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.     

Production of recombinant human osteoprotegrin from Trichoplusia ni cells and Bombyx mori larvae

Human osteoprotegrin (OPG) and its truncated mutant OPG-280 and lengthened mutant OPG-Fc were constructed and successfully expressed in Trichoplusia ni cells and Bombyx mori larvae. Native SDS-PAGE and Western blot analysis revealed that OPG-Fc is present as a homodimer in Tn cells or B. mori larvae compared with OPG and OPG-280. Furthermore, the hypocalcemic effect assay showed that truncation of the C-terminal 100 residues OPG does not abolish the biological activity and Fc can be helpful in forming the OPG homodimer with improved biological activity.     

Differences in susceptibility and physiological fitness of Mexican field Trichoplusia ni strains exposed to Bacillus thuringiensis

The use of different commercial Bacillus thuringiensis (Bt) products in the Bajio guanajuatense area in Mexico began 12 yr ago, and resistance to Bt in this area has been reported for Plutella xylostella (L.) The current study provides a baseline response and resistance potential to Bt in field and laboratory strains of Bajio Trichoplusia ni (Hübner). Differences in susceptibility to Bt among T. ni populations were observed. T. ni neonates collected in Romita, Guanajuato, were more susceptible to Bt than those collected in Salvatierra or San Luis de la Paz, Guanajuato. After five generations of exposure to XenTari in the laboratory, decreased susceptibility was found only in the Salvatierra insects, with an LC50 that was 2.1-fold greater than that of a Mexican laboratory strain. The XenTari-selected San Luis de la Paz strain was from 16- to 87-fold more resistant to CrylA protoxins than U.S. (US) and Mexican laboratory strains. Although CrylAb is not a component of XenTari, this strain also was significantly less susceptible to CrylAb toxin compared with a US strain, with a resistance ratio of 40.4. The larval weights and lengths, pupal lengths, and percentage of pupation were significantly lower for the Salvatierra strain than for all other strains. The relationship of T. ni susceptibilities to Bt Cry toxins and protoxins after several generations of exposure to XenTari and its similarity to P. xylostella behavior.     

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.     

Effect of a nuclepolyhedrovirus of Autographa californica expressing the enhancin gene of Trichoplusia ni granulovirus on T. ni larvae

A recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) strain showing higher virulence against Trichoplusia ni larvae than the wild-type virus was developed. The 'enhancin' (VEF) gene of T. ni granulovirus (TnGV) and the AcMNPV polyhedrin gene were cloned into the baculovirus transfer vector pAcUW31. This plasmid and AcMNPV BacPAK6 DNA were co-transfected into the BTI-Tn5B1-4 cell line. A recombinant AcMNPV strain (BacVEFPol) was purified, amplified, and bioassayed against T. ni first instar larvae. Its estimated LC₅₀ (0.184 OB/mm²) was 2.18 times lower than the LC₅₀ estimated for the wild-type AcMNPV (0.402 OB/mm²). Likewise, an LT₅₀ of 67.7 h was estimated for the recombinant AcMNPV strain while the LT₅₀ of wild-type AcMNPV was estimated at 81.9 h. This indicates a 17.4% reduction of the time required to kill the larvae. The higher virulence of the recombinant strain, evidenced by its LC₅₀ and LT₅₀ values being lower than those of the wild-type strain, indicates that the VEF protein is expressed properly and may be occluded in the OBs.     

Multiple forms of juvenile hormone esterase active sites in the hemolymph of larvae of Trichoplusia ni

Kinetic analysis was performed on the juvenile hormone (JH) esterase activity in the hemolymph of feeding, last instar larvae of Trichoplusia ni (Lepidoptera: Noctuidae). When the results were analyzed by several different graphical and regression procedures, all approaches yielded the same conclusion that at least two forms of JH esterase active sites exist in the hemolymph. The apparent Km for one site for JH I, II and III was 8.5 X 10(-8) M, and 6.6 X 10(-8) M, respectively. The Km for the other site for JH I, II and III was 6.6 X 10(-7) M, 7.6 X 10(-7) M, 40 X 10(-7) M, respectively. When hemolymph JHE activity was subjected to high resolution isoelectric focusing (IEF), two distinct large peaks of JHE activity were observed, with pIs of 5.3 and 5.5, as well as a small peak at pI 5.1. Separate kinetic analysis of the JHE activity in each peak showed that only the higher Km active site for each substrate was present (in the 10(-7) M range). These data necessitate a change in the current model for JHE in T. ni, and some other insects, which states that a single active site is responsible for most or all of the JH esterase activity in vivo. The data also explain the different estimates of the Km of JHE in T. ni obtained by different laboratories. Studies on the purification of, and the development of inhibitors for, JHE esterase must consider the role of both JHE forms and sites in regulation of T. ni metamorphosis.     

Vairimorpha necatrix in Adipose Cells of Trichoplusia ni

Vairimorpha necatrix infected adipose ceiis of the fat body organ of Trichoplusia ni larvae 3–31/2 days after exposure of the larvae to infective spores. During the subsequent 4–6 days, the parasitized adipose cells were hypertrophied in part due to the rapid propagation of V. necatrix schizonts. A calcium-sensitive tubule network developed at the interface of the schizonts and the adipose ceil cytoplasm. The paired nuclei of V. necatrix have pores at the nuclear interface. The pores for each nucleus at this interface are spatially positioned so that they are in conjunction; hence, there is the potential for a channel system between the 2 nuclei.     

Suppression of Plutella xylostella and Trichoplusia ni in cole crops with attracticide formulations

The three key lepidopteran pests of cole, Brassica oleracea L., crops in North America are diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae); cabbage looper; Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae); and imported cabbageworm, Pieris rapae (L.) (Lepidoptera: Pieridae). Two species-specific pheromone-based experimental attracticide formulations were evaluated against these pests: LastCall DBM for P. xylostella and LastCall CL for T. ni. No LastCall formulation was available against P. rapae. Laboratory toxicity experiments confirmed the effectiveness of each LastCall formulations in killing conspecific males that made contact. In replicated small plots of cabbage and collards in central Alabama, over four growing seasons (fall 2003, spring 2004, fall 2004, and spring 2005), an attracticide treatment receiving the two LastCall formulations, each applied multiple times at the rate of 1,600 droplets per acre, was compared against Bacillus thuringiensis. subspecies kursatki (Bt) spray at action threshold and a negative untreated control. Efficacy was measured by comparing among the three treatments male capture in pheromone-baited traps, larval counts in plots, and crop damage rating at harvest. LastCall provided significant reductions in crop damage comparable to Bt in three of the four seasons. Efficacy of LastCall was dependent upon lepidopteran population densities, which fluctuated from season to season. In general, reduction in crop damage was achieved with LastCall at low-to-moderate population densities of the three species, such as typically occurs in the fall in central Alabama, but not in the spring when high P. rapae population pressure typically occurs in central Alabama. Significant reductions in pheromone trap captures did not occur in LastCall plots, suggesting that elimination of males by the toxicant (permethrin), rather than interruption of sexual communication, was the main mechanism of effect.     

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.     

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.     

Rate of increase of Autographa californica nuclear polyhedrosis virus in Trichoplusia ni larvae determined by DNA:DNA hybridization

The rate of increase and doubling time of the HOB clone of Autographa californica nuclear polyhedrosis virus (AcMNPV-HOB) in neonate Trichoplusia ni larvae was determined by measuring the increase in viral DNA through time following inoculation with average doses of 50 or 17,400 occlusion bodies per larva. Changes in total DNA and viral DNA through time were followed by fluorescence spectroscopy and quantitative slot-blot DNA:DNA hybridization, respectively. Total DNA content (i.e., larval DNA and viral DNA) of larvae infected with the intermediate dose lagged behind that of noninfected larvae 30 hr post-inoculation (p.i), reached a maximum at 51 hr p.i., and stayed constant thereafter. The total DNA content of larvae inoculated with the high dose lagged behind that of the control group from 18 hr p.i. and increased slowly until death of the larvae (ca. 48 hr p.i.). The amount of viral DNA in larvae inoculated with the intermediate dose increased exponentially between 15 and 42 hr p.i., reached a maximum at 48 hr p.i., and stayed constant until 68 hr p.i., by which time most larvae had died. The amount of viral DNA in larvae inoculated with the high dose did not increase exponentially; initially the rate of increase was the same as that for larvae inoculated with the intermediate dose but became progressively lower after 13 hr p.i. Calculations of the rate of increase for AcMNPV-HOB in neonate T. ni larvae inoculated with the intermediate dose and incubated at 29 degrees C resulted in a value of 0.264 hr-1 (doubling time: 2.63 hr).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mixed-genotype infections of Trichoplusia ni larvae with Autographa californica multicapsid nucleopolyhedrovirus: Speed of action and persistence of a recombinant in serial passage

Fast-acting recombinant baculoviruses have potential for improved insect pest suppression. However, the ecological impact of using such viruses must be given careful consideration. One strategy for mitigating risks might be simultaneous release of a wild-type baculovirus, so as to facilitate rapid displacement of the recombinant baculovirus by a wild-type. However, at what ratio must the two baculoviruses be released? An optimum release ratio must ensure both fast action, and the eventual competitive displacement of the recombinant virus and fixation of the wild-type baculovirus in the insect population. Here we challenged Trichoplusia ni larvae with different ratios of wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and a derived recombinant, vEGTDEL, which has the endogenous egt gene (coding for ecdysteroid UDP-glucosyltransferase) deleted. Time to death increased with the proportion wild-type virus in the inoculum mixture, although a 1:10 ratio (wild-type: recombinant) resulted in equally rapid insecticidal action as vEGTDEL alone. Five serial passages of three different occlusion body (OB) mixtures of the two viruses were also performed. OBs from 10 larval cadavers were pooled and used to initiate the following passage. Although the wild-type baculovirus was maintained over five passages, it did not go to fixation in most replicates of the serial passage experiment (SPE), and there was no good evidence for selection against the recombinant. Long-term maintenance of a recombinant in serial passage suggests an ecosystem safety risk. We conclude that for assessing ecological impact of recombinant viruses, SPEs in single and multiple larvae are relevant because of potential modulating effects at the between-host level.     

Transgenic tobacco plants carrying a baculovirus enhancin gene slow the development and increase the mortality of Trichoplusia ni larvae

This study was done to assess insect growth and mortality on tobacco plants transformed with baculovirus enhancin genes, as a first step toward the possible use of enhancin transgenes as part of an insect control system. Enhancin genes from Trichoplusia ni or Helicoverpa armigera baculoviruses were introduced into tobacco via Agrobacterium tumefaciens with kanamycin selection. PCR analyses of genomic DNA confirmed the presence of the enhancin genes in the kanamycin-resistant plants; however, the expression of the genes was very low and could be detected only with RT-PCR. Bioassays with Trichoplusia ni larvae showed that larval growth and development was significantly slower on some transgenic lines and that larval mortality was higher. The majority of the enhancin-transgenic plants had little or no inhibitory effect. The low expression of enhancin in plants carrying current expression cassettes and the relevance of these results to pest management are discussed.     

Characterization and cDNA cloning of midgut carboxypeptidases from Trichoplusia ni

Carboxypeptidase A and carboxypeptidase B activities from the midgut of Trichoplusia ni larvae were characterized. In the T. ni larval midgut, the primary digestive carboxypeptidase activity was attributed to carboxypeptidase A, which was eight times more active than carboxypeptidase B. Both the midgut carboxypeptidase A and carboxypeptidase B exhibited maximal activities at pH 8.0-8.5 and were similarly susceptible to inhibition by potato carboxypeptidase inhibitor and phenanthroline. The midgut carboxypeptidase activities were analyzed in T. ni larvae fed on various diet sources and the results indicated that midgut carboxypeptidase activities per milligram of gut were similar regardless of the amount of dietary proteins or amino acids. However, midgut carboxypeptidase A activity was significantly higher in larvae exposed to soybean trypsin inhibitor and was significantly lower in larvae fed on broccoli foliage. From the T. ni larval midgut, five putative carboxypeptidase cDNAs were cloned, demonstrating that midgut carboxypeptidase activities are composed of multiple carboxypeptidase types. Sequence analysis indicated that the midgut carboxypeptidases were produced as secreted proenzymes which could be activated after removal of an N-terminal activation fragment by a trypsin. Two cloned cDNAs are predicted to code for carboxypeptidase A and one cDNA is predicted to code for a putative carboxypeptidase B. The other two cDNAs are highly similar to carboxypeptidase A and carboxypeptidase B in sequences, but their activity was not predictable.