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Juvenile hormone esterases (JHEs) from six insects belonging to three orders (Lepidoptera, Coleoptera, and Diptera) were compared in terms of their deduced amino acid sequence and biochemical properties. The four lepidopteran JHEs showed from 52% to 59% identity to each other and about 30% identity to the coleopteran and dipteran JHEs. The JHE of Manduca sexta was remarkably resistant to the addition of organic co-solvents and detergent; in some cases, it demonstrated significant activation of activity. Trifluoromethylketone (TFK) inhibitors with chain lengths of 8, 10 or 12 carbons were highly effective against both lepidopteran and coleopteran JHEs. The coleopteran JHE remained sensitive to TFK inhibitors with a chain length of 6 carbons, whereas the lepidopteran JHEs were significantly less sensitive. When the chain was altered to a phenethyl moiety, the coleopteran JHE remained moderately sensitive, while the lepidopteran JHEs were much less sensitive. The lepidopteran and coleopteran JHEs did not show dramatic differences in specificity to alpha-naphthyl and rho-nitrophenyl substrates. However, as the chain length of the alpha-naphthyl substrates increased from propionate to caprylate, there was a trend towards reduced activity. The JHE of M. sexta was crystallized and the properties of the crystal suggest a high-resolution structure will follow.

Helicoverpa armigera (Hübner) is the major insect pest of cotton in Africa, Turkey, Asia, India, Indonesia and Australia. Populations recently developed resistance to pyrethroids in West Africa via the overproduction of cytochrome P450 (oxidases) increasing pyrethroid metabolism. One way to overcome pyrethroid resistance is to use compounds that show negative cross-resistance to pyrethroids. Triazophos is one of these compounds: it is slightly more toxic against pyrethroid resistant larvae of H. armigera than against susceptible ones. Overproduced oxidases transform the non active triazophos into its active form, triazophos-oxon, since this form was significantly more often found in larvae from pyrethroid resistant strain (23%) than in susceptible strain (15%). This suggests that oxidases, which provide resistance by degradation of pyrethroids in the resistant individuals, also activate triazophos in its toxic oxon form resulting in a negative cross-resistance.

The insect cell-baculovirus expression vector system, widely used for glycoprotein production, is not ideal for pharmaceutical glycoprotein production due to the characteristics of the N-glycans in the expressed products. Insect cells lack several enzymes required for mammalian-type N-glycan synthesis and contain a specific N-acetylglucosaminidase that stunts the growth of chains and a core alpha-1,3-fucosyltransferase that yields potentially allergenic glycoforms. Current knowledge on N-glycan processing in lepidopteran insect cells is summarized, and strategies to develop better glycoprotein expression systems suitable for pharmaceutical glycoprotein production are discussed.

Based on the results from the use of selective inhibitors and activators, active protein kinase A, protein tyrosine kinase, and protein kinase C (PKC) isoforms decreased the adhesion of larval Galleria mellonella hemocytes to glass slides. The protein kinase A inhibitor at all concentrations increased granular cell adhesion only whereas protein tyrosine kinase elevated both granular and plasmatocyte attachment at the lowest concentration. Active, Ca(2+)- and lipid-dependent PKC isoforms limited plasmatocyte and granular cell adhesion whereas PKC that was inhibited by selected compounds (with differed modes of PKC inhibition) enhanced hemocyte attachment. The granular cells were more sensitive to the PKC inhibitors than were plasmatocytes. Phospholipase C and its diacylglyceride product were necessary to reduce hemocyte adhesion and maintain PKC activity. Extracellular Ca(2+), possibly transported through L-channels, was required for plasmatocyte attachment. In contrast, lowering the levels of cytosolic Ca(2+) was associated with decreased PKC activity and was required for hemocyte adhesion., (Copyright 2003 Wiley-Liss, Inc.)

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.

Although there is a significant knowledge about mammalian metallocarboxypeptidases, the data available on this family of enzymes is very poor for invertebrate forms. Here we present the biochemical characterization of a metallocarboxypeptidase from the insect Helicoverpa armigera (Lepidoptera: Noctuidae), a devastating pest spread in subtropical regions of Europe, Asia, Africa and Oceania. The zymogen of this carboxypeptidase (PCPAHa) has been expressed at high levels in a Pichia pastoris system and shown to display the characteristics of the enzyme purified from the insect midgut. The in vitro activation process of the proenzyme differs significantly from the mammalian ones. The lysine-specific endoprotease LysC activates PCPAHa four times more efficiently than trypsin, the general activating enzyme for all previously studied metalloprocarboxypeptidases. LysC and trypsin independently use two different activation targets and the presence of sugars in the vicinity of the LysC activation point affects the activation process, indicating a possible modulation of the activation mechanism. During the activation with LysC the prodomain is degraded, while the carboxypeptidase moiety remains intact except for a C-terminal octapeptide that is rapidly released. Interestingly, the sequence at the cleavage point for the release of the octapeptide is also found at the boundary between the activation peptide and the enzyme moieties. The active enzyme (CPAHa) is shown to have a very broad substrate specificity, as it appears to be the only known metallocarboxypeptidase capable of efficiently hydrolysing basic and aliphatic residues and, to a much lower extent, acidic residues. Two carboxypeptidase inhibitors, from potato and leech, were tested against CPAHa. The former, of vegetal origin, is the most efficient metallocarboxypeptidase inhibitor described so far, with a Ki in the pm range.

Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.

A novel trypsin inhibitor was purified from the seeds of Peltophorum dubium (Spreng.). SDS-PAGE under reducing conditions showed that the inhibitor consisted of a single polypeptide chain (ca. 20 kDa). The dissociation constants of 4 x 10(-10) and 1.6 x 10(-10) M were obtained with bovine and porcine trypsin, respectively. This constant was lower (2.6 x 10(-7) M) for chymotrypsin. The inhibitory activity was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the P. dubium inhibitor showed a high degree of homology with other Kunitz-type inhibitors. When fed to the insect Anagasta kuehniella, in an artificial diet (inhibitor concentration 1.6%), the inhibitor produced approximately 56% and delayed the development of this lepidopteran. The concentration of inhibitor in the diet necessary to cause a 50% reduction in the weight (ED50) of fourth instar larvae was approximately 1%. The action of the P. dubium trypsin inhibitor (PDTI) on A. kuehniella may involve inhibition of the trypsin-like activity present in the larval midgut, resistance of the inhibitor to digestion by midgut enzymes and bovine trypsin, and association of the inhibitor with a chitin column and chitinous structures in the peritrophic membrane and/or midgut of the insect.

Secreted human placental alkaline phosphatase (SEAP) was produced in a nonengineered Trichoplusia ni insect cell line, Tn-4s, using a recombinant Autographa californica baculovirus expression vector. The effect of culture conditions on SEAP specific yield and glycosylation was studied. When cultured in the high aspect ratio vessel (HARV) or in tissue culture flasks (T-flasks), baculovirus-infected Tn-4s cells produced high levels of SEAP (13 and 23 U/10(6) cells, respectively; 4 days postinfection), but in those conditions SEAP possessed only high mannose, paucimannosidic, and hybrid structures. In spinner flasks, lower SEAP yields were obtained (<4 U/10(6) cells, 3 days postinfection), but in such cultures, sialylation of SEAP could be achieved. Several spinner-flask culture conditions were tested and resulted in different SEAP specific yields and levels of sialylation. The highest level of sialylation (9%) was obtained in the culture with the lowest agitation rate and lowest yield (1.2 U/10(6) cells), suggesting a limiting capacity of the Tn-4s cells to process glycoproteins to sialylation. High specific yield, low passage number Tn5B1-4 cells did not produce SEAP with complex glycosylation when cultured in a low agitation rate spinner-flask. On the basis of these results, we propose that the Golgi apparatus has a limited capacity for processing proteins to complex glycosylation and sialylation and that this capacity is easily overwhelmed by high levels of foreign protein productivity. Selected media additives such as Pluronic F-68, dextran sulfate (MW 12 500) and a lipids premix did not allow improvement of the specific yield of sialylated SEAP when supplemented to spinner-flask cultures.

Several investigators have independently identified membrane-associated aminopeptidases in the midgut of insect larvae as the initial interacting ligand to the insecticidal crystal proteins of Bacillus thuringiensis. Though several isoenzymes of aminopeptidases have been identified from the midgut of an insect and their corresponding cDNA cloned, only one of the isoform has been expressed heterologously and studied for its binding to Cry toxins. Here we report the cloning and expression of two aminopeptidases N from Helicoverpa armigera (American cotton bollworm) (HaAPNs). The full-length cDNA of H. armigera APN1 (haapn1) is 3205 bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116 bp in size and corresponds to a 1012-amino-acid protein. Structurally these proteins show sequence similarity to other insect aminopeptidases and possess characteristic aminopeptidase motifs. Both the genes have been expressed in Trichoplusia ni (cabbage looper) cells using a baculovirus expression vector. The expressed aminopeptidases are membrane-associated, catalytically active and glycosylated. Ligand-blot analysis of both these aminopeptidases with bioactive Cry1Aa, Cry1Ab and Cry1Ac proteins displayed differential interaction. All the three toxins bound to HaAPN1, whereas only Cry1Ac interacted with HaAPN2. This is the first report demonstrating differential Cry-toxin-binding abilities of two different aminopeptidases from a susceptible insect.

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.

Three beta-glycosidases, named betaGly1, betaGly2 and betaGly3, were isolated from midgut tissues of the sugar cane borer, Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae). The three enzymes have similar Mr (58,000; 61,000; 61,000), pI (7.5, 7.4, and 7.4) and optimum pH (6.7, 6.3, and 7.2) and were resolved by hydrophobic chromatography. The beta-glycosidases prefer beta-glucosides to beta-galactosides, have four subsites for glucose binding and hydrolyse glucose-glucose beta-1,3 linkages better than beta-1, 4- or beta-1,6 linkages. betaGly1 and 2 were completely purified, whereas betaGly3 was isolated with a contaminant peptide that has no activity upon beta-glycosides. By using competing substrates, it was shown that betaGly 1 and 3 have one active site, whereas betaGly2 has two, one hydrolyzing natural and the other synthetic substrates. betaGly2 is the only D. saccharalis beta-glycosidase that can efficiently hydrolyse prunasin, the glycoside remaining after glucose removal from the plant glycoside amygdalin and that liberates the cyanogenic mandelonitrile. As shown elsewhere, betaGly2 activity is reduced when D. saccharalis is reared in amygdalin containing diets. From the results, we propose that the physiological role of betaGly 1 and 3 is the digestion of oligo- and disaccharides derived from hemicelluloses and of betaGly2 is glycolipid hydrolysis. Free energy relationships showed that D. saccharalis betaGly3 and Tenebrio molitor (Coleoptera) betaGly1 have active sites that bind similarly the transition states formed with different substrates. The same is also true for the active sites of D. saccharalis betaGly1 and T. molitor betaGly2. This suggests that active sites of similar enzymes are probably homologous, displaying nearly identical bonds between active site amino acids and substrate moieties.

Populations of Choristoneura rosaceana (Harris) from orchards in Ontario were shown to be resistant to azinphos-methyl and to other types of organophosphorus insecticides. Resistance extended to methyl carbamates and to methomyl. The laboratory population used for these assays and selected with azinphosmethyl was also resistant to the pyrethroid, cypermethrin. Resistance was associated with increased esterase activity and was reduced by the addition of the synergist DEF. IEF studies of esterases also indicated increased activity in resistant populations, but did not identify any unique esterases associated with the resistance. Resistance was highly correlated (r = 0.78) with elevated esterases but not with increased glutathione-S transferase activity (r = 0.13). Other mechanisms did not appear to be related.

Chitinase (CfChitinase) cDNA from the spruce budworm, Choristoneura fumiferana, was cloned using reverse transcription PCR and cDNA library screening. The CfChitinase cDNA was determined to be 2856 nucleotides long with the longest open reading frame made up of 1671 nucleotides that encoded a protein that was 557 amino acid long with a predicted molecular mass of 62 kDa. The deduced amino acid sequence showed 76-79% identity with other lepidopteran chitinases. Northern blots revealed that transcripts of CfChitinase appeared prior to each molt and peaked on the day of ecdysis from the second instar to the pupal stage but disappeared immediately after the molt. No transcripts could be detected in the early first instar prior to the spinning of the hibernaculum or in the diapausing second instars or during the intermolt periods of the other instars. Western blot analysis revealed that the protein appeared 12 h prior to ecdysis and disappeared 12 h after ecdysis from the sixth instar to pupal stage. The 20-hydroxyecdysone analog, tebufenozide (RH5992), induced expression of CfChitinase in the early stage of the sixth instar and caused a precocious and incomplete molt into an extra larval stage. During the sixth instar to the pupal molt, transcripts could be detected only in the epidermis and fat bodies, but not in the midgut. Western blots showed that the protein was present in the epidermis and midgut, but not in the fat bodies. The recombinant protein expressed in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) showed high levels of chitinolytic activity with an optimal pH range 6-9. Glycosylation appeared to be necessary for the chitinolytic activity and secretion of the recombinant protein.

Lepidopteran insects use sex pheromones derived from fatty acids in their species-specific mate recognition system. Desaturases play a particularly prominent role in the generation of structural diversity in lepidopteran pheromone biosynthesis as a result of the diverse enzymatic properties they have evolved. These enzymes are homologous to the integral membrane desaturases, which play a primary role in cold adaptation in eukaryotic cells. In this investigation, we screened for desaturase-encoding sequences in pheromone glands of adult females of eight lepidopteran species. We found, on average, six unique desaturase-encoding sequences in moth pheromone glands, the same number as is found in the genome database of the fly, Drosophila melanogaster, vs. only one to three in other characterized eukaryotic genomes. The latter observation suggests the expansion of this gene family in insects before the divergence of lepidopteran and dipteran lineages. We present the inferred homology relationships among these sequences, analyze nonsynonymous and synonymous substitution rates for evidence of positive selection, identify sequence and structural correlates of three lineages containing characterized enzymatically distinct desaturases, and discuss the evolution of this sequence family in insects.

Jasmonate and salicylate are plant-produced signals that activate plant defence genes after herbivory or pathogen attack. Amplification of these signals, evoked by either enemy attack or experimental manipulation, leads to an increase in the synthesis of toxic compounds (allelochemicals) and defence proteins in the plants. Although the jasmonate and salicylate signal cascades activate different sets of plant defence genes, or even act antagonistically, there is substantial communication between the pathways. Jasmonate and salicylate also contribute to protecting plants against herbivores by causing plants that experience insect damage to increase their production of volatile molecules that attract natural enemies of herbivorous insects. In response to plant defences, herbivores increase their production of enzymes that detoxify allelochemicals, including cytochrome P450s (refs 15, 16). But herbivores are potentially vulnerable to toxic allelochemicals in the duration between ingesting toxins and induction of detoxification systems. Here we show that the corn earworm Helicoverpa zea uses jasmonate and salicylate to activate four of its cytochrome P450 genes that are associated with detoxification either before or concomitantly with the biosynthesis of allelochemicals. This ability to 'eavesdrop' on plant defence signals protects H. zea against toxins produced by host plants.

The insect neuropeptides FMRF amide, leucomyosupressin (LMS) and neuropeptide analogues leucosulfakinins (FLSK and LSK II Ser (SO(3)H)), perisulfakinin (PSK), proleucosulfakinin (PLSK), 14A[phi1]WP-I, 542phi1, and 378A[5b]WP-I were assayed for their effects on the release of amylase and protease from the midgut tissue of larvae of Opisina arenosella. In the bioassay, empty midgut tubes ligated at both ends using hair were incubated with insect saline containing neuropeptides/analogues in a bioassay apparatus at 37 degrees C for 30 min. After incubation the contents of the midgut preparations were analyzed for amylase and protease activity. In control experiments, the midgut preparations were incubated in insect saline without neuropeptides. The results of the study reveal that for stimulating amylase release from midgut tissue, the peptides require an FXRF amide (X may be methionine or leucine) sequence at the C-terminal. The presence of HMRF amide at C-terminal of peptides may inhibit the release of amylase. Meanwhile, peptides with both FMRF and HMRF amide sequence at the C-terminal are found to be effective in stimulating protease release. The tetrapeptide segment at the C-terminal probably represent the active core of the neuropeptide.

Lysozymes were purified from the hemolymph of three immunized Lepidopteran larvae, Galleria mellonella, Bombyx mori, Agrius convolvuli to compare their physico-chemical properties and antibacterial activities with those of chicken lysozyme. Four lysozymes including the one from chicken had similar molecular masses and chromatographic behavior on reverse phase-high pressure liquid chromatography. Western blotting analysis using an antibody raised against G. mellonella revealed that lysozyme cross-reacted with two other insect lysozymes but not with commercial chicken lysozyme. Antibacterial activities of lysozymes were measured in two types of tests: radial diffusion assay and colony count assay. Our antibacterial tests revealed that all lysozymes have strong activities against Gram-positive bacteria and three insect lysozymes still retain a little potency against Gram-negative bacteria, while chicken lysozyme has no activity against Gram-negative bacteria. Taken together, we conclude three Lepidopteran lysozymes have a common distinct structure and have an antibacterial activity, which is absent in chicken lysozyme, against Gram-negative bacteria.

A digestive protease from Spilosoma obliqua (Lepidoptera: Arctiidae) fifth instar larval guts was purified and characterized. The protease was purified using ammonium sulfate fractionation, ion-exchange chromatography, and hemoglobin-sepharose affinity chromatography. The purification procedure resulted in a 37-fold increase in the specific activity of the protease. Protease thus obtained was found to be electrophoretically pure under native and denaturing conditions. The purified protease had a molecular mass of 90 kDa as determined by gel filtration, and a pH optimum of 11.0. The purified protease optimally hydrolyzed casein at 50 degrees C. A Km of 2 x10(-6) M was obtained using BApNA as a substrate for the purified alkaline protease. The ability of S. obliqua protease and bovine trypsin to hydrolyze various synthetic substrates (BApNA, BAEE, and BAME), and the inhibition patterns of S. obliqua and bovine trypsin with "classical" trypsin inhibitors are also reported., (Copyright 2002 Wiley-Liss, Inc.)

Four cytochrome P450 genes, CYP6B8, CYP6B9, CYP6B27 and CYP6B28, exist in the Helicoverpa zea genome as two pairs of paralogs that evolved from gene duplication and 5'-polar gene conversion events. RT-PCR gel blot analyses have shown that all of these genes are expressed constitutively in midguts of all larval instars, suggesting that they have primary roles in the detoxification of plant allelochemicals. Among these, CYP6B9 is expressed only in midgut tissue whereas its paralog, CYP6B27, is expressed primarily in midgut and secondarily in fat body and ovary. CYP6B28 is expressed in midgut, fat body and, to lesser extents in ovary and integument whereas its paralog, CYP6B8, is expressed in midgut and to some extent in fat body. Comparison of the expression levels induced by eight plant allelochemicals, one drug (phenobarbital), and an insecticide (alpha-cypermethrin) indicates that, for the most part, the four P450s respond individually to these inducers, with all four induced strongly by chlorogenic acid, a shikimate pathway intermediate and a lignin biosynthesis intermediate present in a wide variety of plants, and indole-3-carbinol, a glucobrassicin breakdown product present in the Brassicaceae. The multiple levels at which these P450 genes are apparently diverging (e.g. transcriptional responses, protein sequences) support the suggestion that gene conversion events facilitate gene evolution by allowing duplicated copies greater time to acquire selectable differences in both coding and promoter sequences.

In vivo and in vitro metabolism of [14C]fipronil was examined in a susceptible European corn borer (Ostrinia nubilalis, Hübner) laboratory strain. [14C]Fipronil penetrated the larval integument slowly, with 71.5% of the applied radioactivity recovered from surface rinses 24 h after topical application. Despite this slow penetration, radioactivity was detected in both the excrement and internal organo-soluble fractions. Radioactivity in the internal aqueous fraction and tissue pellet accounted for less than 0.8% of total radioactivity. The in vivo studies suggest that fipronil oxidation to its sulfone metabolite is the major route of metabolic conversion. In vitro studies were performed using subcellular microsomal fractions isolated from European corn borer larval midguts. Cytochrome P450-dependent monooxygenase activity (methoxyresorufin O-demethylase) was consistently observed in midgut preparations, and formation and detection of the sulfone metabolite in the same midgut preparations was also NADPH-dependent and inhibited by piperonyl butoxide. In vitro metabolism results indicate microsomal monooxygenases are responsible for the conversion of fipronil to its sulfone form in the European corn borer.

Leucokinins are a group of structurally related neuropeptides stimulating gut motility and fluid secretion by Malpighian tubule in insects. For studying effect of neuropeptides on digestive enzyme release, empty midgut tubes of larvae of Opisina arenosella ligated at both ends with hair were incubated with Leucokinins (LK I-VIII), LK analogues and Leucopyrokinin (LPK) in a bioassay apparatus at 37 degrees C for 30 min. The lumen contents were subsequently analyzed for digestive enzyme levels. The neuropeptides LK III, FFSWG amide, 122 A[1] WP-2, LPK and 434 [phi2] WP-1 inhibited the release of digestive enzymes, protease and amylase while LK VIII, unique in having tyrosine residue, stimulated protease release. The minimum sequence of amino acids at the C-terminal required for activity of LK peptides was found to be FXSWGamide (X=Asn, His, Ser, or Trp). The N-terminal pyroglutamate residue and proline at the C-terminal may contribute to the inhibitory effect of LPK on digestive enzyme release. The present study reveals for the first time an inhibitory effect for leucokinins and pyrokinin on the release of digestive enzymes from the insect midgut.

Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin-binding region of each APN isoform. The results suggest that APN isoforms have a common toxin-binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS-PAGE is determined by factors such as expression level in conjunction with differences in binding affinity.

Molecular phylogenetic analysis was conducted using conserved cytoplasmic actin and diversified cytochrome P450 (P450) sequences isolated from Helicoverpa zea and Helicoverpa armigera, two species thought to be closely related based on allozyme analyses. These sequences were compared in turn with published sequences from other insects to gain insight into how different gene families evolve. In Bombyx mori and these Helicoverpa species, cytoplasmic actin genes are present as a pair of tandemly duplicated paralogs with coding sequence identities as high as 95.5% (B. mori), 98.9% (H. zea) and 98.5% (H. armigera) due to recent 5'-polar gene conversions. Phylogeny and interspecies comparisons assign the six actin genes into two orthologous groups: HaA3a/HzA3a/BmA3 and HaA3b/HzA3b/BmA4, which exhibit more similarities between H. zea and H. armigera than between Helicoverpa species and B. mori. Like the actin genes in H. zea, four CYP6B genes exist as two pairs of duplicated paralogs with recent 5'-polar gene conversions. Interspecific comparisons and phylogeny analysis identified three groups of orthologous CYP6B genes: H. zea CYP6B8 or CYP6B28/H. armigera CYP6B7, H. zea CYP6B27/H. armigera CYP6B6, and H. zea CYP6B9/H. armigera CYP6B2/Heliothis virescens CYP6B10. The low degree of divergence in the first two of these groups is comparable to allelic variation within a single species. These orthologous relationships and the high degrees of similarity in both actin and P450 genes strongly indicate that these Helicoverpa species are extremely closely related.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme., (Copyright 2001 Academic Press.)

Brush border membrane vesicles (BBMVs) were prepared from the 2nd instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type delta-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn++ chelating agents 2,2'-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of delta-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60-77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S.

A strong correlation between two mannose phosphate isomerase (MPI) isoenzymes and resistance to Cry1A toxins from Bacillus thuringiensis has been found in a Plutella xylostella population. MPI linkage to Cry1A resistance had previously been reported for a Heliothis virescens population. The fact that the two populations share similar biochemical, genetic, and cross-resistance profiles of resistance suggests the occurrence of homologous resistance loci in both species.

The utility of microplate and electrophoretic assays for detection of biochemical and physiological mechanisms underlying resistance to profenofos in the tobacco budworm, Heliothis virescens (F.), was assessed. Esterase (EST) activities were significantly higher in profenofos-resistant than -susceptible larvae, and activities were highly correlated (r(2) = 0.87) with resistance to profenofos. Both qualitative and quantitative variation was observed in electrophoretic gels stained with alpha- and beta-naphthyl acetates. Staining of ESTs was more intense with resistant larvae than those from a susceptible strain. In addition, a band (designated A') was expressed in larvae from profenofos-resistant strains, but not in larvae from an insecticide-susceptible strain. The frequency of expression of A' increased following selection with profenofos and was detected in 100% of the individuals from a profenofos-selected strain. The appearance of this band coincided with the decreased expression of a second band (designated A). A similar pattern (overexpression of A' and underexpression of A) also was observed in larvae from field-collected strains. Finally, reduction in the activity or the sensitivity of acetylcholinesterase to inhibition by chlorpyrifos oxon was observed in laboratory-selected and field-collected larvae that expressed resistance to profenofos. These results suggest that microplate and electrophoretic assays can be utilized as complementary tools for detecting and monitoring profenofos resistance in H. virescens.

Atrazine (1,000 ppm), endosulfan (1 ppm) or butylated hydroxyanisole (BHA) (1,000 ppm) added to a semi-synthetic diet of Orthosia gothica for 2 days in the last instar did not change the soft tissue cytosolic glutathione-S-transferase (GST) activities towards 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and cumene hydroperoxide (CU). However, all three pesticides changed the GST subunit composition compared with the control as observed by reverse phase high performance liquid chromatography of the isozymes purified by glutathione-Sepharose affinity chromatography. The changes seem to have occurred mainly in the GST class 2 subunit. There is no obvious explanation for the changes, which may be a result of interactions between xenobiotic and GST in the cytosol as well as changes in the level of regulation of synthesis. However, the observation added to our knowledge of the processes involved when pesticides are degraded by GSTs in vivo.

Telomeres of most insects consist of pentanucleotide (TTAGG)n repeats, although the repeats are absent in Diptera and some other insect species, where the telomere regions are perhaps maintained without telomerase. To understand various and unusual telomere formation in insects, we have studied the characteristic features of a putative insect telomerase that has not been previously described. Using a modified telomeric repeat amplification protocol (TRAP), we first detected the telomerase activity in crickets, cockroaches and two Lepidopteran insects. The telomerase from crickets and cockroaches required dATP, dGTP and dTTP but not dCTP as a substrate and sequence analyses of the products of TRAP revealed that the (TTAGG)n repeats are synthesized by telomerase. The cockroach telomerase was detected both in somatic (fat body, muscle and neural tissues) and germ line (testis) cells, suggesting that expression of this enzyme is not regulated in a tissue-specific manner at an adult stage. While we detected high levels of telomerase activity in crickets and cockroaches, we could not detect activity in all tissues and cell cultures of the silkworm, Bombyx mori and in two Drosophila and one Sarcophaga cell lines. This supports the theory that Dipteran insects maintain their telomeres without telomerase.

Some properties of the purified alkaline protease from larvae of the insect Spilosoma obliqua (Lepidoptera) and its potential application as an additive in various bio-formulations are reported. The novel feature of the present study is the use of insect protease. The protease was found to be compatible with some of the commercial detergents tested, and was also effective in cleaving various protein substrates tested, albeit to different extents, implying broader substrate specificity and effectiveness of the protease against a wide variety of stains. This property of the protease can also be exploited by using it as an active component in enzymic debriders in view of its ability to digest various protein substrates. The insect protease appears to be potentially useful as an additive in detergent, stain remover and other bio-formulations.

The parasitic wasp Tranosema rostrale transmits a polydnavirus (PDV) to its host, Choristoneura fumiferana, during oviposition. Last-instar C. fumiferana larvae parasitized by T. rostrale early in the stadium fail to undergo metamorphosis, and injection of the wasp's calyx fluid (CxF; contains PDV) into healthy caterpillars induces a dose-dependent delay in initiation of metamorphosis (D. Doucet and M. Cusson, 1996, Entomol. Exp. Appl. 81, 21-30). In the present work, parasitization and injection of CxF (0.5 female equivalent) on the first day of the last stadium both prevented the rise in hemolymph 20-hydroxyecdysone (20HE) titer observed between day 4 and day 7 in control and saline-injected larvae. Similarly, juvenile hormone esterase (JHE) activity was depressed following parasitization or CxF injection, whereas control larvae displayed a peak on day 4. However, neither parasitism nor injection of CxF on day 1 prevented the JH-producing glands from turning off during the first half of the last stadium. Likewise, low but clearly detectable JH titers were observed in the first hours following the molt but very low titers, at or near the detection limit of our radioimmunoassay, were seen in both control and parasitized larvae on day 4. Prothoracic glands showed no apparent sign of degeneration 4 days after injection of CxF but had significantly smaller cells than saline-injected larvae 7 days postinjection. It is not clear whether this was a direct effect of T. rostrale PDV. Thus, disruption of spruce budworm metamorphosis by T. rostrale CxF involves depression of 20HE titers but is not associated with a measurable increase in the level of JH, as shown for some other host-parasitoid systems. In view of the latter observation, we put forward three hypotheses regarding the functional significance of the observed suppression of JHE activity in developmentally arrested C. fumiferana larvae.

Structural differences in dihydrofolate reductases from different species have been exploited to develop specific inhibitory molecules, such as chemotherapeutic agents, antibiotics or antihelminthics, that show species specificity or selectivity. As dihydrofolate reductase (DHFR) is a crucial enzyme for the synthesis of purines, pyrimidines and some amino acids, and also because developing insects show a remarkably rapid rate of cell division, DHFR is a potentially promising target for the discovery of novel insecticides. We have thus isolated and characterized the enzyme from a serious agricultural pest, Heliothis (Helicoverpa) virescens, the tobacco budworm. Sequencing tryptic peptides of the 35 000-fold purified DHFR allowed the subsequent isolation of a partial cDNA, with the full Dhfr gene sequence obtained from a genomic library. The H. virescens Dhfr spans 4 kb, with three introns, and encodes 185 amino acids. The enzyme shows an overall similarity of approximately 68% with DHFR from other metazoans, which has facilitated the molecular modeling of the protein. DHFRs from insects appear to have strikingly reduced sensitivity to inhibition by methotrexate, compared with the vertebrate enzymes, and this reduction was also reflected in the total binding energy seen after modeling experiments. Four residues that may be characteristic of insect DHFR, as well as a unique cysteine in the H. virescens DHFR active site, offer insight into the nature of inhibitor selectivity and provide suitable target sites for insecticide discovery.

(1) In homogenates of vitellogenic follicles from Hyalophora cecropia, cyclic nucleotides promoted the transfer of label from [gamma32P]-ATP to at least four polypeptides. PKI (6-20) amide, an inhibitor of PKA (cAMP-dependent protein kinase), prevented all four reactions. Quantitative tests using kemptide as a substrate indicated that 80% of the total follicular PKA activity was localized in the follicle cells; labeling at 45, 32, and 27 kDa was particle-associated and also restricted to the follicle cells, while a 58 kDa substrate was labeled only in homogenates of the oocyte. (2) When intact follicles were incubated in [32P]-phosphate and okadaic acid, a protein phosphatase inhibitor, the 32 kDa substrate again exhibited cAMP-dependent labeling. There was thus a physiological relationship between PKA activation and 32 kDa protein phosphorylation, while exposure of at least two of the other three substrates to appropriate kinases required homogenization. The latter was illustrated by phosphorylation of the 42 kDa small subunit of vitellogenin, which occurred only when homogenization mixed the proteins of the yolk bodies with cytoplasmic kinases. (3) PKA activation is known to promote the termination of vitellogenesis, even in the absence of detectable labeling of the 32 kDa substrate. The possibility remains that phosphorylation at 32 kDa concerns later aspects of postvellogenic development that were not tested by the assay system used here.

The phylogenetic relationships between Bombyx mori and Bombyx mandarina species of Bombycidae, and Antheraea yamamai and Antheraea pernyi species of Saturniidae were investigated based on mtDNA RFLP and cytochrome oxidase I gene. The sizes of the mtDNA of all the species were estimated at approximately 16 kbp +/- 500 bp by total length of all the restricted fragments and no variation in size was recognized. Of the fourteen different restriction endonucleases used, BamHI, HindIII, PstI, EcoRI and XbaI showed RFLP. Among these, only HindIII showed RFLP between B. mori and B. mandarina. A comparative analysis of sequences was also conducted with the mitochondrial cytochrome oxidase I genes of each species. The results indicated that B. mori shared a 97%, 85% and 87% sequence identity with B. mandarina, A. yamamai and A. pernyi, respectively. B. mandarina shared a 87% and 88% sequence identity with A. yamamai and A. pernyi, respectively. A. yamamai shared 92% sequence identity with A. pernyi. The results of the phylogenetic analysis exhibited monophyly and confidence limits of more than 99% in all trees for both Bombycidae and Saturniidae.

We screened a midgut cDNA library from diamondback moth, Plutella xylostella, with a probe generated using sequence information from an aminopeptidase N gene from Manduca sexta (MsAPN-1). The sequence recovered (PxAPN-A) encodes a protein of 988 resides with a 60% sequence identity to MsAPN-1. The two proteins share a signal peptide which directs processing by the endoplasmic reticulum, a C-terminal hydrophobic region satisfying the criterion for a GPI anchor and cleavage, and the possibility of an O-glycosylated rigid stalk attached to the GPI anchor. PxAPN-A is more closely related to MsAPN-1 than it is to another aminopeptidase recently reported from P. xylostella. Sequence comparisons with other species suggests that at least one aminopeptidase gene duplication occurred in an ancestral lepidopteran.

A cDNA clone specific for cytochrome b5 was isolated from Helicoverpa armigera. This sequence corresponded to a mRNA of an estimated 544 nucleotides in length excluding the poly A tail. The mRNA contained an open reading frame of 381 nucleotides encoding a protein of 127 amino acid residues with a molecular weight of 14,564 Daltons. The encoded protein sequence showed 51% protein sequence identity with cytochrome b5 from M. domestica and 36-37% identity with mammalian and avian cytochrome b5 sequences. Northern analysis of larval RNA using this cDNA as probe, revealed that cytochrome b5 mRNA expression is tissue specific with the mRNAs being expressed in abundance in the midguts of larvae, at a lower level in fatbody but is not detectable in larval integument. During normal development this mRNA was undetectable in eggs but was present at similar levels from first to fifth instar larvae. The mRNA was expressed at very low levels in pupae and adult moths. The cytochrome b5 mRNA was found to be inducible by treatment with the monoterpene, a-pinene, and to be over-expressed in some individuals of a pyrethroid resistant population of H. armigera. The induction and over-expression patterns were identical to the cytochrome P450, CYP6B7 mRNA. The present data suggests that cytochrome b5 may be involved in CYP6B7 mediated pyrethroid resistance in H. armigera.

Two digestive trypsin-like proteinases from Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae) larvae were purified by benzamidine-Sepharose affinity chromatography. The purified enzymes showed molecular size of 27 (trypsin-I) and 24 KDa (trypsin-II). Amino acid analysis and N-terminal sequencing confirmed their relationship with other trypsins from lepidopteran larvae. However, trypsin-I presented one lysine at position 11, being the first report of this amino acid in the sequence of a lepidopteran digestive trypsin. Trypsin-I had an isoelectric point of 6.0, and a Km of 2.2 x 10(-4) M and 3.9 x 10(-5) M for BApNa and BAEE, respectively. Trypsin-II presented an isoelectric point of 8.7, and Km values of 1.7 x 10(-4) M (BApNa) and 3.8 x 10(-5) M (BAEE). Both enzymes were differentially inhibited by some proteinase inhibitors. In particular, trypsin-I was inhibited by E-64 (ID50 = 6 microM) but not by lima bean trypsin inhibitor (LBI), whereas trypsin-II was inhibited by LBI (ID50 = 1 microM) and poorly by E-64 (ID50 = 85 microM). Changes in the susceptibility of the trypsin-like activity of midgut extracts from different larval instars to these inhibitors suggest that the relative proportion of these two enzymes varied through larval development, being predominant in early instars trypsin-I and in late instars trypsin-II.

In contrast to the vertebrate immune system, nearly nothing is known about the immunological role of nitric oxide (NO) in invertebrates. This study provides evidence of the presence of a NO synthase (NOS) activity in an immune-competent, macrophage-like insect hemocyte line, previously established from larvae of the lepidopteran insect Estigmene acraea. As proven by photometric determination of nitroblue tetrazolium reduction after cell fixation, the E. acraea cells possess NADPH diaphorase (NADPHd) activity. This NADPH diaphorase activity was NADPH dependent, not inhibitable by superoxide dismutase, influenced by extracellular addition of L-arginine, and inhibited in a dose-dependent manner by the specific NOS inhibitor Nomega-monomethyl-L-arginine. Furthermore, the NADPH diaphorase activity was stimulated within 30 min by the addition of insect pathogenic bacteria (Bacillus thuringiensis var. kurstaki, Photorhabdus luminescens), bacterial lipopolysaccharide, and silica beads. In activated E. acraea cell suspensions strongly increased amounts of L-citrulline and enhanced levels of total nitrite/nitrate (as NO derivates) can be determined. This is the first report on stimulable NOS activity in insect hemocytes.

Using synthetic substrates we have characterised carboxypeptidase activity in gut extracts from Helicoverpa armigera larvae. Carboxypeptidase A activity predominates, with only low levels of carboxypeptidase B activity present. Maximum carboxypeptidase A activity occurs over a broad pH range and is inhibited by phenanthroline and potato carboxypeptidase inhibitor. A cDNA clone encoding carboxypeptidase (the first such sequence from a lepidopteran insect) was isolated from a larval gut library. The sequence predicts a secreted polypeptide of Mr 46.6 k with homology to metallocarboxypeptidases from mammalian and invertebrate species. The presence of a serine residue at the active site suggests carboxypeptidase A activity. To further characterise the gene product, the complete cDNA sequence was expressed in insect cells using the baculovirus system. Culture supernatant from these cells contained carboxypeptidase A activity, with no activity against a carboxypeptidase B substrate; the carboxypeptidase B activity in gut extracts must thus be due to a separate enzyme. In agreement with this conclusion, the expressed carboxypeptidase cDNA is a member of a small multigene family. Chronic ingestion of soybean Kunitz trypsin inhibitor by H. armigera larvae results in increased accumulation of carboxypeptidase mRNA in the midgut cells, and an increase in carboxypeptidase A activity detected in gut extract.

Angiotensin converting enzyme (ACE) is Zn2+ metallopeptidase which plays an important role in blood pressure homeostasis in mammals and other vertebrates. Homologues of ACE involved in the biosynthesis of mammalian peptide hormones have also been identified in the insects, Musca domestica, Drosophila melanogaster and Haematobia irritans exigua. In the pursuit of the biological role of insect ACE, this work focused on the tissue and cellular distribution of ACE in several insect species. The localisation of ACE in the central nervous system and reproductive tissues from a number of insect species suggests that ACE is of physiological importance in these tissues. By means of an antiserum to housefly ACE, we found that ACE-like immunoreactivity was abundantly present in the neuropil areas of the brain of all insects investigated, suggesting a role for ACE in the metabolic inactivation of peptide neurotransmitters. Especially in the fleshfly, Neobellieria bullata neuropile staining is abundant. In the cockroach Leucophaea maderae, immunoreactive staining was abundant in the neuronal perikarya as well as in the neuropilar regions. Staining in neurosecretory cells was also observed in the brains of the lepidopteran species, Bombyx mori and Mamestra brassica. The localisation of ACE in neurosecretory cells is consistent with the role as a processing hormone, involved in the generation of active peptide hormones. ACE was found to be co-localised with peptides of the FXPRLamide family in M. brassica and in B. mori, suggesting a role for the biosynthesis of these hormones. Finally, we found ACE-like immunoreactivity in the testis of Locusta migratoria, N. bullata and Leptinotarsa decemlineata, providing additional evidence for its important role in insect reproduction., (Copyright 1998 Elsevier Science B.V.)

A Helicoverpa armigera larval midgut cDNA library from larvae raised on an artificial, protein-rich, inhibitor-free diet contained very large numbers of serine proteinase positive clones. DNA sequencing of six random positive cDNAs and 12 PCR derived products identified trypsin genes classifiable into three families, and chymotrypsin and elastase genes classifiable into a single family each. Genomic blots established that the most highly expressed of the trypsin families contained about 18 genes, and that the chymotrypsin and elastase families contained about 14 and 2 genes respectively. The levels of mRNA corresponding to the highly expressed trypsin and chymotrypsin families were determined following chronic ingestion of four proteinase inhibitors. Compared to insects on an inhibitor-free diet, chymotrypsin mRNA was increased by all inhibitors, and trypsin mRNA levels decreased. This occurred independent of whether the inhibitor was solely a trypsin inhibitor (aprotinin), an inhibitor of both trypsin and chymotrypsin (proteinase inhibitor II, soybean trypsin inhibitor) or predominantly a chymotrypsin inhibitor (proteinase inhibitor I). Changing the protein level of the diet did not affect trypsin mRNA levels, but chymotrypsin mRNA levels decreased with increasing dietary protein.

Two kinds of cDNA clones encoding prophenoloxidases (ProPO; zymogen of phenoloxidase (monophenol, L-dopa: oxygen oxydoreductase, EC 1.14.18.1)) were isolated by polymerase chain reaction (PCR) followed by screening of cDNA library that was prepared from whole larvae of the fall webworm, Hyphantria cunea (Lepidoptera, Arctiidae). The cDNAs encode 681 and 697 amino acids with molecular masses of 78.2 and 80.2 kDa, respectively. Deduced amino acid sequence homology between the two H. cunea ProPOs are only 49% whereas the homology against other insect ProPOs ranged from about 40 to 72%. The phylogenic analysis showed that the insect ProPOs are grouped mainly into two families. A putative proteolytic cleavage site for enzyme activation was identical to other insect ProPOs. The conserved copper binding sites were 84-62% homologous to arthropod ProPOs. Two additional highly conserved regions were found in the carboxy terminal. Furthermore, like other insect prophenoloxidases, hydrophobic signal peptide sequences were absent in the deduced ProPOs from H. cunea. Southern blot analysis indicated that the H. cunea ProPO1 is present as a single copy in the genome. Northern blot analysis showed that the expression of the ProPO genes were concentrated in mid-instar larvae, but were much lower in other developmental stages.

A protein trypsin inhibitor from seeds of snail medic (Medicago scutellata), MsTI, has been purified by ion-exchange chromatography, gel-filtration chromatography and reverse-phase HPLC. The protein inhibits the catalytic activity of bovine beta-trypsin, with an apparent Kd of 1.8 x 10(-9), but exhibits no activity towards bovine alpha-chymotrypsin. Moreover, MsTI inhibits the trypsin-like proteinase activity present in larvae of the crop pests Adoxophyes orana, Hyphantria cunea, Lobesia botrana and Ostrinia nubilalis. The complete amino acid sequence of MsTI consists of 62 residues corresponding to a M(r) of 6925. Sequence comparison shows that MsTI exhibits significant similarity to other proteins belonging to the Bowman-Birk trypsin inhibitor family, and the closest identity (81%) with the wound-induced trypsin inhibitor from Medicago sativa leaves.

The N,N'-diacetyllactosediamine (lacdiNAc) pathway of complex-type oligosaccharide synthesis is controlled by a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalac-tesaminyltransferase (beta 4-GalNAcT) that acts analogously to the common UDP-Gal:GlcNAc beta-R beta 1-->4-galactosyltransferase (beta 4-GalT). LacdiNAc-based chains particularly occur in invertebrates and cognate beta 4-GalNAcTs have been identified in the snail Lymnaea stagnalis, in two schistosomal species, and in several lepldopteran insect cell lines. Because of the similarity in reactions catalyzed by both enzymes, we investigated whether L. stagnalis albumen gland beta 4-GalNAcT would share with mammalian beta 4-GalT the property of interacting with alpha-lactalbumin (alpha-LA), a protein that only occurs in the lactating mammary gland, to form a complex in which the specificity of the enzyme is changed. It was found that, under conditions where beta 4-GalT forms the lactose synthase complex with alpha-LA, the snail beta 4-GalNAcT was induced by this protein to act on Glc with a > 100-fold increased efficiency, resulting in the formation of the lactose analog GalNAc beta 1-->4Glc. This forms the second example of a glycosyltransferase, the specificity of which can be altered by a modifier protein. So far, however, no protein fraction could be isolated from L. stagnalis that could likewise interact with the beta 4-GalNAcT. Neither had lysozyme c, a protein that is homologous to alpha-LA, an effect on the specificity of the enzyme. These results raise the question of how the capability to interact with alpha-LA has been conserved in the snail enzyme during evolution without any apparent selective pressure. They also suggest that snail beta 4-GalNAcT and mammalian beta 4-GalT show similarity at a molecular level and allows the identification of the beta 4-GalNAcT as a candidate member of the beta 4-GalT family.

Mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides. A key aim of much recent work has been to increase the speed of kill and so improve commercial efficacy of these biocontrol agents. This might he achieved by adding insecticidal genes to the fungus, an approach considered to have enormous potential for the improvement of biological pesticides. We report here the development of a genetically improved entomopathogenic fungus. Additional copies of the gene encoding a regulated cuticle-degrading protease (Pr1) from Metarhizium anisopliae were inserted into the genome of M. anisopliae such that Pr1 was constitutively overproduced in the hemolymph of Manduca sexta, activating the prophenoloxidase system. The combined toxic effects of Pr1 and the reaction products of phenoloxidase caused larvae challenged with the engineered fungus to exhibit a 25% reduction in time of death and reduced food consumption by 40% compared to infections by the wild-type fungus. In addition, infected insects were rapidly melanized, and the resulting cadavers were poor substrates for fungal sporulation. Thus, environmental persistence of the genetically engineered fungus is reduced, thereby providing biological containment.

Lysozyme is an important antibacterial protein in the insect defense system. Lysozyme was isolated from hemolymph of Heliothis virescens larvae using gel filtration and ion-exchange chromatography. Heliothis lysozyme had a molecular mass of 16,000 daltons by SDS-PAGE. Using acid gel electrophoresis, Heliothis lysozyme migrated faster than egg white lysozyme. The pI of Heliothis lysozyme was estimated as greater than 9.5. Heliothis lysozyme had specific bactericidal activity against three Gram-positive bacteria but no activity against Escherichia coli. The bactericidal activity was stable at 100 degrees C at pH 3.0 after 60 min incubation, but was labile at 100 degrees C at pH 6.8 after 60 min incubation. Heliothis lysozyme was an inducible protein that increased 9 times when comparing unvaccinated with vaccinated larvae. Lysozyme from H. virescens was more similar in molecular mass, heat sensitivity and pH sensitivity to lysozyme isolated from Galleria mellonella and Bombyx mori than to lysozyme isolated from Hyalophora cecropia.

Neural membrane fractions, prepared from brain-subesophageal ganglion complexes of the adult lepidopteran Lymantria dispar, contain at least two peptidases capable of metabolizing locust adipokinetic hormone-I in vitro. The initial fragments, pGlu1-Leu2-Asn3 and Phe4-Thr5-Pro6-Asn7-Trp8-Gly9-Thr10, result from the action of an endopeptidase with properties similar to those reported for neutral metalloendopeptidase in Schistocerca gregaria and mammalian endopeptidase 24.11. The heptapeptide is further degraded by an aminopeptidase that exhibits kinetic properties similar to those described for aminopeptidase 3.4.11.2. These enzymes appear to be responsible for the first two steps in AKH catabolism in L. dispar.

The transmembrane sector of V-ATPases is involved in proton conduction across the membrane where a 15-17 kDa proteolipid forms a putative proton channel. An affinity-purified rabbit polyclonal antibody was developed to an antigenic and putatively extracellular region of a cloned 17 kDa proteolipid. In larval tissue sections, this antibody labeled the midgut goblet cell apical membrane in Heliothis virescens (Lepidoptera: Noctuidae) and the apical membrane in Malpighian tubules from H. virescens and Manduca sexta (Lepidoptera: Sphingidae). The antibody also recognized the 17 kDa protein in an immunoblot of H. virescens Malpighian tubule homogenate. Northern blot analysis revealed the presence of two transcript sizes in the midgut (1.9 and 1.2 kb) and Malpighian tubules (2.2 and 1.9 kb). Our results strongly support the hypothesis that the 17 kDa protein is a component of the V-ATPase, where it is thought to be the proton-conducting subunit. This polyclonal antibody may provide a powerful tool for V-ATPase regulation studies, while the use of the anti-peptide antibody approach may be helpful for the immunolocalization of other ductins.