Sardar S. Sohi

Spruce budworm (Choristoneura fumiferana) juvenile hormone esterase: hormonal regulation, developmental expression and cDNA cloning.

We have used the differential display of mRNAs technique to identify Choristoneura fumiferana genes that are induced by juvenile hormone I (JH I). Of the six PCR products identified, one bound to a 2.8-kb mRNA from CF-203 cells whose abundance increased when the cells were grown in the presence of JH I. The same 2.8-kb mRNA decreased to undetectable levels when the CF-203 cells were grown in the presence of 20-hydroxyecdysone (20E). The PCR fragment probe also detected a 2.8-kb mRNA in the C. fumiferana larval tissues. This 2.8-kb mRNA was present on the first day of the first, third, fourth, fifth and sixth larval and pupal stadia, but was conspicuously absent on the first day of the second larval stadium, as well as during the intermolt periods of the first to fifth instar larval stages. In the sixth instar larvae the 2.8-kb mRNA was detected in the fat body, epidermis and midgut during the intermolt period. The PCR fragment was used as a probe to screen a cDNA library. The deduced amino acid sequence of this 2.8-kb cDNA clone showed similarity with the deduced amino acid sequences of Heliothis virescens juvenile hormone esterases (HvJHE). The deduced amino acid sequence of the cDNA clone contained all five functional motifs that are present in most of esterases, proteases and lipases. The cDNA clone was expressed in the baculovirus expression system, producing a protein that showed JHE activity.

Studies on two ecdysone receptor isoforms of the spruce budworm, Choristoneura fumiferana.

A full-length cDNA clone corresponding to the Choristoneura fumiferana ecdysone receptor-A isoform (CfEcR-A) was isolated. The deduced amino acid sequence of CfEcR-A differed from CfEcR-B in the NH2-terminal region of the A/B domain. The CfEcR-A-specific region showed high amino acid identity with EcR-A isoforms of Manduca sexta, Bombyx mori, Drosophila melanogaster and Tenebrio molitor. Isoform-specific probes were used to study the expression of EcR-A and EcR-B mRNAs. Both probes detected 6 kb mRNAs that were present in second-sixth larval instars and in the pupae. Both EcR-A and EcR-B mRNA levels increased during the molting periods. In the sixth instar larvae, the increase in EcR-A and EcR-B mRNA levels were more pronounced in the midgut than in epidermis and fat body. Both EcR-A and EcR-B mRNAs were induced in CF-203 cells (a cell line developed from C. fumiferana midgut) grown in the presence of 4 x 10(-6) M 20E. EcR-B specific mRNAs were induced within 1 h of exposure to 20E, but EcR-A specific mRNAs were induced only after 3 h of exposure to 20E. Induction of mRNAs for both isoforms was unaffected by the presence of a protein synthesis inhibitor, cyclohexamide, in the culture medium. RH-5992, a stable ecdysone agonist, caused a similar induction pattern of EcR-A and EcR-B mRNAs in the midgut, epidermis and fat body of sixth instar larvae. In vitro translated CfEcR-A, CfEcR-B and CfUSP proteins were used to study the DNA binding and ligand binding properties of EcR-A/USP and EcR-B/USP protein complexes. The Kd values indicated that both complexes have similar binding affinities for ecdysone response elements and ponasterone A.

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression

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.

Cloning and developmental expression of Choristoneura hormone receptor 75: A homologue of the Drosophila E75A gene

Cloning and characterization of a cDNA of the spruce budworm, Choristoneura fumiferana, that showed high amino acid similarity with the deduced amino acid sequences of E75 cDNAs cloned from Manduca sexta, Galleria melonella, and Drosophila melanogaster are described. Initially, a cDNA fragment and then a full length cDNA were cloned from C. fumiferana. The longest open reading frame of this cDNA had 690 codons and its deduced amino acid sequence had all five domains typical of a steroid hormone nuclear receptor. The deduced amino acid sequence of this cDNA showed the highest identity with the deduced amino acid sequence of E75A cDNAs cloned from M. sexta, G. melonella, and D. melanogaster, and is therefore named Choristoneura hormone receptor 75A (CHR75A). The CHR75A cDNA probe detected a 2.6 kb mRNA that was abundant at the time of the ecdysteroid peaks during molting in the embryonic, larval and pupal stages. In the sixth instar larvae, CHR75 mRNA was detected in the epidermis, fat body and midgut, and maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CHR75 mRNA was induced in ecdysone treated CF-203 cells and in the midgut, fat body and epidermis of larvae that were fed the non-steroidal ecdysteroid agonist, RH-5992. In vitro transcription and translation of the CHR75A cDNA yielded a 79 kDa protein that bound to the retinoic acid receptor related orphan receptor response element (RORE).

The gene encoding the capsid protein P82 of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus: sequencing, transcription and characterization by immunoblot analysis.

A gene encoding a capsid-associated viral structural protein has been identified and sequenced in the genome of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV). The gene has a 1872 nucleotide open reading frame (ORF) encoding 624 amino acids with a predicted molecular mass of 71.4 kDa. Transcription, which appeared to be initiated from a conserved GTAAG motif of baculovirus late genes, was detected at 12 h, reached a maximum at 48 h and declined at 72 h post-infection (p.i.). Part of the ORF was cloned in frame into a prokaryotic expression vector, pMAL-c2, and the fusion protein was used to generate antibodies in rabbits. It was shown, with the aid of the polyclonal antiserum, that this viral protein was detectable at 24 h p.i. in infected cells. The protein appeared as an 82 kDa band in occlusion-derived virus and as an 82 kDa band and a 72 kDa band in budded virus. Amino acid sequence comparisons revealed that this ORF had high homology with the ORF p87 (77% similarity) of Orgyia pseudotsugata (Op) MNPV and the ORF p80 (60% similarity) of Autographa californica (Ac) MNPV. Immunoblots confirmed that the CfMNPV protein had antigenic similarities to the P87 protein of OpMNPV, but not to the P80 of AcMNPV.

Comparison of the response of midgut epithelial cells and cell lines from lepidopteran larvae to CryIA toxins from Bacillus thuringiensis

The cytotoxic responses of midgut epithelial cells (MEC) from spruce budworm (SBW), gypsy moth (GM) and silkworm (SW) larvae were compared with the cytotoxic response of lepidopteran cell lines (SF-9, SE-1a, and CF-1) to CryIA toxins from Bacillus thuringiensis. The MEC from SBW, SW and GM had binding proteins for CryIA(a,b,c) toxins, whereas the lepidopteran cell lines had binding proteins for CryIA(c). Single MEC exposed to CryIA(a,b,c) toxins in a qualitative lawn assay were equally susceptible to the toxins with a threshold response at about 1ng. The cell lines were not susceptible to CryIA(a,b) toxins in the dose range tested, but had threshold responses for CryIA(c) of 3.4ng for SF-9, 50.2ng for SE-1a and 5.9ng for CF-1. In the quantitative Live/Dead assay, MEC were equally susceptible to CryIA(a,b,c) toxins with a threshold effect at about 1ng and a maximum effect at about 10ng. CF-1 was most sensitive to CryIA(c) with a threshold effect at 0.39ng and a maximal effect at about 1ng. In contrast, a 25-50 times greater dose of CryIA(a) or CryIA(b) was required to elicit a similar response as CryIA(c) for CF-1. SF-9 and SE-1a were most susceptible to CryIA(c) with a threshold effect observed at about 0.5ng and maximal effects at about 2ng. SF-9 cells have a threshold and maximum response to CryIA(a,b) of about 10ng and 20ng, respectively. SE-1a cells have a threshold and maximal response to CryIA(a,b) of 5ng and 10ng, respectively. Intact midgut epithelium exposed to CryIA(a,b,c) toxins had a threshold dose of 2ng for CryIA(b), 10-30ng for CryIA(a) and 2-30ng for CryIA(c). This study has shown that MEC are affected by a broader spectrum of toxins compared to the lepidopteran larvae and insect cell lines.

Molecular analysis of the p48 gene of Choristoneura fumiferana multicapsid nucleopolyhedroviruses CfMNPV and CfDEFNPV

Attempts were made to linearize the DNA of Choristoneura fumiferana (Cf) multicapsid nucleopolyhedrovirus (MNPV), in order to improve the efficiency of generation of recombinant viruses after transfection. A unique site for the restriction enzyme Sse83871 was found in ORF p48. The requirement for this ORF during virus replication was investigated by molecular analyses including sequencing, transcriptional analysis and inactivation by insertion of marker genes. Sequence analysis showed that ORF p48 consists of 1233 nucleotides encoding a potential protein of 47.88 kDa. The proteins encoded by ORF p48 from CfMNPV and Orgyia pseudotsugata MNPV contain 411 amino acids while that from CfDEFNPV (a virus that is defective for infection by the per os route) is slightly smaller, at 408 amino acids. Transcriptional and primer extension analyses showed that the mRNA is initiated from a typical baculovirus late gene ATAAG motif. The mRNA was detected at 24 h post-infection (p.i.), reached maximum levels at 48 h p.i. and declined by 96 h p.i., which confirmed the late property of the gene. Inactivation of the gene was attempted by inserting a cassette containing either the gene encoding beta-galactosidase or that encoding green fluorescent protein. Blue or fluorescent green plaques of infected cells were observed after transfection. Attempts to generate a plaque-purified virus were not successful. Restriction enzyme analysis showed that the marker genes were inserted randomly at positions other than the p48 locus. This indicated that the gene may be needed for virus replication. The gene is relatively well conserved among baculoviruses but its function remains unclear.

Biochemical and biological mode of action of ecdysone agonists on the spruce budworm

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.

Molecular modifications of Baculoviruses for the control of forest insect pests

Publisher Summary This chapter describes some of the more recent research in the areas of gene cloning and recombinant viruses, genome sequencing of spruce budworm viruses, and development of cell lines. The insect communities in the forests often contain a rich complex of natural enemies of the insects, such as parasites, predators, bacteria, fungi, and viruses. These natural enemies can be used as control agents and are attractive alternatives to chemical pesticides, because they are not only naturally occurring but also in many instances host specific with little or no adverse effects on the environment. Many insect viruses are attractive as environmentally benign control agents, because their host range is generally quite narrow, infecting only a few species or genera. This narrow host range permits the use of insect viruses in integrated pest management of forests. Although the viruses eventually kill the insects, because they take a long time to act, the pest insect often causes extensive defoliation before they die. The forest ecosystem is an environmentally sensitive region and is, especially suited for the use of environmentally safe baculoviruses as control agents. Recombinant DNA technology has provided a powerful tool to genetically manipulate the viruses and make it possible to enhance the control potential of baculoviruses.

Molecular cloning of a female-specific cDNA with unique repeat sequences from the fat body of the adult locust, Locusta migratoria.

A cDNA clone encoding a 25-kDa protein (25K) was isolated from a cDNA library made from RNA isolated from the adult fat body and ovaries of the locust, Locusta migratoria. The longest open reading frame of this cDNA clone encodes a 225-amino acid polypeptide, the N-terminal end of which was similar to the 21-kDa and 19-kDa juvenile hormone induced proteins identified in the locust hemolymph, but the C-terminal end was different. The C-terminal end of the 25K cDNA contained seven unique repeat elements of 10 amino acids each, most of which are polar residues. Expression of the 25K mRNA was tissue-, development- and sex-specific. A 1.2-kb mRNA was detected using the 25K cDNA as a probe only in the fat body of adult females. The mRNA started to appear at day 4 after the insect molted to the adult and rapidly increased by day 6. The mRNA was absent in the ovarian follicle cells and fat body of adult males. In vitro transcription and translation of the 25K cDNA produced a protein that migrated around 32 kDa on sodium dodecyl sulfate polyacrylamide gels. The 25K cDNA was expressed in a baculovirus expression system and the protein produced also migrated around 32 kDa.