Temporal, spatial and induced expression of Choristoneura fumiferana chitinase (CfChitinase) was studied using immunohistochemistry and Western blots. CfChitinase was detected in the integument, the midgut peritrophic membrane, the cuticular lining of the trachea, the spiracle, and salivary glands. The enzyme was expressed as larvae were preparing to molt from one instar to the next. The spatial and temporal expression patterns are consistent with its function in degrading chitin during the molting process. The 20-hydroxyecdysone agonist, tebufenozide (RH5992), induced the expression of the CfChitinase gene in the early stage of the sixth-instar larvae and the enzyme was detected in the epidermis and molting fluid 24 h post treatment.

The effect of RH-5992 (tebufenozide), a non-steroidal ecdysone agonist, on adult development of the spruce budworm, Choristoneura fumiferana, was investigated by administering the compound intrahemocoelically to pupae on days 1-6 after pupal ecdysis. At concentrations of 200ng/pupa there was significant mortality but at doses of 50-100ng/pupa, the emerging adults displayed wing deformities which reduced their ability to mate and oviposit. Light microscopy of the pupal wings revealed that there was degeneration of the epithelial cells, reduction in the number of veins, precocious cuticle formation and inhibition of growth of normal wing scales. Injection of RH-5992 into pupae resulted in a dose dependent induction of mRNA for ecdysone-induced transcription factor, Choristoneura hormone receptor 3 (CHR3). These results suggest that the pupae respond to RH-5992 in a manner similar to larvae. However, the effects are not expressed overtly and are camouflaged by the pharmacological effects.

Spruce budworm larvae (Choristoneura fumiferana) upon ingesting tebufenozide (RH-5992) stop feeding and go into a precocious, incomplete molt, leading eventually to death. Like 20-hydroxyecdysone (20E), tebufenozide also acts at the receptor level and transactivates the expression of up-regulated genes but, because of its persistence, the down-regulated genes that are normally expressed in the absence of 20E are not expressed. While tebufenozide is lepidopteran-specific, an analog, RH-5849, is effective on dipterans. This is reflected in the respective effects of the two compounds on Cf-203 (C. fumiferana--203), a lepidopteran cell line and Dm-2 (Drosophila melanogaster--2), a dipteran cell line. Cf-203 cells accumulated [14C]tebufenozide and expressed CHR3 (Choristoneura hormone receptor 3), but Dm-2 cells excluded the material and did not express DHR3 (Drosophila hormone receptor 3). Using yeast ABC (ATP binding cassette) transporter mutants, we determined that PDR5 (pleiotropic drug resistance 5) was responsible for the exclusion. We discovered recently that older instars of the white-marked tussock moth (Orgyia leucostigma) are resistant to tebufenozide, perhaps as a result of such an exclusion system. We are currently cloning PDR5 (pleiotropic drug resistance 5), which is an essential step in studying the resistance mechanism.

Publisher Summary Diapause is a genetically determined, hormonally mediated state of suppressed development, and is an important adaptive mechanism for insect survival during unfavourable environmental conditions, such as low winter temperatures. The spruce budworm, Choristonuera fumiferana, overwinters as a diapausing second-instar larva. Molecular analyses of larvae in prediapause, diapause, and postdiapause stages led to the identification of sugars and proteins that are related to diapause or overwintering. C . fumiferana produces the disaccharide trehalose and proteins, such as diapause associated proteins 1&2 and glutathione S-transferase that are diapause related. The glycerol and the defensin-like protein that the larvae produce appear to be related to the overwintering process. Glycerol production and synthesis of defensin-like protein are clearly overwintering related events, and these happen only after the larvae are exposed to cold treatment for certain lengths of time. If the larvae are not exposed to the cold treatment, they fail to synthesize glycerol or defensin-like protein. On the other hand, increases in trehalose levels and synthesis of CfDAPl and CfDAP2, as well as increased synthesis of CfGST, occur as soon as the larvae hatch and start preparing for diapause. They also start spinning hibemacula at this time. These are probably diapause-related events. Absence of ecdysteroids in the diapausing second instars prevents molting and metamorphosis, allowing them to overwinter in this stage. This chapter describes the simultaneous occurrence of obligatory diapauses and the overwintering process in the second-instar larvae of the spruce budworm, C. fumiferana . However, these two physiological events can be distinguished from each other on the basis of some of the biochemical changes that occur in these larvae during the obligatory overwintering diapause.

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.

The mode of action of RH-5992 (tebufenozide), a non-steroidal ecdysone agonist, on the eastern spruce budworm, Choristoneura fumiferana, was investigated. This diacyl hydrazine compound, upon ingestion, initiates a precocious incomplete molt that is lethal in most lepidopteran larvae including the spruce budworm. This was found to be induced when the larvae ingested the compound early in the stadium prior to the appearance of the ecdysone peak in the hemolymph. The larvae stopped feeding within 8h post feeding (PF) and remained quiescent just as they do in preparation for a normal molt. Head capsule slippage started at 12h PF, became pronounced by 24h, and by 48h an untanned new head capsule was visible behind the old one. The lack of tanning of the new cuticle was due to the failure of dopadecarboxylase gene expression. Although the old cuticle was loose around the entire body, indicating that apolysis had occurred, there was no evidence of ecdysis of the old cuticle, suggesting that eclosion hormone was probably not released. The transcription factor, Choristoneura hormone receptor 3 (CHR3), which is normally expressed at the onset of the hemolymph ecdysone peak, was expressed in the epidermis 1h PF of RH-5992 confirming that this analogue acts through the ecdysone receptor system. This unique mode of action at the molecular level of this ecdysone agonist and its effectiveness as an environmentally benign control agent for the spruce budworm are described.

Choristoneura fumiferana nucleopolyhedrovirus(C f MNPV) is a baculovirus that infects the spruce budworm, Choristoneura fumiferana(Cf) , and a few other related species. To study the infection process of this virus within the spruce budworm, we have constructed a recombinant virus (C f MNPV-GFP) expressing the green fluorescence protein(GFP) under the control of the polyhedrin promoter of Autographa californica nucleopolyhedrovirus. Each larva was fed 10,000 C f MNPV-GFP occlusion bodies(OB) applied to diet plugs and various tissues were dissected from the larvae at 24 hr intervals and examined for the presence of the OB and/or GFP using a fluorescence microscope. Larvae were also fixed and embedded for light and electron microscopy. GFP-specific fluorescence was first detected as a few spots at the posterior end of the midgut at 72 hr post feeding(PF). By 96 hr PF, the fluorescence had expanded to include adjacent cells and some hemocytes.

Spruce budworm larvae produce large quantities of two proteins (Choristoneura fumiferana diapause associated proteins 1 and 2, CfDAP1 and CfDAP2) that are diapause related. These proteins appeared soon after hatching and increased in abundance, reaching maximum levels by four days into the 1st instar, and they remained at high levels until three days after the termination of diapause. These two proteins were purified to homogeneity and their NH2-terminal sequences were obtained. Oligonucleotide primers designed on the basis of these NH2-terminal sequences were used in RT-PCR to isolate the cDNA fragments coding for these proteins. These PCR fragments were then used as probes to isolate the cDNAs that contained the complete coding region. The 2.5kb mRNAs coding for these proteins started to appear 24hr after hatching and large quantities of these mRNAs were detected in 1st instar and 2nd instar larvae until the 2nd instar larvae entered diapause. Low levels of these 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 that 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 stages. The mRNAs reappeared 24hr after the 5th instar larvae molted into the 6th instar and increased to reach maximum levels by 60hr after ecdysis. The mRNA levels remained high until 156hr after ecdysis into the 6th instar (36–48hr before pupal ecdysis), after which they disappeared once again. Immunocytochemical analyses showed that CfDAP1 protein 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 the first time before C. fumiferana larvae entered diapause and for a 2nd time before pupation.

Morphological and molecular changes produced byAutographa californicanuclear polyhedrosis virus (AcMNPV) infection in a permissive cell line, IPLB-SF-21AE (SF-21), ofSpodoptera frugiperdaand a nonpermissive cell line, FPMI-CF-203 (CF-203), ofChoristoneura fumiferanaare described. CF-203 cells inoculated withAcMNPV showed a DNA ladder and morphological changes such as plasma membrane granulation, blebbing, and nuclear fragmentation, which are characteristic of apoptosis. Typical virus replication and occlusion body (OB) production were seen in SF-21 cells inoculated withAcMNPV and no apoptosis-like symptoms were observed. mRNA for the apoptosis suppressor gene p35 was detected 9 hr later inAcMNPV-inoculated CF-203 cells than in SF-21 cells. Only a trace amount of mRNA for theAcMNPV-inhibitor of apoptosis homologue (Ac-iap) gene and no mRNAs for the late genes,AcMNPV-polyhedrin (Ac-polh) andAcMNPV-p10 (Ac-p10), were detected inAcMNPV-inoculated CF-203 cells. Inoculation of CF-203 cells withCfMNPV at least 12 hr prior to inoculation withAcMNPV prevented apoptosis-like cell death, and mRNAs forAc-iap, Ac-polh,andAc-p10genes were expressed resulting in successful virus replication and OB production.

The non-steroidal ecdysone agonist, RH-5992, is effective in inducing an incomplete molt in the spruce budworm, Choristoneura fumiferana (Clemens), only when it is fed to the larvae prior to the appearance of the endogenous ecdysteroid peak. When this compound is administered after the ecdysteroid peak, the larvae molt normally into the next stage. However, because of its persistence, the agonist induces an incomplete molt in the subsequent larval stage. The effect does not appear to be due to ecdysone responsive tissues becoming refractory after the ecdysteroid peak. Choristoneura hormone receptor 3 (CHR3), a homologue of Manduca hormone receptor 3 (MHR3), is induced in the epidermis, fat body and midgut of 6th instar larvae treated with RH-5992 during all days of the 6th stadium. Possible reasons for this age specific effect are discussed.