Abstract
Enzymes play a key role in insect-plant relationships. For a better understanding of these interactions, we analyzed Tuta absoluta digestive enzymes. Here, we describe a detailed protocol for the detection of trypsin and papain-like enzymes in Tuta absoluta larvae by enzyme histochemistry. This assay uses frozen and unfixed samples to avoid the loss of enzymatic activity. We also describe a protocol for the quantification of trypsin and papain-like enzymes in the larvae of Tuta absoluta at different developmental instars.
Keywords: Tuta absoluta, Trypsin, Papain, Cryostat, Histochemistry
Background
Plants and insects have coexisted for million years and evolved a set of interactions which affect both organisms at different levels. Insects manage to develop different physiological and morphological adaptations to overcome plants defense mechanisms. Therefore, a better understanding of their vital functions would facilitate their targeted control. Different physiological functions in insects rely on enzymes: digestive, respiratory, circulatory, muscular, nervous, reproductive and endocrine. Several enzymes participate in the digestion. Proteases such as trypsin, chymotrypsin, pepsin or carboxypeptidases are responsible for protein digestion, which is a source of amino acids for the insect. Thus, digestive protease inhibitors have been successfully used to improve plant resistance to insects (Smigocki et al., 2013; Quilis et al., 2014; Hamza et al., 2018). In order to design such approaches, it is important to identify the target insect digestive enzymes. Enzyme histochemistry is a useful method for the localization of active enzymes in tissue sections of an organism. However, few protocols have been described for insects. One of the main issues of this method is the sensibility of the enzymes to fixatives. In a previous study, Erban and Hubert (2011) visualized the digestive enzymes in the body of the acarid mite Lepidoglyphus destructor after feeding them with chromogenic and fluorescent substrates taking advantage of their transparent body. This approach is not suitable for larvae of bigger insects such as Tuta absoluta. These larvae do not have transparent body allowing the visualization of the fluorescence and are reared with fresh tomato leaves instead of artificial diet and thus cannot be supplemented with enzymatic substrates. In our work, we designed a protocol for the detection of serine proteases and papain-like proteases in cryosections of larvae of Tuta absoluta without tissue fixation. We also quantified these enzymes in larvae at different instars using chromogenic substrates.
Materials and Reagents
Equipment
Procedure
Data analysis
Recipes
Acknowledgments
Rim Hamza acknowledges fellowships from the Tunisian Ministry for Higher Education and Scientific Research and from the Erasmus Mundus EMMAG program of the European Union. This work was partly supported by grants BIO2013-40747-R and AGL2014-55616-C3 from the Spanish Ministry of Economy and Competitiveness (MINECO). We wish to thank Drs. Alberto Urbaneja and Meritxell Pérez-Hedo (Instituto Valenciano de Investigaciones Agrarias, Centro de Protección Vegetal y Biotecnología, Unidad Asociada de Entomología, UJI-IVIA) for providing Tuta absoluta larvae and to Marisol Gascón (IBMCP) for technical assistance in sample processing. This protocol has been described in the publication: Hamza et al. (2018).
Competing interests
The authors declare that there are no conflicts of interest related with this work.
References
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