# Volatiles Emission by _Crotalaria nitens_ after Insect Attack
## 1 Introduction  
The coevolutionary process has allowed plants to biosynthesize a great diversity of secondary metabolites that reduce the attack of herbivores [1, 2]. Alkaloids, phenylpropanoids, and digestion inhibitors directly repel phytophagous insects [3, 4], while herbivory-induced plant volatiles (HIPV) play an important role in the indirect defense of plants, attracting antagonistic natural enemies of herbivores [5, 6]. Interactions at different trophic levels are becoming relevant as a phenomenon that shapes the distribution and abundance of plants and insects in natural ecosystems [7, 8, 9]. Plants of the genus _Crotalaria_ (Fam. Fabaceae) or rattlepods, grow as weeds in plains and roadsides of the inter-Andean valleys [10], they produce pyrroligidine alkaloids as a direct defense against generalist herbivores [11]. In 1982, [PERSON] reported for the first time that pyrrolidine alkaloids from _Crotalaria pallida_ were related to the chemical defense of the moth _Utetheisa ornatrix_ ([PERSON]) (Lepidoptera: Erebidae) [12]. The insect can sequester, transform, and use these alkaloids without negative effects on the fitness of _U. ornatrix_[13, 14, 15]. Many specialist herbivores have physiological adaptations that allow them to tolerate toxins [16, 17]. Previous reports [18, 19] showed that, under laboratory experiments, _U. ornatrix_ caterpillars sequestered and accumulated more pyrroligidine alkaloids from _Crotalaria nitens_ Kunth than other species evaluated. However, the attackof the caterpillar had no effect on the concentration of these alkaloids in the leaves of _C. nitens_[19]. This result led to the hypothesis that volatiles may be involved in the interaction of _C. nitens_ with _U. ornatrix_, and to our knowledge this appearance has not been evaluated in any species of the genus _Crotalaria_. Knowledge about volatiles in plants, their regulation, biosynthesis, and ecological importance in ecosystems has increased considerably since they were first reported around thirty years ago [8; 9; 20], especially due to their potential use as biological control tools in agricultural systems [21] by manipulating the behavior of insect pests or their natural enemies [22; 23; 24]. Therefore, it requires the development of sensitive extraction and analytical methods for their identification and analysis. The most common sampling method is the purge and trap (P&T) of the volatiles in ad/absorbent materials [25; 26]. However, solid-phase microextraction (SPME), developed by [PERSON] et al. [27] in the early '90s, has proven to be a valuable tool for the extraction of volatile compounds due to its sensitivity and simplicity of handling without the use of solvents [28]. The technique has recently been applied to the detection of HIPV in plant-insect interaction experiments [29]. Here, we compared the two methods--P&T and SPME--for the extraction and identification of HIPV emitted by _C. nitens_ leaves. HIPVs can generate contrasting effects, repelling, or attracting conspecific adult herbivores. For instance, the lepidopteran moths _Heliothis virescens_ ([PERSON]) (Nocutiidae) [30], _Tricholusai ni_ ([PERSON]) (Nocutiidae) [31], and _Manduca sexta_ ([PERSON]) (Sphingidae) [32] are repelled by HIPVs released by the attacks of their caterpillars, while other conspecific adult species are attracted to infested plants, such as the lepidoptera _Epiphyas postvitatana_ ([PERSON]) (Tortricidae) [33], _Busseala fusca_ ([PERSON]) (Nocutiidae), _Sesamia calamistis_ ([PERSON]) (Nocutiidae), and _Chilo parrellus_ (C. [PERSON]) (Crambidae) [34]. However, it is unknown whether the moths of the specialist insect _U. ornatrix_ are attracted to or repelled by the blend of HIPVs emitted by _C. nitens_. The objective of this work was to determine the composition and temporal dynamics of HIPVs release in _C. nitens_ leaves attacked by _U. ornatrix_ caterpillars or injured by mechanical means.