Lack of fitness costs in dsRNA-resistant Leptinotarsa decemlineata ([Coleoptera]: [Chrysomelidae]).

The Colorado potato beetle, Leptinotarsa decemlineata (Say) ([Coleoptera]: [Chrysomelidae]), is the most important defoliator of solanaceous plants worldwide. This insect displays a notorious ability in adapting to biological and synthetic insecticides, although in some cases this adaptation carries relevant fitness costs. Insecticidal gene silencing by RNA interference is a novel mode of action pesticide against L. decemlineata that is activated by ingestion of a double stranded RNA (dsRNA) targeting a vital L. decemlineata gene. We previously reported laboratory selection of a > 11,000-fold resistant strain of L. decemlineata to a dsRNA delivered topically to potato leaves. In this work, we tested the existence of fitness costs in this dsRNA-resistant colony by comparing biological parameters to the parental strain and an additional susceptible reference strain. Biological parameters included length of egg incubation period, number of eggs per clutch, egg viability, larval viability, length of larval and pupal periods, adult emergence, number of eggs laid per day, sex ratio, and adult longevity. Comparisons between the 3 beetle strains detected no fitness costs associated with resistance to dsRNA. This information is important to guide effective insect resistance management plans for dsRNA insecticides against L. decemlineata applied topically to potato leaves.

Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests.

Cry1Ac protoxin (the active insecticidal toxin in both Bollgard and Bollgard II cotton [Gossypium hirsutum L.]), and Cry2Ab2 toxin (the second insecticidal toxin in Bollgard II cotton) were bioassayed against five of the primary lepidopteran pests of cotton by using diet incorporation. Cry1Ac was the most toxic to Heliothis virescens (F.) and Pectinophora gossypiella (Saunders), demonstrated good activity against Helicoverpa zea (Boddie), and had negligible toxicity against Spodoptera exigua (Hübner) and Spodoptera frugiperda (J. E. Smith). Cry2Ab2 was the most toxic to P. gossypiella and least toxic to S. frugiperda. Cry2Ab2 was more toxic to S. exigua and S. frugiperda than Cry1Ac. Of the three insect species most sensitive to both Bacillus thuringiensis (Bt) proteins (including H. zea), P. gossypiella was only three-fold less sensitive to Cry2Ab2 than Cry1Ac, whereas H. virescens was 40-fold less sensitive to Cry2Ab2 compared with CrylAc. Cotton plants expressing Cry1Ac only and both Cry1Ac and Cry2Ab2 proteins were characterized for toxicity against H. zea and S.frugiperda larvae in the laboratory and H. zea larvae in an environmental chamber. In no-choice assays on excised squares from plants of different ages, second instar H. zea larvae were controlled by Cry1Ac/Cry2Ab2 cotton with mortality levels of 90% and greater at 5 d compared with 30-80% mortality for Cry1Ac-only cotton, depending on plant age. Similarly, feeding on leaf discs from Cry1Ac/Cry2Ab2 cotton resulted in mortality of second instars of S.frugiperda ranging from 69 to 93%, whereas exposure to Cry1Ac-only cotton yielded 20-69% mortality, depending on plant age. When cotton blooms were infested in situ in an environmental chamber with neonate H. zea larvae previously fed on synthetic diet for 0, 24, or 48 h, 7-d flower abortion levels for Cry1Ac-only cotton were 15, 41, and 63%, respectively, whereas for Cry1Ac/Cry2Ab2 cotton, flower abortion levels were 0, 0, and 5%, respectively. Cry1Ac and Cry2Ab2 concentrations were measured within various cotton tissues of Cry1Ac-only and Cry1Ac/Cry2Ab2 plants, respectively, by using enzyme-linked immunosorbent assay. Terminal leaves significantly expressed the highest, and large leaves, calyx, and bracts expressed significantly the lowest concentrations of Cry1Ac, respectively. Ovules expressed significantly the highest, and terminal leaves, large leaves, bracts, and calyx expressed significantly (P < 0.05) the lowest concentrations of Cry2Ab2. These results help explain the observed differences between Bollgard and Bollgard II mortality against the primary lepidopteran cotton pests, and they may lead to improved scouting and resistance management practices, and to more effective control of these pests with Bt transgenic crops in the future.

Improving the insecticidal activity of Bacillus thuringiensis subsp. aizawai against Spodoptera exigua by chromosomal expression of a chitinase gene.

A transcriptionally fused gene comprising the P19 gene from Bacillus thuringiensis subsp. israelensis fused with a chitinase gene (chiBlA) from B. licheniformis was integrated into the B. thuringiensis subsp. aizawai BTA1 genome by homologous recombination. The resulting B. thuringiensis subsp. aizawai strain (INT1) showed growth and sporulation comparable with that of the wild-type strain. INT1 produced four chitinases of different molecular masses (i.e., 66, 55, 39, 36 kDa). Three of these (66, 55, 36 kDa) were derived from the cloned chiBlA gene, whereas the 39-kDa chitinase originated from BTA1. Using surface contamination bioassays, the 50% lethal concentration of lyophilized whole culture broth of INT1 against Spodoptera exigua neonate larvae was 12.2 microg/cm2, compared with 30.8 microg/cm2 for BTA1. Bioassays using filtered culture supernatant of INT1 (110 microg/cm2) together with trypsin-activated purified Cry1C protein of B. thuringiensis (1,280 ng/cm2) showed 75.0% mortality, compared with 56.7% mortality for Cry1C combined with BTA1 at the same concentration. Using scanning electron microscopy, clear perforations were observed in S. exigua fifth instar peritrophic membranes incubated with either crude or purified chitinase, or isolated from fifth instar S. exigua fed purified chitinase since the first instar. These results show that chitinase can increase the activity of B. thuringiensis subsp. aizawai against S. exigua. This is the first documentation of expressing a chimeric chitinase gene on the chromosome of B. thuringiensis; and chromosomal integration might be used as a potential technique for strain improvement.

Pyrethroid resistance and cross-resistance in the German cockroach, Blattella germanica (L).

A German cockroach (Blatella germanica (L)) strain, Apyr-R, was collected from Opelika, Alabama after control failures with pyrethroid insecticides. Levels of resistance to permethrin and deltamethrin in Apyr-R (97- and 480-fold, respectively, compared with a susceptible strain, ACY) were partially or mostly suppressed by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF), suggesting that P450 monooxygenases and hydrolases are involved in resistance to these two pyrethroids in Apyr-R. However, incomplete suppression of pyrethroid resistance with PBO and DEF implies that one or more additional mechanisms are involved in resistance. Injection, compared with topical application, resulted in 43- and 48-fold increases in toxicity of permethrin in ACY and Apyr-R, respectively. Similarly, injection increased the toxicity of deltamethrin 27-fold in ACY and 28-fold in Apyr-R. These data indicate that cuticular penetration is one of the obstacles for the effectiveness of pyrethroids against German cockroaches. However, injection did not change the levels of resistance to either permethrin or deltamethrin, suggesting that a decrease in the rate of cuticular penetration may not play an important role in pyrethroid resistance in Apyr-R. Apyr-R showed cross-resistance to imidacloprid, with a resistance ratio of 10. PBO treatment resulted in no significant change in the toxicity of imidacloprid, implying that P450 monooxygenase-mediated detoxication is not the mechanism responsible for cross-resistance. Apyr-R showed no cross-resistance to spinosad, although spinosad had relatively low toxicity to German cockroaches compared with other insecticides tested in this study. This result further confirmed that the mode of action of spinosad to insects is unique. Fipronil, a relatively new insecticide, was highly toxic to German cockroaches, and the multi-resistance mechanisms in Apyr-R did not confer significant cross-resistance to this compound. Thus, we propose that fipronil could be a valuable tool in integrated resistance management of German cockroaches.

Exchange of domain I from Bacillus thuringiensis Cry1 Toxins Influences protoxin stability and crystal formation.

Influence of domain I exchange on the stability and production of Bacillus thuringiensis Cry1 protoxins as well as on the shape of inclusion and toxicity to Spodoptera exigua and Plutella xylostella larvae was investigated. Chimeric genes were prepared by exchanging the regions coding for domain I between Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The AcCC chimera accumulated into bipyramidal inclusion bodies, whereas CEE produced round-shaped inclusion bodies, and ECC and AaEE protoxins produced small granules. AbEE and EAaAa did not produce any inclusion body and were visualized by immunodetection only. AcCC, CEE, ECC, and AaEE were stable to trypsin, whereas AbEE and EAaAa were not. Bioassays showed that the chimeras were not toxic in vivo. However, S. exigua larvae fed with the activated AcCC toxin displayed a lower growth rate.

Immunological analysis of phloem sap of Bacillus thuringiensis corn and of the nontarget herbivore Rhopalosiphum padi (Homoptera: Aphididae) for the presence of Cry1Ab.

Phloem sap of transgenic Bacillus thuringiensis (Bt) corn expressing a truncated form of the B. thuringiensis delta-endotoxin Cry1Ab, sap sucking aphids feeding on Bt corn and their honeydew were analysed for presence of Cry1Ab using ELISA. Phloem sap of Bt and non-Bt corn was collected using a newly developed technique with a microcapillary being directly inserted into the phloem tubes. Using this technique, no Cry1Ab was detected in the phloem sap. In contrast, measurable concentrations of Cry1Ab in the range of 1 ppb were detected when phloem sap of pooled leaf samples was extracted using EDTA buffer. This was probably because of Cry1Ab toxin released from damaged cells. When analysing apterous adults of Rhopalosiphum padi L. and their honeydew, no Cry1Ab could be detected. In contrast, Cry1Ab was clearly detected in both larvae of the leaf chewing herbivore Spodoptera littoralis (Boisduval) and their faeces, showing that Cry1Ab is detectable after ingestion and excretion by herbivores. These results suggest that R. padi ingests or contains no or only very low concentrations of Cry1Ab in the range of the detection limit. In consequence it is hypothesized that R. padi as an important prey for beneficial insects in corn is unlikely to cause any harm to its antagonists due to mediating Bt toxin.

Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals.

In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways, a problem that is compounded by variable expression levels. In order to accomplish expression of multiple genes in a single transformation event, we have introduced several genes into the chromoplast genome. We confirmed stable integration of the cry2Aa2 operon by PCR and Southern blot analyses in T(0) and T(1) transgenic plants. Foreign protein accumulated at 45.3% of the total soluble protein in mature leaves and remained stable even in old bleached leaves (46.1%), thereby increasing the efficacy and safety of transgenic plants throughout the growing season. This represents the highest level of foreign gene expression reported in transgenic plants to date. Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves. Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals. Formation of crystals of foreign proteins (due to hyperexpression and folding by the putative chaperonin, ORF 2) provides a simple method of purification by centrifugation and enhances stability by protection from cellular proteases. Demonstration of expression of an operon in transgenic plants paves the way to engineering new pathways in plants in a single transformation event.

Expression of orf1 from the Bacillus thuringiensis NRD-12 cry2Aa1 operon.

The 5' untranslated region and the orf1 sequence from the cry2Aa1 operon from Bacillus thuringiensis subsp. kurstaki NRD-12 were sequenced and compared to that from strain HD-1. The start codon described in HD-1 does not yield in NRD-12 a protein of the expected size of 20 kDa, but a 10-amino acid peptide. A second, highly conserved start codon is located 25 bp downstream from the first one and corresponds to an open reading frame of the same size in all known orf1-related sequences. Expression of lacZ gene fusions created at the level of the first ATG, second ATG, and stop codon of the NRD-12 orf1 sequence showed that orf1 is translated from the second ATG. The expected protein is 19 kDa in size. The expression starts at t2, which is in agreement with the presence of a BtI promoter in the cry2Aa1 operon.

Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects.

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5, 000-10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.

Differential effects of helper proteins encoded by the cry2A and cry11A operons on the formation of Cry2A inclusions in Bacillus thuringiensis.

To compare the differential effects of cry2A operon orf2 (29-kDa protein gene) and Cry11A operon orf3 (20-kDa protein gene) on Cry2A synthesis and inclusion formation, we expressed the cry2A gene along with either the 29-kDa gene, 20-kDa gene, or both genes. Constructs containing 20-kDa, in the presence or absence of 29-kDa, produced more Cry2A than constructs which lacked this gene. Cry2A synthesis was also higher when the 29-kDa gene was included with 20-kDa in the construct. However, even in the presence of increased Cry2A synthesis facilitated by the 20-kDa gene, typical Cry2A crystals did not form if the 29-kDa gene was not included in the construct. These results suggest that the 29-kDa and 20-kDa proteins have different functions, with the 20-kDa protein acting like a molecular chaperone to enhance net Cry2A synthesis, and the 29-kDa protein likely serving as a template for the stabilization of Cry2A molecules and their organization into the rectangular inclusion characteristic of wild-type Cry2A crystals.

Synergism between Bacillus thuringiensis Spores and Toxins against Resistant and Susceptible Diamondback Moths (Plutella xylostella).

We studied the effects of combinations of Bacillus thuringiensis spores and toxins on the mortality of diamondback moth (Plutella xylostella) larvae in leaf residue bioassays. Spores of B. thuringiensis subsp. kurstaki increased the toxicity of crystals of B. thuringiensis subsp. kurstaki to both resistant and susceptible larvae. For B. thuringiensis subsp. kurstaki, resistance ratios were 1,200 for a spore-crystal mixture and 56,000 for crystals without spores. Treatment of a spore-crystal formulation of B. thuringiensis subsp. kurstaki with the antibiotic streptomycin to inhibit spore germination reduced toxicity to resistant larvae but not to susceptible larvae. In contrast, analogous experiments with B. thuringiensis subsp. aizawai revealed no significant effects of adding spores to crystals or of treating a spore-crystal formulation with streptomycin. Synergism occurred between Cry2A and B. thuringiensis subsp. kurstaki spores against susceptible larvae and between Cry1C and B. thuringiensis subsp. aizawai spores against resistant and susceptible larvae. The results show that B. thuringiensis toxins combined with spores can be toxic even though the toxins and spores have little or no independent toxicity. Results reported here and previously suggest that, for diamondback moth larvae, the extent of synergism between spores and toxins of B. thuringiensis depends on the strain of insect, the type of spore, the set of toxins, the presence of other materials such as formulation ingredients, and the concentrations of spores and toxins.

Simultaneous production of the 34-kDa and 40-kDa proteins from Bacillus thuringiensis subsp. thompsoni is required for the formation of inclusion bodies.

Cooperation of two crystal proteins from Bacillus thuringiensis subsp. thompsoni. strain HnC was shown to be essential for the formation of inclusion bodies. Expression of the operon containing the 34-kDa and 40-kDa protein genes from HnC in a B. thuringiensis crystal minus strain resulted in the formation of inclusion bodies identical to those from strain HnC. Interruption of one of the genes in the operon led to the lack of inclusion body and to low production of the remaining protein. Absence of inclusion body and low rate of protein production were also observed when both genes were simultaneously expressed but on different vectors. To show a cooperative effect in the formation of the inclusion body, both proteins must be produced from the same transcript.

Toxicity of Bacillus thuringiensis Spore and Crystal Protein to Resistant Diamondback Moth (Plutella xylostella).

A colony of Plutella xylostella from crucifer fields in Florida was used in mortality bioassays with HD-1 spore, CryIA(a), CryIA(b), CryIA(c), CryIB, CryIC, CryID, CryIE, or CryIIA. The data revealed high levels of field-evolved resistance to HD-1 spore and all CryIA protoxins and no resistance to CryIB, CryIC, or CryID. CryIE and CryIIA were essentially not toxic. When HD-1 spore was combined 1:1 with protoxin and fed to susceptible larvae, spore synergized the activity of CryIA and CryIC 5- to 8-fold and 1.7-fold, respectively, and did not synergize the mortality of CryIIA. When fed to Florida larvae, spore failed to synergize the activity of all three CryIA protoxins, synergized the activity of CryIC 5.3-fold, and did not synergize the mortality for CryIIA. Binding studies with CryIA(b), CryIB, and CryIC were performed to determine possible mechanisms of resistance. The two techniques used were (i) binding of biotinylated toxin to tissue sections of larval midguts and (ii) binding of biotinylated toxin to brush border membrane vesicles prepared from whole larvae. Both showed dramatically reduced binding of CryIA(b) in resistant larvae compared with that in susceptible larvae but no differences in binding of CryIB or CryIC.

Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae).

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 4.30 (mu)g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.

Reversal of resistance to Bacillus thuringiensis in Plutella xylostella.

Continued success of the most widely used biopesticide, Bacillus thuringiensis, is threatened by development of resistance in pests. Experiments with Plutella xylostella (diamondback moth), the first insect with field populations resistant to B. thuringiensis, revealed factors that promote reversal of resistance. In strains of P. xylostella with 25- to 2800-fold resistance to B. thuringiensis compared with unselected strains, reversal of resistance occurred when exposure to B. thuringiensis was stopped for many generations. Reversal of resistance was associated with restoration of binding of B. thuringiensis toxin CryIA(c) to brush-border membrane vesicles and with increased biotic fitness. Compared with susceptible colonies, revertant colonies had a higher proportion of extremely resistant individuals. Revertant colonies responded rapidly to reselection for resistance. Understanding reversal of resistance will help to design strategies for extending the usefulness of this environmentally benign insecticide.

Insecticidal activity of the CryIIA protein from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki expressed in Escherichia coli and Bacillus thuringiensis and in a leaf-colonizing strain of Bacillus cereus.

A 4.0-kb BamHI-HindIII fragment encoding the cryIIA operon from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki was cloned into Escherichia coli. The nucleotide sequence of the 2.2-kb AccI-HindIII fragment containing the NRD-12 cryIIA gene was identical to the HD-1 and HD-263 cryIIA gene sequences. Expression of cryIIA and subsequent purification of CryIIA inclusion bodies resulted in a protein with insecticidal activity against Heliothis virescens, Trichoplusia ni, and Culex quinquefasciatus but not Spodoptera exigua. The 4.0-kb BamII-HindIII fragment encoding the cryIIA operon was inserted into the B. thuringiensis-E. coli shuttle vector pHT3101 (pMAU1). pMAU1 was used to transform an acrystalliferous HD-1 strain of B. thuringiensis subsp. kurstaki and a leaf-colonizing strain of B. cereus (BT-8) by using electroporation. Spore-crystal mixtures from both transformed strains were toxic to H. virescens and T. ni but not Helicoverpa zea or S. exigua.

Resistance to Toxins from Bacillus thuringiensis subsp. kurstaki Causes Minimal Cross-Resistance to B. thuringiensis subsp. aizawai in the Diamondback Moth (Lepidoptera: Plutellidae).

Repeated exposure in the field followed by laboratory selection produced 1,800- to >6,800-fold resistance to formulations of Bacillus thuringiensis subsp. kurstaki in larvae of the diamondback moth, Plutella xylostella. Four toxins from B. thuringiensis subsp. kurstaki [CryIA(a), CryIA(b), CryIA(c), and CryIIA] caused significantly less mortality in resistant larvae than in susceptible larvae. Resistance to B. thuringiensis subsp. kurstaki formulations and toxins did not affect the response to CryIC toxin from B. thuringiensis subsp. aizawai. Larvae resistant to B. thuringiensis subsp. kurstaki showed threefold cross-resistance to formulations of B. thuringiensis subsp. aizawai containing CryIC and CryIA toxins. This minimal cross-resistance may be caused by resistance to CryIA toxins shared by B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai.

Broad-spectrum resistance to Bacillus thuringiensis toxins in Heliothis virescens.

Evolution of pest resistance to insecticidal proteins produced by Bacillus thuringiensis (Bt) would decrease our ability to control agricultural pests with genetically engineered crops designed to express genes coding for these proteins. Previous genetic and biochemical analyses of insect strains with resistance to Bt toxins indicate that (i) resistance is restricted to single groups of related Bt toxins, (ii) decreased toxin sensitivity is associated with changes in Bt-toxin binding to sites in brush-border membrane vesicles of the larval midgut, and (iii) resistance is inherited as a partially or fully recessive trait. If these three characteristics were common to all resistant insects, specific crop-variety deployment strategies could significantly diminish problems associated with resistance in field populations of pests. We present data on Bt-toxin resistance in Heliothis virescens, a major agricultural pest targeted for control with Bt-toxin-producing crops. A laboratory strain of H. virescens developed resistance in response to selection with the Bt toxin CryIA(c). In contrast to other cases of Bt-toxin resistance, this H. virescens strain exhibits cross-resistance to Bt toxins that differ significantly in structure and activity. Furthermore, the resistance in this strain is not accompanied by significant changes in toxin binding, and resistance is inherited as an additive trait when larvae are treated with high doses of CryIA(c) toxin. These findings have important implications for Bt-toxin-based pest control.

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.

Impact of UV radiation on activity of linear furanocoumarins andBacillus thuringiensis var.Kurstaki againstSpodoptera exigua: Implications for tritrophic interactions.

Acidic fogs with a pH of 2.0 and duration of 2 hr did not reduce the efficacy ofBacillus thuringiensis var.Kurstaki (Berliner). Therefore, the impact of UV radiation was investigated on the interactions between (1) levels of the antibacterial linear furanocoumarins psoralen, bergapten, and xanthotoxin inApium graveolens (L.) occurring following a 2.0 pH acidic fog episode, (2) the noctuidSpodoptera exigua (Hübner), and (3) a sublethal dosage of the microbial pathogenB. thuringiensis var.Kurstaki. Mean time to pupation in the absence of UV radiation (survival was too low to conduct this analysis for insects exposed to UV) was significantly extended by the addition of either psoralens orB. thuringiensis. Larvae developing on diets containingB. thuringiensis plus psoralens required nearly 40% longer to pupate than controls, but their effects were additive as the interaction was not significant. Although the mean times to adult emergence were significantly different, time spent in the pupal stage did not vary significantly between treatments, indicating that increases in larval developmental time were responsible for the observed decrease in developmental rate. Mean time to mortality, a weighted average time of death, was not significantly affected by any of the treatments. In a 2 × 2 × 2 factorial analysis, all main effects (linear furanocoumarins.B. thuringiensis, UV radiation) reduced survival significantly, as did the three-way interaction. Thus, antagonistic interactions with psoralens that would reduce the effectiveness ofB. thuringiensis in the field were not observed. When pairs of main effects were nested within the two levels (presence and absence) of the third factor, several two-way interactions were found. Interestingly, the activity ofB. thuringiensis and the psoralens, individually or in combination, was enhanced by exposure to UV radiation. Implications of this research are discussed for both natural and agricultural ecosystems.