In vitro secretion of digestive phospholipase A(2) by midguts isolated from tobacco hornworms, manduca sexta

Abstract

We report on secretion of phospholipase A(2) (PLA(2)) by in vitro preparations of midguts isolated from tobacco hornworms, Manduca sexta. This enzyme is responsible for hydrolysis of fatty acids from the sn-2 position of phospholipids, a necessary step in fatty acid absorption. The in vitro midgut preparations are competent to secrete PLA(2) into incubation buffer. Secretion began within the first 30 min of incubation and increased to a maximum at 8 h. We selected 2 h incubations because substantial loss of tissue integrity was observed after 8 h incubations. Using 2 h incubations, we recorded increased secretion of digestive PLA(2) from midguts incubated in buffer amended with diet or with yeast as a component of the diet. We also recorded small increases in secretion of PLA(2) from midguts incubated in buffer amended with a specific phospholipid, phosphatidylcholine. Midguts incubated in buffer amended with increased concentrations of phospholipid did not yield higher levels of PLA(2) activity. Lepidopteran midguts can be divided into three regions, and we recorded the highest secretion of PLA(2) from the middle region and lowest secretion from the anterior region. Because isolated midguts responded to food chemicals with increased secretion of digestive PLA(2), we suggest that secretion of digestive enzymes in tobacco hornworms is regulated by a prandial and/or paracrine mechanism, as suggested for digestive proteases in other insect species. Arch. Insect Biochem. Physiol. 42:179-187, 1999.Copyright 1999 Wiley-Liss, Inc.