Plant resistance and its effect on the peritrophic membrane of southwestern corn borer (Lepidoptera: Crambidae) larvae.

The southwestern corn borer, Diatraea grandiosella Dyar (Lepidoptera: Crambidae), is a serious pest of corn, Zea mays L., in the southern United States. Corn germplasm lines with conventional genetic leaf-feeding resistance to this pest, the fall armyworm, Spodoptera frugiperda (J.E. Smith), and other lepidopterans have been released to the public by USDA-ARS scientists located in Mississippi. Recent studies suggest the insect resistant lines disrupt the integrity of the peritrophic membrane of the fall armyworm. The objectives of this study were to investigate any morphological differences in the structure of the peritrophic membrane of southwestern corn borer larvae feeding on resistant and susceptible corn hybrids and to quantify the damage. Larvae were reared under field and laboratory conditions on three corn hybrids (two resistant and one susceptible). Scanning electron microscopy was used to examine the peritrophic membrane for abnormalities such as holes or tears and to count the holes or tears in the membrane. Differences in the degree of damage to peritrophic membrane of larvae fed on resistant and susceptible plants were not detected. Up to five distinct layers of the membrane were observed in each larva. Variation in the amounts of damage to the peritrophic membrane observed from larvae feeding on all plant material was high. Plant resistance adversely affects growth and development of southwestern corn borer larvae, and further investigations are needed to explain the role of plant resistance and its relation to peritrophic membrane in southwestern corn borer larvae.

Degradation of the S. frugiperda peritrophic matrix by an inducible maize cysteine protease.

A unique 33-kDa cysteine protease (Mir1-CP) rapidly accumulates at the feeding site in the whorls of maize (Zea mays L.) lines that are resistant to herbivory by Spodoptera frugiperda and other lepidopteran species. When larvae were reared on resistant plants, larval growth was reduced due to impaired nutrient utilization. Scanning electron microscopy (SEM) indicated that the peritrophic matrix (PM) was damaged when larvae fed on resistant plants or transgenic maize callus expressing Mir1-CP. To directly determine the effects of Mir1-CP on the PM in vitro, dissected PMs were treated with purified, recombinant Mir1-CP and the movement of Blue Dextran 2000 across the PM was measured. Mir1-CP completely permeabilized the PM and the time required to reach full permeability was inversely proportional to the concentration of Mir1-CP. Inclusion of E64, a specific cysteine protease inhibitor prevented the damage. The lumen side of the PM was more vulnerable to Mir1-CP attack than the epithelial side. Mir1-CP damaged the PM at pH values as high as 8.5 and more actively permeabilized the PM than equivalent concentrations of the cysteine proteases papain, bromelain and ficin. The effect of Mir1-CP on the PMs of Helicoverpa zea, Danaus plexippus, Ostrinia nubilalis, Periplaneta americana and Tenebrio molitor also was tested, but the greatest effect was on the S. frugiperda PM. These results demonstrate that the insect-inducible Mir1-CP directly damages the PM in vitro and is critical to insect defense in maize.

Rapid qualitative protease microassay (RPM).

A rapid qualitative protease microassay (RPM) was developed as an alternative to conventional assays of cysteine protease activity in HPLC fractions. Using this technique protease activity in samples could be visually determined within 5 min. The method was sensitive to 3.3x10(-7) U/mL of papain and detected cysteine protease activity in dilute HPLC fractions with activity of 5.4x10(-5) U/mL. Because the method monitors the decolorization of Coomassie Brilliant Blue stained substrate, it can be modified to detect other classes of proteases.

Heterologous expression of maize (Zea mays L.) Mir1 cysteine proteinase in eukaryotic and prokaryotic expression systems.

Several heterologous expression systems were tested for their ability to express a unique maize cysteine proteinase Mir1. A baculovirus-based expression system using Trichoplusia ni larvae as host resulted in the expression of Mir1 that was correctly processed and exhibited proteinase activity. Expression in Escherichia coli resulted in accumulation of Mir1, but it had limited solubility and enzymatic activity. Large quantities of Mir1 were produced when Pichia pastoris was used as the host, but the enzyme was insoluble and inactive.

An automated rotisserie system for processing Western blots.

An apparatus to automate completely the processing of Western blots is described. The prototype is based on a popular rotisserie system design. The incubation chamber consists of an inner cylinder that rotates inside an outer cylinder (incubation chamber). The blot is contained in the inner cylinder. Two magnets are mounted at one end of the inner cylinder, and rotation of the inner cylinder is effected by two magnets mounted on a motor drive outside the incubation chamber. Movement of chemicals into and out of the incubation chamber is driven pneumatically, and the entire process is controlled by a computer. Processing a blot for chemiluminescent detection takes 7 h to complete without human intervention. The quality of the resulting image is comparable to or better than a blot using manual processing. In addition, the prototype is capable of re-collecting all three antisera for future use.

Characterizing the Hez-PBAN gene products in neuronal clusters with immunocytochemistry and MALDI MS.

Methods to characterize pheromone biosynthesis activating neuropeptide (PBAN) and other PBAN gene encoded neuropeptides (PGN) from individual subesophageal ganglion neuronal clusters of the corn earworm moth, Helicoverpa zea, were developed. Individual antisera against the N-terminal sequence to PBAN and each of the three PGNs from the Hez-PBAN prohormone were developed, and their specificity determined. In all cases, each antiserum stains the same three groups of subesophageal ganglion ventral midline neurons-the mandibular, maxillary and labial neurons-in both adult females and males. These results were confirmed using matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) of individual subesophageal ganglion neuronal clusters. Using mass spectrometry, the amidated PGN-24 was not detected but an N-terminally extended form is observed that is two amino acids longer. Other peptides resulting from the processing of the Hez-PBAN prohormone were detected. Using both the specific antisera and the cellular profiling abilities of MALDI MS, the roles of individual members of the Hez-PBAN prohormone derived peptides can now be explored.

Cloning and functional expression of a cDNA encoding a metabolic acyl-CoA delta 9-desaturase of the cabbage looper moth, Trichoplusia ni.

Acyl-CoA delta 9-desaturases play essential roles in fatty acid metabolism and the regulation of cell membrane fluidity. In this research, a cDNA sequence was obtained from Trichoplusia ni adult fat body mRNA by using RT-PCR with degenerate primers based on other characterized delta 9-desaturase sequences. The remainder of the sequence was amplified using 3'- and 5'-RACE. A 1439 bp cDNA reconstructed from three overlapping PCR products contains an ORF encoding a 353-amino acids (aa) protein that shows clear homology (greater than 50% aa identity and greater than 65% aa similarity to characterized insect and vertebrate desaturases). The ORF of this cDNA was subcloned into an expression vector, which relieved the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient yeast strain following genetic transformation. The newly characterized desaturase from T. ni produced fatty acids delta 9-16 and delta 9-18 in a 1:6 ratio, compared to a 5:1 ratio, respectively, with the yeast delta 9 desaturase. A Northern blot hybridization and a RT-PCR experiment showed that temporal and tissue-specific patterns of expression of the corresponding mRNA are distinct from those of the delta 11-desaturase mRNA present in the pheromone glands of adult females. Based on its homology to other desaturases, the widespread distribution of its corresponding mRNA in various tissues, and its functional assay, we conclude that this cDNA encodes the apoprotein corresponding to the desaturase component of the metabolic delta 9-desaturase complex of T. ni.

Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Delta11-desaturase of the cabbage looper moth, Trichoplusia ni.

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Delta9-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Delta9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Delta11Z-desaturation mechanism. The largest ORF of the approximately 1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Delta11Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Delta9-desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Delta9Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an approximately 1,250-nt PDesat-Tn Delta11Z mRNA that is consistent with the spatial and temporal distribution of Delta11-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Delta11Z resulted in complementation of the strain's fatty acid auxotrophy and the production of Delta11Z-unsaturated fatty acids.

Baculovirus expression of an insect gene that encodes multiple neuropeptides.

Sex pheromone production in the corn earworm, Helicoverpa zea, is regulated by a 33-amino-acid neuropeptide named Hez-PBAN (pheromone biosynthesis activating neuropeptide). Hez-PBAN is encoded in a preprohormone that also contains four other structurally related peptides. Two recombinant baculoviruses that contain two different sequences of Hez-PBAN cDNA under the control of a strong polyhedrin promotor were constructed. The first virus, AcWT-PBAN, contains the entire prepro-Hez-PBAN coding sequence. The second virus, AcBX-PBAN, contains a synthetic chimera gene encoding a bombyxin signal peptide sequence fused to a pro-Hez-PBAN sequence. Cell extracts, culture medium of BTI-TN-5B1-4 cells, and hemolymph from 4th instar Trichoplusia ni larvae, all infected with AcBX-PBAN, showed a high level of pheromonotropic activity. Pheromonotropic activity was not detected in the cells infected with AcWT-PBAN. Results of chromatographic and immunochemical studies showed that some of the potential processing sites in the expressed pro-Hez-PBAN sequence were not used during posttranslational processing in the AcBX-PBAN-4-infected BTI-TN-5B1-4 cells and 4th instar T. ni larvae. However, the processing pattern of the recombinant pro-Hez-PBAN in AcBX-PBAN-infected 4th instar T. ni larvae was similar to that exhibited in the central nervous system of H. zea adult females, since a PBAN-like immunoreactive-peptide-band was found in the hemolymph of Ac-BX-PBAN-4-infected 4th instar T. ni larvae. In a droplet feeding assay, neonate and 3rd instar T. ni larvae infected with AcBX-PBAN-4 showed a significant reduction in survival time (26% and 19%, respectively) when compared to control larvae that were infected with a polyhedrin-deficient virus, Ac-E10.

Structural organization of the Helicoverpa zea gene encoding the precursor protein for pheromone biosynthesis-activating neuropeptide and other neuropeptides.

Sex pheromone biosynthesis in a number of moth species is induced by a conserved 33-amino acid amidated neuropeptide PBAN (pheromone biosynthesis-activating neuropeptide). We have isolated and characterized the Helicoverpa zea PBAN cDNA corresponding to a 766-nucleotide mRNA that is expressed in the subesophageal ganglion of adult moths. This mRNA is encoded on a transcription unit comprising 6 exons. The longest open reading frame of the cDNA encodes a 194-amino acid precursor protein that contains the PBAN peptide sequence. Proteolytic processing of this protein, which has structural features consistent with its being a preprohormone, is predicted to generate Hez-PBAN and four additional neuropeptides having a common C-terminal pentapeptide motif, Phe-Xaa-Pro-(Arg or Lys)-Leu (Xaa = Gly, Ser, or Thr), which is also found in insect pyrokinin and myotropin peptide families.

Experimental verification of modified synthetic discriminant function filters for rotation invariance.

A modified binary synthetic discriminant function filter designed to recognize objects over a range of rotated views has been verified on a laboratory optical correlator. A binary synthetic discriminant function filter has been previously described that will produce a specified correlation response for a set of training images. [See D. A. Jared and D. J. Ennis, "Inclusion of Filter Modulation in Synthetic-Discriminant-Function Construction," Appl. Opt. 28, 232-239 (1989).] In the filter design, the modulation characteristics of the device onto which the filter is mapped are included in the synthesis equations. The system of nonlinear equations is then solved using an iteration procedure based on the Newton-Raphson algorithm. The development of the filter-SDF (fSDF) method was driven by the practical concern to make currently available spatial light modulators with limited modulation capabilities functional for distortion invariant pattern recognition. This technique is used to synthesize filters for a binary magnetooptic spatial light modulator (MOSLM), the Sight-MOD produced by Semetex. Two MOSLMs are used in the laboratory correlator, one in the filter plane and one in the input plane. We demonstrate that a single filter produces equal correlation peaks for a sample object (a Shuttle Orbiter in these tests) over in-plane and out-of-plane rotation ranges up to 75 degrees . The correlator is able to track dynamically the shuttle as it moves along a curved path across the input field. Views of the object in between those in the training set are also recognized when training images are sufficiently close in angle (~5 degrees apart).