Progress 10/01/09 to 09/30/14
Outputs
Target Audience: The USDA Forest Service, USDA-APHIS-PPQ and the MA DCR benefited from the lure and trap development studies for Asian longhorned beetle (ALB) detection and are incorporating this approach into their eradication program. Residents of Massachusetts, Ohio, and New York who are living with ALB in quarantined areas also benefitted from this work. Permission from local residents had to be obtained to hang traps on private property and this provided an opportunity to improve relations between the eradication program and local residents. All states infested with hemlock woolly adelgid (HWA) will benefit from the biological control studies on Scymnus camptodromus, including federal, state, and local governments in the Eastern U.S such as the Pennsylvania Bureau of Forestry. The scientific community as well as ALB managers will benefit from the studies on mechanisms of plant resistance against ALB and impacts on its gut microbial community. The U.S. Forest Service was also the target audience for studies on the baculovirus that infects the gypsy moth. Changes/Problems: Studies on predator-prey interactions between S. camptodromus and HWA could not be conducted in the 2013-14 season because of very high adelgid mortality due to the polar vortex. What opportunities for training and professional development has the project provided? The technician responsible for rearing ALB attending two Insect rearing workshops, students and postdocs had numerous opportunities to give presentations at meetings, and received training in molecular techniques, chemical ecology, and bioinformatics. Several students contributed to reviewing manuscripts, writing grants, and new methods development. How have the results been disseminated to communities of interest? Project results were published in peer review journals, presented at scientific meetings, and at the USDA Interagency Research Forum. Meetings were also held with state and federal regulatory authorities regarding HWA biocontrol and ALB trapping. Annual reports were submitted to funding agencies. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Numerous experiments were conducted to evaluate the potential for improving potency of the gypsy moth virus LdMNPV by inserting a gene that blocks apoptosis (p35), but no changes in potency or time to death were noted. Recombinants of LdMNPV were constructed in which site directed mutagenesis was used at the zinc binding site of the purported metalloproteases in the viral genome that enhance potency of the virus to determine if these proteins act through digestion of peritrophic matrix proteins. These viruses were used in bioassays and in vitro digestability experiments showing that the enhanced viral potency conferred by the two enhancin genes has a different mechanism than reported for all other enhancins. These genes in LdMNPV do not appear to degrade the peritrophic matrix and are likely involved in midgut cell entry. However because the double deletion recombinant was unstable despite numerous attempts to attach GFP to a duplicated viral capsid protein (vp39), virus entry could not be studied. Studies on interactions between ALB and its gut microbiota were conducted using sequencing and analysis of the larval midgut transcriptome and the metagenome and metatranscriptome of the gut microbes. We found extensive opportunities for nutrient exchange between the insect and its gut microbes in collaboration to break down lignin and plant cell wall carbohydrates, releasing fermentable sugars that are converted into essential nutrients by the gut microbiota. The beetle was found to produce cellulases from 3 different glycoside hydrolase families, and a diverse array of β-glucosidases and xylanases. The beetle also produces a significant collection of detoxification enzymes (including 67 unique carboxylesterases), many of which likely play pivotal roles in detoxifying tree defensive compounds. However, ALB largely lacks the capacity to synthesize essential nutrients or degrade lignin polymers. In contrast, the taxonomically diverse gut microbiota is rich in laccases, which could play key roles in lignin depolymerization, and encodes enzymes involved in the fermentation of polysaccharides (e.g. glucose and xylose) and pathways for the conversion of fermentation products into essential nutrients. We also detected several microbial genes and transcripts that are involved in nitrogen cycling and in nitrogen fixation. These gene functions were also corroborated using stable isotopes of nitrogen, with conversion of fixed and recycled nitrogen into insect biomass and essential amino acids. The beetle can make its own contributions to its nitrogen economy through recycling nonessential amino acids. Interactions between ALB and its gut community likely contribute to this beetle’s expansive host range and its ability to thrive in woody tissue. The impacts of ALB larval feeding in Chinese white poplar (a resistant host) and Eastern cottonwood (a susceptible host) were compared to determine how plant defenses affected larval development, midgut expression of genes from the beetle itself and genes from the gut microbiota. Differential expression was noted for numerous beetle and microbial genes when larvae fed in the resistant host. Larval development was delayed considerably in larvae fed on diets containing ground wood from the resistant tree, while extraction of phenolics restored normal larval development. Trapping studies for ALB to improve the pheromone and kairomone components of the lure were conducted in Worcester, MA and in China to evaluate ratios and release rates, which led to optimization of the lure components. Work is ongoing to improve the trap design to take advantage of the beetles' natural preference for walking rather than flying.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Scully, ED, Hoover, K, Carlson, JE, Tien, M, and Geib, SM. 2013. Midgut transcriptome profiling of Anoplophora glabripennis, a lignocellulose degrading, wood-boring cerambycid. BMC Genomics, 14(850): doi:10.1186/1471-2164-14-850.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Metatranscriptome analysis of microbes associated with the Asian longhorned beetle midgut: insights into lignocellulose digestion, symbiosis, and nutritional ecology. BMC Genomics, (In Press).
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Meng, P., R.T. Trotter, M.A. Keena, T. Baker, S. Yan, E. Schwartzberg, and K. Hoover. Effects of pheromone and plant volatile release rates and ratios on trapping Anoplophora glabripennis in China. Environ. Entomol. 43:1379-1388.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Ayayee, P., C. Rosa, J.G. Ferry, G.W. Felton, M. Saunders and K. Hoover. 2014. Gut microbes contribute to nitrogen provisioning in a wood-feeding cerambycid. Environ. Entomol., 43(4):903-912.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Limbu, S., M.A. Keena, D. Long, N. Ostiguy and K. Hoover. Scymnus camptodromus larval development and predation of hemlock woolly adelgid. Environ. Entomol., (In Press).
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Nehme, M.A., R.T. Trotter, M.A. Keena, C. McFarland, J. Coop., H.M. Hull-Sanders, P. Meng, C.M. DeMoraes, M. Mescher, and K. Hoover. Development and evaluation of a trapping system for Anoplophora glabripennis in the United States. Environ. Entomol., 43(4):1034-1044.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The USDA Forest Service, USDA-APHI-CPHST and the MA DCR benefited from the lure and trap development studies and are incorporating our trapping findings into their eradication program. The public who live in ALB quarantined areas also benefitted from this work. Permission from local residents had to be obtained to hang traps on private property and this provided an opportunity to educate local residents about the project. Federal, state, and local governments in the Eastern U.S. with hemlock woolly adelgid infestation will benefit from the predator studies. The scientific community will benefit from the metagenome and transcriptome data on ALB and its microbial gut community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Four graduate students and two undergraduates were trained on this project. Graduate students attended scientific conferences where they reported their work. All students have received mentoring from the scientists on the project and have been involved in experimental design, learning new techniques, analyzing data, and writing publications. How have the results been disseminated to communities of interest? Results of trapping studies have been reported to the USDA-APHIS eradication program. Every time a beetle is trapped in the quarantined area of Worcester, MA it is reported to program personnel, usually leading to a find of previously missed infested trees. All findings have or are being prepared for submission to scientific journals. Results have also been disseminated at regional and national meetings of the Entomological Society of America, the USDA Interagency Research Forum on Invasive Species, and the Hemlock Woolly Adelgid biocontrol working group. Flyers were prepared and distributed to local residents in Worcester, MA about the trapping project for ALB. What do you plan to do during the next reporting period to accomplish the goals? We plan to deploy traps for ALB in a newly discovered infestation in Farmingdale, NY where beetle pressure is still high to obtain higher trapper catches to improve our ability to distinguish the best lure components statistically. Followup trapping studies will be conducted in Hebei, China in 2014 where ALB populations are higher and host trees have not been treated with insecticides. Additional studies on the impacts of host plant resistance on beetle gut microbes and diigestive physiology will be conducted. Mass rearing procedures for Scymnus camptodromus will be continued to be developed to provide sufficient predators for field releases and an EA will be submitted for review for field release. Ongoing studies on predator functional and numerical responses will be performed this winter season.
Impacts
What was accomplished under these goals?
A second year of trapping for Asian longhorned beetle (ALB) was conducted in Harbin, China in July and August of 2013. This year the purpose of the experiment was to evaluate a higher release rate of a 3-plant volatile blend and the addition of a fourth plant volatile, linalool oxide. Testing of lures made by two different companies was also done in China. The goal of this evaluation was to find the best formulation for stabilizing the aldehyde component of the pheromone lure, which releases initially in a burst with the remaining chemical oxidizing and cross linking, reducing the release rate after just 3-4 weeks. Also, volatile collections were made and tested by GC against internal standards. The lures from one company outperformed the other. Results form the field tests in China indicated that the 10-fold higher plant volatile release rate of linalool, cis-3-hexenol, and trans-caryophyllene enhanced attraction of the pheromone lure. In the USDA quarantined infestation in Worcester, MA, 210 traps were deployed using different lure combinations, including the higher kairomone release rate. At this time, the traps are still in the field and will be taken down for evaluation in early October. Bioassays of a four-component female-produced trail pheromone discovered in collaboration with USDA scientists showed that this pheromone is highly attractive to males, but repels females. It is not produced until the female completes sexual maturation following adult eclosion. Over 9,000 insect-derived transcripts from putative protein coding regions were assembled from the ALB midgut using a combination of 454 shotgun and Illumina paired-end reads. The most dominant transcripts predicted to encode digestive-related enzymes were trypsin, carboxylesterase, and beta-glucosidases. Furthermore, 180 transcripts were predicted to encode carbohydratases, including several GH 5, 45, and 48 cellulases, GH 1 xylanases, and beta-glucosidases. In addition, transcripts predicted to encode enzymes involved in detoxification were detected, including a substantial number classified as cytochrome P450s (CYP6B) and carboxylesterases, which are hypothesized to play pivotal roles in detoxifying host tree defensive chemicals and likely contribute to ALB’s expansive host range. No transcripts predicted to encode enzymes required for lignin degradation or synthesis of essential amino acids or sterols were detected, suggesting that microbes associated with the beetle gut likely fill these roles. Bacteria with urea hydrolysis activity were identified in the larval ALB gut and in eggs, indicating these bacteria are vertically transmitted. Genes and transcripts for urea hydrolysis were detected in all life stages of the larvae. Routing of recycled nitrogen into essential and non-essential amino acids in larval biomass was shown using stable isotope studies. Confined release studies of a predator of hemlock woolly adelgid (HWA), Scymnus camptodromus (Sc), were conducted with both predator larvae and adults showing that Sc consumed a significant number of HWA eggs over a 6-week period. A degree-day model for Sc development was produced from development data as a function of temperature.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
von Dohlen, C., U. Spaulding, K. Shields, N. P. Havill, C. Rosa, K. Hoover. 2013. Diversity of proteobacterial endosymbionts in hemlock woolly adelgid (Adelges tsugae) (Hemiptera: Adelgidae) from its native and introduced range. 2013. Environ. Microbiol. http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12102/full
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Levitt, A.L., R. Singh, D. L. Cox-Foster, E. Rajotte, N. Ostiguy, K. Hoover, E. C. Holmes. 2013. Evidence for cross-species transmission and active replication of honey bee viruses in associated arthropods. Virus Research 176: 232-240. http://dx.doi.org/10.1016/j.virusres.2013.06.013.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2013
Citation:
Scully ED, Geib S, Hoover K, Tien M, Tringe SG, Barry KW, Herr J & Carlson JE. 2013. Comparative Metagenomic Profiling Reveals Lignocellulose Degrading Systems in Microbial Communities Associated with Wood-Feeding Insects. PLoS One (in press)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
Hoover, K., M.A. Keena, M. Nehme, S. Wang, P. Meng and A. Zhang. 2013.Sex-specific trail pheromone mediates complex mate finding behavior in Anoplophora glabripennis (Coleoptera: Cerambycidae). Journal of Chemical Ecology (in review).
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Studies on plant volatiles of 6 different species of maples, in combination with the Asian longhorned beetle (ALB) male-produced pheromone, were conducted to assess enhancement of attraction of the pheromone. Three plant volatiles from Acer mono (Chinese maple) were identified at a particular ratio that was field tested during the 2012 field season in OH (new outbreak of ALB in collaboration with USDA, CPHST), Worcester, MA, and Harbin, China. In Worcester, 390 traps were deployed using four different lure mixtures in radial transects to assist survey teams in delimiting the quarantine boundaries. MA Department of Conservation of Resources deployed 1,000 traps (no controls) in collaboration with PSU and the US Forest Service. In China different ratios of the two pheromone components and different release rates were tested using a constant rate/ratio of plant volatiles. A higher release rate of pheromone (2.5-4 mg/day) and the 1:1 ratio of the 2 pheromone components trapped the most beetles, which were mostly virgin females. Ten members of the Sentinel Plant Network of the American Public Gardens Association also deployed traps to monitor for ALB in public arboreta in several state in the Northeast and Midwest of the U.S. using PSU's lure and trap combination, after attending a workshop at Longwood Gardens taught by PSU and US Forest Service collaborators. Functional annotation of shotgun metagenome data was completed, predicting functions for over 1 million gene tags generated through 454 pyrosequencing of ALB gut contents from larvae reared in a preferred host. Results of taxonomic classification detected the presence of 166 bacterial taxa. Within the most dominant family (Leuconostocaceae), several genera of bacteria were detected that have been previously implicated in lignocellulose, hemicellulose, and other aromatic hydrocarbon degrading roles and may participate in these processes in the beetle gut. The fungal community is considerably less diverse, containing approximately 7 distinct taxa, exclusively belonging to the phylum Ascomycota. Annotation of a set of insect midgut transcripts generated through a combination of 454 FLX and Illumina sequencing was also completed. The impact of ALB feeding in a resistant species of poplar, 'Populus tomentosa', on the larval midgut microbiota was evaluated in comparison to that of larvae fed in the susceptible P. nigra and the most preferred host, sugar maple. After 2 weeks of feeding in the different tree species, weight and instar were determined, branches from each tree were collected for phenolic glycoside analysis, and guts from insects were dissected and used for DNA and RNA extractions and protein preparations were made to examine the impact of resistance on digestive enzyme activity. The host tree showed tremendous impacts on the gut microbial community. On average, control insects and individuals reared in P. nigra had more diverse gut bacterial communities, containing approximately 285 and 241 OTUs on average. In contrast, individuals reared in P. tomentosa had relatively less diverse bacterial communities, containing 130 OTUs on average. Gene expression studies are in progress. PARTICIPANTS: Collaborators at Penn State on the ALB gut microbial community include Ming Tien in the Department of Biochemistry & Molecular Biology; John Carlson, School of Forest Resources; Erin Scully, graduate student in the Intercollege Program in Genetics, and Paul Ayayee, graduate student in Entomology. C.J. Tsai from the University of Georgia provided analysis of phenolic glycosides from poplar species used in this study. The ALB pheromone study collaborators include Aijun Zhang, a chemist at the USDA, ARS in Beltsville, MD; Melody Keena of the US Forest Service in Hamden, CT, and Consuelo De Moraes and Mark Mescher of the Department of Entomology at Penn State. Trapping in OH and large scale trap deployment in Worcester, MA was performed by the USDA, CPHST group from Buzzards Bay, MA and the MA Department of Conservation Resources with PSU's cooperation. Other cooperators included Don Eggen of the PA Bureau of Forestry, and Sentinel Plant Network members from MD, NY, NJ, OH, PA, FL and IL. TARGET AUDIENCES: The scientific community was served by this project through publications and presentations at scientific meetings. The USDA Forest Service, USDA CPHST and the MA DCR benefited from the lure and trap development studies and are incorporating these findings into their eradication program. In addition, outreach was conducted on the trapping study in Worcester, MA to inform the public about the economic and ecological concerns of ALB and the purpose of our trapping studies. Permission from local residents had to be obtained to hang traps on private property and this provided an opportunity to improve relations between the eradication program and local residents. Findings from the ALB gut symbionts studies will be of interest to the biofuels industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results from this year's ALB trapping studies will lead to several improvements in ALB lure and trap design. Working with the pheromone production company, improvements in lure release rates will be undertaken over the off-season to prepare for the following field season. Continued cooperation between PSU, the USDA Forest Service, the USDA, CPHST group, and MA DCR will facilitate a more concerted approach to detection and control of ALB in U.S. infestations. No beetles were trapped by participating arboreta, which are outside the currently known ALB infestations. Results from China were critical to confirming that the pheromone components are most effective at a 1:1 ratio and that higher release rates of the pheromone are more effective. The upper limit of effectiveness was also determined. New information obtained through plant volatile collection and bioassays provided leads on additional plant volatiles to evaluate to further improve lure performance, particularly to pull in males in addition to females. Through the study of ALB gut symbionts, important insight was gained into the ability of the gut microbiota to help ALB overcome the nutritional and digestive challenges associated with feeding in healthy host trees, including digestion of lignin, digestion of cellulose, hemicellulose, and other prominent plant well polysaccharides, detoxification of plant secondary metabolites and allelochemicals, and acquisition of essential nutrients absent from woody tissue, including nitrogen, amino acids, proteins, vitamins, fatty acids and sterols. Many genes were discovered that produce enzymes that have potential application to bioprocessing of lignocellulose to cellulosic ethanol. Through functional annotations of gene tags, the gut community has the metabolic potential to break many of the major linkages in polymeric lignin. The community also produces a number of glycoside hydrolases, which liberate glucose from cellulose and pentameric (xylose and arabinose) and hexameric sugars (mannose, rhamnose, glucose, and their corresponding uronic acids) from hemicellulose. The gut microbiota have the machinery to convert both xylose and arabinose into pyruvate and acetyl coA, which could be assimilated and utilized by ALB as carbon sources. Also produced are enzymes that neutralize toxic plant metabolites, fix atmospheric nitrogen, and recycle nitrogenous waste products, such as uric acid and urea, converting them back into ammonia where they can be reintegrated into nucleotides and amino acids. Results confirmed that a resistant host tree, Chinese maple, which co-evolved with ALB in China, markedly influences the gut microbiota, reducing diversity of taxa. Studies underway on gene expression and enzyme activities, if impacted as predicted, will reveal that host plant resistance can act through major impacts on a symbiotic gut community that mediates insect fitness. Ultimately, the findings from this study may lead to novel targets for ALB control.
Publications
- Uzunovic, A., B. Gething, A. Coelho, A. Dale, J. J. Janowiak, R. Mack, and K. Hoover. 2012. Lethal temperature for pinewood nematode, 'Busaphelenchus xylophilus', in infested wood using radio frequency (RF) energy. J. Wood Sci. http://dx.doi.org/10.1007/s10086-012-1306-2.
- Geib, S., E. D. Scully, M. del Mar Jimenez-Gasco, J. E. Carlson, M. Tien, and K. Hoover. 2012. Phylogenetic analysis of 'Fusarium solani' associated with the Asian longhorned beetle, 'Anoplophora glabripennis'. Insects 3(1):141-160.
- Scully, E. D., K. Hoover, J. Carlson, M. Tien, and S. Geib. 2012. Proteomic analysis of 'Fusarium solani' isolated from the Asian longhorned beetle, 'Anoplophoria glabripennis'. PLoS ONE. 7(4):e32990. http://dx.doi.org/10.1371/journal.pone.0032990.
- van Houte, S., V. I. D. Ros, T. Mastenbroek, K. Hoover, and M. M. van Oers. 2012. Protein tyrosine phosphatase-induced hyperactivity is a conserved strategy of a subset of baculoviruses to manipulate lepidopteran host behavior. PLoS ONE. 7(10):e46933. http://dx.doi.org/10.1371/journal.pone.0046933.
- Harrison, R. and K. Hoover. 2012. Baculoviruses. In: Insect Pathology and Microbial Pest Control. Second edition. Chapter 4. E. F. Vega and H.K. Kaya (eds). Elsevier, Inc. Amsterdam. pp. 73-131.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: The gene that causes gypsy moth larvae to die at elevated positions was discovered and reported in the journal Science. The egt gene was found to induce larvae to die at elevated positions, while deletion of egt produced the opposite effect. The p35 gene of Autographa californica multiple nucleopolyhedrovirus, which is a potent inhibitor of apoptosis, was inserted into the genome of the gypsy moth baculovirus, Lymantria dispar MNPV, but expression of this gene did not improve the potency of the virus as hypothesized. Recombinants of LdMNPV with alterations in the active site of the two enhancin genes were constructed for the single enhancin deletion mutants. Construction of the double-deletion mutant with active site modification is underway. Methods were developed for testing these recombinant viruses for their impact on binding of virions to the midgut epithelial cells of the gypsy moth. Primary midgut cell cultures were prepared from gypsy moth larvae. GFP signals could be visualized under microscopy as virions bound to midgut cells and the signal remained in the endosomal membrane as expected. This approach will be used to evaluate the possible function of enhancins encoded by LdMNPV in facilitating virus entry. Oviposition into 'Populus tomentosa' trees by Asian longhorned beetles (ALB) resulted in early mortality of larvae with intense tree responses. Larval galleries were filled with black, oxidized material, which killed larvae within a few weeks. To obtain larvae fed in 'P. tomentosa' long enough to evaluate the effects of tree resistance on the larval gut symbionts, beetles were confined to sugar maple trees for several weeks of oviposition. These larvae will be removed from sugar maples and inserted into poplar trees to permit further investigation into the effects of tree resistance as outlined in our objectives. Non-target testing of Scymnus camptodromus, an introduced predator from China for control of hemlock woolly adelgid, is nearly complete. Predators did not feed or oviposit on elongate hemlock scale or woolly apple aphid. The final test against woolly alder aphid is underway. Among other adelgid species, the predator only fed appreciably on pine bark adelgid, but could not complete development on this species. Efficacy of S. camptodromus against HWA has been tested in a lab setting so far and was found to be a voracious predator of HWA eggs and of small nymphs. Studies on plant volatiles, in combination with the ALB male-produced pheromone, were conducted. Three plant volatiles from Acer mono were especially effective at increasing attraction of females and, to some extent, males. In Worcester, MA, 500 traps were deployed using four different lure mixtures for 3 months using radial transects in an effort to point survey teams in the direction of infested trees that were not detected in earlier surveys. PARTICIPANTS: Dr. Cristina Rosa and Kim Duong participated in the baculovirus studies. Dr. James Slavicek of the U.S. Forest Service in Delaware, OH collaborated on and funded the gypsy moth-baculovirus studies and constructed the recombinant viruses used in experiments. Collaborators at Penn State on the ALB gut microbial community include Ming Tien in the Department of Biochemistry & Molecular Biology; John Carlson, School of Forest Resources; Erin Scully, graduate student in the Intercollege Program in Genetics, and Paul Ayayee, graduate student in Entomology. The ALB pheromone study collaborators include Aijun Zhang, a chemist at the USDA, ARS in Beltsville, MD; Melody Keena of the US Forest Service in Hamden, CT, and Consuelo De Moraes and Mark Mescher of the Department of Entomology at Penn State. Personnel for the trapping study were also provided by the USDA, APHIS group from Buzzards Bay, MA. TARGET AUDIENCES: The scientific community was served by this project through publications and presentations at scientific meetings. The USDA Forest Service and APHIS received research findings from this project on trapping studies of ALB and biological control studies on hemlock woolly adelgid. In addition, outreach was conducted on the trapping study in Worcester, MA to inform the public about the economic and ecological concerns of ALB and the purpose of our trapping studies. Permission from local residents had to be obtained to hang traps on private property. The general public was served by numerous news stories and radio interviews on the gypsy moth and manipulation of its behavior by the baculovirus that infects this insect in nature. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Finding a viral gene that manipulates the behavior of its host was the first discovery of the genetic basis for the extended phenotype. The fact that the enzyme encoded by this gene manipulates the host's hormones may point other scientists toward examining pathogen or parasite genes that affect hormones in their hosts as possible mechanisms of controlling host behavior. Populus tomentosa was known to be resistant to Asian longhorned beetle, but the response of the tree to this insect was more intense than has been reported before for any wood-borer. This serendipitous finding will be explored further and may lead to new targets for resistance to other insects as well. Data gathered on S. camptodromus will be used to prepare an environmental assessment report to permit field testing of this predator for control of HWA. Given the importance of hemlocks to forest ecosystems, and riparian ecosystems in particular, this project will contribute to the USDA's national effort to preserve hemlocks. Development of an effective lure/trap combination for early detection of ALB is very important to the USDA-APHIS' eradication effort. The discovery of a new infestation of ALB in Ohio this summer has increased the urgency of finding a better way to detect this invasive insect. Every insect trapped this summer was conveyed to APHIS personnel, allowing climbers to locate infested trees that were missed in earlier surveys.
Publications
- Hoover, K., M. Grove, M. Gardner, D. P. Hughes, J. McNeil, and J. Slavicek. 2011. A gene for an extended phenotype. Science 333:1401.
- Schortemeyer, M., K. Thomas, R. A. Haack, A. Uzunovic, K. Hoover, J. A. Simpson, and C. A. Grgurinovic. Appropriateness of Probit-9 in the development of quarantine treatments for timber and timber commodities. 2011. J Econ Entomol. 104:717-731.
- Haack, R. A., A. Uzunovic, K. Hoover, and J. A. Cook. 2011. Seeking alternatives to probit 9 when developing treatments for wood packaging materials under ISPM No. 15. OEPP/EPPO Bulletin 41:39-45.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Several recombinant baculoviruses of LdMNPV were generated during the past year that are designed to test the hypothesis that the enhancin genes of this virus, specific to the gypsy moth, have a role beyond degradation of peritrophic membrane proteins to gain entry to the midgut cell environment. For each enhancin gene, either the active site of the metalloprotease was deleted or site-directed mutagenesis was used to alter a key amino acid in the zinc-binding domain using a homologous amino acid to maintain the three-dimensional conformation of the enzyme. Also, an enhanced gfp gene fused to the capsid protein vp39 of the virus was inserted in these recombinant viruses. These viruses are currently undergoing plaque purification and will be used in experiments to examine binding and fusion of virus to the midgut cells under fluorescent microscopy. In addition, the potent anti-apoptosis gene p35 of 'Autographa californica' NPV was inserted into LdMNPV to determine if this protein could be used to improve potency of LdMNPV in gypsy moth, given that the caterpillar battles infection by this virus using apoptosis. This recombinant was bioassayed in second instar gypsy moths, but was found to have drastically reduced potency. The integrity of the recombinant virus is currently being examined to determine if other mutations occurred during transfection or if addition of p35 is indeed responsible for the reduced potency. After several years of effort, sufficient high quality RNA was isolated from the gut of Asian longhorned beetle larvae fed in sugar maple trees. Meta-transcriptome sequencing is in progress. The data from this sequencing project will establish a baseline for comparison with gut meta-transcriptome data from beetle larvae fed in resistant (Populus tomentosa) vs. susceptible (P. nigra) tree species. The metagenomic sequence data on the microbial gut community of ALB produced by the Joint-Genome Institute is being assembled and annotated. These data will facilitate functional analysis of the transcriptome sequences, particularly when combined with the proteomic profiles produced last year from the ALB gut. A second season of trapping for ALB was conducted in Worcester, MA and at several sites in New York using a lure of ALB male-produced pheromone combined with a blend of plant volatiles. Female ALB were again trapped at the Worcester site, but to date no beetles have been trapped in New York where it is unknown if any infested trees still remain. Non-target testing of 'Scymnus camptodromus' adults was initiated. Although this predatory beetle consumed more of the target pest eggs (hemlock woolly adelgid) in a choice test than it did of non-target adelgid eggs, it is able to feed to some extent on other adelgids. In no choice tests on pine bark adelgid, the predator fed and developed to an adult, but it was deformed and lived less than 24 hours. In preparation for mass rearing 'S. camptodromus', a hoop house was constructed at Rock Springs over about 20 planted hemlocks that are in the process of being infested with hemlock woolly adelgid to feed the predatory beetles. PARTICIPANTS: Diana Cox-Foster in the Department of Entomology at Penn State participated in the baculovirus studies. As a postdoctoral researcher, James McNeil conducted the studies on anti-viral defenses in gypsy moth to the baculovirus. James Slavicek of the U.S. Forest Service in Delaware, OH collaborated on and funded the gypsy moth-baculovirus interaction studies and constructed the recombinant viruses used in the experiments. Collaborators at Penn State on the ALB gut microbial community include Ming Tien in the Department of Biochemistry & Molecular Biology; John Carlson, School of Forest Resources; and Maria Del Mar Jimenez-Gasco from the Dept. of Plant Pathology. Carlson is a molecular genetics scientist serving as both lead PI on the ALB gut community metagenomics project and as co-advisor of a Ph.D. student in Genetics, Erin Scully, who is working on the project. Tien and Jimenez-Gasco served on Scott Geib's dissertation committee. Geib completed his postdoctoral work on the gut microbial symbionts of ALB and is now working as a scientist for the USDA, ARS in Hilo, HI. The ALB gut symbionts project is currently funded by a USDA-NRI grant and the Alphawood Foundation. The metagenome sequencing was performed by the DOE-JGI under a proposal submitted by Carlson as lead PI. The ALB pheromone study collaborators include Aijun Zhang, a chemist at the USDA, ARS in Beltsville, MD; Melody Keena of the US Forest Service in Hamden, CT, and Vic Mastro, Director of the Methods Development Lab, Otis ANGB, MA. The graduate student who conducted the pheromone work was Dr. Maya Nehme who completed her Ph.D. in May 2009 and is now working on various projects funded through USAID in Lebanon. Katie Cassidy is a graduate student working on the hemlock woolly adelgid project funded through a collaborative agreement with the USDA, Forest Service with Melody Keena. TARGET AUDIENCES: The baculovirus project serves the US Forest Service and will ultimately serve the general public when improved performance of Gypcheck becomes possible since it avoids non-target effects of pesticides and Bt for control of gypsy moth. The ALB gut symbionts project may benefit the biofuels industry, the scientific community, and the public if results can be used to control the beetle. The target audience for the pheromone studies is the USDA to provide a tool for early detection of ALB, which will ultimately benefit the general public by protecting their shade trees. The studies on 'S. camptodromus' as a predator of hemlock woolly adelgid benefits the US Forest Service and the public by helping to save hemlock trees, which are an important part of the ecosystem, particularly in riparian areas. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The knowledge base for understanding the co-evolutionary interactions between the gypsy moth and its host specific baculovirus continues to mount. Studies are continuing that will ultimately lead to a more potent virus for control of this invasive species. Proof of concept of the utility of the ALB pheromone combined with plant volatiles was provided by a second year of successful trapping in an area in the U.S. with a known infestation, although the beetle population is now at lower levels than it was last year due to cutting down of infested trees. These trapping data are being used, along with follow-up surveys in collaboration with USDA/APHIS, to determine the potential range of the lure. It was also found that application of liquid Teflon painted black (Fluon) to the panel traps improved trap catches. Sequencing results on the ALB gut microbial community has revealed important candidate genes for study, including genes that are likely involved in lignocellulose degradation and nitrogen fixation. These data have implications for mining the beetle gut for candidate enzymes for biofuels production and for beetle control. Data collected on non-target effects of 'S. camptodromus' will be used in an environmental assessment report to permit release of this predator for hemlock woolly adelgid control. Development of mass rearing procedures will be required for biological control using this predator, and these procedures are underway at Rock Springs on the Penn State campus.
Publications
- Nehme, M. E., M. A. Keena, A. Zhang, T. C. Baker, Z. Xu, and K. Hoover. 2010. Evaluating the use of male-produced pheromone components and plant volatiles in two trap designs to monitor 'Anoplophora glabripennis'. Environ. Entomol. 39:169-176.
- Geib, S. M., M. Tien, and K. Hoover. 2010. Identification of proteins involved in lignocellulose degradation using in gel zymogram analysis combined with mass spectroscopy-based peptide analysis of gut proteins from larval Asian longhorned beetles, Anoplophora glabripennis. Insect Science 17:253-264.
- Georgievska, L., N. Joosten, K. Hoover, J. S. Cory, J. M. Vlak, and W. van der Werf. 2010. Effects of single and mixed infections with wildtype and genetically modified H. armigera NPV on movement behaviour of cotton bollworm larvae. Entomol. Exp. Appl. 135:56-67.
- McNeil, J., J. Slavicek, S. Thiem, M. Gardner, D. Cox-Foster, and K. Hoover. 2010. Pathogenesis of 'Lymantria dispar' multiple nucleopolyhedrovirus (LdMNPV) in 'L. dispar' and mechanisms of developmental resistance. J Gen. Virol. 91:1590-1600.
- Georgievska, L., J. S. Cory, D. Munoz, P. Caballero, K. Hoover, W. van der Werf, and J. M. Vlak. 2010. Dose dependency of time to death in single and mixed infections with a wildtype and egt deletion strain of H. armigera NPV. J. Invertebr. Pathol. 104:44-50.
- Hoover, K., M. A. Humphries, A. R. Gendron, and J. M. Slavicek. 2010. Impact of viral enhancin genes on potency of 'Lymantria dispar' multiple nucleopolyhedrovirus in 'L. dispar' following disruption of the peritrophic matrix. J. Invertebr. Pathol. 104:150-152.
- McNeil, J., D. Cox-Foster, J. Slavicek, and K. Hoover. 2010. Contributions of immune responses to developmental resistance in 'Lymantria dispar' challenged with baculovirus. J. Insect Physiol. 56:1167-1177.
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