Insect Biochem. Vol. 14, No. 6, pp. 713-717, 1984 0020-1790/84 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright ~.') 1984 Pergamon Press Lid ACTIVATION OF THE SECRETION OF SPECIFIC PROTEINS FROM FAT BODY FOLLOWING INJURY TO THE BODY WALL OF SARCOPHAGA PEREGRINA LARVAE HARUO TAKAHASHI, HIROTO KOMANO, NOBUAKi KAWAGUCHI, MASUO OBINATA and SHUNJI NATORI Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received 22 September 1983; revised 10 December 1983 and 29 February 1984) Abstract--When the body wall of Sarcophaga peregrina larvae was injured, the fat body was transiently activated to synthesize and secrete 70k and 27k proteins. It was found that genes for these proteins were activated at the time of the injury. The amount of resulting mRNA was too small to detect a translation product in vitro, using the reticulocyte lysate system, but the mRNA seemed to be translated quite efficiently in vivo, suggesting translational control of these mRNA. Key Word Index: Sarcophaga peregrina, fat body, defence mechanism, haemolymph protein, trans- lational control INTRODUCTION Previously, we reported the induction of three bacte- ricidal proteins and one lectin in the haemolymph of Sarcophaga peregrina (flesh-fly) larvae when the body wall was injured with a hypodermic needle (Komano et al., 1980; Okada and Natori, 1983). The lectin was shown to be synthesized in the fat body (Komano et al., 1983). The bactericidal proteins may also be synthesized in this tissue and eventually secreted into the haemolymph as observed with cecropins, which are antibacterial proteins in the silkworm Hyalophora cecropia (Faye and Wyatt, 1980). Stevenson and Wyatt (1962) observed stimulation of fat body pro- tein synthesis after integrnentary injury to diapausing saturniid silkmoths pupae. There seems to be three critical steps in the regu- lation of the induction of these serum proteins which are responsible for the defence mechanism of this insect: transcription, translation and secretion. How- ever, it is now know how stimuli from the injury are transmitted to the fat body and which of the three steps are activated by the stimuli to cause production of specific serum proteins. This paper reports results showing that fat body prepared from injured larvae synthesizes and secretes 70k and 27k proteins in vitro, whereas that from normal larvae apparently does not. The content of these proteins in haemolymph from injured larvae was not high, but its synthesis and secretion were markedly activated by injury to the body wall with 3-6 hr. MATERIAIAg;AND METHODS Animals; culture of fat body in vitro Third instar larvae of Sarcophagaperegrina, kept at 25°C in a plastic container with a small amount of water, were used throughout the experiment. Prior to body wall injury, the larvae were anaesthetized on an ice-cold glass plate for a few minutes and then the posterior part of the body wall was pricked with a hypodermic needle. To label the protein, a fat body was removed under a binocular microscope and cultured in 300 ~ul of methionine-free Grace's insect medium (Grace, 1962) for 60 min at 25°C in the presence of 35/~Ci of [35 S]methionine ( 1360 Ci/mmol, Amersham). Injection of ~t-amanitin The larvae were anaesthetized by keeping them on ice for a few minutes and then 5/~1 of 1 mg/ml of 0t-amanitin solution was injected into the posterior part of the abdom- inal cavity of each larva with a microsyringe under a binocular microscope. SDS-polyacrylamide gel electrophoresis Protein samples were prepared from the fat body and the culture medium. After being labelled, the fat body was rinsed well with phosphate buffered saline (PBS, 130 mM NaCI, 3 mM KCI in 10mM phosphate buffer, pH 7.4), homogenized in 0.5 ml of PBS containing 0.1"/o SDS, and then centrifuged at 10,000g for 10 min at 4'C. Then 200 ~ul of the clear supernatant were mixed with the .same volume of 10~o (w/v) TCA, and the resulting precipitate was col- lected by centrifugation at 3000g for 10rain at 4~C. The precipitate was washed with I ml of diethyl ether, dissolved in 3% (w/v) sodium dodecyl sulfate solution containing 10"/o (v/v) glycerol and 5% (v/v) fl-mercaptoethanol, and then subjected to electrophoresis. For analysis of the proteins in the culture medium, 250~ul of 10~o TCA were added to the same volume of the culture medium and the resulting precipitate was treated in the same way as that from the fat body. SDS-polyacrylamide gel electrophoresis (12.5% acryl- amide) was carried out as described by Laemmli (1970). For autoradiography, gels were dried onto a sheet of filler paper and placed in contact with Kodak X-Omat AR film with an intensifying screen. After a suitable period of exposure the film was developed. Isolation of poly(A)-containing RNA and its translation in vitro The total RNA was extracted from 50 to 60 fat bodies and poly(A)-containing RNA was prepared from the total RNA by chromatography on oligo(dT)-cellulose as described previously (Tahara et al., 1982). Translation of poly(A)- containing RNA was carried out in a rabbit reticulocyte lysate, as described by Pelham and Jackson (1976). 713