# Volatiles Emission by _Crotalaria nitens_ after Insect Attack
## 3 Discussion  
The secondary metabolites produced by plants are important for their survival in all ecosystems, for adaptation to environmental changes and for ecological interactions with other organisms [4]. However, the identification and release dynamics of the volatiles emitted in _Crotalaria_ genus plants in response to attack by herbivorous insects are still unknown [9]. In the present investigation, it was shown that _C. nitens_ plants recognized the wound of the specialist caterpillar _U. ornatrix_ and emitted HIPVs in response to its attack. To our knowledge, it is the first time that HIPVs are identified for a species of _Crotalaria_. The volatiles emitted by the leaves of _C. nitens_ modified the behavior of _U. muratrix_ moths, significantly reducing their oviposition on previously attacked plants and preferring undamaged plants. The volatiles released by plants play an important role in interactions with other organisms [2], therefore sensitive extraction methods are needed that accurately represent the mixture of compounds released [25; 42]. In this work, we studied the volatiles emitted by _C. nitens_ leaves by sampling with SPME and with a conventional P&T system. Twenty-one volatile compounds released by the _C. nitens_ leaves were identified, belonging to the GLVs, terpenoid, homoterpene, aldoxime, and aromatic compound families. The sampling with the PDMS/DVB fiber was more sensitive and managed to isolate some terpene compounds that could not be detected in the extracts obtained by the P&T extraction. The reason is that the SPME fiber allowed the analytes to be concentrated on the fiber and then desorbed directly into the injection port of the chromatographic system, whereas with the P&T method, the compounds trapped in the sorbent were diluted with the extracting solvent before their analysis. The volatile compounds extraction using SPME was simpler to operate and did not require the use of solvents and therefore eliminated  
Figure 4: Oviposition preference of _U. ornatrix_ female moths on _C. nitens_ leaves. (**A**) On both sides of one carboard box were placed one undamaged plant. (**B**) On one side of a second box an undamaged plant was used and, on the other side, a plant that had been attacked by six third-instar caterpillars was included. (**C**) A third box contained two damaged plants. [PERSON]’s two sample _t_-test (**_p_ < 0.01). Values are the mean \(\pm\) standard error (_n_ = 7). Each plant pair was used only once. Numbers included in bars correspond to the total number of eggs laid by moths across all replicates. possible interferences and cross-contamination compared to the P&T method. However, during the collection of HIPVs by SPME, the container accumulates moisture, and this condition can interfere with the physiological processes of the plant or the insect [25]. This problem did not occur with the P&T method, due to the injection of filtered air into the system from the outside. However, the constant air flow increased the adsorption of pollutants that could not be retained by the activated carbon filters installed at the inlet of the system. The _C. nitens_ leaves emitted a wide variety of volatiles in response to the attack by the specialist caterpillar _U. ornatrix_. GLVs were the first compounds emitted by _C. nitens_ leaves [35; 43]. Subsequently, the terpenoids, the homoterpene DMNT, aldoximes, and aromatic compounds were released with a maximum emission at six hours after the attack. When the _C. nitens_ leaves were subjected to mechanical damage, large amounts of GLVs were detected, but after two hours, the volatile emissions decreased dramatically compared to those generated upon insect attack. This result indicates that the _U. ornatrix_ caterpillars were responsible for generating a specific response in _C. nitens_ leaves. The ability of plants to distinguish types of damage, allows them to reduce the expenditure of valuable resources for defense in situations where it is not necessary.