SYNTHESIS OF THE SAME 2 PROTEINS PRIOR TO LARVAL DIAPAUSE AND PUPATION IN THE SPRUCE BUDWORM, CHORISTONEURA-FUMIFERANA

Spruce budworm larvae produce large quantities of two proteins (Choris toneura fumiferana diapause associated proteins 1 and 2, CfDAP1 and Cf DAP2) that are diapause related. These proteins appeared soon after ha tching and increased in abundance, reaching maximum levels by four day s into the 1st instar, and they remained at high levels until three da ys after the termination of diapause. These two proteins were purified to homogeneity and their NH2-terminal sequences were obtained. Oligon ucleotide primers designed on the basis of these NH2-terminal sequence s were used in RT-PCR to isolate the cDNA fragments coding for these p roteins. These PCR fragments were then used as probes to isolate the c DNAs that contained the complete coding region. The 2.5 kb mRNAs codin g for these proteins started to appear 24 hr after hatching and large quantities of these mRNAs were detected in 1st instar and 2nd instar l arvae until the 2nd instar larvae entered diapause. Low levels of thes e mRNAs were detected in the 2nd instar larvae that were preparing to enter diapause, in those that were in diapause as well as in those tha t terminated diapause. Low levels of CfDAP1 mRNA were also detected on days 1 and 2 after ecdysis to the 3rd instar. However, no CfDAP1 and CfDAP2 mRNAs could be detected during the 4th and 5th instar larval st ages. The mRNAs reappeared 24 hr after the 5th instar larvae molted in to the 6th instar and increased to reach maximum levels by 60 hr after ecdysis, The mRNA levels remained high until 156 hr after ecdysis int o the 6th instar (36-48 hr before pupal ecdysis), after which they dis appeared once again. Immunocytochemical analyses showed that CfDAP1 pr otein was present in 2nd and 6th instar larval fat body but not in 5th instar larval fat body. Thus, the same two genes were expressed for t he first time before C. fumiferana larvae entered diapause and for a 2 nd time before pupation. Crown Copyright (C) 1998 Published by Elsevie r Science Ltd. All rights reserved.