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Accueil du site → Doctorat → Belgique → Enzymatic and transcript analysis of the protease inhibitor induced regulation of digestion in two locust species, Schistocerca gregaria and Locusta migratoria

KU Leuven (2014)

Enzymatic and transcript analysis of the protease inhibitor induced regulation of digestion in two locust species, Schistocerca gregaria and Locusta migratoria

Spit, Jornt

Titre : Enzymatic and transcript analysis of the protease inhibitor induced regulation of digestion in two locust species, Schistocerca gregaria and Locusta migratoria

Onderzoek naar de protease inhibitor geïnduceerde regulatie van de vertering op enzymatisch en transcript niveau in twee sprinkhaansoorten, Schistocerca gregaria en Locusta migratoria

Auteur : Spit, Jornt

Université de soutenance : KU Leuven

Grade : Doctor 2014

Résumé
An important source of alternative insect control proteins is the plant defence system. By millions of years of co-evolution, plants have evolved (some very effective) counter measures to predation by herbivorous insects. Many different plant defensive compounds have been shown to possess some toxic or anti-metabolic effect on insects and hence are considered as possible candidates for the genetic modification of crop plants. Protease inhibitors (PIs) form a group of candidate proteins that have received attention for a long time and are amongst the most advanced in terms of development. PIs target the digestive proteolytic enzymes in the gut of insects. However, frequently, insect adaptation to these anti-nutritional proteins is observed. One of the reasons for this is the large variety of digestive protease genes encoded by insects that can be transcriptionally regulated, thereby compensating for the loss of digestive activity. Little information exists on the digestive enzymes of locusts, especially at the transcript level. Furthermore, to date, our knowledge on insect compensation for PI ingestion is strictly based on studies in a limited number of lepidopteran and coleopteran species. Therefore, the general aim of this thesis was to identify possible PI induced compensatory responses in the gut of the desert locust, Schistocerca gregaria,and the migratory locust, Locusta migratoria, both at the enzyme activity and transcript level. We were able to show that both locust species possess the ability to compensate for the ingestion of PI. ExistingEST databases for L. migratoria were screened for serine protease-like sequences. A total of 5 putative trypsins and 15 putative chymotrypsins could be characterized. The relative gene expression levels of representative members were determined under different feeding conditions. Transcript levels for all measured serine proteases were strongly reduced after starvation. On the other hand, rapid initial upregulation was observed for all tested transcripts after PI ingestion.A microarray based approach was used to identify transcripts that were specifically transcribed in gut, brain, or both tissues. Gut specific transcripts were functionally annotated, and were found to be enriched in transcripts with putative functions closely related to the physiological functions of the gut as a muscular digestive organ, and as the first barrier against micro-organisms and a wide range of toxins. In addition, a second microarray experiment with a selected subset of sequences was conducted, which aimed at revealing underlying transcriptional changes after initial detection of inhibitor presence in the locust midgut. We discovered that several transcripts involved in energy metabolism were quickly altered after PI ingestion. Apart from upregulating digestive proteases, transcripts involved in carbohydrate and lipid metabolism were found to be differentially expressed. In addition, the results suggest that during the physiological challenge of PIingestion, fewer resources are invested in defence, stress responses, and in the maintenance of structural integrity. Furthermore, several transcripts encoding juvenile hormone binding proteins were found to be upregulated. Their involvement in PI compensation in L. migratoria was further assessed using RNA interference. In conclusion, the findings in this thesis contribute to a better understanding of protease inhibitor induced compensatory responses in insects. The fact that also orthopteran insects were able to compensate quickly for ingestion of dietary inhibitors is especially interesting, and suggests that the basic mechanisms responsible for the changes in proteolytic enzyme expression may have arose early in insect evolution. Our results further indicate that for PI to be successful as biopesticides, the insects’ adaptive mechanisms have to be circumvented, or that a combined approach, simultaneously targeting the digestive system and other vulnerable physiological systems might be preferred

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Page publiée le 8 décembre 2014, mise à jour le 24 novembre 2018