Occurrence and accumulation of viruses of Trichoplusia ni in treated field plots

Concentrations of the nuclear-polyhedrous virus (T. ni NPV) and the granulosis virus (T. ni GV) of the cabbage looper, Trichoplusia ni, in soil and on foliage were monitored up to 4 years after treatment.A single application of T. ni NPV to soil in August or 5 foliar applications of the virus at 10-day intervals in August and early September maintained substantial concentrations of the virus on foliage and high concentrations of the virus accumulated in soil. With development of natural epizootics of the virus disease in populations of the host larvae in September and October, substantial concentrations of the virus accumulated in soil and on foliage in nontreated plots, eventually becoming equal in amount with the virus in virus-treated plots. The virus accumulated more slowly in plots treated with chemical insecticides or Bacillus thuringiensis because few host larvae survived to support late-season epizootics of the disease. Small quantities of T. ni NPV were detected in heads of cabbage harvested from the plots in October.Long-term studies in which nontreated plots and plots treated with T. ni NPV or T. ni GV were replanted for up to 4 years after treatment showed that concentrations of T. ni NPV in surface soil remained constant during the winter but were reduced by dilution during cultivation preparatory to planting in the spring. T. ni NPV accumulated during the late summer and autumn with development of epizootics of the disease in populations of host larvae. Increased concentrations of the virus in soil coincided with increased concentrations on leaves in each year. T. ni GV did not persist on leaves or in soil following application and only small amounts were found 2 years after application.T. ni NPV disease was prevalent in September and October in populations of host larvae in plots in which substantial residues of the virus were found. These epizootics contributed substantially to late-season control of the looper after completion of spraying.     

Indirect plant-mediated effects on insect immunity and disease resistance in a tritrophic system

Host plant quality can significantly influence the growth and condition of phytophagous insects, and consequently their susceptibility to pathogens. This study examined the relationship between host plant quality, insect condition, immune responsiveness and resistance to pathogens in the cabbage looper, Trichoplusia ni. Two baseline and induced immune parameters were estimated, haemocyte numbers and haemolymph phenoloxidase (PO) activity, for larvae on two host plants, broccoli and cucumber. Haemolymph protein concentration was assessed as an indication of insect condition, and the susceptibility of larvae to T. ni single nucleopolyhedrovirus (SNPV) was used as a measure of disease resistance. T. ni growth, survival and condition was much higher on broccoli than cucumber. Haemocyte numbers were significantly higher in broccoli-reared larvae, whereas PO activity was not. An immune challenge induced significantly elevated numbers of haemocytes for larvae reared on both host plants, but did not affect PO activity or protein concentrations. Susceptibility to T. ni SNPV was markedly higher in larvae reared on cucumber than on broccoli. These results clearly indicate that host plant quality can affect both immune response and disease resistance of T. ni larvae and that bottom-up effects could be important in interactions between insects and entomopathogens.     

Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism.     

Susceptibility of larvae of Trichoplusia ni and Anticarsia gemmatalis to intrahemocoelic injections of conidia and blastospores of Nomuraea rileyi

The LD₅₀ for larvae of Trichoplusia ni injected with blastospores of Nomuraea rileyi was 4.30 ± 1.16 hyphal bodies/larva; the LD₅₀ for injected conidia was ca. 25,000 conidia/larva. The dose-mortality regression line for blastospores was Y = 4.6504 + 0.5487 X. Larval mortalities of Anticarsia gemmatalis and T. ni at 100 blastospores/larva were 0.4 ± 0.5% and 96.7 ± 1.9%, respectively. At a dosage of 25,000 conidia/larva, larval mortalities for A. gemmatalis and T. ni were 0.4 ± 0.5% and 43.1 ± 8.7%, respectively. Thus, larvae of A. gemmatalis were > 100 times and >200 times more resistant to injected conidia and blastospores, respectively, than were larvae of T. ni. Resistance of A. gemmatalis to N. rileyi may not be solely at the integumental barrier, as is often believed, but may also be a function of an internal physiological response.     

Sequence variation and differential splicing of the midgut cadherin gene in Trichoplusia ni

The insect midgut cadherin serves as an important receptor for the Cry toxins from Bacillus thuringiensis (Bt). Variation of the cadherin in insect populations provides a genetic potential for development of cadherin-based Bt resistance in insect populations. Sequence analysis of the cadherin from the cabbage looper, Trichoplusia ni, together with cadherins from 18 other lepidopterans showed a similar phylogenetic relationship of the cadherins to the phylogeny of Lepidoptera. The midgut cadherin in three laboratory populations of T. ni exhibited high variability, although the resistance to Bt toxin Cry1Ac in the T. ni strain is not genetically associated with cadherin gene mutations. A total of 142 single nucleotide polymorphisms (SNPs) were identified in the cadherin cDNAs from the T. ni strains, including 20 missense mutations. In addition, insertion and deletion polymorphisms (indels) were also identified in the cadherin alleles in T. ni. More interestingly, the results from this study reveal that differential splicing of mRNA also occurs in the cadherin gene expression. Therefore, variation of the midgut cadherin in insects may not only be caused by cadherin gene mutations, but could also result from alternative splicing of its mRNA regulated by factors acting in trans. Analysis of cadherin gene alleles in F2, F3 and F4 progenies from the cross between the Cry1Ac resistant and the susceptible strain after consecutive selections with Cry1Ac for three generations showed that selection with Cry1Ac did not result in an increase of frequencies of the cadherin alleles originated from the resistant strain.     

Differential susceptibility of parasitized and nonparasitized larvae of Trichoplusia ni to a nuclear polyhedrosis virus

Nonparasitized second-instar larvae of Trichoplusia ni were twice as susceptible (at the LD₅₀ level) to the singly enveloped T. ni nuclear polyhedrosis virus as those parasitized by Hyposoter exiguae (Hymenoptera: Ichneumonidae). The LD₅₀ values for nonparasitized and parasitized larvae were 1.58 × 10³ and 3.16 × 10³ polyhedra/ml of diet, respectively. The LD₉₅ value for parasitized larvae was approximateely 5 times higher than that for nonparasitized larvae. The slopes (b values) were 1.2 for parasitized larvae and 1.7 for nonparasitized larvae. The LT₅₀ values for parasitized larvae also were significantly longer than those for nonparasitized larvae. No significant difference was found between the food consumption of parasitized and nonparasitized T. ni larvae.     

Mechanism of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Greenhouse Population of the Cabbage Looper, Trichoplusia ni

The cabbage looper, Trichoplusia ni, is one of only two insect species that have evolved resistance to Bacillus thuringiensis in agricultural situations. The trait of resistance to B. thuringiensis toxin Cry1Ac from a greenhouse-evolved resistant population of T. ni was introgressed into a highly inbred susceptible laboratory strain. The resulting introgression strain, GLEN-Cry1Ac-BCS, and its nearly isogenic susceptible strain were subjected to comparative genetic and biochemical studies to determine the mechanism of resistance. Results showed that midgut proteases, hemolymph melanization activity, and midgut esterase were not altered in the GLEN-Cry1Ac-BCS strain. The pattern of cross-resistance of the GLEN-Cry1Ac-BCS strain to 11 B. thuringiensis Cry toxins showed a correlation of the resistance with the Cry1Ab/Cry1Ac binding site in T. ni. This cross-resistance pattern is different from that found in a previously reported laboratory-selected Cry1Ab-resistant T. ni strain, evidently indicating that the greenhouse-evolved resistance involves a mechanism different from the laboratory-selected resistance. Determination of specific binding of B. thuringiensis toxins Cry1Ab and Cry1Ac to the midgut brush border membranes confirmed the loss of midgut binding to Cry1Ab and Cry1Ac in the resistant larvae. The loss of midgut binding to Cry1Ab/Cry1Ac is inherited as a recessive trait, which is consistent with the recessive inheritance of Cry1Ab/Cry1Ac resistance in this greenhouse-derived T. ni population. Therefore, it is concluded that the mechanism for the greenhouse-evolved Cry1Ac resistance in T. ni is an alteration affecting the binding of Cry1Ab and Cry1Ac to the Cry1Ab/Cry1Ac binding site in the midgut.     

Host suitability and fitness-related parameters of Campoletis sonorensis (Hymenoptera: Ichneumonidae) as a parasitoid of the cabbage looper,Trichoplusia ni (Lepidoptera: Noctuidae)

The seven age-classes of Trichoplusia ni (Hübner) larvae evaluated in this study as hosts of Campoletis sonorensis indicates that early 2nd larval instar (3–5 day-old larvae) of T. ni represents the most suitable host stage for the development of the larval endoparasitoid C. sonorensis. The higher suitability of early 2nd larval instar of T. ni resulted in more parasitised larvae, a higher rate of successful parasitoid emergence, a higher rate of female progeny, and a lower rate of immature parasitoid mortality. The fitness gain of C. sonorensis on late 1st larval instar (2 day-old larvae) and late 2nd larval instar –early 3rd instars (6–8 day-old larvae) stages of T. ni is negatively affected by the trade-offs between the different physiological and behavioral characteristics influencing their suitability as hosts of C. sonorensis.     

Aerosol infectivity of a Baculovirus to Trichoplusia ni larvae: An alternative larval inoculation strategy for recombinant protein production

The baculovirus–insect expression system is a popular tool for recombinant protein production. The standard method for infecting insect larvae with recombinant baculovirus for protein production involves either feeding occlusion bodies or injecting budded virus into the cuticle. In this study, we showed that the recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) at titers >10⁸ pfu/mL efficiently infected Trichoplusia ni (T. ni) larvae through aerosol inoculation of budded virus at a pressure of 5.5 × 10⁴ Pa. The dipping T. ni larvae in virus‐containing solution efficiently infected them. These results indicate that surface contamination, either by aerosol or dipping, lead to infection via spiracles. The aerosol infection route for AcMNPV was restricted to T. ni and Plutella xylostella larvae, whereas Spodoptera litura and Helicoverpa armigera larvae were resistant to this inoculation process. The yields of the reporter proteins DsRed and EGFP from T. ni larvae following aerosol infection were nearly identical to those following oral feeding or injection. This alternative baculovirus infection strategy facilitates recombinant protein and virus production by insect larvae. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Incidence and influence on baculovirus activity

A small RNA virus infectious to Trichoplusia ni larvae (TRV) was observed as a contaminant of several Autographa californica nuclear polyhedrosis virus preparations (AcMNPV). The extent of contamination in various AcMNPV preparations was studied by means of serial enrichment passages through T. ni larvae and enzyme-linked immunosorbent assay (ELISA). TRV could not be detected by ELISA in the original preparation of AcMNPV polyhedra prepared in 1968 even after five enrichment passages. Antibody inactivation offers a possible prophylactic method against TRV but temperature inactivation (55°C) does not. Although TRV reduced larval weight, it had little or no effect on bioassays of AcMNPV to T. ni and Heliothis virescens.     

Biochemical comparison of polyhedral protein from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

Polyhedral protein preparations from five nuclear polyhedrosis viruses isolated from four closely related host insects of the noctuid subfamily Plusiinae were characterized by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), high voltage paper electrophoresis, and amino acid analysis. The viruses were Autographa california multiple-embedded virion type (MEV), Pseudoplusia includens singly embedded virion type (SEV), Rachiplusia ou MEV, Trichoplusia ni MEV, and T. ni SEV. Each was produced in its own host; A. californica MEV was also produced in T. ni larvae to determine possible host influence on polyhedral protein chemistry. Each test revealed minor, reproducible differences among most isolates. In SDS-PAGE, the major protein component ranged from 26,700 to 28,300 MW among the isolates. Differences were confined to minor protein bands or to band intensity. Peptide maps showed differences among most isolates in numbers of acidic and basic peptide spots, but all had an identical number of neutral spots. Migration patterns also differed among most isolates. The amino acid compositions of the six polyhedral inclusions were very similar, with aspartic and glutamic acids being the predominant residues. The greatest differences were found between the MEV and SEV groups, with lesser differences within each group. In all analyses, A. californica MEV produced in A. californica was indistinguishable from virus produced in T. ni.     

Novel Isolate of Bacillus thuringiensis subsp. thuringiensis That Produces a Quasicuboidal Crystal of Cry1Ab21 Toxic to Larvae of Trichoplusia ni

A new isolate (IS5056) of Bacillus thuringiensis subsp. thuringiensis that produces a novel variant of Cry1Ab, Cry1Ab21, was isolated from soil collected in northeastern Poland. Cry1Ab21 was composed of 1,155 amino acids and had a molecular mass of 130.5 kDa, and a single copy of the gene coding for this endotoxin was located on a ~75-kbp plasmid. When synthesized by the wild-type strain, Cry1Ab21 produced a unique, irregular, bipyramidal crystal whose long and short axes were both approximately 1 μm long, which gave it a cuboidal appearance in wet mount preparations. In diet incorporation bioassays, the 50% lethal concentrations of the crystal-spore complex were 16.9 and 29.7 μg ml⁻¹ for second- and fourth-instar larvae of the cabbage looper, Trichoplusia ni, respectively, but the isolate was essentially nontoxic to larvae of the beet armyworm, Spodoptera exigua. A bioassay of autoclaved spore-crystal preparations showed no evidence of β-exotoxin activity, indicating that toxicity was due primarily to Cry1Ab21. Studies of the pathogenesis of isolate IS5056 in second-instar larvae of T. ni showed that after larval death the bacterium colonized and subsequently sporulated extensively throughout the cadaver, suggesting that other bacteria inhabiting the midgut lumen played little if any role in mortality. As T. ni is among the most destructive pests of vegetable crops in North America and has developed resistance to B. thuringiensis, this new isolate may have applied value.     

Resistance of Trichoplusia ni Populations Selected by Bacillus thuringiensis Sprays to Cotton Plants Expressing Pyramided Bacillus thuringiensis Toxins Cry1Ac and Cry2Ab

Two populations of Trichoplusia ni that had developed resistance to Bacillus thuringiensis sprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. The T. ni colonies resistant to Bacillus thuringiensis serovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistant T. ni strains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in the T. ni populations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptible T. ni strain. The resistance to Cry2Ab in resistant T. ni was an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops.     

Behavior of a Recombinant Baculovirus in Lepidopteran Hosts with Different Susceptibilities

Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae of M. brassicae. Both viruses produced a greater yield in M. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.     

In vivo and in vitro host range of Autographa californica nuclear polyhedrosis virus and Spodoptera frugiperda nuclear polyhedrosis virus

Baculoviruses from Autographa californica (AcNPV-E2) and Spodoptera frugiperda (SfNPV-2) were titered in five insect cell lines: IAL-PID2, IAL-SFD1, IPLB-SF-21AE, TN-368, and IAL-TND1. AcNPV-E2 replicated in all the cell lines while SfNPV-2 did not replicate in the lines TN-368 and IAL-TND1. Further in vivo studies of SfNPV-2 showed the virus was not infectious when fed to Trichoplusia ni larvae per os or when injected into the hemocoel. These data suggest that the barrier to SfNPV-2 infectivity in T. ni is at the cellular level, as opposed to the midgut.     

Comparison of nuclear polyhedrosis virus replication in five lepidopteran cell lines

Comparative studies were performed on the replication of the Autographa californica nuclear polyhedrosis virus in cell lines from Estigmene acrea, BTI-EAA; Lymantria dispar, IPLB-LD64BA; Mamestra brassicae, IZD-MB0503; Spodoptera frugiperda, IPLB-SF1254; and Trichoplusia ni, TN-368. Significant differences were observed in the amount of virus obtained from the cell lines, with M. brassicae and T. ni producing more polyhedra than the other lines. These two cell lines also produced nonoccluded virus most rapidly, followed by S. frugiperda, E. acrea, and L. dispar. Sensitivities of the cell lines to infection by the virus, as determined by plaque formation, followed the same pattern, with M. brassicae being most sensitive and L. dispar least so. The T. ni cell line produced polyhedra which were more pathogenic to T. ni larvae than those from the other cells. These differences have important implications in the application of cell cultures in the development of microbial insecticides.     

Midgut cysteine protease-inhibiting activity in Trichoplusia ni protects the peritrophic membrane from degradation by plant cysteine proteases

The action of plant cysteine proteases on the midgut peritrophic membrane (PM) of a polyphagous herbivorous lepidopteran, Trichoplusia ni, was studied. Proteins in PMs isolated from T. ni larvae were confirmed to be highly resistant to the serine proteinases trypsin and chymotrypsin, but were susceptible to degradation by plant cysteine proteases, which is consistent with the known molecular and biochemical characteristics of the T. ni PM proteins. However, the PM proteins were not degraded by plant cysteine proteases in larvae or in the presence of larval midgut fluid in vitro. With further biochemical analysis, cysteine protease-inhibiting activity was identified in the midgut fluid of T. ni larvae. The cysteine protease-inhibiting activity was heat resistant and active in the tested pH range from 6.0 to 10.0, but could be suppressed by thiol reducing reagents or reduced by treatment with catalase. In addition to T. ni, cysteine protease-inhibiting activity was also identified from two other polyphagous Lepidoptera species, Helicoverpa zea and Heliothis virescens. In conclusion, results from this study uncovered that herbivorous insects may counteract the attack of plant cysteine proteases on the PM by inhibiting the potentially insecticidal cysteine proteases from plants in the digestive tract. However, the biochemical identity of the cysteine protease-inhibiting activity in midgut fluid has yet to be identified.     

Genetic variation in fitness parameters associated with resistance to Bacillus thuringiensis in male and female Trichoplusia ni

Resistance of insects to insecticides is often associated with reduced fitness in the absence of selection. We examined fitness trade-offs associated with resistance to the microbial insecticide, Bacillus thuringiensis (Bt), across full-sib families in a resistant population of Trichoplusia ni. Significant genetic variation in and heritability of susceptibility to Bt occurred among the full-sib families. Male pupal weight was positively correlated with Bt susceptibility, indicating a potential fitness cost, but no such correlation occurred for females. Significant heritability for pupal weight was present for males but not females. A significant negative genetic correlation existed between development time and Bt susceptibility, indicating that resistant larvae developed more slowly than susceptible larvae. Selection for Bt resistance in T. ni resulted in changes in life-history traits that affected males more than females.