Successful establishment, spread, and impact of the introduced parasitoid Spathius galinae (Hymenoptera: Braconidae) on emerald ash borer (Coleoptera: Buprestidae) populations in postinvasion forests in Michigan.

Spathius galinae is a larval parasitoid native to the Russian Far East that was approved for release in the United States in 2015 for biological control of the emerald ash borer (EAB), Agrilus planipennis, an invasive beetle from Asia responsible for widespread mortality of ash trees (Fraxinus spp.) in North America. From 2015 to 2017, 1,340-1,445 females of S. galinae along with males were released into each release plot, paired with a nonrelease control plot (1-12.5 km apart), at 6 postinvasion forested sites containing abundant pole-sized ash trees in Michigan. By 2018, S. galinae had spread to all but one control plot. Based on the first year that S. galinae was found in trees in each control plot and the distances of those trees to the parasitoid release point within each site, we estimated that S. galinae spread at 3.7 (±1.9) km per year after its initial releases in 2015. The proportion of sampled trees with S. galinae broods, brood densities within sampled trees, and parasitism of EAB larvae increased sharply in both control and release plots after the last field releases in 2017, with the highest parasitism rates (42.8-60.3%) in 2020. Life table analysis showed that S. galinae alone reduced EAB's net population growth rate by 35-55% across sites from 2018 to 2020. These results demonstrate that S. galinae has established an increasing population in Michigan and now plays a significant role in reducing EAB populations in the area.

Postrelease assessment of Oobius agrili (Hymenoptera: Encyrtidae) establishment and persistence in Michigan and the Northeastern United States.

The emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is an invasive woodboring pest of ash trees (Fraxinus sp.) in North America. Among the Asiatic parasitoids being released for the management of EAB in North America, Oobius agrili Zhang and Huang (Hymenoptera: Encyrtidae) is the only EAB egg parasitoid. To date, more than 2.5 million O. agrili have been released in North America; however, few studies have examined its success as a biological control agent of EAB. We conducted studies to assess O. agrili establishment, persistence, spread, and EAB egg parasitism rates in Michigan at the earliest release sites (2007-2010), as well as at more recent release sites (2015-2016) in 3 Northeastern United States (Connecticut, Massachusetts, New York). In both regions, we documented successful O. agrili establishment at all but one release site. In Michigan, O. agrili has persisted at release sites for over a decade and spread to all control sites located 0.6-3.8 km from release sites. Overall, EAB egg parasitism ranged from 1.5% to 51.2% (mean of 21.4%) during 2016-2020 in Michigan and from 2.6% to 29.2% (mean of 16.1%) during 2018-2020 in the Northeastern states. Future research efforts should focus on factors affecting the spatiotemporal variation in EAB egg parasitism rates by O. agrili, as well as its potential range in North America.

A Phenology Model for Simulating Oobius agrili (Hymenoptera: Encyrtidae) Seasonal Voltinism and Synchrony With Emerald Ash Borer Oviposition.

In North America, the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), continues to spread, and its egg parasitoid, Oobius agrili Zhang and Huang (Hymenoptera: Encyrtidae), is being released for emerald ash borer biocontrol well beyond their endemic climatic ranges in China. We developed a multiple cohort rate summation model to simulate O. agrili F0, F1, and F2 generations, and emerald ash borer oviposition for examining host-parasitoid synchrony across a north-south gradient from Duluth, MN (latitude 46.8369, longitude -92.1833) to Shreveport, LA (latitude 32.4469, longitude -93.8242). Temporal occurrences of critical day length for O. agrili diapause induction were integrated into the model. We used O. agrili and emerald ash borer trapping data from south central and northwestern Lower Michigan for model validation. Simulations demonstrated that 1) F0 adult emergence consistently occurred 2-5 d before emerald ash borer oviposition began; 2) F1 adult emergence was most synchronized with peak emerald ash borer oviposition compared with other generations; and 3) emerald ash borer oviposition was complete, or near so, when F2 adult emergence was predicted across the north-south gradient. Comparison of O. agrili trap captures with model simulations demonstrated that primarily two adult O. agrili generations (F0 and F1) emerged per year in Michigan and almost all F2 larvae entered diapause despite day lengths longer than critical day length in south central Michigan. Critical day length varied temporally across the north-south gradient during emergence of O. agrili generations. Determining day lengths perceived by O. agrili larvae in the field should improve model realism for examining spatiotemporal variation in O. agrili population dynamics.

Effects of Extreme Low Winter Temperatures on the Overwintering Survival of the Introduced Larval Parasitoids Spathius galinae and Tetrastichus planipennisi: Implications for Biological Control of Emerald Ash Borer in North America.

Climate change has been linked to shifts in the distribution and phenology of species although little is known about the potential effects that extreme low winter temperatures may have on insect host-parasitoid interactions. In late January 2019, northern regions of the United States experienced a severe cold wave caused by a weakened jet stream, destabilizing the Arctic polar vortex. Approximately 3 mo later at six study sites in southern Michigan and three in southern Connecticut, we sampled the overwintering larvae of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), and two larval parasitoids, Spathius galinae (Hymenoptera: Braconidae) and Tetrastichus planipennisi (Hymenoptera: Eulophidae), that are being introduced as emerald ash borer biocontrol agents in North America. At these nine study sites, emerald ash borer-infested ash trees and/or saplings were debarked and each overwintering emerald ash borer and parasitoid larva was then examined for cold-induced mortality, as indicated by a brown coloration, flaccid, and watery consistency. In early spring in Michigan, we found 4.5-26% of emerald ash borer larvae, 18-50% of S. galinae larvae, and 8-35% of T. planipennisi larvae were killed by cold. In Connecticut where temperatures were more moderate than in Michigan during the 2019 cold wave, <2% of the larval hosts and parasitoids died from cold injury. Our findings revealed that cold-induced mortality of overwintering larvae of emerald ash borer and its larval parasitoids varied by location and species, with higher mortality of parasitoid larvae in most Michigan sites compared to host larvae. The potential impacts of our findings on the management of emerald ash borer using biocontrol are discussed.

Two new species of Oobius Trjapitzin (Hymenoptera, Encyrtidae) egg parasitoids of Agrilus spp. (Coleoptera, Buprestidae) from the USA, including a key and taxonomic notes on other congeneric Nearctic taxa.

Oobius Trjapitzin (Hymenoptera, Encyrtidae) species are egg parasitoids that are important for the biological control of some Buprestidae and Cerambycidae (Coleoptera). Two species, Oobiusagrili Zhang & Huang and Oobiuslongoi (Siscaro), were introduced into North America for classical biocontrol and have successfully established. Two new native North American species that parasitize eggs of Agrilus spp. (Buprestidae) are described and illustrated from the USA: Oobiusminusculus Triapitsyn & Petrice, sp. n. (Michigan), an egg parasitoid of both Agrilussubcinctus Gory on ash (Fraxinus spp.) and Agrilusegenus Gory on black locust (Robiniapseudoacacia L.) trees, and Oobiuswhiteorum Triapitsyn, sp. n. (Pennsylvania), an egg parasitoid of Agrilusanxius Gory on European white birch (Betulapendula Roth). A taxonomic key and notes on the Nearctic native and introduced Oobius species are also included.

Establishment and abundance of Tetrastichus planipennisi (Hymenoptera: Eulophidae) in Michigan: potential for success in classical biocontrol of the invasive emerald ash borer (Coleoptera: Buprestidae).

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 approximately 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.

Susceptibility of two hymenopteran parasitoids of Agrilus planipennis (Coleoptera: Buprestidae) to the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales).

Emerald ash borer (EAB), Agrilus planipennis Fairmaire, native to Asia, is killing ash trees (Fraxinus spp.) across 15 states and southeastern Canada. Integrated pest management using biological control is the only viable long-term approach for controlling the spread of EAB outside of host resistance. Three hymenopteran parasitoids, Spathius agrili Yang, Tetrastichus planipennisi Yang, and Oobius agrili Zhang and Huang were discovered attacking EAB in China and were approved for release in the United States in 2007. The objective of this study was to assess susceptibility of the larval parasitoid species S. agrili and T. planipennisi, relative to that of EAB, to Beauveria bassiana, an entomopathogenic fungus that infects and kills EAB adults when sprayed on ash bark or foliage. Adult EAB and parasitoids were exposed to B. bassiana inoculated ash twigs for 2 h and then monitored daily for death and signs of infection for up to 10 days. All EAB adults exposed to B. bassiana were fatally infected while mean survival for control EAB was 77%. Average survival in the treatment groups for T. planipennisi and S. agrili were 99% and 83%, respectively, indicating these parasitoids are relatively unaffected by exposure to B. bassiana. This research elucidates interactions between a fungal pathogen and two parasitoids of EAB, and provides data necessary to developing a successful multi-stage integrated management approach to control of EAB.

Role of emerald ash borer (Coleoptera: Buprestidae) larval vibrations in host-quality assessment by Tetrastichus planipennisi (Hymenoptera: Eulophidae).

The biological control agent Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae) is a gregarious larval endoparasitoid of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), an invasive cambium-feeding species responsible for recent, widespread mortality of ash (Fraxinus spp.) in North America. T. planipennisi is known to prefer late-instar emerald ash borer, but the cues used to assess host size by this species and most other parasitoids of concealed hosts remain unknown. We sought to test whether vibrations produced by feeding emerald ash borer vary with larval size and whether there are any correlations between these cues and T. planipennisi progeny number (i.e., brood size) and sex ratio. The amplitudes and rates of 3-30-ms vibrational impulses produced by emerald ash borer larvae of various sizes were measured in the laboratory before presenting the larvae to T. planipennisi. Impulse-rate did not vary with emerald ash borer size, but vibration amplitude was significantly higher for large larvae than for small larvae. T. planipennisi produced a significantly higher proportion of female offspring from large hosts than small hosts and was shown in previous work to produce more offspring overall from large hosts. There were no significant correlations, however, between the T. planipennisi progeny data and the emerald ash borer sound data. Because vibration amplitude varied significantly with host size, however, we are unable to entirely reject the hypothesis that T. planipennisi and possibly other parasitoids of concealed hosts use vibrational cues to assess host quality, particularly given the low explanatory potential of other external cues. Internal chemical cues also may be important.

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

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.

Suitability and accessibility of immature Agrilus planipennis (Coleoptera: Buprestidae) stages to Tetrastichus planipennisi (Hymenoptera: Eulophidae).

Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae), a gregarious larval endo-parasitoid, is one of three biocontrol agents from Asia currently being released in the United States to combat the invasive emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae). The current protocol for rearing T. planipennisi involves presenting the wasps with artificially infested ash sticks made by placing field-collected larvae into shallow grooves beneath flaps of bark. Although third and fourth instars are readily accepted by T. planipennisi in these exposures, the suitability of younger or older developmental stages, which are often more readily available in the field, has not been tested. In this study, we used both artificially infested ash sticks and naturally infested ash logs to test which emerald ash borer developmental stages (second to fourth instars, J larvae [preprepupae], prepupae, and pupae) are most suitable for rearing T. planipennisi. T. planipennisi parasitized all stages except for pupae, but parasitized fewer J larvae and prepupae in naturally infested logs than in artificially infested ash sticks. This is probably because, in naturally infested ash logs, these stages were confined to pupal chambers excavated in the sapwood and may have been largely beyond the reach of ovipositing T. planipennisi. The number of T. planipennisi progeny produced was positively correlated (logarithmic) with host weight, but this relationship was stronger when J larvae and prepupae were excluded from the data set. Fourth instars yielded the most parasitoid progeny, followed by, in approximately equal numbers, J larvae, prepupae, and third instars. Second instars yielded too few parasitoid progeny to benefit rearing efforts.

Measuring the impact of biotic factors on populations of immature emerald ash borers (Coleoptera: Buprestidae).

Cohorts of emerald ash borer larvae, Agrilus planipennis Fairmaire, were experimentally established in July of 2008 on healthy green ash (Fraxinus pennsylvanica) trees in two wooded plots at each of three sites near Lansing, MI, by caging gravid emerald ash borer females or placing laboratory-reared eggs on trunks (0.5-2 m above the ground) of selected trees. One plot at each site was randomly chosen for release of two introduced larval parasitoids, Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae) and Spathius agrili Yang (Hymenoptera: Braconidae), whereas the other served as the control. Stage-specific mortality factors and rates were measured for all experimentally established cohorts and for associated wild (i.e., naturally occurring) emerald ash borer immature stages via destructive sampling of 2.5 m (above the ground) trunk sections of cohort-bearing trees in the spring and fall of 2009. Host tree defense was the most important mortality factor, causing 32.0 to 41.1% mortality in the experimental cohorts and 17.5 to 21.5% in wild emerald ash borer stages by spring 2009, and 16.1 to 29% for the remaining experimental cohorts, and 9.9 to 11.8% for wild immature emerald ash borer stages by fall 2009. Woodpecker predation was the second most important factor, inflicting no mortality in the experimental cohorts but causing 5.0 to 5.6% mortality to associated wild emerald ash borer stages by spring 2009 and 9.2 to 12.8% and 3.2 to 17.7%, respectively, for experimental cohorts and wild emerald ash borer stages by fall 2009. Mortality from disease in both the experimental and wild cohorts was low (<3%) in both the spring and fall sample periods. In the fall 2009 samples, ≈ 1.5% of experimental cohorts and 0.8% of the wild emerald ash borer stages were parasitized by T. planipennisi. While there were no significant differences in mortality rates because of parasitism between parasitoid-release and control plots, T. planipennisi was detected in each of the three release sites by the end of the study but was not detected in the experimental cohorts or associated wild larvae in any of the three control plots.

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 (OTUs(0.03)) in the larval, prepupal, and adult stages, respectively, and a total of 132 OTUs(0.03) when data from the three stages are pooled. The larval gut community shared all its OTUs(0.03) with either the adult or the prepupal gut community, and the adult and prepupal gut communities shared 27 OTUs(0.03). Twenty-two OTUs(0.03) 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.

Nondormancy in Entomophaga maimaiga azygospores: effects of isolate and cold exposure.

Azygospores (resting spores) of the fungal pathogen Entomophaga maimaiga are produced in later larval instars of the gypsy moth Lymantria dispar and normally enter constitutive dormancy. In the laboratory cadavers of recently dead larvae containing immature azygospores were placed on 1.0% water agar at 20 C for 2 wk after host death, allowing time for azygospore maturation. We found that some azygospores produced in this way did not enter dormancy. To investigate nondormancy, suspensions of azygospores from individual cadavers were transferred to moist, sterile soil at 15 C. Groups of gypsy moth larvae were exposed sequentially to azygospores for consecutive 4 d periods for 196 d. Infections first were seen among larvae exposed 24-28 d after bioassays began, and infection potential continued 196 d. Germination tests confirmed azygospore germination. Additional cadavers containing azygospores produced under the same conditions were maintained at 4 C for 1-8 mo, and each month new sequential bioassays were initiated. There was a general trend of earlier initiation of infection with longer durations of 4 C exposure; after 6-8 mo at 4 C first infections occurred 6-10 d after bioassays began. With 5-8 mo at 4 C infection levels declined after 96 d of sequential bioassays. Activity of azygospores differed by the individual larval cadaver in which they were produced; azygospores from 29.2% of cadavers yielded only 0-0.3% infection. Infection from nondormant azygospores did not differ among three Japanese isolates and two North American isolates, although azygospores from a third North American isolate caused no infections in 84 d, suggesting that dormancy had not been prevented.

Susceptibility of Agrilus planipennis (Coleoptera: Buprestidae) to Beauveria bassiana and Metarhizium anisopliae.

The susceptibility of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) to selected strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin was evaluated through bioassays with direct immersion or foliar exposure under laboratory conditions. Results showed that A. planipennis adults were susceptible to B. bassiana and M. anisoplae. Significant time-mortality response was found for each isolates. Isolate B. bassiana GHA killed A. planipennis adults at a faster rate compared with other isolates tested, with the lowest average time-to-death values. The LC50 values estimated under direct immersion method ranged from 1.7 x 10(5) to 1.9 x 10(7), 3.5 x 10(4) to 5.3 x 10(5), and 4.1 x 10(3) to 2.9 x 10(5) conidia/ml for B. basissiana and from 3.2 x 10(6) to 1.1 x 10(7), 4.5 x 10(3) to 4.5 x 10(5), and 1.4 x 10(2) to 1.2 x 10(5) conidia/ml for M. anisopliae at 4, 5, and 6 d after treatment, respectively. By days 5 and 6, B. bassiana GHA outperformed all other isolates tested except ARSEF 7234, followed by ARSEF 7152, 6393, and 7180. Significant concentration-mortality response was also observed for two B. bassiana GHA formulations, BotaniGard ES and Mycotrol O, and M. anisopliae F52 when insects were treated through foliar exposure. The LC50 values ranged from 114.5 to 309.6, 18.4 to 797.3, and 345.3 to 362.0 conidia/cm2 for BotaniGard, Mycotrol, and M. anisopliae F52, respectively. Based on the results of these bioassays, the efficacy of both B. bassiana GHA formulations and M. anisopliae F52 were similar against adult A. planipennis. The potential use of entomopathogenic fungi for management of A. planipennis in North America is discussed.

Field evaluations of systemic insecticides for control of Anoplophora glabripennis (Coleoptera: Cerambycidae) in China.

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), a pest native to China and Korea, was discovered in North America in 1996. Currently, the only reliable strategy available for eradication and control is to cut and chip all infested trees. We evaluated various doses of the systemic insecticides azadirachtin, emamectin benzoate, imidacloprid, and thiacloprid for control of A. glabripennis in naturally infested elms (Ulmus spp.), poplars (Populus spp.), and willows (Salix spp. ) in China between 2000 and 2002. Significantly more dead A. glabripennis adults were found beneath elm and poplar trees treated with imidacloprid (in 2000 and 2001) or thiacloprid (in 2001) and beneath willow trees injected with imidacloprid or thiacloprid (in 2002) compared with control trees. In 2000, 4 mo after injection, the density of live A. glabripennis was significantly reduced in poplar trees treated with imidacloprid (90%) and in willow trees treated with imidacloprid (83%) or emamectin benzoate (71%) compared with controls. In 2001, 9 mo after injection, the density of live A. glabripennis was significantly reduced in poplar (76%) and willow (45%) trees treated with imidacloprid compared with control trees. Similarly, percentage mortality of all life stages of A. glabripennis feeding within trees was significantly higher on poplar trees 4 mo after injection with imidacloprid (64%) in 2000 and on elms (55%) and poplars (63%) 9 mo after injection with imidacloprid in 2001 compared with control trees. Imidacloprid residue levels in leaves and twigs collected at various times from 1 d to 9 mo after injection ranged from 0.27 to 0.46 ppm. Injecting A. glabripennis-infested trees with imidacloprid can result in significant mortality of adults during maturation feeding on leaves and twigs and of all life stages feeding within infested trees. Imidacloprid is translocated rapidly in infested trees and is persistent at lethal levels for several months. Although, injection with imidacloprid does not provide complete control of A. glabripennis, systemic insecticides may prove useful as part of an integrated eradication or management program.

Laboratory evaluation of the toxicity of systemic insecticides for Control of Anoplophora glabripennis and Plectrodera scalator (Coleoptera: Cerambycidae).

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.

Effect of entomopathogenic nematodes on Plectrodera scalator (Fabricius) (Coleoptera: Cerambycidae).

Entomopathogenic nematodes were screened for efficacy against the cottonwood borer, Plectrodera scalator (Fabricius). Steinernema feltiae SN and S. carpocapsae All killed 58 and 50% of larvae, respectively, in filter paper bioassays but less than 10% in diet cup bioassays. S. glaseri NJ, S. riobrave TX, and H. indica MG-13 killed less than 10% of larvae in both assays. H. marelata IN was ineffective in the diet cup bioassay and killed 12.9% of larvae in a filter paper bioassay. The nematode isolates we tested are not suitable for use as biological control agents against P. scalator.

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.