Progress 02/01/09 to 01/31/13
Outputs
OUTPUTS: As a baseline for our studies, we sequenced and analyzed the transcriptome of the ALB larval gut to detect gene transcripts produced by the beetle without contributions from microbes. ALB makes its own cell wall degrading enzymes and can use components of plant chloroplasts to make fatty acids. The insect can partially synthesize sterols and mediate interactions with microbes. There were more types of chitinases relative to any other insect transcriptome. Although the beetle transcriptome contains cellulases, it only produces 9 types. There were also more carboxylesterases than any other insect transcriptome. There were transcripts with diverse detoxification functions. Other detoxification transcripts detected were 80 cytochrome P450s, GSTs/peroxidases, and UDP-glucurosyl transferases. ALB larvae can incorporate ammonia into different types of amino acids and produce many proteinases and other nitrogen-scavenging enzymes. ALB does not appear to have the ability to break down lignin; it can produce one extracellular laccase. Taxonomic and functional characterization of the ALB gut microbiota through amplicon and shotgun metagenome sequencing was followed by a large-scale comparison with the metagenomes in other herbivore-associated communities. This analysis distinguished the ALB gut microbiota from the gut microbiota of other herbivores, including termite hindgut metagenomes. The ALB gut microbial metagenome clustered with a fungal gallery community associated with Sirex noctilio that has lignin degrading potential. We identified candidate lignin degrading genes including laccases, dye-decolorizing peroxidases, and β-etherases found in association with most wood-feeding insect microbiota. Novel peroxidases and hydrogen-peroxide generating enzymes were found in high abundances relative to other herbivore-associated communities. We found genes that code for enzymes that detoxify allelochemicals and assist the insect with acquiring essential nutrients not found in woody tissue, including nitrogen, amino acids, proteins, vitamins, fatty acids and sterols. We classified microbes in association with ALB as it feeds in a preferred host using phylogenetic marker genes through targeted sequencing of the 16S/18S region and phylogenetic binning of predicted coding regions. We found 166 bacterial taxa in 7 different phyla in the gut microbiome, while 4 taxa could not be assigned. The fungal community was less diverse, containing approximately 7 distinct taxa, all within phylum Ascomycota. Fusarium solani is always found in the gut. Incorporation of fixed and recycled nitrogen in insect biomass was shown in larvae using stable nitrogen isotopes. ALB larvae fed in the resistant host P. tomentosa showed a significant reduction in the number and types of microbes that reside in the gut. Within a few weeks, feeding in diet with incorporation of P. tomentosa wood significantly reduced larval growth, while this did not occur with incorporation of wood from P. nigra. Larvae in the resistant trees that hatched from eggs died within a few weeks. We have identified 3 types of phenolic glycosides that are significantly higher in P. tomentosa than P. nigra. PARTICIPANTS: Kelli Hoover, Ming Tien and John Carlson are co-PI's on the project at Penn State University and participated in all experimental design, interpretation of results, and publication preparation. Erin Scully and Paul Ayayee are doctoral students being trained through this project; together they conducted most of the experiments and Erin did the annotation of the ALB gut metagenome and transcriptome. Scott Geib of the USDA, ARS in Hilo, HI cooperated on the project by providing expertise and access to his computing capabilities at his facility in HI. C.J. Tsai of the University of Georgia analyzed the phenolic glycosides from resistant vs. susceptible trees. Josh Herr is a doctoral student who worked on a portion of the fungal genome of the gut symbiotic fungus in the beetle gut. TARGET AUDIENCES: Target audiences for this project include other researchers in plant-insect interactions, insect physiology, microbiology, genetics, biofuels, development of resistant trees, and forest pest management. Groups that will benefit include the United States Department of Agriculture, DOE-Joint Genome Institute, arborists, state agricultural departments and state departments of natural resources. Market segments include plant production companies that grow trees, landscape management companies, and the biofuels industries. Community audiences include homeowners and environmental groups interested in alternative energy sources. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Wood is a highly indigestible food source and is devoid of many key nutrients required for growth and development, yet ALB successfully colonizes and thrives in this environment. In woody plants, carbohydrate and protein reservoirs are protected by lignin, a large structural polymer comprised of over 12 types of chemical bonds, many of which are difficult to disrupt. Thus, overcoming the lignin barrier a key challenge to wood feeding insects. Most of the carbohydrate sugar resources are present in cellulose, a polysaccharide requiring three separate enzymes for conversion to glucose. While animals and insects can produce 2 of the 3enzymes, only bacteria and fungi can produce all 3. Our findings validate that both lignin and cellulose degradation occur efficiently within the gut of ALB and that a fungal species, F. solani, that is consistently associated with the larval gut is likely instrumental in lignin and cellulose digestion and nutrient procurement. While little is known about the contribution of F. solani symbionts to insect physiology, our results suggest that F. solani provides ALB with fungal lignin- and cellulose- digesting enzymes that allow insects to circumvent the lignin barrier and extract nutrients from trees. Several novel enzymes were discovered that are likely involved in lignin degradation, suggesting that the ALB larval gut should be further studied for enzymes that can break down core lignin in the acidic environment of this insect gut. The bacterial community produces many enzymes the insect requires but cannot produce without their gut symbionts. These findings suggest that ALB has developed an intimate relationship with F. solani and a diverse bacterial community that is not limited by geographic range or host tree. Because protein is scarce and nitrogen fixation requires energy, recycling of protein waste products by gut bacteria and conversion to compounds that can be reused by the beetle is important. Taken together the nature of the symbiotic relationships between ALB and gut fungi/bacteria is likely obligate for the beetle. This is the first time a member of the F. solani species complex has been found living in an insect gut. These data were then used for examining the impact of feeding in resistant host Populus tomentosa on the ALB gut symbionts to determine if the mechanism of resistance involves antimicrobial properties. The reduction in microbial taxa and bacterial populations in insects fed on P. tomentosa indicate that resistance is acting through impacts on the gut microbiota, but does not rule out that resistance acts through direct impacts on the insect as well. Higher levels of 3 phenolic glycosides in P. tomentosa point to a possible mechanism of resistance. Yet, because the beetle transcriptome contains more carboxylesterases than any other insect transcriptome with diverse detoxification functions, it's possible that these enzymes are important for conferring phenolic glycoside resistance and likely contribute to the broad host range of ALB, suggesting that resistance may very well occur through antibiotic effects on the gut symbionts.
Publications
- No publications reported this period
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Progress 02/01/11 to 01/31/12
Outputs
OUTPUTS: The consistency of the relationship between the fungal isolate Fusarium solani and the ALB gut was examined by sampling insects reared on 4 different host tree species from the Penn State quarantine colony, insects collected from 3 different host tree species from an infestation site in Brooklyn, NY, and insects collected from a single tree species from the Worcester, MA infestation and performed targeted sequencing of a genetic marker (translation elongation factor alpha) used for F. solani strain identification, which can be used to distinguish these fungi at the subspecies level. Insects reared in our colony as well as insects collected from NYC consistently harbored the same F. solani strain in their guts regardless of the host tree that they were reared in. However, insects from the Worcester population and a very small subset of the insects reared in our colony harbored a different F. solani strain, which was not detected in the NYC population. To provide further support that the insects within the same geographic population consistently harbor the same F. solani strain, we sequenced two additional genetic markers that are also routinely used for F. solani strain identification, including the large ribosomal subunit and ITS region. The results of this analysis confirmed our suspicion that individuals within the NYC population all harbored the same F. solani strain, while individuals from the Worcester population all harbored the same F. solani strain (distinct from the NYC strain). We also characterized the proteome of F. solani's ability grown on wood chip to assess this fungal isolate's ability to degrade lignin and cellulose and to extract other nutrients from woody tissue. This F. solani isolate was grown on a wood-based substrate and over 400 proteins were sequenced. Through this approach, we detected proteins responsible for cellulose digestion, including proteins belonging to 28 glycosyl hydrolase families and several other cell wall degrading enzymes. Proteinases with broad substrate specificities and ureases were observed, indicating that this isolate has the capability to scavenge protein from plant cell walls and can recycle nitrogen under periods of nutrient limitation. Functional biochemical assays were conducted to corroborate the sequencing results and confirmed that cellulose -degrading and accessory enzymes associated with lignin degradation were actually active in culture. We also performed random sequencing of 1 million gene fragments obtained from the ALB gut bacterial community. The functions of these gene fragments were ascertained through database comparisons. Several bacterial enzymes that can break a small fraction of the bonds present in lignin were detected. Many enzymes involved in mediating detoxification were also discovered, including cytochrome p450s and glutathione-S-transferases. Pathways involved in vitamin synthesis, fatty acid, and hormone synthesis were identified. Several nitrogen-fixing and nitrogen recycling genes were also discovered. These results were published this year and reported at several scientific conferences. PARTICIPANTS: Co-PIs include Ming Tien (biochemist with expertise in lignin degradation) and John Carlson (expertise in genetics). Collaborators include Dr. Scott Geib at USDA, ARS in Hilo, HI who provided expertise and access to high level computing power and C.J. Tsai at University of Georgia who provided analysis of the phenolic glycosides in the poplar species used in this study. Two students were trained on this project and worked on the project full-time: Erin Scully and Paul Ayayee. TARGET AUDIENCES: Target audiences for this project include other researchers in plant-insect interactions, insect physiology, microbiology, genetics, biofuels, and forest pest management. Groups that will benefit include the United States Department of Agriculture, DOE-Joint Genome Institute, arborists, state agricultural departments and state departments of natural resources. Market segments include plant production companies that grow trees, landscape management companies, and the biofuels industries. Community audiences include homeowners and environmental groups interested in alternative energy sources. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our findings validate that both lignin and cellulose degradation occur efficiently within the gut of the Asian longhorned beetle and that a fungal species, Fusarium solani, is consistently associated with the larval stage and could be instrumental in both lignin and cellulose digestion and nutrient procurement. While the nature of the symbiosis with F. solani is not fully resolved and little is known about the contribution of F. solani symbionts to insect physiology, our results indicate that F. solani provides ALB with fungal lignin- and cellulose- digesting enzymes that allow insects to circumvent the lignin barrier and extract nutrients from trees. Further, ALB has evolved to harbor an isolate of F. solani that is not limited by geographic range or host tree. This is the first time a member of the F. solani species complex has been found living in an insect gut. Finally, because protein is scarce and nitrogen fixation is not efficient, recycling of protein waste products (conversion to compounds that can be reused by the beetle) could be beneficial for the beetle. Thus, it was discovered that protein can also be efficiently recycled by members of this community as a number of enzymes involved in converting urea to compounds that can be reintegrated into proteins or other compounds requiring nitrogen were identified. Taken together the nature of the symbiotic relationships between ALB and gut fungi/bacteria is likely obligate for the beetle. These data will be used as the baseline for studying the impact of feeding in the resistant host Populus tomentosa on the ALB gut symbionts to determine if the mechanism of resistance involves antimicrobial properties of the phenolic glycosides found in P. tomentosa.
Publications
- Scully, E.D., K. Hoover, J. Carlson, M. Tien, and S. M. Geib. 2012. Proteomic analysis of Fusarium solani isolated from the Asian longhorned beetle, Anoplophora glabripennis. PLoS ONE (In Press).
- Geib, S., E. D. Scully, M. 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.
- Schortemeyer, M., K. Thomas, R. A. Haack, A. Uzunovic, K. Hoover, J. A. Simpson, and C. A. Grgurinovic. 2011. Appropriateness of Probit-9 in the development of quarantine treatments for timber and timber commodities. J. Econ. Entomol. 104:717-731.
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Progress 02/01/10 to 01/31/11
Outputs
OUTPUTS: Phylogenetic and functional characterization of the ALB gut community was conducted to gain an understanding of how gut symbionts contribute to digestive physiology. Through our collaboration with the Department of Energy-Joint Genome Institute, 1.25 million reads were generated from metagenomic DNA extracted from the ALB gut microbiota using 454 Titanium chemistry. Assembly and annotation of the microbial community revealed the presence of phylogenetically diverse taxa; the bacterial fraction was dominated by Actinobacteria and Proteobacteria, while the fungal community was dominated by Ascomycota. About 40% of sequences were functionally annotated by BlastX comparison to the non-redundant protein database and the most dominant COG category was associated with carbohydrate metabolism. Within this pathway, the majority of the reads were classified as xylanases and cellulases, confirming that the community is integrally involved in polysaccharide metabolism. Genes associated with nitrogen fixation, amino acid and vitamin production, sterol and fatty acid biosynthesis, detoxification of plant secondary metabolites, and oxidative cleavage of small lignin subunits were detected, indicating that the ALB gut microbial consortium provides key enzymes that enable survival in wood. To focus future community profiling studies on protein coding regions, selective sequencing of transcriptionally active regions of the microbial community (meta-transcriptome) is underway. Total RNA was extracted from ALB larval luminal gut contents to selectively sample microbial transcripts. Isolated RNA was pooled and amplified using NuGen Ovation RNA-Seq prior to sequencing. A 2 x 150 paired end Illumina library with 175 bp inserts was constructed and is currently being sequenced on Illumina GAIIx platform. Phylogenetic and functional profiling of the meta-transcriptome will be performed using the same pipeline we are using to analyze the metagenomic data. Comparative phytochemical profiling of resistant (Chinese white poplars) and susceptible (Lombardy poplar) trees revealed that Chinese poplar produces significantly higher concentrations of two biochemically-reactive phenolic glycosides, salicin and tremulacin, but there were no significant differences in constitutive levels of total phenolics, condensed tannins, or hydrolyzable tannins. In addition, "in vitro" cellulose digestibility was compared using crude enzymatic extracts from healthy ALB larvae reared in sugar maple to determine if cellulase inhibitors were present in resistant tree tissue or if structural properties (such as higher lignin content) may limit access to cellulose. Glucose release was quantified and total glucose concentrations determined; more glucose was released from Chinese poplar wood, indicating that cellulase inhibitors do not contribute to resistance. To verify that the fungal isolate of Fusarium solani harbored by the ALB gut is involved in lignin degradation, secretome analysis revealed cellulase, xylanase, and lignin peroxidase activities. NifH genes were detected in the ALB gut and nitrogen-fixing bacteria were cultured anaerobically to identify the bacterial species involved. PARTICIPANTS: Trainees on this project include: Erin Scully who is a doctoral student funded on this project in the Intercollege Graduate Program in Genetics. Erin conducted most of the experiments reported here, including preparation of all samples for transcriptome sequencing and phytochemical extractions. Erin has been assembling and annotating the metagenome data from the ALB gut and is learning scripting during this process. Graduate student Paul Ayayee in Entomology conducted all experiments on nitrogen fixation and is responsible for beetle colony and greenhouse maintenance. The undergraduate students trained on this project are Karen Bingham, Jessica Shilladay, and Andrew Roth. They each performed various tasks in colony and greenhouse maintenance and assisted with experiments. Co-investigators on the project include: Dr. Ming Tien of the Dept. of Biochemistry and Molecular Biology in the Eberly College of Science at Penn State who consulted on all aspects of the project and is working on the enzyme analyses of the beetle gut fungus. He is also the co-advisor for Paul Ayayee. Dr. John Carlson, Professor in the School of Forest Resources and Huck Institutes of the Life Sciences and Director of The Louis W. Schatz Center for Tree Molecular Genetics at Penn State, was involved in experimental design and is the lead investigator on the metagenome sequencing project by JGI. He is co-advisor of graduate student Erin Scully. Dr. Kelli Hoover of the Department of Entomology at Penn State is lead investigator on this project and was involved in all aspects, as well as serving as co-advisor of all students on the project. Dr. C.J. Tsai at the University of Georgia is our collaborator working on the identification and quantification of phytochemicals that may be involved in host plant resistance. TARGET AUDIENCES: Target audiences for this project include other researchers in plant-insect interactions, insect physiology, microbiology, genetics, biofuels, plant pathology and forest pest management. Groups that will benefit include the United States Department of Agriculture, DOE-Joint Genome Institute, arborists, state agricultural departments and state departments of natural resources. Market segments include plant production companies that grow trees, landscape management companies, and the biofuels industries. Community audiences include homeowners and environmental groups interested in alternative energy sources. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In collaboration with DOE-JGI, we analyzed the results of our metagenomic study indicating that ALB harbors a phylogenetically diverse microbial community dominated by taxa that are capable of efficient lignocellulose and hemicellulose digestion and nitrogen fixation. Furthermore, members of this community have the metabolic capacity to help ALB overcome other challenges associated with living in wood and could synthesize vitamins, amino acids, sterols and fatty acids, and compounds that degrade xenobiotics or other plant secondary metabolites. These observations suggest that the gut community enables ALB larvae to grow and develop in the heartwood of healthy host trees and that disruption of the community could have profound deleterious phenotypic consequences on the insect. Furthermore, the results of our phytochemical profiling indicate that Chinese white poplar (resistant tree) constitutively produces significantly higher levels of two phenolic glycosides, salicin and tremulacin. Salicin has documented antimicrobial activity against a broad diversity of fungal and bacterial isolates and has the potential to negatively impact the gut microbiota "in vivo." Although its antimicrobial properties have not been investigated previously, tremulacin has been implicated in resistance to defoliating lepidopteran pests and it could have negative impacts on ALB and its gut community. Disrupting the gut microbiota in the ALB gut would represent a cost-effective and parsimonious method of pest control. Preparing microbial enriched metatrancriptomic samples virtually free of insect transcripts for functional profiling of transcriptionally active microbes is an arduous task. Not many groups have the technical skills and expertise to produce a high quality RNA sample from luminal contents containing wood (due to presence of nucleases and other wood polysaccharides that often co-purify with nucleic acids) and as a result, metatranscriptomic studies are usually limited to environments where removing host RNA is not a major consideration. However, we are currently sequencing an RNA sample that we believe is enriched with microbial transcripts. Demonstrating that we can attain high quality sequence data and successfully annotate transcripts from this harsh environment may inspire others to pursue similar studies in guts of other organisms. Resources that contribute to output include the sequencing facilities at DOE-Joint Genome Institute, Genomics Core Facility at Pennsylvania State University, and DNA Sequencing and Genotyping Center at the University of Delaware.
Publications
- Geib, S.M., M. Tien, and K. Hoover. 2010. Biochemical analysis of secreted gut proteins from larval Asian longhorned beetle, Anoplophora glabripennis, with potential application to the biofuels industry. Insect Science 17: 253-264.
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Progress 02/01/09 to 01/31/10
Outputs
OUTPUTS: To establish a baseline of the "normal" microbiota and transcripts produced by the Asian longhorned beetle (ALB) gut when it is feeding in its most highly preferred host, transcriptome analysis of RNA purified from whole insect guts was performed using larvae fed in sugar maple trees. Once sufficient concentration of high quality RNA was obtained, the sample was enriched for mRNA followed by 454-FLX sequencing at Penn State University. Preliminary assembly and annotation indicated the presence of a number of contigs that share a strong degree of sequence homology with insect-derived endoglucanases and beta-glucosidases, which are involved in biochemical pathways responsible for cellulose digestion. Furthermore, a number of contigs that encode hydrolytic and proteolytic domains were identified, which could be involved in digestion. In order to explore the potential role of gut microbiota in this insect gut system, microbial metagenomic DNA was extracted from insect gut contents and shotgun sequencing performed using 454-Titanium pyrosequencing through collaboration with the US Department of Energy Joint Genome Institute. Analysis indicated the presence of genes from many bacterial and fungal clades including, Alpha-, Beta-, and Gamma-Proteobacteria, and ascomycota-like fungi. From this, specific microbial species will be targeted and mined for specific enzymes that are critical in this gut system for digestion of lignocellulose and/or fixing nitrogen. To begin to assess the role of host plant resistance to the ALB gut microbial community in larvae fed in "Populus tomentosa" (resistant host tree) vs. "Popular nigra" (susceptible host tree), phenolic extracts from both leaf and bark material of these tree species were analyzed with HPLC to characterize the phenolic profiles of these samples and identify compounds that are unique to either of the tree species that may be conferring resistance. Fresh leaf and bark tissue was extracted twice in methanol and the combined extract was analyzed using HPLC measuring UV absorbance at 280 nm in replicates. Separation was performed on a Waters RP18 Xterra 4.6 X 150 mm column. Currently these extracts are being further analyzed by liquid chromatography tandem mass spectroscopy (LC-MS/MS) with searching against an in house plant metabolomic database. This will lead to identification of tree chemicals that may be driving resistance in this species. The goal is to correlate host tree chemistry with gut gene expression and microbial diversity, so that the mechanisms of resistance can be identified and understood at a physiological level. In addition, using the acetylene reduction assay, nitrogen fixation by the ALB larval gut community was demonstrated and quantified in comparison with a lower termite species. Further experiments are planned to identify the microbial species and genes involved in nitrogen fixation. These data were presented at the USDA Interagency Research Forum in Annapolis, MD, the USDA AFRI workshop in Washington, D.C. and at the User's Workshop of the DOE-Joint Genome Institute in Walnut Creek, CA. PARTICIPANTS: Trainees on this project include: Dr. Scott Geib, a postdoctoral fellow in Biochemistry and Molecular Biology, and Erin Scully, a doctoral student funded on this project in the Intercollege Graduate Program in Genetics. Scott and Erin conducting most of the experiments reported here, including preparation of all samples for metagenome and transcriptome sequencing and phytochemical extractions and preliminary analyses. Graduate student Paul Ayayee in Entomology conducted all experiments on nitrogen fixation and was responsible for beetle colony and greenhouse maintenance. The undergraduate students trained on this project are Karen Bingham, Jessica Shilladay, Owen Bellis and Steve Rosinski. They each performed various tasks in colony and greenhouse maintenance and assisted with experiments. Co-investigators on the project include: Dr. Ming Tien of the Dept. of Biochemistry and Molecular Biology in the Eberly College of Science at Penn State who consulted on all aspects of the project and supervised Dr. Geib. He is also the co-advisor for Paul Ayayee. Dr. John Carlson, Professor in the School of Forest Resources and Huck Institutes of the Life Sciences and Director of The Louis W. Schatz Center for Tree Molecular Genetics at Penn State, was involved in experimental design and is the lead investigator on the metagenome sequencing project by JGI. He is co-advisor of graduate student Erin Scully. Dr. Kelli Hoover of the Department of Entomology at Penn State is lead investigator on this project and was involved in all aspects, as well as serving as co-advisor of all students on the project. Dr. Pierluigi (Enrico) Bonello, Dept. of Plant Pathology at Ohio State University, is our collaborator working on the identification and quantification of phytochemicals that may be involved in host plant resistance. TARGET AUDIENCES: Target audiences for this project include other researchers in plant-insect interactions, insect physiology, microbiology, genetics, biofuels, plant pathology and forest pest management. Groups that will benefit include the United States Department of Agriculture, DOE-Joint Genome Institute, arborists, state agricultural departments and state departments of natural resources. Market segments include plant production companies that grow trees, landscape management companies, and the biofuels industries. Community audiences include homeowners and environmental groups interested in alternative energy sources. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results generated so far indicate that the ALB gut microbial community produces a number of enzymes that are likely critical to digestion of lignocellulose and are capable of fixing nitrogen. The most novel finding of these studies to date is the unique fungus "Fusarium solani" that is harbored by the ALB gut. "Fusarium solani" has been reported to be a mycangial symbiont in ambrosia beetles (specialized structures on the outside of the body that carries symbiotic fungi), assisting with degradation of lignocellulose when inoculated into the tree. Our findings show that ALB has evolved to carry this symbiont within its gut. This fungus, in conjunction with key bacterial species in the beetle gut, has considerable promise for applications in biofuels production. In addition, understanding the role of microbial symbionts in a wood-boring insect that lives deep in healthy trees will make significant contributions to our understanding of the physiological contributions of these symbionts to digestive physiology and how host plant resistance affects the function of these symbionts. Techniques developed from this project include methods for extracting and enriching bacterial DNA and RNA from a wood-feeding insect gut that contains bacteria, fungi, wood, and insect tissue. Development of this technique is important because few researchers have done this successfully; thus, these aspects of our project were of great interest at the Joint Genome Institute User's Workshop. Resources that contributed to these outputs include the metagenomic sequencing facilities of the DOE-Joint Genome Institute and the Huck Institute of Life Sciences shared facilities at Penn State University.
Publications
- Geib, S. M., M. Jimenez-Gasco, J. E. Carlson, M. Tien, and K. Hoover. 2009. Microbial community profiling to investigate transmission of bacteria between life stages of the wood-boring beetle, Anoplophora glabripennis. Micro. Ecol. 58:199-211.
- Geib, S. M., M. Jimenez-Gasco, M. Tien, J. Carlson and K. Hoover. 2009. Effect of host tree species on cellulase activity and bacterial community composition in the Anoplophora glabripennis gut. Environ. Entomol. 38:686-699.
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