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Rice (Oryza sativa var. Nipponbare) was transformed with an artificial avidin gene. The features of this construct are as follows: (1) a signal peptide sequence derived from barley alpha amylase was added at the N-terminal region, (2) codon usage of the gene was optimized for rice, and (3) the gene was driven by rice glutelin GluB-1, an endosperm-specific promoter. Avidin was produced in the grain of the transgenic rice but not in the leaves. The concentration of avidin in the kernels was about 1,800 ppm. All larvae of the confused flour beetle (Tribolium confusum) and Angoumois grain moth (Sitotroga cerealella) died when fed transgenic avidin rice powder or kernels, respectively, whereas most of the test insects developed into adults when they were fed a nontransgenic rice control diet. Avidin extracted from the transgenic rice kernel lost most biotin-binding activity after 5 min heating at 95 degrees C.

In insects, storage proteins or hexamerins accumulate apparently to serve as sources of amino acids during metamorphosis and reproduction. Two storage protein-like cDNAs obtained from a cDNA library prepared from fourth instar larvae of the Indianmeal moth (Plodia interpunctella) were cloned and sequenced. The first clone, PinSP1, contained 2431 nucleotides with a 2295 nucleotide open reading frame (ORF) encoding a protein with 765 amino acid residues. The second cDNA, PinSP2, consisted of 2336 nucleotides with a 2250-nucleotide ORF encoding a protein with 750 amino acid residues. PinSP1 and PinSP2 shared 59% nucleotide sequence identity and 44% deduced amino acid sequence identity. A 17-amino acid signal peptide and a molecular mass of 90.4 kDa were predicted for the PinSP1 protein, whereas a 15-amino acid signal peptide and a mass of 88 kDa were predicted for PinSP2. Both proteins contained conserved insect larval storage protein signature sequence patterns and were 60-70% identical to other lepidopteran larval storage proteins. Expression of mRNA for both larval storage proteins was determined using the quantitative reverse transcription polymerase chain reaction method. Only very low levels were present in the second instar, but both mRNAs dramatically increased during the third instar, peaked in the fourth instar, decreased dramatically late in the same instar and pupal stages, and were undetectable during the adult stage. Males and females exhibited similar mRNA expression levels for both storage proteins during the pupal and adult stages. The results support the hypothesis that P. interpunctella, a species that does not feed after the larval stage, accumulates these two storage proteins as reserves during larval development for subsequent use in the pupal and adult stages.

Two cDNA fragments encoding full-length trypsinogen-like proteins were cloned from larvae of two strains (RC688s and HD198r) of the Indianmeal moth, Plodia interpunctella (Hübner), which differed in their sensitivity to Bacillus thuringiensis protoxins. One cDNA fragment contained 874 nucleotides, including a 780-nucleotide open reading frame that encoded a trypsinogen-like protein (PiT2b). Another cDNA fragment amplified from both P. interpunctella strains contained 864 nucleotides including a 780 bp open reading frame encoding a second trypsinogen-like protein (PiT2c). The cDNA sequence of PiT2b shared 89% sequence identity with PiT2a, a trypsinogen-like protein cloned previously from this species. The cDNA sequences of PiT2a and PiT2c shared 83% identity. The cDNA sequence identity between PiT2b and PiT2c was 80%. The cDNA for PiT2b from strain RC688s was different at six nucleotide positions from that of PiT2b from strain HD198r. Five nucleotide replacements occurred in the open reading frame leading to amino acid changes at all five positions. There were five nucleotide differences in the cDNAs for PiT2c trypsinogen-like proteins from the two strains. Two nucleotide substitutions in the open reading frame resulted in replacements of two amino acid residues in the deduced protein sequences. Amino acid sequences for PiT2a and PiT2b shared 84% identity, but only 50% identity was observed between PiT2c and the other two trypsinogen-like proteins. The deduced amino acid sequences for PiT2b and PiT2c included both signal and zymogen activation peptides and amino acid sequence motifs which are conserved in seven homologous trypsinogen-like proteins from other insects. Typical features of the putative trypsinogen-like proteins from P. interpunctella included the serine proteinase active site triad (His(81), Asp(133), and Ser(233)), three pairs of cysteine residues for disulfide bridges, and three residues, Asp(227), Gly(250), and Gly(260), that help to confer trypsin-like specificity to the enzymes. Quantitative RT-PCR analyses showed that, in fourth instar larvae, RC688s had 1.6-fold higher PiT2a trypsinogen-like mRNA than did HD198r. Expression of PiT2b mRNA was 3.4-fold higher in HD198r than in RC688s. Expression of PiT2c mRNA was 2.8-fold higher in RC688s than in HD198r. Mean accumulation levels of mRNAs for all three trypsinogen-like proteins were slightly higher in RC688s than in HD198r based on total RNA, and 1.3-fold higher in RC688s than in HD198r based on wet weight of larval body tissues.

Aminopeptidase N has been reported to be a Bacillus thuringiensis (Bt) Cry1A toxin-binding protein in several lepidopteran insects. cDNAs of aminopeptidase-like proteins from both Bt-susceptible RC688s and Bt-resistant HD198r strains of the Indianmeal moth, Plodia interpunctella, were cloned and sequenced. They contain 3345 and 3358 nucleotides, respectively, and each has a 3048 bp open reading frame that encodes 1016 amino acids. Putative protein sequences include 10 potential glycosylation sites and a zinc metal binding site motif of HEXXH, which is typical of the active site of zinc-dependent metallopeptidases. Sequence analysis indicated that the deduced protein sequences are most similar to an aminopeptidase from Heliothis virescens with 62% sequence identity and highly similar to three other lepidopteran aminopeptidases from Plutella xylostella, Manduca sexta, Bombyx mori with sequence identities of 51-52%. Four nucleotide differences were observed in the open reading frames that translated into two amino acid differences in the putative protein sequences. Polymerase chain reaction (PCR) confirmed an aminopeptidase gene coding difference between RC688s and HD198r strains of P. interpunctella in the PCR amplification of a specific allele (PASA) using preferential primers designed from a single base substitution. The gene mutation for Asp185-->Glu185 was also confirmed in two additional Bt-resistant P. interpunctella strains. This mutation is located within a region homologous to the conserved Cry1Aa toxin binding regions from Bombyx mori and Plutella xylostella. The aminopeptidase-like mRNA expression levels in the Bt-resistant strain were slightly higher than those in the Bt-susceptible strain. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF034483 for susceptible strain RC688s and AF034484 for resistant strain HD198r).

Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two trypsin-like proteinases, which is found in both the Bt-susceptible and -resistant strains of the Indianmeal moth. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF064525 for the RC688 strain and AF064526 for HD198).

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control.

Two Bacillus thuringiensis (Bt)-resistant strains of the Indianmeal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins. The absence of this enzyme is genetically linked to larval survival on Bt-treated diets. When considered with previous data supporting the existence of receptor-mediated insect resistance to Bt, these results provide evidence that insect adaptation to these toxins occurs through multiple physiological mechanisms, which complicate efforts to prevent or manage resistance to Bt toxins in insect control programs.

The ability of proteinases in gut extracts of the Indianmeal moth, Plodia interpunctella, to hydrolyze Bacillus thuringiensis (Bt) protoxin, casein, and rho-nitroanilide substrates was investigated. A polyclonal antiserum to protoxin CryIA(c) was used in Western blots to demonstrate slower protoxin processing by gut enzymes from Bt subspecies entomocidus-resistant larvae than enzymes from susceptible or kurstaki-resistant strains. Enzymes from all three strains hydrolyzed N-alpha-benzoyl-L-arginine rho-nitroanilide, N-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, and N-succinyl-ala-ala-pro-leucine rho-nitroanilide. Zymograms and activity blots were used to estimate the apparent molecular masses, number of enzymes, and relative activities in each strain. Several serine proteinase inhibitors reduced gut enzyme activities, with two soybean trypsin inhibitors, two potato inhibitors, and chymostatin the most effective in preventing protoxin hydrolysis.

Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of the Indianmeal moth, Plodia interpunctella (Hubner), resistant to B. thuringiensis subspecies entomocidus (HD-198) was slower than that by midgut proteinases from the susceptible parent strain or a strain resistant to B. thuringiensis subspecies kurstaki (HD-1, Dipel). Midgut extracts from entomocidus-resistant insects exhibited five-fold lower activity toward the synthetic substrate alpha-N-benzoyl-DL-arginine rho-nitroanilide than extracts from susceptible or kurstaki-resistant insects. Midgut enzymes from susceptible or kurstaki-resistant insects converted the 133 kDa CryIA(c) protoxin to 61-63 kDa proteins, while incubations with entomocidus-resistant enzymes resulted in predominantly products of intermediate size, even with increased amounts of midgut extract. The 61-63 kDa proteins were only produced by entomocidus-resistant midgut extracts after long term incubations with the protoxin. The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B. thuringiensis.

Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation. The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a protein of 554 amino acids with a molecular weight of 62 kDa. Several regions of the amino acid sequence in this protein are similar to sequences in yeast, cucumber and bacterial endo-beta-N-acetylglucosaminidases. Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid. Southern blot analysis indicated that one or two genes related to the cDNA clone are encoding chitinases in the Manduca genome. The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest on c. days 5-7 during the fifth larval instar. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens caused about a 10-fold increase in mRNA levels in both epidermis and gut, and topical application of the juvenile hormone mimic, fenoxycarb, suppressed the ecdysteroid-induced accumulation of chitinase RNA.

Dopamine and its conjugates are widely distributed among biological species and are utilized for a variety of functions. Insects metabolize dopamine for cuticle melanization and sclerotization. Among the most abundant dopamines found in the larval and pupal development stages of Manduca sexta, the tobacco hornworm, are N-acetyldopamine and N-beta-alanyldopamine. In addition, glycosylated derivatives of these dopamines are found mainly in the hemolymph just prior to cuticulogenesis. The 1H and 13C NMR resonances of dopamine, its 3-O-methyl, 4-O-methyl, N-acetyl, and N-beta-alanyl derivatives, norepinephrine, 4-O-(beta-D-glucuronopyranosyl)dopamine, and the glycosylated products of N-beta-alanyldopamine and dopamine have largely been assigned. Assignments were based on one- and two-dimensional NMR analyses of the above compounds combined with that of specifically enriched [C7-13C]dopamine. 1H NMR showed that the major glycosylated natural product isolated from M. sexta pupal hemolymph was a 3-O-glycosyl derivative of N-beta-alanyldopamine. 13C NMR confirmed that the carbohydrate was D-glucose probably in a beta-linkage. 1H NMR of the aromatic ring protons provided the most definitive method to distinguish 3-O- from 4-O-derivatives of dopamine. In addition, the 3-O-glucosyl conjugate of N-beta-alanyldopamine had unique chemical shifts and coupling patterns compared to those of 4-O-(beta-D-glucuronosyl)- and 3-O-(beta-D-glucopyranosyl)dopamine.

Cross-polarization, magic-angle spinning 13C NMR spectra of skeletal components of individual colonies of the New Zealand black coral, Antipathes fiordensis, have a marked similarity to spectra of the sclerotized exoskeleton of the adult tobacco hornworm, Manduca sexta. NMR analysis estimates the organic content of the load-bearing skeletal base of A. fiordensis as 70% protein, 10% chitin, 15% diphenol, and 5% lipid by weight, and that of M. Sexta moth cuticle as 60% protein, 20% chitin, 15% diphenol, and 5% lipid. The younger pinnules or tips of A. fiordensis are less than 3% diphenol by weight. The only diphenols extracted from coral skeleton by hydrochloric acid are 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) and 3,4-dihydroxybenzaldehyde (DOBAL), while the predominant diphenols in acid extracts of insect cuticles are N-acyldopamines. More DOPA is found in the base than in the tips of A. fiordensis and it appears to be a peptidyl component of coral skeletal protein. The oxidation of DOPA and DOBAL to quinones may provide mechanical stabilization of the coral skeleton by cross-linking of structural proteins to other proteins or to chitin.

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.

A method for determining the toxicity of Bacillus thuringiensis subsp. kurstaki parasporal crystal to the tabocco hornworm, Manduca sexta, is described. The use of both mortality and weight loss data have provided a highly sensitive and reproducible bioassay that can be used to compare relative toxicities of crystals from other subspecies as well as toxic components contained therein.

In the brain of adult specimens of the tobacco hornworm moth, Manduca sexta (L), cells immunoreactive for several kinds of neuropeptides were localized by means of the PAP procedure, by use of antisera raised against mammalian hormones or hormonal peptides. In contrast, no such neurosecretory cells were found in the corpora cardiaca and corpora allata (CC/CA); in the CC/CA, however, immunoreactive nerve fibres were observed, reaching these organs from the brain. The neurosecretory cells found in the brain were immunoreactive with at least one of the following mammalian antisera, namely those raised against the insulin B-chain, somatostatin, glucagon C-terminal, glucagon N-terminal, pancreatic polypeptide (PP), secretin, vasoactive intestinal polypeptide (VIP), glucose-dependent insulinotropic peptide (GIP), gastrin C-terminus, enkephalin, alpha- and beta-endorphin, Substance P, and calcitonin. No cells were immunoreactive with antisera specific for detecting neurons containing the insulin A-chain, nerve growth factor, epidermal growth factor, insulin connecting peptide (C-peptide), polypeptide YY (PYY), gastrin mid-portion (sequence 6-13), cholecystokinin (CCK) mid-portion (sequences 9-20 and 9-25), neurotensin C-terminus, bombesin, motilin, ACTH, or serotonin. All the neuropeptide-immunoreactive cells observed emitted nerve fibers passing through the brain to the CC and in some cases also to the CA. In CC these immunoreactive nerve fibers tended to accumulate near the aorta. It was speculated that neuropeptides are released into the circulating haemolymph and act as neurohormones.

The frontal ganglion of the adult forms of the tobacco hornworm, Manduca sexta, was investigated immunocytochemically for the occurrence of the gastro-entero-pancreatic (GEP) neurohormonal peptides, namely insulin, nerve growth factor, epidermal growth factor, insulin C-peptide, somatostatin, glucagon, glicentin, pancreatic polypeptide (PP), polypeptide YY (PYY), secretin, vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP), gastrin, cholecystokinin (CCK), enkephalin, alpha- and beta-endorphins, substance P, neurotensin, bombesin, motilin, ACTH, serotonin, and calcitonin. Among all the antisera tested, positive immunostaining was obtained with anti-insulin B-chain serum only. The insulin B-chain immunoreactivity was localized in 4-6 large (30-40 microns) neurons, in the neuropile, and in the recurrent nerve. It is speculated that the insulin-like immunoreactive material may be transported to the neurohaemal organ (corpora cardiaca) through the nervi cardiaco-somatogastrici.