the pipettes. Control stimulation was made at the beginning and the end of each series of EAG experiments. The test essential oils were then applied randomly at intervals of 60 seconds. EAG amplitudes in response to the essential oils were expressed in relation to the responses to the control (hexane), because of the large differences in overall sensitivity between individual antennae, and to compensate the decline in antennal sensitivity during a measuring session. In this normalization procedure, the responses to the control were defined as 100 %. The values obtained between two calibration references (controls) were calculated by linear interpolation between those references values. The Syntech EAG software (Syntech ® , Hilversum, The Netherlands) calculated the normalized values automatically. The essential oils were tested on 10 antennae each of female and male T. arnobia. The mean normalized responses of the different compounds were submitted to ANOVA for statistical analysis and compared by the Tukey test (P < 0.05).
The GC-EAD coupling consisted of a Shimadzu 17-A chromatograph equipped with a flame ionization detector and a Syntech electroantennography system -EAG (Syntech ® , Hilversum, The Netherlands). The column effluent (carrier gas hydrogen) was mixed with a nitrogen make-up gas flow (12 mL min -1 ) before it was split to the FID and the heated transfer capillary leading to the antennal preparation of the EAD. The separated compounds were eluted from the transfer capillary into a humidified and purified air stream (1.2 L min -1 ), which led them directly to the antennal preparation. To synchronise the retention times monitored by the FID and the EAD, the lengths of the capillaries after the splitting were exactly equal. The same chromatographic conditions as for the GC-MS analyses were used for the chromatographic separation. The FID was kept at 280 °C, whereas the temperature of the transfer capillary was maintained at 290 °C to avoid condensation.

Results and Discussion
Secondary metabolites in plants of Myrtaceae are dominated by the presence of mixtures of C 10 and C 15 terpenoids that are generally called essential oils and can be found in many plant families. The essential oil constituents of the genus Eucalyptus (Myrtaceae) have been well characterized. 10 Eucalyptus species produce numerous volatile compounds in large amounts, especially isoprenoids (here referred to as terpenes), which are accumulated in glands abundantly distributed throughout the leaf parenchyma and bark. 18 Quantitative data on the essential oils of seven eucalypts are shown in Table 1. E. citriodora furnished the  Figures 1 and 2. Electroantennographic responses in both sexes were significantly higher than that for the control (hexane). These results indicate that T. arnobia adults recognize some volatile compound in the Eucalyptus essential oils; that is, in the antennae of both sexes there are neuron receptors (chemoreceptors), present in sensillas, which detected compounds in the essential oils. Usually, receptors for plant volatiles are basiconic sensillas that are generalist neuron receptors. 22 The mean responses of the males to the seven oils ranged from 1.21 ± 0.28 mV (E. saligna) to 0.82 ± 0.25 mV (E. maculata), the control giving 0.42 ± 0.11 mV, while in the females response ranged from 0.73 ± 0.082 mV (E. urophylla) to 0.46 ± 0.09 mV (E. maculata) and 0.26 ± 0.04 mV (control). The normalized peak responses of males and females to the essential oils are in Figures 1 and 2, respectively. The number, location and also size and form of olfactory sensillas present in the male antenna could explain the higher sensitivity of the T. arnobia male antennae. The male antenna flagellum, which has a bipinnate structure, is more specialized than the female antenna, which is filiform (threadlike). Further studies have to be conducted to prove whether this hypothesis is true.
E. grandis essential oil was selected for the coupled gas chromatography-electroantennographic detection (GC-EAD) experiment because it caused the largest depolarization signal in both the male and female antennae in the EAG. Furthermore, this is the species most prevalent in commercial forestation in Brazil.
The composition of E. grandis essential oil shown by our results is given in Table 4. Component relative concentrations were calculated from GC peak areas and   they were arranged in order of GC elution. Forty compounds were found in this essential oil, thirty-two of which were identified, these corresponding to 95.9 % of the total eluted peaks in the chromatogram of the essential oil (Table 4). It was notable that the chromatographic peaks from the oil analysis consisted largely of terpenes. The major component was α-pinene (40.5 %), and the other two monoterpenes present in substantial amounts were γ-terpinene (16.2 %) and p-cimene (13.1 %).
coeluted, but elicited three electrophysiological responses (peaks). Further studies will be conducted in order to identify the other active compounds.
The fact that twenty-eight Eucalyptus essential oil components elicited responses from T. arnobia female and male antennae in this study is strongly indicative that GC-EAD analysis may be a useful method of screening active plant extracts for compounds that contribute to the observed behavior of this moth. 38 The results obtained by EAG and EAD also suggest that this species uses several volatile cues to find a host. Given the promising results of this study, it remains to be determined what behavioral roles is played by these allelochemicals, and whether compounds based on these active compounds will be sufficiently attractive and specific in the field to form the basis of population monitoring or control of the Brazilian Eucalyptus brown looper.