ARS | Publication request: Transepithelial flux of an allotostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro

Submitted to: Peptides
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 24, 2007
Publication Date: February 15, 2008
Citation: Audsley, N., Matthews, J., Nachman, R.J., Weaver, R.J. 2008. Transepithelial flux of an allotostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro. Peptides. 29:286-294.

Interpretive Summary: Because of problems with the development of resistance to conventional pesticides, there is a critical need for new concepts and alternative approaches in controlling insect pests. The basic premise of this research is that neuropeptides (short chains of amino acids) serve as potent messengers in insects to regulate vital functions. Nevertheless, neuropeptides in and of themselves hold little promise as pest control agents because of susceptibility to being degraded in the target pest, and inability to pass through the outside skin (cuticle) and/or digestive tract. Neuropeptide mimics must be designed that resist degradation by enzymes in the digestive tract and blood of pest insects and interact with the active site within agricultural or medical pests by over-activating or blocking critical, neuropeptide-regulated life functions. We report on the ability of biostable analogs of the allatostatin neuropeptide family to penetrate the gut of immature forms of the tobacco hawkmoth. The mimic also demonstrates greatly enhanced stability to enzymes that degrade the natural regulatory neuropeptide. The allatostatin class of neuropeptides is implicated in regulation of developmental and reproductive processes. This work represents an important milestone and lead in the development of practical neuropeptide-like substances that will effectively control insect pests in an environmentally friendly fashion.

Technical Abstract: The transepithelial transport of cydiastatin 4 and analogues across flat sheet preparations of the anterior midgut of larvae of the tobacco hawkmoth moth, Manduca sexta, was investigated using a combination of reversed-phase high performance liquid chromatography (RP-HPLC), enzyme linked immunosorbent assay (ELISA) and matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). The lumen to haemolymph (L-H) flux of cydiastatin 4 was dose and time dependent, with a maximum rate of transport of c. 178 pmol/sq-cm/h measured after a 60 minute incubation with 100 µmol/l of peptide in the lumen bathing fluid. The rates of transport, L-H and H-L, across the isolated gut preparations were not significantly different. These data suggest that transport across the anterior midgut of larval M. sexta is via a paracellular route. Cydiastatin 4 was modified to incorporate a hexanoic acid (Hex) moiety at the N-terminus, the N-terminus extended with 5 P residues and/or the substitution of G7 with Fmoc-1-amino-cyclopropylcarboxylic acid (Acpc). The incorporation of hexanoic acid enhanced the uptake of these amphiphilic analogues compared to the native peptide. Analogues were also more resistant to enzymes in haemolymph and gut preparations from larval M. sexta. A modified N-terminus gave protection against aminopeptidase-like activity and incorporation of Acpc inhibited carboxypeptidase-like activity. Although analogues were stable in the haemolymph, they were susceptible to amidase-like activity in the gut, which appears to convert the C-terminal amide group to a free carboxylic acid, identified by an increase in 1 mass unit of the peptide analogue.