PURIFICATION AND CHARACTERIZATION OF AN EARLY-EXPRESSED POLYDNAVIRUS-INDUCED PROTEIN FROM THE HEMOLYMPH OF MANDUCA-SEXTA LARVAE PARASITIZEDBY COTESIA-CONGREGATA

Parasitism of Manduca sexta by Cotesia congregata causes major qualita tive and quantitative changes in the host's hemolymph proteins, with s everal novel parasitism-specific proteins being expressed in newly inf ected larvae. This report describes evidence for the production of sev eral early-induced proteins (EP's) all of which have subunit molecular weights in the range of 30,000-40,000 and appear in the hemolymph of M. sexta within a few hours following parasitization. Injection of pur ified polydnavirus into non-parasitized larvae induced production of s everal EP proteins, suggesting that onset of their synthesis is virall y mediated. Following analysis of hemolymph proteins on 2-dimensional gels, N-terminal sequence data were obtained for three EP proteins and one was selected for further study, which was designated EP1. This pr otein was purified to homogeneity using gel filtration and chromatofoc using; treatment of native purified protein with glycosidases showed t hat EP1 was heavily glycosylated with polysaccharides of the high-mann ose type and the denatured, deglycosylated protein had a subunit molec ular weight of c. 33,000. The native molecular weight of EP1 was estim ated by gel filtration and native gradient polyacrylamide gel electrop horesis (PAGE) to be c. 190,000. Western blots of hemolymph from paras itized larvae showed that the EP1 antisera detected proteins only in p arasitized larvae; the antibodies showed no cross-reactivity with othe r proteins present in the hemolymph of non-parasitized larvae. The Wes tern blots indicated the early protein(s) were immunologically detecta ble in the hemolymph from 2 to 144 h following parasitization. Slot bl ots using a labeled internal restriction fragment from an early protei n-specific cDNA (believed to be EP1 cDNA) as probe showed that transcr ipts from fat body of parasitized larvae were detectable from 0.5 to 9 6 h post-oviposition, suggesting that synthesis of transcripts rapidly occurs. At 24 h post-oviposition, these transcripts were detected pri marily in fat body and hemocytes; lower amounts were also-detected in Malpighian tubules and midgut tissues.