# Volatiles Emission by _Crotalaria nitens_ after Insect Attack
## 2 Results  
### GC/MS Analysis of Volatile Compounds Isolated by SPME and P&T  
Many plant species emit volatiles in response to biotic and abiotic factors. The volatiles emitted by _C. nitens_ leaves attacked by _U. ornatrix_ caterpillars were sampled by two methods, one with a PDMS/DVB fiber of 65 um (SPME) and the other using the P&T system with 25 mg of PorapakTM adsorbent. The GC/MS analysis of the volatiles allowed the identification of 21 compounds ( >0.05%) (Table 1 and Figure 1) belonging to the families of C\({}_{6}\)-fatty acid derivatives called green leaf volatiles (GLV), terpenoids, aromatic, and aldoximes. The temporal release dynamics of HIPVs in _C. nitens_ leaves was determined every hour during the first two hours of insect attack and every two hours, up to 10 h, after removing the caterpillars (Figure 2). At the start of the attack, GLVs (_cis_-hex-3-enal, hexyl acetate, _cis_-hex-3-enyl acetate, and _cis_-hex-3-en-1-ol) and the homoterpene _trans_-4,8-dimethyl-nonatriene (DMNT) were mainly detected [35]. During this time, the SPME fiber extracted 86.9% of GLVs and 8.9% of terpen, while 97.6% of the compounds were GLVs and only 1.4% were terpenoids, using the P&T system. The SPME fiber isolated some terpenoids, such as, _trans_-\(\beta\)-ocimene (2.9%), linalool (0.4%), \(\alpha\)-humulene (0.09%), and germacrene D (0.15%), not registered in the extract of the P&T system. Figure 1: Chromatographic profiles of HIPVs emitted by _C. nitens_ leaves and the chemical structure of the main volatile compounds. SPME (PDMS/DVB, 65 μm) and P&T (PorapakTM-Q) sampling. GC/MS analysis (DB-WAX column, 60 m). Table 1 contains peak identification; istd, methyl undecanoate. Figure 2: HIPV emission by _C. nitens_ leaves attacked by _U. ornatrix_ caterpillars. The caterpillars were fed for the first two hours, then removed from the plant. Volatiles were extracted using SPME. The ratio between the sum of areas of the group of compounds of interest A\({}_{\text{i}}\) and the area of the internal standard A\({}_{\text{istd}}\) (methyl undecanoate) is reported. Values are the mean \(\pm\) standard error (\(n=7\)). \begin{table}
\begin{tabular}{c c c c c c c c c}  & \multicolumn{3}{c}{**DB-WAX**} & \multicolumn{3}{c}{**DB-SMS**} & \multicolumn{3}{c}{**Relative CC Peak Area (\%, Mean \(\pm\) SE, n = 7)**} \\
**Peak N\({}^{*}\) Figure 1** & **Compound** & **LBI** & **LBI** & **LBI** & **LBI** & **Dosing the Attack Q\({}_{\text{B}}\)** & **After the Attack (\% h)** \\  & & **Experiment. ** & **Repported** & **Experiment. ** & **Repported** & **SPME** & **PRT** & **SPME** & **  
After two hours of attack, the release of GLVs was reduced to 1.9% and 1.2% measured by SPME and P&T, respectively; however, the emission of terpenoids, aromatics, aldoximes, and other oxygenated compounds increased reaching a maximum at six hours. At this time, the proportion of terpenoids (mainly _trans_-\(\beta\)-csimeme, linalool, _trans_-\(\beta\)-caryophyllene, \(\alpha\)-humulene, _trans_, _trans_-\(\alpha\)-farmesene, and _trans_-nerolidol), including the homoterpene DMNT, raised their concentration to 77.2% and 63.7% extracted by SPME and P&T methods, respectively. The aromatic compounds (methyl salicylate, methyl anthranilate and indole) were detected at 2.7% (SPME) and 22.2% (P&T). The aldoximes isobutyraldoxime and 2-methylbutylradoloxime were extracted by the two methods at 11.5% (SPME) and 8.7% (P&T). Such relatively simple oxime structures have been reported in approximately 22 different plant species [41]. Their identification was confirmed by mass spectra analysis and comparison of experimental data with those from the database (NIST, 2017) as shown in Figures S1 and S2. At six hours after the attack, the SPME fiber extracted other compounds, e.g., _allo_-ocimene (0.18%), 3-octanol (0.14%), and germacrene D (0.6%), that were not detected with the P&T method.