Control of the release of digestive enzymes in the cricket Gryllus bimaculatus and the fall armyworm, Spodoptera frugiperda
This thesis investigates control of the release of digestive enzymes in the cricket Gryllus bimaculatus and the fall armyworm, Spodoptera frugiperda. 1. Control of enzyme release in the cricket G. bimaculatus. Using flat-sheet preparations of the caecum, digestive enzyme release was investigated. More trypsin, aminopeptidase and amylase are secreted in the caecum of fed crickets than in unfed crickets, but basal levels of certain enzymes are released continuously even in unfed animals. A variable ratio of nutrients in ingested food leads to a different ratio of digestive enzyme release, but a high nutrient component in the food does not necessarily induce a high digestive enzyme release for that component. Maltose and glucose elevate amylase release from the tissues into the incubation medium, but starch does not. Bovine serum albumin (BSA), peptone and a mixture of amino acids have almost no effect on the release of aminopeptidase, and only low concentrations of peptone increase trypsin release. In crickets, the continuous release of proteases is sufficient to meet the needs for growth, and only moderate stimulation of trypsin results from feeding. Carbohydrates are used for energy, and the release of amylase is adjusted to the amount of food ingested. The neuropeptide allatostatin Grybi-AS 5 elevates the release of amylase in fed females, but not of trypsin or aminopeptidase, however, both amylase and trypsin release are stimulated by AS 5 in unfed crickets. Fed crickets have sufficient trypsin to obtain needed amino acids, but unfed do not, therefore the AS stimulation of trypsin release in unfed crickets makes sense. 2. Control of enzyme release in the larvae of S. frugiperda. A flat-sheet preparation of the ventriculus was used to test the release of amylase, trypsin and aminopeptidase in response to specific nutrients in the food and to specific neuropeptides. The epithelial secretion rate of amylase and trypsin was about 20% of the amount of enzyme present in the ventricular lumen, which, considering the efficient counter-current recycling of enzymes, suggests that the secretion rate is adequate to replace egested enzymes. Dietary carbohydrates are used for energy, and larvae adjust amylase activity to carbohydrate ingestion. Amylase activity is 5-times higher in fed compared to unfed larvae, and sugars in the incubation medium induces more than a 300 % increase in amylase release. Plants contain a low level of protein, but larvae need proteins for growth, thus the larvae can not afford to lose proteins by egestion. Therefore, trypsin activity remains high even in unfed larvae. As a result, proteins and amino acids have little or no effect on trypsin or aminopeptidase release in incubated tissues. The control of enzymes release in response to food is most likely mediated through neurohormones. Indeed, an allatostatin (Spofr-AS A5) inhibits amylase and trypsin release, and allatotropin (Manse- AT) stimulates amylase and trypsin release. Spofr-AS A5 also inhibits ileum myoactivity and Manse- AT stimulates myoactivity. 3. Inhibition of enzyme release in the larvae of S. frugiperda. Exogenous inhibitors are produced by plants, and are ingested by the insect. Endogenous inhibitors are produced by the gut epithelial cells themselves. A dose-dependent inhibition of endogenous enzymes occur in the lumen after feeding L6 larvae with the exogenous serine protease inhibitor from soybean (SBTI), the specific trypsin inhibitor TLCK, an aminopeptidase inhibitor (bestatin), and an amylase inhibitor from wheat. Inhibition in tissue extracts is seen only with higher doses of SBTI and TLCK. Inhibition of enzyme release into the incubation medium is apparent only with very high doses of SBTI. Inhibition in the tissues and inhibition of release indicate a direct cellular response to an inhibitor present in the lumen. The elevation of aminopeptidase activity in response to ingested trypsin inhibitors indicates a cellular synthesis in response to the product of a digestive enzyme. The enzymes investigated are irreversibly inactivated by 10 min at 90°C, but the corresponding inhibitors are not, therefore endogenous inhibitors could be identified. Endogenous inhibitors are present in the ventricular cells and in the lumen. We suggest that the endogenous protease inhibitors may protect the epithelium by inactivation of the trypsin in underfed larvae. This is the first explanation of how insects are able to secrete an active trypsin.
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