Enhanced toxicity of Bacillus thuringiensis Cry3A delta-endotoxin in coleopterans by mutagenesis in a receptor binding loop.

We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 350-354. Two mutant toxins, A1 (R(345)A,Y(350)F,Y(351)F) and A2 (R(345)A,DeltaY(350), DeltaY(351)), showed significantly improved toxicity against Tenebrio molitor (yellow mealworm). The mutant toxin A1 was also more potent against both Leptinotarsa decemlineata (Colorado potato beetle) and Chrysomela scripta (cottonwood leaf beetle), while A2 displayed enhanced toxicity only in L. decemlineata. Competitive binding assays of L. decemlineata brush border membrane vesicles (BBMV) revealed that binding affinities for the A1 and A2 mutant toxins were ca. 2.5-fold higher than for the wild-type Cry3 toxin. Similar binding assays with C. scripta BBMV revealed a ca. 5-fold lower dissociation rate for the A1 mutant as compared to that of Cry3A.

Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence.

The insecticidal activity of Bacillus thuringiensis strains toxic to coleopterous insects is due to Cry3 proteins assembled into small rectangular crystals. Toxin synthesis in these strains is dependent primarily upon a promoter that is active in the stationary phase and a STAB-SD sequence that stabilizes the cry3 transcript-ribosome complex. Here we show that significantly higher yields of Cry3A can be obtained by using dual sporulation-dependent cyt1Aa promoters to drive the expression of cry3Aa when the STAB-SD sequence is included in the construct. The Cry3A yield per unit of culture medium obtained with this expression system was 12.7-fold greater than that produced by DSM 2803, the wild-type strain of B. thuringiensis from which Cry3Aa was originally described, and 1.4-fold greater than that produced by NB176, a mutant of the same strain containing two or three copies of cry3Aa, which is the active ingredient of the commercial product Novodor, used for control of beetle pests. The toxicities of Cry3A produced with this construct or the wild-type strain were similar when assayed against larvae of the cottonwood leaf beetle, Chrysomela scripta. The volume of Cry3A crystals produced with cyt1Aa promoters and the STAB-SD sequence was 1.3-fold that of typical bipyramidal Cry1 crystals toxic to lepidopterous insects. The dual-promoter/STAB-SD system offers an additional method for potentially improving the efficacy of insecticides based on B. thuringiensis.

Interactions between a Nosema sp. (Microspora: nosematidae) and nuclear polyhedrosis virus infecting the gypsy moth, Lymantria dispar (Lepidoptera: lymantriidae).

Simultaneous and sequential per os inoculations of gypsy moth larvae with the Lymantria dispar nuclear polyhedrosis virus (LdNPV) and a Nosema sp. from Portugal demonstrated that the interaction of two pathogens during coinfection was variable, ranging from synergistic to antagonistic. Susceptibility of gypsy moth larvae to viral infection was unaffected by simultaneous and subsequent microsporidian infection. This resulted from the comparatively slow pathogenesis of the microsporidium when compared to the virus. Viral infectivity, however, increased 10-fold when larvae were preinfected with Nosema sp. per os, or through transovarial infection. Time to death decreased for larvae infected with both pathogens compared to larvae infected with the virus alone. Polyhedron production was significantly reduced by microsporidian infection preceding viral infection. In this infection sequence, larvae died at an earlier stage and were less than half the mass of cadavers infected with virus alone. The biological significance of these results on gypsy moth population dynamics and the implication for use of this Nosema sp. from Portugal in gypsy moth biological control are discussed in the context of viral epizootiology.

The insecticidal CryIB crystal protein of Bacillus thuringiensis ssp. thuringiensis has dual specificity to coleopteran and lepidopteran larvae.

The crystals found in sporulation extracts of Bacillus thuringiensis (Berliner) contain proteins that are highly toxic to insects. Different crystal proteins exhibit distinct specificities for restricted groups of insects. An uncharacterized strain of B. thuringiensis (BtS2), derived from China, was found to carry several crystal protein genes and to be toxic to a wide variety of insects, including some coleopterans. Surprisingly, the coleopteran toxicity was traced to a CryIB-class protein. The previously cloned CryIB protein from B. thuringiensis ssp. thuringiensis strain HD-290-I, which was believed to be lepidopteran-specific, was also found to be toxic to at least two species of coleopteran larvae under certain conditions. In contrast to CryIB toxicity toward lepidopterans, the coleopteran activity of CryIB is enhanced by solubilization and by truncation with trypsin prior to administration. The magnitude of this effect varies with the host species and is reversed for the one lepidopteran tested. These results suggest that, for at least some insects, the apparent host specificity of CryIB may depend both on differences in midgut environment and on differences in toxin-receptor interaction. The results of insect toxicity experiments with a series of deletion mutants allowed definition of a CryIB protein fragment of ca. 65 kDa as the smallest peptide that retains bioactivity against both lepidopteran and coleopteran larvae. Deletions smaller than this resulted in the production of a protein that was nontoxic to both lepidopteran and coleopteran larvae.

Characterization of the pH-mediated solubility of Bacillus thuringiensis var. san diego native delta-endotoxin crystals.

Native crystals of Bacillus thuringiensis var. san diego, a coleopteran-specific delta-endotoxin, were metabolically labelled with [35S]methionine. Specific activity was 82,000 CPM/micrograms (2.44 Ci/mmol). Using a universal buffer formulated with the same ionic strength at every pH, we determined that native crystals dissolve above pH 10 and below pH 4. At the acidic pH, the rate of solubilization was substantially slower than at the alkaline pH. Recrystallization rates for the toxin were similar regardless of solubilization conditions. The banding patterns in denatured polyacrylamide gel electrophoresis were unaffected by solubilization conditions. Toxicity was higher for soluble toxin compared to crystal toxin, but virtually identical for the acidic and alkaline produced solutions. Acid solubilization is significant because of the acidic midgut of susceptible Coleoptera.

Nutritional physiology of the eastern spruce budworm, Choristoneura fumiferana, infected with Nosema fumiferanae, and interactions with dietary nitrogen.

Female eastern spruce budworm larvae, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae), inoculated with a medium lethal spore dosage of the microsporidium Nosema fumiferanae (Thomson) exhibited significant reductions in consumptive index (CI), nitrogen consumptive index (NCI), relative growth rate (RGR), and gross (ECI) and net (ECD) production effeciencies when compared to microsporidian-free larvae. Diseased larvae also exhibited significant increases in approximate digestibility (AD), N utilization efficiency (NUE), and larval moisture content. Both healthy and diseased insects were reared on 2.5% N and 4.5% N diets. Those on the 2.5% N diet showed significant increases in CI, although NCI was still lower than NCI measured for larvae reared on 4.5% N. NUE was also higher on the 2.5% N diet. Diseased cohorts reared on 2.5% N diet had significantly greater mortality than those reared on 4.5% N diet. Pupal weight and development time of infected individuals did not respond to dietary N concentration. However, healthy insects achieved greater pupal weights in a shorter time on the 4.5% N diet than those on the 2.5% N diet. Mortality of healthy insects was unaffected by dietary N.