Leah S. Bauer

Field-cage methodology for evaluating climatic suitability for introduced wood-borer parasitoids: Preliminary results from the emerald ash borer system

Abstract n Field-cage methods were developed to evaluate the abilities of Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae) and Spathius agrili Yang (Hymenoptera: Braconidae), biocontrol agents of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), to parasitize, develop and overwinter following three late-season releases at both a northern (Michigan) and a southern (Maryland) location within the current North American range of A. planipennis. In August, September and October of 2009, five young green ash trees were selected at each location. Tetrastichus planipennisi and S. agrili were each randomly assigned to one of two cages attached to each tree, surrounding separate sections of trunk in which late-instar A. planipennis had been inserted. The following April, the caged trunk sections were dissected to determine the fate of each A. planipennis larva and the developmental stages of all recovered parasitoid progeny. At both locations, T. planipennisi and S. agrili were able to parasitize hosts and successfully overwinter (i.e., reach adulthood the following spring). For T. planipennisi, successful parasitism (i.e., parasitoid progeny reached adulthood) occurred for all caged releases in Maryland, but only for the August and September releases in Michigan. At both locations, percent parasitism by T. planipennisi was higher in August and September than in October. For S. agrili, successful parasitism occurred for all caged releases in Maryland, but only for the August release in Michigan. In Maryland, percent parasitism by S. agrili in August and September was higher than in October. The caging method described here should be useful in determining the climatic suitability of other regions before proceeding with large-scale releases of either species and may have utility in other wood-borer parasitoid systems as well.

RESISTANCE: A THREAT TO THE INSECTICIDAL CRYSTAL PROTEINS OF BACILLUS THURINGIENSIS

Insecticidal crystal proteins (also known as d-endotoxins) synthesized by the bacterium Bacillus thuringiensis Berliner (Bt) are the active ingredient of various environmentally friendly insecticides that are 1) highly compatible with natural enemies and other nontarget organisms due to narrow host specificity, 2) harmless to vertebrates, 3) biodegradable in the environment, and 4) highly amenable to genetic engineering. The use of transgenic plants expressing Bt d-endotoxins has the potential to greatly reduce the environmental and health costs associated with the use of conventional insecticides. The complex mode of action of Bt is the subject of intensive research. When eaten by a susceptible insect d-endotoxin crystals are solubilized in the midgut; proteases then cleave protoxin molecules into activated toxin which binds to receptors on the midgut brush border membrane. Part of the toxin molecule inserts into the membrane causing the midgut cells to leak, swell, and lyse; death results from bacterial septicemia. Insecticides formulated with Bt account for less than 1% of the total insecticides used each year worldwide because of high cost, narrow host range, and comparatively low efficacy. Environmental contamination, food safety concerns, and pest resistance to conventional insecticides have caused a steady increase in demand for Bt-based insecticides. The recent escalation of commercial interest in Bt has resulted in more persistent and efficacious formulations. For example, improved Bt-based insecticides have allowed management of the diamondback moth, Plutella xylostella (L.). Unfortunately this has resulted in the evolution of resistance to d-endotoxins in P. xylostella populations worldwide. The recent appearance of Bt resistance in the field, corroborated by the results of laboratory selection experiments, demonstrates genetically-based resistance in several species of Lepidoptera, Diptera, and Coleoptera. The genetic capacity to evolve resistance to these toxins is probably present in all insects, and the heritability, fitness costs, and stability of the resistance trait are documented in several insect populations. In two strains of Bt-resistant lepidopteran species, mechanisms of resistance involve reductions in the binding of toxin to midgut receptors. Research on other resistant strains suggests that other mechanisms are also involved. Unfortunately, the high stability of the resistance trait, as well as broad spectrum cross-resistance to other d-endotoxins, undermines many potential options for resistance management. Genetically engineered plants, expressing d-endotoxin continuously and at ultrahigh doses, ensure intense and rapid selection of the target insect population. The efficacy of transgenic plants can be preserved only by developing an integrated pest management program that is designed specifically to reduce selection pressure by minimizing exposure to Bt and increasing other mortality factors, thereby slowing the rate of pest adaptation to Bt.

Two New Species of Egg Parasitoids (Hymenoptera:Encyrtidae) of Wood-Boring Beetle Pests from China

Oobius agrili sp.n. andAvetianella xystrocerae sp.n. (Hymenoptera: Encyrtidae) are described from China. Morphological characters of the new species are illustrated.O. agrili is an egg parasitoid of the emerald ash borerAgrilus planipennis Fairmaire (Coleoptera: Buprestidae) andA. xystrocerae is an egg parasitoid of the wood borerXystrocera globosa (Olivier) (Coleoptera: Cerambycidae). The two new species are potential biocontrol agents of economically important pest insects. The type specimens are deposited in the Institute of Zoology, Chinese Academy of Sciences, Beijing (IZCAS).

Gut microbiota of an invasive subcortical beetle, Agrilus planipennis Fairmaire, across various life stages.

We characterized gut microbial communities in the emerald ash borer, Agrilus planipennis Fairmaire, an invasive phloem-feeding and wood-boring beetle that has caused extensive mortality to urban and forest ash trees. Analyses included both 16S rRNA gene–based and culture-based approaches. We estimated that the emerald ash borer gut harbors 44, 71, and 49 operational taxonomic units (OTUs0.03) in the larval, prepupal, and adult stages, respectively, and a total of 132 OTUs0.03 when data from the three stages are pooled. The larval gut community shared all its OTUs0.03 with either the adult or the prepupal gut community, and the adult and prepupal gut communities shared 27 OTUs0.03. Twenty-two OTUs0.03 were shared among the three life stages. Rarefaction analyses suggest that these gut microbial communities are close to being completely sampled at the phylum level. Culture-independent techniques yielded a higher diversity of bacteria than did culturing. Three species of bacteria inhabiting guts of emerald ash borer showed cellulolytic activity. The diverse, dynamic, and presumably multifunctional microbial community associated with emerald ash borer guts suggests that invasive insects should be viewed as multispecies complexes and that such an interpretation can improve our ability to develop more effective management approaches.

Changes in protease activity and Cry3Aa toxin binding in the Colorado potato beetle: implications for insect resistance to Bacillus thuringiensis toxins

Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound approximately 60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of (125)I-Cry3Aa to BBMV did not reveal any differences in binding affinity (K(d) approximately 0.1 microM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.

Characterization of the pH-Mediated solubility of Bacillus thuringiensis var. san diego native δ-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.

Establishment and Abundance of Tetrastichus planipennisi (Hymenoptera: Eulophidae) in Michigan: Potential for Success in Classical Biocontrol of the Invasive Emerald Ash Borer (Coleoptera: Buprestidae)

ABSTRACT n Tetrastichus planipennisi Yang is a gregarious larval endoparasitoid native to China and has been introduced to the United States since 2007 for classical biological control of the invasive emerald ash borer, Agrilus planipennis Fairmaire, an exotic beetle responsible for widespread ash mortality. Between 2007–2010, T. planipennisi adults (3,311–4,597 females and ≈1,500 males per site) were released into each of six forest sites in three counties (Ingham, Gratiot, and Shiawassee) of southern Michigan. By the fall of 2012, the proportion of sampled trees with one or more broods of T. planipennisi increased to 92 and 83% in the parasitoid-release and control plots, respectively, from 33 and 4% in the first year after parasitoid releases (2009 fall for Ingham county sites and 2010 for other sites). Similarly, the mean number of T. planipennisi broods observed from sampled trees increased from less than one brood per tree in the first year after parasitoid releases to 2.46 (at control plots) to 3.08 (at release plots) broods by the fall of 2012. The rates of emerald ash borer larval parasitism by T. planipennisi also increased from 1.2% in the first year after parasitoid releases to 21.2% in the parasitoid-release plots, and from 0.2 to 12.8% for the control plots by the fall of 2012. These results demonstrate that T. planipennisi is established in southern Michigan and that its populations are increasing and expanding. This suggests that T. planipennisi will likely play a critical role in suppressing emerald ash borer populations in Michigan.

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.

Population dynamics of an invasive forest insect and associated natural enemies in the aftermath of invasion: implications for biological control

Summary 1. Understanding the population dynamics of exotic pests and associated natural enemies is important in developing sound management strategies in invaded forest ecosystems. The emerald ash borer (EAB) Agrilus planipennis Fairmaire is an invasive phloem-feeding beetle that has killed tens of millions of ash Fraxinus trees in North America since first detected in 2002. 2. We evaluated populations of immature EAB life stages and associated natural enemies over a 7-year period (2008–2014) in six stands of eastern deciduous forest in southern Michigan, where Tetrastichus planipennisi Yang and two other Asian-origin EAB parasitoids were released for biological control between 2007 and 2010. 3. We observed 90% decline in densities of live EAB larvae in infested ash trees at both parasitoid-release and control plots from 2009 to 2014 and found no significant differences in EAB density or mortality rates by parasitoids, avian predators or other undetermined factors between parasitoid-release and control plots. The decline in EAB larval density in our study sites was correlated with significant increases in EAB larval parasitism, first by native parasitoids, then by T. planipennisi. 4. Life table analyses further indicated that parasitism by the introduced biocontrol agent and the North American native parasitoids contributed significantly to the reduction of net EAB population growth rates in our study sites from 2010 to 2014. 5. Synthesis and applications. Our findings indicate that successful biocontrol of emerald ash borer (EAB) may involve suppression of EAB abundance both by local, generalist natural enemies (such as Atanycolus spp.) and by introduced specialist parasitoids (such as T. planipennisi). Biological control programmes against EAB in the aftermath of invasion should focus on establishing stable populations of T. planipennisi and other introduced specialist parasitoids for sustained suppression of low-density EAB populations. Moreover, we recommend releasing the introduced specialist biocontrol agents as soon as possible to prevent the outbreak of EAB populations in both newly infested and aftermath forests when EAB densities are still low.