Prophenoloxidases 1 and 2 from the spruce budworm, Choristoneura fumiferana: Molecular cloning and assessment of transcriptional regulation by a polydnavirus

Abstract

Immune challenge in arthropods is frequently accompanied by melanization of the hemolymph, a reaction triggered by the activation of prophenoloxidase (PPO). Because their immature stages are spent inside the hemocoel of insect larvae, endoparasitoids have evolved strategies to escape or counter melanin formation. Very little molecular information is available on these endoparasitoid counterstrategies. We have sought to shed light on the inhibition of melanization in the spruce budworm, Choristoneura fumiferana, by the parasitic wasp Tranosema rostrale, by cloning two host PPO homologs and studying their transcriptional regulation after parasitization. The two polypeptides are encoded by transcripts of approximately 3.3 kb (for CfPPO1) and 3.0 kb (for CfPPO2) and possess structural features typical of other insect PPOs. While there appears to be a single CfPPO2 gene in the C. fumiferana genome, we detected three CfPPO1 mRNA variants displaying insertions/deletions in the 3' untranslated region, suggesting that there may be more than one CfPPO1 gene copy. Both CfPPO1 and CfPPO2 were expressed at high levels in C. fumiferana 6th instars, and parasitization by T. rostrale had no apparent impact on the level of their transcripts. Injection of a large dose (0.5 female-equivalent) of polydnavirus-laden calyx fluid extracted from T. rostrale, which is known to inhibit melanization in C. fumiferana, only caused a transient decrease in CfPPO1 and CfPPO2 transcript accumulation at 2-3 d post injection. It thus appears that transcriptional downregulation of C. fumiferana PPO by T. rostrale plays a minor role in the inhibition of hemolymph melanization in this host-parasitoid system.

Proteome changes in the plasma of Papilio xuthus (Lepidoptera: Papilionidae): effect of parasitization by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

“…Our results suggest that the transcription or translation of proPO and MasSPH can be altered by parasitization. Studies on other parasitoid-host systems have found that the transcription of melanization-related genes such as proPO, serine proteases, and serpins is regulated by parasitization (Wertheim et al, 2005;Doucet et al, 2008). In accordance with these studies, we found that the expressions of proPO and MasSPH were altered following parasitization, suggesting that disruption of melanization would be a way for P. puparum to modulate P. xuthus immune reaction.…”

“…As a key defense mechanism against intruding organisms, melanization is the result of the proPO activation cascade regulated by serine proteinases, which leads to the formation of melanin and other toxic phenolic compounds (Ashida and Brey, 1998;Vass and Nappi, 2000). Among the known mechanisms to fend off parasitoid, defensive melanization is perhaps the most ubiquitous and best characterized among humoral response (Doucet et al, 2008). Thus a reduced melanization caused by the inhibition of phenoloxidase (PO) activity and proPO-activating enzyme serine protease (PPAE) is often observed of parasitization (Lavine and Beckage, 1995;Asgari, 2006).…”

Sequencing and characterization of two cDNAs putatively encoding prophenoloxidases in the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Yponomeutidae)

“…This is similar to results from previous studies of H. cunea (Park et al 1997), H. virescens (Shelby and Popham 2008), and C. fumiferana (Doucet et al 2008).…”

“…In A. subalbatus at least five distinct PPOs have been identified and the genomes of Drosophila melanogaster and Manduca sexta have three PO genes. However, only two cDNAs encoding PPOs have been reported in most of other lepidopteran insects, for example Hyphantria cunea (Park et al 1997), B. mori (Asano and Ashida 2001), H. virescens (Shelby and Popham 2008) and C. fumiferana (Doucet et al 2008). In our study, by using native-PAGE and coloration with standard substrate L-dopa, we found that P. xylostella also has two PPO isoenzymes, PxPPO1 and PxPPO2, which was further proved by sequencing of their cDNA.…”

“…The cDNA encoding PO III from Armigeres subalbatus has been sequenced and proved to be associated with morphogenesis of mosquitoes (Tsao et al 2009). Doucet et al (2008) cloned PPO 1 and 2 from the spruce budworm, Choristoneura fumiferana, and investigated their transcriptional regulation after parasitization.…”