Purification and characterization of an early-expressed polydnavirus-induced protein from the hemolymph of Manduca sexta larvae parasitized by Cotesia congregata

Abstract

Parasitism of Manduca sexta by Cotesia congregata causes major qualitative and quantitative changes in the host's hemolymph proteins, with several novel parasitism-specific proteins being expressed in newly infected larvae. This report describes evidence for the production of several 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 purified polydnavirus into non-parasitized larvae induced production of several EP proteins, suggesting that onset of their synthesis is virally 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 protein was purified to homogeneity using gel filtration and chromatofocusing; treatment of native purified protein with glycosidases showed that EP1 was heavily glycosylated with polysaccharides of the high-mannose type and the denatured, deglycosylated protein had a subunit molecular weight of c. 33,000. The native molecular weight of EP1 was estimated by gel filtration and native gradient polyacrylamide gel electrophoresis (PAGE) c. 190,000. Western blots of hemolymph from parasitized larvae showed that the EP1 antisera detected proteins only in parasitized larvae; the antibodies showed no cross-reactivity with other proteins present in the hemolymph of non-parasitized larvae. The Western blots indicated the early protein(s) were immunologically detectable in the hemolymph from 2 to 144 h following parasitization. Slot blots using a labeled internal restriction fragment from an early protein-specific cDNA (believed to be EP1 cDNA) as probe showed that transcripts from fat body of parasitized larvae were detectable from 0.5 to 96 h post-oviposition, suggesting that synthesis of transcripts rapidly occurs. At 24 h post-oviposition, these transcripts were detected primarily in fat body and hemocytes; lower amounts were also detected in Malpighian tubules and midgut tissues.