Deborah L. Miller

Enhanced toxicity of Bacillus thuringiensis Cry3A δ-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 (R345A,Y350F,Y351F) and A2 (R345A,ΔY350,ΔY351), 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.

Laboratory Evaluation of the Toxicity of Systemic Insecticides for Control of Anoplophora glabripennis and Plectrodera scalator (Coleoptera: Cerambycidae)

Abstract Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) is one of the most serious nonnative invasive forest insects discovered in North America in recent years. A. glabripennis is regulated by federal quarantines in the United States and Canada and is the subject of eradication programs that involve locating, cutting, and chipping all infested trees. Other control methods are needed to aid in eradication and to form an integrated management program in the event eradication fails. We conducted laboratory bioassays to determine the toxicity of two systemic insecticides, azadirachtin and imidacloprid, for potential control of A. glabripennis and the cottonwood borer, Plectrodera scalator (F.) (Coleoptera: Cerambycidae), a closely related native cerambycid. Larvae of both cerambycid species were fed artificial diet with dilutions of azadirachtin or imidacloprid for 14 wk. Both insecticides exhibited strong antifeedant effects and some toxicity against A. glabripennis and P. scalator larvae. For A. glabripennis, the highest larval mortality at the end of the bioassay was 60% for larvae fed artificial diet treated with azadirachtin (50 ppm) or imidacloprid (1.6 ppm). For P. scalator, the highest larval mortality at the end of the bioassay was 100% for larvae fed artificial diet treated with azadirachtin (50 ppm) or imidacloprid (160 ppm). At 14 wk, the LC50 values for P. scalator were 1.58 and 1.78 ppm for azadirachtin and imidacloprid, respectively. Larvae of both species gained weight when fed diet treated with formulation blanks (inert ingredients) or the water control but lost weight when fed diet treated with increasing concentrations of either azadirachtin or imidacloprid. In a separate experiment, A. glabripennis adults were fed maple twigs treated with high and low concentrations of imidacloprid. A. glabripennis adult mortality reached 100% after 13 d on twigs treated with 150 ppm imidacloprid and after 20 d on twigs treated with 15 ppm imidacloprid. There was no visible feeding by A. glabripennis adults on twigs treated at the higher imidacloprid rate, and feeding was significantly reduced for adults placed on twigs treated at the low imidacloprid rate compared with adults on untreated twigs. In summary, imidacloprid and azadirachtin had both antifeedant and toxic effects against A. glabripennis and P. scalator and have potential for use in management programs. Based on our results, the delivery of high and sustained insecticide concentrations will be needed to overcome the antifeedant effects and lengthy lethal time for both larvae and adults exposed to these insecticides.

Monitoring the establishment and flight phenology of parasitoids of emerald ash borer (Coleoptera: Buprestidae) in Michigan by using sentinel eggs and larvae

Abstract The emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is an important invasive pest of ash (Fraxinus) trees in North America. Two larval parasitoid species, Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae) and Spathius agrili Yang (Hymenoptera: Braconidae), and 1 egg parasitoid species, Oobius agrili Zhang and Huang (Hymenoptera: Encyrtidae), were introduced into the United States in 2007 as part of a classical biological control program. We conducted field studies to assess the flight phenology of introduced and native parasitoids of emerald ash borer in central Michigan from 2011 to 2013 by using sentinel logs. Parasitism rates of sentinel A. planipennis eggs by O. agrili fluctuated throughout the season from 0 to 22% in 2011 and 0 to 6% in 2012. Flight phenology of O. agrili adults varied between years, and discrete generations were not apparent. Rather, O. agrili adults were generally continually present over a 3 mo period each year. Parasitism rates of sentinel A. planipennis larvae by T. planipennisi and the North American native Atanycolus spp. (Hymenoptera: Braconidae), respectively, ranged from 0 to 5% and 33 to 77% in 2011, from 0 to 69% and 0 to 27% in 2012, and from 0 to 53% and 0 to 46% in 2013. Phenology of adult flight of both T. planipennisi and Atanycolus spp. was inconsistent between years. Development of nondestructive methods to determine when stages of A. planipennis suitable for parasitism are present in combination with the use of sentinel logs to observe parasitoid phenology as described here will enhance the ability to evaluate the impacts of parasitoids on emerald ash borer.

GENETIC TRANSFORMATION OF POTATO WITH BACILLUS THURINGIENSIS HD 73 CRYIA(C) GENE AND DEVELOPMENT OF INSECT RESISTANT PLANTS

Solanum tuberosum L. was transformed using an Agrobacterium tumefaciens binary vector containing DNA encoding a translational fusion between the Bacillus thuringiensis var. kurstaki (B.t.k.) HD-73 δ-endotoxin and neomycin phosphotransferase II (NPT II). Two hunded forty-three shoots were regenerated from 50 inoculated leaf explants. Putatively transformed shoots were selected based on their ability to root in Murashige and Skoog medium supplemeted with 50 mg/1 kanamycin. DNA sequences encoding NPT II were detected using the polymerase chain reaction in several plants which formed roots in the selection medium. Southern blot hybridization of total DNA isolated from greenhouse grown transformed plant demonstrated integration of the translational fusion into the plant genome. In addition, transcription of the gene fusion in the transgenic potato plant was detected by reverse transcription of mRNA followed by amplification using the polymerase chain reaction (PCR). Insect assays, using tobacco hornworm (Manduca sexta) neonate larvae, indicated a significant reduction of leaf consumption in the transgenic plant when compared to a control potato plant (Cheng et ah., 1992). Second generation transgenic plants were grown from tubers and bioassayed against potato tubermoth (Phthorimaea operculella) and European corn borer (Ostrinia nubilalis). Ten percent mortality mortality of potato tubermoth was observed in transgenic plants but leaf consumption of surviving larvae was almost the same as in control plants. European corn borer larvae were significantly less capable of surviving in transgenic potato than in control plants as alternative host. Preference test showed that leaf disks of transgenic plants were less preferred than the control plants by 3rd instar European corn borer after 24 hours of exposure.