Trichoplusia Ni Larvae Research Articles

Functional characterization of black widow spider neurotoxins synthesised in insect cells.

alpha-latrotoxin, alpha-latroinsectotoxin and the low-molecular-mass protein from black widow spider venom were synthesised in insect cells using the baculovirus expression system. SDS/PAGE analysis of recombinant-virus-infected cells revealed novel proteins that migrated with sizes similar to those of the neurotoxins from spider venom. The identities of these proteins as alpha-latrotoxin, alpha-latroinsectotoxin or the low-molecular-mass protein were confirmed by immunoblot analysis of infected cells with anti-(alpha-latrotoxin), anti-(alpha-latroinsectotoxin) or anti-(low-molecular-mass protein) IgG. Neither the low-molecular-mass protein nor alpha-latrotoxin were toxic upon injection into Trichoplusia ni larvae or upon virus-derived synthesis directly in the cytoplasm of the target tissue. Analysis of the biological activity of the recombinant virus encoding alpha-latroinsectotoxin, however, revealed a strong toxic effect on the T. ni larvae. These data indicate that the toxic effect of the native insectotoxin may be promoted by the alpha-latroinsectotoxin subunit alone and provides evidence that the mechanism of action of alpha-latroinsectotoxin may be mediated by internalisation of part of the neurotoxin alpha-subunit molecule.

Field Studies of the Co-Occlusion Strategy with a Genetically Altered Isolate of the Autographa californica Nuclear Polyhedrosis Virus

The first field study of a genetically altered virus in the United States was performed with an isolate of the Autographa californica nuclear polyhedrosis virus (AcMNPV), which lacks a polyhedrin gene. In the first year of the study, three applications of 7.4 × 1011 AcMNPV polyhedra containing 48% genetically altered and 52% wild-type virus particles (co-occluded) were made on a O.1-ha circular plot of cabbage plants. The application area was surrounded by a 0.7-ha circular buffer zone. Before each application, the plants in the application area were infested with 4,500 third-ins tar Trichoplusia ni (Hübner) larvae. After each application, 100% of the T. ni test larvae sampled 5 d after infection were infected with AcMNPV and produced progeny polyhedra containing an average of 42 ± 17.6% genetically altered virus particles. At the end of the 1st yr, the progeny polyhedra population in the application area was estimated at 1.6 × 1013 polyhedra. In the 2nd yr, the application and buffer sites were replanted with cabbage plants. At four times during the growing season, the plants were seeded with T. ni larvae or eggs. Less than 2% of the test larvae became infected with AcMNPV. Polyhedra were extracted from soil samples collected in the application and buffer areas. Using neonate larval bioassays with the soil extracts, it was estimated that the soil in the application and buffer areas contained an average of 1,652 ± 3,370 and 832 ± 2,539 biologically active polyhedra per gram dry weight, respectively. Seventy-five larvae infected with polyhedra extracted from application area soil samples produced progeny polyhedra containing a mean of 9 ± 19% genetically altered virus particles. In the 3rd yr, the application area soil samples contained an average of 1,671 ± 3,274 biologically active polyhedra per gram dry weight. Eighty-four progeny polyhedral samples contained a mean of 6 ± 14% genetically altered virus particles. The co-occlusion strategy did not alter the environmental persistence of the polyhedra containing both wild-type and polyhedrin-minus virus particles. However, the data show a decline in the percent of polyhedrin-minus particles in the polyhedra and demonstrate that the persistence of a polyhedrin-minus virus in a cycling virus population is limited by the co-occlusion process. The environmentally desirable attributes of using the co-occlusion process for genetically enhanced baculovirus pesticides and possible problems are discussed.

Human Group II Phospholipase A 2 Expressed in Trichoplusia ni Larvae-Isolation and Kinetic Properties of the Enzyme

Human secreted synovial fluid/platelet-type group II phospholipase A 2 (sPLA 2) was expressed in Trichoplusia ni (cabbage looper) larvae and cultured Sf9 insect cells by infection with a recombinant baculovirus. Active sPLA 2, with correct N-terminal proteolytic processing, was not secreted by Sf9 cells in culture. The enzyme was isolated from their homogenate without any need for refolding or renaturation of the protein. The enzyme was extracted from the 5000 g pellet with 1 M KBr and isolated by chromatography on a cation exchange column followed by reverse-phase chromatography on a Butyl Aquapore column. The yield of active enzyme (25 μg/g insect) was comparable to yields obtained in CHO cells or Escherichia coli by other investigators. The recombinant enzyme had the correct N-terminal sequence, expected molecular weight, and reacted with antisera raised against peptides inferred from the cDNA sequence of the natural enzyme. Monoclonal antibodies were raised against the recombinant sPLA 2 and they permitted the isolation of the natural enzyme from human serum by immunoaffinity. The recombinant sPLA 2 showed a preference for substrate yesides with a net negative charge. The baculovirus expression system provided active sPLA 2 that can be produced economically in insects, purified simply, had well-defined kinetic properties, and should be useful in studies of inflammatory disorders.

Baculovirus expression of the maize mitochondrial protein URF13 confers insecticidal activity in cell cultures and larvae.

The URF13 protein, which is encoded by the mitochondrial gene T-urf13, is responsible for cytoplasmic male sterility and pathotoxin sensitivity in the Texas male-sterile cytoplasm (cms-T) of maize. Mitochondrial sensitivity to two host-specific fungal toxins (T toxins) is mediated by the interaction of URF13 and T toxins to form pores in the inner mitochondrial membrane. A carbamate insecticide, methomyl, mimics the effects of T toxins on isolated cms-T mitochondria. URF13 was expressed in Spodoptera frugiperda (fall army-worm) cells (Sf9) in culture and in Trichoplusia ni (cabbage looper) larvae with a baculovirus vector. In insect cells, URF13 forms oligomeric structures in the membrane and confers T toxin or methomyl sensitivity. Adding T toxin or methomyl to Sf9 cells producing URF13 causes permeabilization of plasma membranes. In addition, URF13 is toxic to insect cells grown in culture without T toxins or methomyl; even a T-toxin-insensitive mutant form of URF13 is lethal to cell cultures. Baculoviruses expressing URF13 are lethal to T. ni larvae, at times postinjection comparable to those obtained by injecting a baculovirus expressing an insect neurotoxin. This result suggests that URF13 could be useful as a biological control agent for insect pests. Our data indicate that URF13 has two independent mechanisms for toxicity, one that is mediated by T toxin and methomyl and one that is independent of these toxins. Similarly, male sterility and toxin sensitivity in cms-T maize may be due to independent mechanisms.

Suppression of the Colorado Potato Beetle (Coleoptera: Chrysomelidae) with Augmentative Releases of Predaceous Stinkbugs (Hemiptera: Pentatomidae)

Field cage tests in 1987 established that releases of 5-10 Perillus bioculatus (F.) and Podisus maculiventris (Say) per plant were able to reduce high density populations (≈450 per plant) of Colorado potato beetle by ≈50%. P. bioculatus provided greater foliage protection than P. maculiventris . In 1988, in similar cage tests, P. bioculatus released at rates of 2-8 per plant reduced Colorado potato beetle densities of ≈100 per plant by ≈8.5% per predator released. Field plot tests in 1988 confirmed cage results with P. bioculatus and demonstrated that 1 and 3 predators per plant suppressed populations of Colorado potato beetle by 30 and 62%, respectively. Three P. bioculatus per plant significantly reduced defoliation and increased yield 65% over the untreated control. Progeny production of a laboratory colony of P. bioculatus maintained on Trichoplusia ni (Hubner) larvae was equivalent to that for a colony maintained on Colorado potato beetle larvae. Our data indicate that inoculative releases of stinkbug predators for suppression of Colorado potato beetle populations could be an important component of an integrated management program.

Localization of juvenile hormone esterase during development in normal and in recombinant baculovirus-infected larvae of the moth Trichoplusia ni

The pathogenesis and cellular localization of juvenile hormone esterase (JHE) was examined in larvae of the moth Trichoplusia ni, infected with a recombinant baculovirus ( Autographa californica nuclear polyhedrosis virus: AcNPV) engineered to produce high levels of JHE (JHE virus). The course of JHE localization in the recombinant virus infected larvae was compared with that of both wild type AcNPV infected, and uninfected larvae, using immunogold electron microscopy. In the JHE virus infected insects, high levels of JHE were observed in the endoplasmic reticulum of all cells showing evidence of viral structures in the nucleus, except for gut cells which showed only background JHE levels. Tracheole cells and haemocytes appeared to play a role in the dissemination of infection. In uninfected larvae, fat body and epidermis were the major tissues staining for JHE, which was only detectable at peak times of JHE activity during the fifth instar: lower levels at other times could not be distinguished from background. JHE was also present in lysosomes of granular haemocytes: these lysosomes increased in number in the fifth instar compared to the fourth instar. Similar lysosome-like granules in the pericardial cells did not become highly positive for JHE antigen until the fifth instar.

Biologically active and amidated cecropin produced in a baculovirus expression system from a fusion construct containing the antibody-binding part of protein A

A synthetic antibody-binding part derived from protein A from Staphylococcus aureus was used as a fusion partner in a eukaryotic expression system employing Autographa californica nuclear polyhedrosis as a vector. This, in conjunction with an efficient signal sequence, facilitated the purification of the antibacterial peptide cecropin A from the medium of Spodoptera frugiperda cells infected with a recombinant virus. In order to increase further the concentrations of fusion protein, Trichoplusia ni larvae were used as host. Cecropin A could be obtained after cleavage of the fusion protein with CNBr. Biological activity as well as the correct structure including the C-terminal amide group was shown using electrophoresis with detection of antibacterial proteins and mass spectroscopy.

Molecular characterization of immune inhibitor A, a secreted virulence protease from Bacillus thuringiensis

The gene for the secreted neutral metalloprotease, immune inhibitor A (InA), from Bacillus thuringiensis var. alesti has been cloned and sequenced. The deduced amino acid sequence has been confirmed by partial amino acid sequencing. The central part of the amino acid sequence showed similarity to the active site in thermolysin. Southern and Western blots show that InA-related sequences are common among other B. thuringiensis subspecies. In Western blots, 17 out of 25 tested species gave a positive signal. Culture filtrates from subspecies expressing InA were toxic when injected in Trichoplusia ni larvae, whereas filtrate from a strain negative in Western blot had no effect when injected. The LD50 dose of purified InA protein injected in T. ni larvae was 12.5 +/- 2.5 ng per mg of larval body weight.

A Nuclear Polyhedrosis Virus of the Yellowstriped Armyworm (Lepidoptera: Noctuidae)

A multiple-embedded nuclear polyhedrosis virus (MNPV) was isolated and characterized from a field-collected, fourth-instar yellowstriped armyworm, Spodoptera ornithogalli Guenee, in Boone County, Missouri. The LC50 value of this virus was determined to be 0.42 occlusion bodies (OB)/mm2 for first instar and 7.0 OB/mm2 for third instar. Heliothis zea (Boddie), S. frugiperda (J. E. Smith), and Plutella xylostella (L.) larvae were not susceptible to this MNPV. Mortality was observed in Trichoplusia ni (Hubner) larvae challenged with high concentrations; however, no occlusion bodies were observed in or recovered from tissue of survivors or cadavers, and liquefaction of cadavers did not occur. This MNPV appears to be specific for S. ornithogalli ; further studies against other Spodoptera species may be justified.

Construction of genetically engineered baculovirus insecticides containing the Bacillus thuringiensis subsp. kurstaki HD-73 delta endotoxin

The delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki HD-73 was inserted into Autographa californica nuclear polyhedrosis virus (AcMNPV) using two transfer vector systems. In the first, the delta-endotoxin gene was placed under the control of the polyhedrin gene promoter in lieu of the polyhedrin coding sequences, thus deriving a polyhedrin-negative virus. In the second, it was inserted under the control of a copy of the AcMNPV p10 promoter positioned upstream of the polyhedrin gene to produce a polyhedrin-positive virus. Analysis of infected cell extracts showed that the delta-endotoxin was expressed in insect cells as 130K, 62K and 44K proteins, with peak syntheses at 18 h post-infection. Each of these products reacted with antisera specific for the complete protoxin and the cleaved, active form. When extracts from the cells infected with the polyhedrin-negative virus were fed to Trichoplusia ni larvae, feeding by the insects was inhibited and deaths occurred that were inconsistent with virus infection. This effect was also observed after the inoculum had been treated with detergents to inactivate virus particles prior to feeding to the larvae. These data indicate that the expression of the B. thuringiensis delta-endotoxin gene by a baculovirus in insect cells produces material with insecticidal activity. The biological activities of the two recombinant viruses were assessed in conventional bioassay tests by feeding virus particles or occlusion bodies to the insects. The polyhedrin-negative virus preparation appeared to be contaminated with endotoxin which inhibited feeding of the insects and prevented determination of the LD50 value. The polyhedrin-positive virus had an LD50 value about twofold higher than that of unmodified AcMNPV. The significance of these data for the genetic engineering of virus insecticides is discussed.

Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular adenosine deaminase mRNA, protein, and enzymatic activity were detected. The recombinant human adenosine deaminase represented 10% of the total cellular protein and exhibited a specific activity of 70 units/mg of protein in crude homogenate. This specific activity is 70-350 times greater than that exhibited by the enzyme in homogenates of the two most abundant natural sources of human adenosine deaminase, thymus and leukemic cells. When the recombinant virus was injected into insect larvae, the maximum recombinant enzyme was produced 4 days postinfection and represented about 2% of the total insect protein with a specific activity of 10-25 units/mg of protein. The recombinant human adenosine deaminase was purified to homogeneity from both insect cells and larvae and demonstrated to be identical to native adenosine deaminase purified from human cells with respect to molecular weight, interaction with polyclonal anti-adenosine deaminase antibody, and enzymatic properties. A pilot purification yielded 8-9 mg of homogeneous enzyme from 22 larvae. The production of large quantities of recombinant human adenosine deaminase in insect larvae is inexpensive and rapid and eliminates the need for specialized facilities for tissue culture. This method should be applicable to large-scale production of many recombinant proteins.

Another version of the human insulin receptor kinase domain: expression, purification, and characterization.

We have overexpressed another insulin receptor kinase molecule, which consists of residues 941-1343 inclusive of the human insulin receptor, by using the baculo-virus expression vector pVL941. Unlike the two previous preparations of insulin receptor kinase in this expression system, this molecule contains the complete unmodified sequence of the cytoplasmic domain of the human insulin receptor beta subunit. Our construct allows high-level expression of the recombinant protein in cultured Sf9 cells and in cabbage looper (Trichoplusia ni) larvae. An improved purification procedure yields greater than or equal to 95% pure protein in 55% yield from the cells. The specific activity of this purified protein is 3.5-fold greater than from the previously described baculovirus insulin receptor kinase (that included residues 946-1343 from the proreceptor). Like the latter molecule, the insulin receptor kinase molecule reported here sediments as a monomer, and its autophosphorylation occurs by an intramolecular process. Preliminary data about the spectroscopic features of the cytosolic domain of the human insulin receptor are presented.

Transition Probabilities for Trichoplusia ni (Lepidoptera: Noctuidae) Larvae on Cabbage as a Function of Microclimate

To identify factors affecting the spatial dynamics of Trichoplusia ni (Hubner) on cabbage, the movements of individual larvae were monitored. Larvae were marked with 32p and released individually, one per plant, on cabbage plants in research plots. Their movements were monitored by recording their position on the plant daily, locating them with a Geiger counter. In the same plots, hourly measurements of temperature and relative humidity were recorded in three different parts of the crop canopy. Transition probabilities for the larvae from and to each of five vertical plant strata were modeled as definite integrals of the Beta probability density function (pdf). The shape parameters for these Beta pdf's were modeled as a function of microclimate, and the necessary parameters to do so were estimated by the method of maximum likelihood. This model predicted the data well and was more efficient than logistic regression. The model predicts that under high temperatures and low vapor pressure deficit (vpd), larvae move down to the shaded, cooler, lower parts of the plant; whereas under low temperatures and high vpd, they moved upward toward the economically important wrapper leaves and cabbage heads.

Alteration of a lepidopteran peritrophic membrane by baculoviruses and enhancement of viral infectivity

The peritrophic membrane (PM), which lines the midgut of many insect species, has several functions. In particular, it may serve as a mechanical barrier to invading microorganisms. The protein composition of the PM from healthy and baculovirus-treated Trichoplusia ni (cabbage looper) larvae was analyzed by polyacrylamide gel electrophoresis. A specific interaction took place between baculoviruses and the PM of susceptible T. ni larvae. A 68-kDa glycoprotein of the PM disappeared within 15 min postinoculation with occlusion bodies of either Autographa californica nuclear polyhedrosis virus (AcMNPV) or T. ni nuclear polyhedrosis virus (TnSNPV). In contrast, inoculation of larvae with a T. ni granulosis virus (TnGV) resulted in the disappearance of three distinct major glycoproteins with molecular weights of 253, 194, and 123 kDa. PMs of virus-treated larvae were very fragile compared with those of untreated controls, indicative of a physical/chemical change in their structure. T. ni larval bioassays showed that a factor, present in the TnGV granulin or AcMNPV polyhedrin, enhanced the infectivity of AcMNPV. These data showed that a factor present in the occlusion bodies of three distinct baculoviruses can cause specific biochemical and structural changes in the PM. The biological significance of these observations in relation to increased larval infection is not known at this time.

Effects of infection with a granulosis virus on larval growth, development and ecdysteroid production in the cabbage looper, Trichoplusia ni

ABSTRACT. Granulosis virus‐infected Trichoplusia ni (Hûbner) larvae exhibited an increased larval life span with no supernumerary moult and no pupation. Weight gain was not affected. Insects infected shortly after hatching were slower in reaching the fourth and fifth stadia than were control insects. Haemolymph ecdysteroid titres were lower in virus‐infected insects than control insects, but these differences were only significant (P<0.05) in the fifth stadium. Electron microscopic examination of the pro thoracic glands revealed extensive granulosis virus infection, and glands from virus‐infected insects produced no RIA‐detectable ecdysteroids in vitro. Injection of 20‐OII‐ecdysone into virus‐infected larvae at various concentrations and times did not induce pupation.

Hemolymph juvenile hormone binding proteins of lepidopterous larvae: Enantiomeric selectivity and photoaffinity labeling

Juvenile hormone binding proteins (JHBP) from the hemolymph of late instar larvae of Heliothis virescens (Noctuidae), Lymantria dispar (Lymantriidae), Manduca sexta (Sphingidae), and Trichoplusia ni (Noctuidae) were purified by gel filtration by monitoring [ 3 H](10R,11S) JH I binding using a charcoal assay. Relative binding affinities of the four partially purified JHBPs for JH homologs and analogs were determined in competition experiments using unlabeled (10R,11S) JH I, (10S,11R) JH I, (10R,11S) JH II and (10S,11R) JH II, (±)JH III and (±)epoxyfarnesyl diazoacetate (EFDA) against the natural 3H-labeled JH I enantiomer. The gel-filtered proteins were photoaffinity labeled by irradiation at 254 nm in the presence of [ 3H]EFDA with and without protection by JH I. JH-specific binding proteins were located by fluorescence autoradiography. The versatility of [ 3H]EFDA as a photolabeling agent for insect JHBP is emphasized by its selective photoattachment to proteins of different JH homolog selectivity. This is the first report of enantiomeric selectivity for these proteins in which both the radioinert and radiolabeled hormones are optically active.

Standardization of HD-1-S-1980: U.S. Standard for Assay of Lepidopterous-active Bacillus thuringiensis

A new HD-1 standard, HD-1-S-1980, has replaced HD-1-S-1971, whose supply has been exhausted. In comparative bioassays by five laboratories, using HD-1-S-1971 and E-61 as standards, the potency of HD-1-S-1980 was determined to be 16,000 IU/mg. Unlike HD-1-S-1971, HD-1-S-1980 is homologous with normal HD-1's in respect to its relative activity towards Trichoplusia ni (Hubner) and Heliothis virescens (F.) larvae.

Nitrogen elimination in Trichoplusia ni parasitized by the insect parasite, Hyposoter exiguae

1. 1. The fecal uric acid concentration and total uric acid excreted per mg fresh body weight were sharply increased in Trichoplusia ni larvae parasitized by the insect parasite, Hyposoter exiguae. 2. 2. This result was consistent with previous studies that indicated an elevated rate of gluconeogenesis in parasitized individuals. 3. 3. In contrast, pair-fed larvae had lower levels of excreted uric acid than their unparasitized counterparts.

The importance of nerve terminal depolarization in pyrethroid poisoning of insects

The effects of several pyrethroids and DDT were examined on neuromuscular preparations from larvae of Musca domestica, Culex quinquefasciatus, Anopheles stephensi, and Trichoplusia ni, in order to determine the importance of the type I effect (repetitive firing) and the type II effect (increased miniature excitatory postsynaptic potential (mepsp) rate, indicating nerve terminal depolarization) in the poisoning process. α-Cyano pyrethroids were very potent in increasing mepsp rate, and although DDT and non-α-cyano compounds were more potent at inducing repetitive firing, all these compounds increased mepsp rate at higher concentrations. A variety of evidence showed that the mepsp rate-increasing activity could be an important factor in toxicity: among all of the compounds, there was a positive correlation between toxicity and mepsp rate-increasing activity; mepsp rate-increasing activity had a negative temperature dependence; nerves of kdr larvae in vitro were resistant to the mepsp rate-increasing activity, commensurate with resistance level; and finally, increased mepsp rate and neuromuscular block were observed in poisoned insects, associated with paralysis. Among type I compounds, repetitive firing activity was correlated negatively with toxicity, but positively with knockdown activity.

Influence of Soybean Leaf Extracts on Ester Cleavage in Cabbage and Soybean Loopers (Lepidoptera: Noctuidae)

Midgut-mediated ester cleavage of p-nitrophenyl acetate increased in Trichoplusia ni (Hübner) larvae that were fed on diets containing extracts from resistant and susceptible soybean, Glycine max (L.) Merr., varieties. Activity ranged as follows: control < susceptible < resistant variety. The opposite trend was noted for ester cleavage by midguts of Pseudoplusia includens (Walker) larvae fed on the same diets, such that activity ranged as follows: control > susceptible > resistant variety. The use of the inhibitors piperonyl butoxide and S,S,S-tri-n-butyl phosphorotrithioate indicated that ester cleavage was primarily mediated by hydrolytic esterases.