Proteolytic breakdown of the Neb-trypsin modulating oostatic factor (Neb-TMOF) in the hemolymph of different insects and its gut epithelial transport

The degradation of the unblocked hexapeptide, trypsin modulating oostatic f actor of the flesh fly Neobellieria (Sarcophaga) bullata (Neb-TMOF) was stu died in vitro in the hemolymph of the lepidopteran Spodoptera frugiperda, t he orthopteran Schistocerca gregaria and the dictyopteran Leucophaea madera e. The half-life in the different species varied from similar to3 min in L. maderae to similar to 25 min in S. gregaria. Purification of the degradati on products and ESI-Qq-oa-Tof mass spectrometry revealed the fragments Asn- Pro-Thr-Asn, Leu-His and Asn-Pro, which were the same in the hemolymph of a ll species. Except in Leucophaea, Neb-TMOF was cleaved in dipeptides starti ng from the C-terminus and the reaction could be, at least partially, inhib ited by captopril. These observations suggest that a dipeptidase, which has very similar enzymatic properties as mammalian angiotensin converting enzy me (ACE) and which circulates in the hemolymph, apparently is involved in t he breakdown of Neb-TMOF and might be a common but not a universal enzyme i n insect hemolymph. The introduction of Neb-TMOF into the gut of S. gregaria with the help of a capillary tube (intubation) demonstrated that the intact peptide is able t o cross the gut epithelium and to appear in the hemolymph compartment. Sinc e [H-3]-inulin, which is too large to cross cell membranes, was found to pe netrate the gut walls at a measurable rate, the paracellular pathway might be also permeable to smaller peptides. There was indeed a clear correlation between the molecular weight of inulin, Neb-TMOF, and inositol and the rat e of penetration of these compounds through the gut epithelium to the hemol ymph. These are promising findings in view of a potential use of such pepti des for insect control purposes. (C) 2001 Elsevier Science Ltd. All rights reserved.