Effects of catechin polyphenols and preparations from the plant-parasitic nematode Heterodera glycines on protease activity and behaviour in three nematode species.

Protease activities in preparations from the plant-parasitic nematodes Heterodera glycines and Meloidogyne incognita and the free-living nematode Panagrellus redivivus were inhibited by exposure to a series of eight catechin polyphenol analogues, (+)-catechin, (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-gallocatechin gallate (GCG), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) (1 mm each), and by a preparation from H. glycines cysts. General protease activity detected with the FRET-peptide substrate QXL520-KSAYMRF-K(5-FAM)a and proteasome chymotrypsin-like (CTL) activity detected with succinyl-LLVY-AMC were each inhibited significantly more (P < 0.05) by the gallated form of the polyphenol than by the corresponding non-gallated form. Species differences in response to inhibition across all analogues were revealed with the CTL substrate, but CG was a consistently potent inhibitor across all three species and with each substrate. A heat-stable component (CE) from H. glycines cysts inhibited M. incognita CTL activity by 92.07 ± 0.68%, significantly less (P < 0.05) in H. glycines (52.86 ± 2.77%), and by only 17.24 ± 0.55% (P < 0.05) in P. redivivus preparations. CTL activity was, however, inhibited more than 60% in all preparations by the proteasome-specific inhibitor MG-132. Hatching of M. incognita infective juveniles exposed to 1 mm CG, ECG, GCG or EGCG was reduced by 83.88 ± 4.26%, 69.98 ± 9.14%, 94.93 ± 1.71% and 87.93 ± 2.89%, respectively, while hatching of H. glycines was reduced less than 25% by each analogue. CE had no effect on nematode hatch, but did cause a 60% reduction in mobility of H. glycines infective juveniles exposed overnight to CE in vitro, which was more (P < 0.05) than the reduction of M. incognita infective juvenile mobility (20%).

A comparison of the FMRFamide-like peptide proteolytic activities of preparations from two plant-parasitic nematodes (Heterodera glycines and Meloidogyne incognita): possible targets for novel control.

Proteolytic activities in extracts from the plant-parasitic nematodes Heterodera glycines and Meloidogyne incognita were examined for their abilities to digest three FRET-modified peptide substrates representing members of the large FMRFamide-like peptide (FLP) family in nematodes. Included were sequences distributed across all nematode species (KSAYMRFa and KHEYLRFa) and a sequence confined to a narrow range of plant-parasitic nematodes (KHEFVRFa). Species variations were observed among substrate affinities, reaction rates and effect of protease inhibitors. K m values for KHEYLRFa (1.48 ± 0.34 µm) and KSAYMRFa (2.13 ± 0.24 µm) in H. glycines were each lower (P< 0.05) than those for the same substrates in M. incognita (5.26 ± 1.30 µm and 3.90 ± 0.61 µm, respectively). The K m of KHEFVRFa was lower (P< 0.05) in M. incognita (5.83 ± 0.36 µm) than in H. glycines (11.01 ± 1.26 µm). Reaction rates (V max/min/µg) for KHEYLRFa were the same for both species, but KSAYMRFa and KHEFVRFa digestion rates were each nearly twofold higher (P< 0.05) in M. incognita than in H. glycines. Digestion of KSAYMRFa was strongly inhibited in both species by 4-(2-aminoethyl)-benzenesulfonyl-fluoride-HCl (AEBSF) and EDTA, but M. incognita was more sensitive (P< 0.05) to inhibition. AEBSF and EDTA (both at 1 mm) inhibited M. incognita activity 62.3% and 36.6% more, respectively, than H. glycines activity. Serine protease inhibition differed significantly (P< 0.05) between the two species. Maximum inhibition of M. incognita (76%) occurred at 1.85 mm AEBSF while maximum inhibition of H. glycines was 40% at 1.19 mm AEBSF.

In vitro proteolysis of nematode FMRFamide-like peptides (FLPs) by preparations from a free-living nematode (Panagrellus redivivus) and two plant-parasitic nematodes (Heterodera glycines and Meloidogyne incognita).

Proteolytic activities in extracts from three nematodes, the plant parasites Heterodera glycines and Meloidogyne incognita, and the free-living Panagrellus redivivus, were surveyed for substrate preferences using a battery of seven FRET-modified peptide substrates, all derived from members of the large FMRF-amide like peptide (FLP) family in nematodes. Overall protease activity in P. redivivus was four- to fivefold greater than in either of the parasites, a result that might reflect developmental differences. Digestion of the M. incognita FLP KHEFVRFa (substrate Abz-KHEFVRF-Y(3-NO2)a) by M. incognita extract was sevenfold greater than with H. glycines extract and twofold greater than P. redivivus, suggesting species-specific preferences. Additional species differences were revealed upon screening 12 different protease inhibitors. Two substrates were used in the screen, Abz-KHEFVRF-Y(3-NO2)a and Abz-KPSFVRF-Y(3-NO2)a), which was digested equally by all three species. The effects of various inhibitor, substrate and extract source combinations on substrate digestion suggest that M. incognita differs significantly from P. redivivus and H. glycines in its complement of cysteine proteases, particularly cathepsin L-type protease.

In vitro comparison of protease activities in preparations from free-living (Panagrellus redivivus) and plant-parasitic (Meloidogyne incognita) nematodes using FMRFa and FMRFa-like peptides as substrates.

Extracts prepared from the microbivorous nematode Panagrellus redivivus and the plant-parasitic nematode Meloidogyne incognita were used to provide general protease activities for peptide substrate screening and species comparisons. Each extract was evaluated for its ability to degrade a broad range of nematode FMRFamide-like peptides (FLPs), key regulatory messengers governing nematode growth and development. Clear quantitative differences between the two extracts were observed using FMRFamide as a substrate. Extract potency assessed at EC50 (µg/µ l extract protein for 50% substrate digestion) was 1.8-fold greater for P. redivivus than for M. incognita, and potency assessed at EC90 was 2.5-fold greater. An overall potency difference was also present when screening the digestion of 17 nematode FLPs, but it was not universal. The mean percentage digestion of eight of the 17 FLPs was greater (P < 0.02) with P. redivivus extract (76.3 ± 8.2) than with M. incognita extract (38.1 ± 8.7), but the means for the other nine FLPs were not different. Three FLPs (KPSFVRFa, AQTFVRFa, RNKFEFIRFa) were degraded extensively by the extracts of both species, and two FLPs (SAPYDPNFLRFa, SAEPFGTMRFa) were degraded 2.9-fold and 5.3-fold greater, respectively, with M. incognita extract than with P. redivivus extract. The ability of each extract to degrade FMRFa and KSAYMRFa was significantly reduced by using peptide analogues containing single d-amino acid substitutions, and the substitution effects were positional. Both FMRFa and KSAYMRFa were competitive substrates for aminopeptidases in each extract, but only the competitive ability of FMRFa was reduced by d-amino acid substitution. The variety and complexity of nematode FLP degradation by preparations representing phylogenetically and developmentally different nematode sources are discussed.

Digestion of invertebrate neuropeptides by preparations from the free-living nematode Panagrellus redivivus.

Proteases in the soluble fraction of homogenates prepared from the free-living nematode Panagrellus redivivus hydrolysed the amidated invertebrate neuropeptides FMRFa and FLRFa, and nematode FMRFa-like peptides (FLPs) KPNFLRFa (FLP-1-H), APKPKFIRFa (FLP-5-A), KNEFIRFa (FLP-8), KPSFVRFa (FLP-9), RNKFEFIRFa (FLP-12) and KHEYLRFa (FLP-14) in vitro. Results were assessed by analysing reaction components with RP-HPLC, UV detection at 210 nm and peak integration. Based upon substrate peak size, more than 90% of most of the peptide substrates was consumed after 1 h at 27 degrees C, but digestion was not complete even with a crude protease mixture. Two peptides, FLP-12 and FLP-14, were significantly less susceptible to digestion than the others. FLP-12 was the least susceptible of all sequences (71% loss; P < 0.0001), while FLP-14 was digested less (84% loss; P < 0.0004) than all but FLP-12. Product peak digestion patterns of FLP-12, a second nonapeptide (FLP-5-A), and FMRFa, incubated with aminopeptidase (amastatin) and serine endoprotease (AEBSF) inhibitors, demonstrated highly specific behaviours of each sequence to protease cleavage. Amastatin significantly (P < 0.03) reduced digestion of FLP-12 (54% loss) and FMRFa (61% loss; P < 0.0005), but had no effect on FLP-5-A. AEBSF had no protective effect on FMRFa but significantly decreased hydrolysis of FLP-5-A (77% loss; P < 0.0001) and FLP-12 (59% loss; P < 0.03). The combination of both inhibitors had additive effects only for FMRFa (34% loss; P < 0.0005). Further analysis of FMRFa digestion using peptides with D-amino acid substitutions demonstrated nearly complete protection of FdMRFa (2% loss; P < 0.0001) from all proteolytic digestion, whereas digestion of FMRdFa was complete. Results suggest that in addition to aminopeptidase and serine proteases, both deamidase and aminopeptidase P participate in neuropeptide metabolism in P. redivivus.

Responses of Heterodera glycines and Meloidogyne incognita to exogenously applied neuromodulators.

Biogenic amines regulate important behaviours in nematodes and are associated with pharyngeal activity in plant-parasitic nematodes. A robust behavioural assay based upon nematode body movements was developed to expand the study of these and other neuroregulators in plant-parasitic nematodes. Dopamine, octopamine and serotonin each had significant but differing effects on the behaviour of soybean cyst nematode Heterodera glycines and root-knot nematode Meloidogyne incognita juveniles. Body movement frequency was increased twofold in H. glycines by 5 mM dopamine (P = 0.0001), but decreased by 50 mM dopamine in H. glycines (88%) and M. incognita (53%) (P < 0.0001). Movement frequency in both species was increased by 50-70% (P < 0.0001) by 50 mM octopamine, and 5 mM octopamine increased M. incognita movement frequency more than twofold (P < 0.0001). Movement frequency in each species was reduced by more than 90% by 5 mM serotonin (P < 0.0001). While amplitude of body movement in H. glycines was unaffected by any amine, it was significantly reduced in M. incognita by all amines (P < 0.0006). Stylet pulsing frequencies in either species were unaffected by dopamine or octopamine, but 5 mM serotonin stimulated pulsing in H. glycines by nearly 13-fold (P < 0.0001) and in M. incognita by more than 14-fold (P < 0.0001). The invertebrate neuropeptide FLRFamide (N-Phe-Leu-Arg-Phe) increased M. incognita body movement frequency 45% (P = 0.02) at 1 mM but did not affect stylet activity. Finally, H. glycines egg hatch was completely suppressed by 50 mM serotonin, and partially suppressed by 50 mM dopamine (75%; P < 0.0001) and 50 mM octopamine (55%; P < 0.0001).

Characterization of Aminopeptidase in the Free-living Nematode Panagrellus redivivus: Subcellular Distribution and Possible Role in Neuropeptide Metabolism.

Aminopeptidase was detected in homogenates of the free-living nematode Panagrellus redivivus with the aminoacyl substrate L-alanine-4-nitroanilide. Subcellular distribution of activity was 80% soluble and 20% membrane-associated. Aminopeptidases in the two fractions differed in affinity for Ala-4-NA, with Km's of 0.65 mM (soluble) and 2.90 mM (membrane). Specific activities (units/mg) at pH 7.8, 27 degrees C were 9.10 (soluble) and 14.30 (membrane). Each enzyme was competitively inhibited by amastatin (90% at 100 muM inhibitor, IC(50) = 3.7 muM) and inhibited by puromycin (30% at 500 muM) and 1,10-phenanthroline (IC(50's:); 148 muM, soluble; 89 muM, membrane). Activity was restored by Zn(++), with maximum recoveries of 50% (soluble) and 90% (membrane), each at 23 muM ZnCl(2). Estimated molecular masses for each were approximately 150 kDa. FMRFamide-like neuropeptides behaved as competitive inhibitors. Modification of the N-terminal F of FMRFamide weakened inhibition by 95%, suggesting that the N-terminus is essential for binding to the enzyme. Two nematode FMRFamides, APKPFIRFa and RNKFEFIRFa, were the most potent tested. This is the first biochemical characterization of aminopeptidase in a free-living nematode other than Caenorhabditis elegans and demonstrates the high selectivity of the P. redivivus enzymes for neuropeptide substrates.

Changes in FaRP-like peptide levels during development of eggs from the plant-parasitic cyst nematode, Heterodera glycines.

The plant-parasitic cyst nematode Heterodera glycines requires a host plant to complete its life cycle, which involves hatching of infective juveniles that parasitize through root entry. A laboratory population of H. glycines grown on soybean, Glycine max, undergoes a sharp increase in maturity between 5 and 6 weeks in culture, as measured by the proportion of eggs containing well developed pre-hatch juveniles (late development eggs) versus eggs without visible juveniles (early development eggs). The median percent of eggs classified as late development, representing all samples taken from 4 to 7 weeks in culture, was 61%. For all samples taken up to 5 weeks, 80% scored below the median. In samples taken after 5 weeks, 15% scored below the median. This shift in population maturity was accompanied by a significant increase (P < 0.01) in the number of hatched juveniles present in each sample. There was also a significant increase (P < 0.02) in amount of FaRP-like peptide detected by specific ELISA. Total FaRP levels increased from 0.18 +/- 0.07 fMol FLRFamide equivalents per ng protein in early development eggs to 0.40 +/- 0.17 in late development eggs. The level remained high in hatched juveniles. HPLC/ELISA detected as many as nine potential FaRPs in H. glycines, two of which were specifically increased (P < 0.005) in hatched juveniles. The association of FaRPs with maturing eggs and the possible involvement of these neuropeptides with juvenile hatching and motility are discussed.

Molecular Characterization of Aldolase from Heterodera glycines and Globodera rostochiensis.

Fructose-bisphosphate aldolase (EC 4.1.2.13) is a key enzyme in glycolysis. We have characterized full-length coding sequences for aldolase genes from the cyst nematodes Heterodera glycines and Globodera rostochiensis, the first for any plant-parasitic nematode. Nucleotide homology is high (83% identity), and the respective sequences encode 40 kDa proteins with 89% amino acid identity. Genomic sequences contain six introns located at identical positions in both genes. Intron 4 in the H. glycines gene is >500 bp. Partial genomic sequences determined for seven other cyst nematode species reveal that the large fourth intron is characteristic of Heterodera but not Globodera aldolase genes. Total aldolase-like specific activity in homogenates from H. glycines was 2-fold lower than in either Caenorhabditis elegans or Panagrellus redivivus (P = 0.001). Activity in H. glycines samples was higher in juvenile stages than in adults (P = 0.003). Heterodera glycines aldolase has Km = 41 microM and is inhibited by treatment with carboxypeptidase A or sodium borohydride.

In vitro metabolism of an insect neuropeptide by homogenates of the nematode Caenorhabditis elegans.

The cytosolic fraction of homogenates from the free-living soil nematode Caenorhabditis elegans is capable of metabolizing the insect neuropeptide adipokinetic hormone, a decapeptide blocked at the N-terminus by a pGlu residue. Analysis of digests by RP-HPLC and LC-MS revealed that an initial endoproteolytic cleavage step produced a heptapeptide with an unblocked N-terminus that can serve as a substrate for aminopeptidases. The aminopeptidase activity is depressed in the presence of the inhibitor amastatin; the initial product of the endoproteolytic step accumulates during incubation, and expected aminopeptidase product peptides are reduced in amount, as assessed by chromatographic peak size. The absence of some expected peptide fragments in the reaction mixtures suggests that multiple proteases contribute to short peptide half-lives. Comparison of the adipokinetic hormone digestion in C. elegans to that reported previously for insects reveals the same general pattern of peptide fragment production.

Aminopeptidases in Caenorhabditis elegans and Panagrellus redivivus: detection using peptide and non-peptide substrates.

Aminopeptidase activities were detected in extracts of the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus using the aminoacyl substrate L-alanine-4-nitroanilide. The activities exhibited similarities in Km (C. elegans = 2.22 mM; P. redivivus = 2.09 mM) and specific activity (C. elegans = 1.38 +/- 0.43 mAU min(-1) x g(-1); P. redivivus, 1.23 +/- 0.18m AU min(-1) microg(-1). Each is inhibited competitively by amastatin (C. elegans IC50 = 0.46 microM; P. redivivus IC50 = 15.90 microM) and non-competitively by leuhistin (C. elegans IC50 = 3.00 microM; P. redivivus IC50 = 37.35 microM). The bioactive peptides adipokinetic hormone and substance P decrease the apparent aminopeptidase activities of each extract suggesting that the peptides compete with the Ala-pNA as substrates. With each extract, adipokinetic hormone appeared to be the more effective substrate. Digestion of adipokinetic hormone by C. elegans and P. redivivus extracts in the presence and absence of 1 mM amastatin produced distinct chromatographic profiles that suggest different digestion patterns for the two species. However, amastatin had clear effects on chromatographic profiles from each species indicating that an aminopeptidase is involved in the digestion of the peptide substrates. The data presented indicate that extracts of free-living nematodes are capable of metabolizing peptide hormones, and that this metabolism involves substrate-selective aminopeptidases.

Aminopeptidase-like activities in Caenorhabditis elegans and the soybean cyst nematode, Heterodera glycines.

Aminopeptidase-like activities in crude whole body extracts of the free-living nematode Caenorhabditis elegans and the plant parasitic soybean cyst nematode Heterodera glycines were examined. General characteristics including pH optima, heat lability, and inactivation of enzyme by organic solvent were the same for the two species. All developmental stages of H. glycines exhibited activity. In older females, activity was present primarily in the eggs. Affinity for the substrate L-alanine-4-nitroanilide was the same regardless of the stage examined, and was similar for the two species (m for C. elegans and m for H. glycines). Nearly all (>95%) of C. elegans aminopeptidase-like activity was present in the soluble fraction of the extract, while H. glycines activity was distributed between the soluble and membrane fractions. Specific activities of the soluble enzymes were highest in C. elegans and H. glycines juveniles. The C. elegans enzyme was susceptible to a number of aminopeptidase inhibitors, particularly to amastatin and leuhistin, each of which inhibited aminopeptidase-like activity more than 90% at 90 microm. In H. glycines, aminopeptidase-like activity was inhibited 39% by amastatin at 900 microm. The apparent molecular weight of the soluble C. elegans enzyme is 70-80 kDa. Some activity in H. glycines is present in the 70-80 kDa range, but most activity (80-90%) is associated with a very high molecular weight (>240 kDa) component.

Comparison of FaRP immunoreactivity in free-living nematodes and in the plant-parasitic nematode Heterodera glycines.

The family of FMRFamide-related peptides (FaRPs) is widely distributed among invertebrates, where the peptides serve as neuromodulators. Published reports indicate that numerous FaRP sequences exist in free-living and animal parasitic nematodes. Using a FMRFamide ELISA, FaRP immunoreactivity was detected in extracts of the soybean cyst nematode, Heterodera glycines, in both sexes and at all developmental stages. HPLC-ELISA results revealed a number of immunoreactive components in H. glycines preparations, and a comparison with extracts of the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus showed significant qualitative differences in FaRP immunoreactivity between the plant parasite and the two free-living nematodes. Total and specific immunoreactivities varied during H. glycines development, with the highest specific activity in juveniles and males, and the highest total activity in mature females. Total female immunoreactivity was located primarily within the mature eggs. A significant portion, however, was associated with the female body, perhaps with egg laying.

FMRFamide-like Immunoactivity in Heterodera glycines (Nemata: Tylenchida).

Material antigenically related to the neuromodulatory peptide FMRFamide was detected and examined in preparations of the soybean cyst nematode, Heterodera glycines, and in the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. FMRFamide-related peptides were quantified by an enzyme-linked immunosorbent assay. Specific activities were remarkably similar among all of the vermiform members of the three species. FMRFamide-related peptide immunoactivity was present in both sexes and all stages of H. glycines examined. The highest specific activity was present in second-stage juveniles and in males, and the lowest in white and yellow females. Total FMRFamide-related peptide level per individual was highest in brown females, with 90% of the activity associated with the eggs. Peptide levels in these eggs and in second-stage juveniles were comparable and increased in adults, especially in females. Chromatographic analysis of FMRFamide-related peptide preparations from H. glycines juveniles, C. elegans, and P. redivivus revealed distinct qualitative differences between the infective plant parasite and the free-living nematodes.

Detection and Partial Characterization of Egg Polypeptides from Heterodera glycines.

The presence of two major egg polypeptides was demonstrated in the plant-parasitic nematode Heterodera glycines. The polypeptides were present in equal amounts in, and were most abundant in, eggs from yellow females. They were also present in brown females but were not detected in second-stage juveniles (J2). The two major egg polypeptides, MEP-I and MEP-II, accounted for more than 50% of the total protein in egg extracts evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. During development of females from the yellow stage to the brown stage, the levels of MEP-I and MEP-II declined at twice the rate as total protein. MEP-I and MEP-II had estimated molecular masses of 190 kD and 180 kD, respectively, similar to those reported for female-specific proteins, vitellins, from free-living nematodes.

In vitro metabolism of an insect neuropeptide by neural membrane preparations from Lymantria dispar.

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.

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm, Manduca sexta, and the corn earworm, Helicoverpa zea.

The tritium-labeled bis-norleucine analog of Helicoverpa zea pheromone biosynthesis-activating neuropeptide ([3H]NLPBAN) was incubated in vitro with hemolymph from Manduca sexta or H. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [3H]NLPBAN concentration of 0.9 microM the degradation proceeded at a very slow but physiologically plausible rate (2-10 fmol/min/microliters hemolymph). The primary [3H]NLPBAN degradation reaction in M. sexta hemolymph was not inhibited by 20 microM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph of M. sexta and H. zea contains peptidase(s) capable of inactivating circulating PBAN.

A novel low molecular weight ecdysiotropin in post-diapause, pre-hatch eggs of the gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae).

Extracts of post-diapause, pre-hatch eggs of the gypsy moth, Lymantria dispar L. were examined for prothoracicotropic hormone (PTTH)-like activity using an in vitro assay involving last-instar prothoracic glands (PGs). The eggs were extracted in water, eluted from a low-pressure C18-silica cartridge in 60% acetonitrile, and fractionated on a high-performance, size-exclusion column. The primary ecdysiotropic activity eluted with an estimated molecular weight of 2.1 kDa far below the 4-7 kDa size determined for the low molecular weight PTTHs (bombyxins). Dose-response analysis revealed that the maximum activation was reduced by 75% by organic solvent extraction, but the remaining activity retained the ability to maximally activate the PGs 10-fold in vitro. At least some of the ecdysiotropic activity in the post-diapause, pre-hatch egg is localized in the brain of the pharate larva, and this activity increases dramatically prior to hatch when eggs are incubated at 25 degrees C.

Overexpression of Bombyx mori prothoracicotropic hormone using baculovirus vectors.

Recombinant baculoviruses were constructed that express the cDNA encoding the prothoracicotropic hormone (PTTH) of Bombyx mori. This hormone stimulates the production of ecdysteroids by the insect's prothoracic glands. Two groups of viruses were constructed, expressing either the entire cDNA encoding prepro-PTTH, or a synthetic chimeric gene encoding a signal peptide fused to the mature PTTH subunit. In both cases, the genes were expressed in wild-type Autographa californica nuclear polyhedrosis virus (AcMNPV) and in vEGTDEL, an ACMNPV mutant that lacks a functional egt gene. The egt gene is required for viral-mediated inactivation of host ecdysteroids. High levels of functional PTTH were produced only by viruses expressing the mature subunit cDNA. This recombinant PTTH resembled the native hormone by all criteria examined. The overproduction of B. mori PTTH induced higher than normal levels of haemolymph ecdysteroids but had no observable effects on the development of infected Spodoptera frugiperda larvae. However, expression of PTTH by AcMNPV was found to inhibit the pathogenicity of the virus. This effect was particularly marked in the case of viruses lacking a functional egt gene.

Complete base sequence for the mitochondrial large subunit ribosomal RNA of the gypsy moth Lymantria dispar (L.).

A 1355 bp sequence (accession number L32141) isolated from a gypsy moth (Lymantria dispar) cDNA library showed 68-74% sequence identity to mitochondrial large subunit ribosomal RNA (mt IrRNA) sequences of Locusta migratoria, Apis mellifera, Aedes albopictus, Anopheles gambiae and two Drosophila species. A comparison of the primary sequences of the mt IrRNAs from the above insects in four orders and from Esherichia coli demonstrated regions of conservation which presumably correspond to regions of functional and/or structural homology. A secondary structure for the gypsy moth mt IrRNA sequence was derived based on the proposed secondary structures of Drosophila yakuba and Aedes albopictus mt IrRNAs (Gutell & Fox, Nucleic Acid Res 16 (Suppl.), r175-r269, 1988). This sequence was found to hybridize to about 10-15% of the clones in several (eleven) gypsy moth cDNA libraries.