Source: UNIVERSITY OF FLORIDA submitted to
IDENTIFICATION AND CHARACTERIZATION OF NOVEL TARGET SITES FOR INNOVATIVE TERMITICIDES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0215662
Grant No.
(N/A)
Project No.
FLA-ENY-004757
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2008
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Project Director
Scharf, M. E.
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
ENTOMOLOGY & NEMATOLOGY
Non Technical Summary
This research will integrate bioassay, genomic, biochemistry, physiology, toxicology and chemical ecology approaches to define termite molecular physiology, and subsequently apply this information in designing novel pest management tools. The rationale that underlies this research is that, once the nature of highly specific aspects of termite caste differentiation and gut function are known, this information will significantly improve our understanding of termite development, endocrinology, symbiosis, and lignocellulose digestion/ assimilation. This information, in turn, is expected to lead to (i) a greater ability to target termites with highly specific control agents, (ii) an improved ability to protect structures and urban landscapes from damage by termites, and (iii) enhanced protection of natural and urban landscapes from damage caused by overuse of non-specific, highly toxic soil termiticides. For example, hundreds of thousands of gallons of liquid soil termiticides are applied pre-construction across the Southeastern US each year. The majority of these applications may be made un-necessarily, and could possibly be causing irreparable environmental damage. In this respect, this research has the potential to lead to more environmentally-benign alternatives for termite IPM.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1330650104020%
1363110105020%
7114010113020%
7214050115020%
7235220118020%
Knowledge Area
133 - Pollution Prevention and Mitigation; 136 - Conservation of Biological Diversity; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 721 - Insects and Other Pests Affecting Humans; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
5220 - Pesticides; 0650 - Wood and wood products; 4010 - Bacteria; 3110 - Insects; 4050 - Protozoa;

Field Of Science
1130 - Entomology and acarology; 1180 - Pharmacology; 1050 - Developmental biology; 1040 - Molecular biology; 1150 - Toxicology;
Goals / Objectives
Broad Goals: To use an integrative approach involving genomics, biochemistry, physiology, toxicology and chemical ecology to identify and define target sites for innovative, environmentally-friendly termiticides. Target sites that receive major focus will be tied to caste differentiation and lignocellulose digestion. --------------------- Specific Objectives: 1.Caste differentiation target sites: a.Identify and characterize novel hormones and semiochemicals involved in termite caste differentiation. b.Identify suites of developmental genes that respond to active hormones and semiochemicals. c.Functionally characterize key developmental genes using RNA interference. d.Design and test novel control agents targeted to protein products encoded by highly influential caste regulatory genes. ----------------- Specific Objectives: 2.Target sites in the gut: a.Use genomic approaches to sequence from the termite gut transcriptome, and identify a large number of lignocellulose digestion/assimilation-related genes as candidate termiticide target sites. b.Use metagenomic approaches to sequence from the termite gut symbiont metagenome (prokaryotic and eukaryotic) and identify a large number of potential termiticide target sites. c.Use RNA interference and protein/enzyme biochemistry approaches to functionally characterize relevant genes from the termite gut and gut symbionts. d.Design and test prototype termiticides that are specifically targeted to protein products encoded by key lignocellulose digestion/ assimilation genes. ------------------- Expected Outputs: This research is expected to reveal important genes involved in termite caste differentiation, lignocellulose digestion, and nutrition, as well as new strategies for termite pest management that involve disruption of chemical communication, caste homoestasis, and expression of developmental and nutrition-related genes. Pest management technologies resulting from this work will include environmentally-friendly, termite-specific chemicals and interfering RNAs that specifically silence termite genes.
Project Methods
OBJECTIVE 1a. The working hypothesis in this objective is that termite soldier head extracts work synergistically with JH to induce phenotypic caste differentiation, and that only a subset of extract components will have impacts on caste differentiation. This hypothesis will be tested by using a combination of model caste-differentiation bioassays, thin layer chromatography and gas chromatography - mass spectrometry analysis to fractionate and identify active soldier head extract components. OBJECTIVE 1b. This objective will be met using model bioassays to expose worker termites to active semiochemicals, followed by determination of gene expression levels in workers at precise time intervals during the bioassays. OBJECTIVE 1c. This hypothesis will be met by injecting double-stranded RNAs (dsRNAs) corresponding to specific gene sequences into worker termites. After injection, workers will be subjected to model caste differentiation bioassays for observation of developmental impacts. OBJECTIVE 1d. This objective will met using approaches that include model bioassays, gene expression characterization, and other biochemistry and physiological approaches to examine hormone titers, hormone binding, and relevant enzyme biochemistry. -------------------- OBJECTIVE 2a. This objective is based upon the working hypothesis that a R. flavipes gut-specific cDNA library will contain a robust sampling of termite-genome-derived genes that collaborate to accomplish lignocellulose digestion and assimilation. This objective will be accomplished using a high quality normalized cDNA library (already made) representing symbiont-free termite gut tissues, then by sequencing several thousand expressed sequence tags (ESTs) from this library. OBJECTIVE 2b. Justification for the approach taken in this objective is that (i) the termite hindgut fermentation chamber contains numerous species of unculturable bacteria and protozoa that interact in lignocellulose processing, and (ii) decades of one-dimensional biochemistry research has yielded few leads on collaborative lignocellulose processing in termites. New pyrosequencing technology available at the UF-ICBR will be used. OBJECTIVE 2c. The goal of this sub-objective is to functionally characterize relevant genes identified under Objectives 2a and 2b that encode potentially vulnerable target sites for novel termiticides. The hypothesis to be tested under this objective is that relevant target genes will play critical and quantifiable roles in termite digestion and nutrient assimilation. OBJECTIVE 2d. Using information from Objective 2c, the goal of this objective is to design and test candidate materials as novel termiticides. Our hypothesis here is that prototype toxins that are targeted to the most vulnerable target sites will cause pronounced disruptions in termite feeding and/or survival.

Progress 07/01/08 to 09/30/12

Outputs
OUTPUTS: Outputs from 2010 were again in both project areas of: (1) termite caste differentiation and (2) the termite gut. With respect to caste differentiation, (1a) novel semiochemicals have been purified and characterized, (1b) dozens of candidate developmental genes that respond to semiochemicals and hormones have been identified, (1c) critical responsive genes have been characterized using functional/ translational genomics and RNA interference, and (1d) steps to develop prototype termiticides have been initiated. With respect to the termite gut, (2a,2b) sequence verification from the termite gut and symbiont meta-transcriptomes has continued, and (2c) functional characterizations of gut genes using biochemistry approaches and RNA interference have continued. Results have been disseminated through peer-reviewed publications, extension materials / presentations, mass media interviews, and direct contacts with the pest control industry. PARTICIPANTS: Faculty, post-docs, graduate students, and undergraduate students who have contributed to the project are listed below. Those who received training and underwent professional development include the three graduate students and four undergraduate interns. The graduate students conducted research as part of dissertation projects, whereas the undergraduate interns participated as part of an undergraduate research experience course at the University Of Florida (ENY4905). Faculty / post-doctoral associates Michael E. Scharf (PD) - associate professor; 10% effort Xuguo Zhou (co-PD) - post-doctoral associate; 100% effort (2006-2007) Monique R. Coy (Ph.D.) - post-doctoral associate; 100% effort (2008-2009) Faith M. Oi (Ph.D.) - assistant extension scientist; project participant Drion G. Boucias (Ph.D.) - professor; project participant Graduate Students Matthew R. Tarver (Ph.D. student) - graduate research assistant; 100% effort Marsha M. Wheeler (M.S. student) - graduate research assistant; project participant Andres Sandoval-Mojica (Ph.D. student) - graduate research assistant; project participant Undergraduate Interns Caitlin E. Buckspan (undergraduate student) - REU / intern project participant Traci L. Grzymala (undergraduate student) - REU project participant Matthew Carroll (undergraduate student) - REU project participant Steven Hazim (undergraduate student) - REU project participant TARGET AUDIENCES: The target audiences of this research include the urban pest control community, the termite research community, pesticide research and development researchers and companies, bioenergy technology development and biorefinery industries. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Findings from 2010 contributed directly to the development of termite control technologies, and secondarily, to the bioenergy / renewable energy area. Control technologies include RNAi termiticides and enzymatic inhibitors. Bio/ renewable energy technologies include novel pre-treatment enzymes that contribute to biomass breakdown (i.e., pre-treatment/ delignification) and release of fermentable sugars (i.e., saccharification). Progress has remained steady and on-track, and objectives were not in need of adjustment. Resources supported personnel, research costs and supplies/expenses directly associated with attainment of project objectives.

Publications

  • Tarver M.R., E.A. Schmelz, M.E. Scharf. 2010. Soldier caste influences on gamma cadinene and cadinene-aldehyde levels in termite workers and impacts on juvenile hormone-dependent caste differentiation. Journal of Experimental Biology In Review (JEXBIO/2010/050351).
  • Schwinghammer, M.A., Zhou, X., Kambhampati, S., Bennett, G.W., Scharf, M.E. 2010. A novel gene from the takeout family involved in termite trail-following behavior. Gene In Review (GENE-D-10-00295).
  • Coy, M.R., Salem T.Z., Denton J.S., Kovaleva E., Liu Z., Barber D.S., Campbell J.H., Davis D.C., Buchman G.W., Boucias D.G., Scharf M.E. 2010. Phenol-oxidizing laccases from the termite gut. Insect Biochemistry and Molecular Biology In Press.
  • Scharf, M.E., Kovaleva, E.S., Jadhao, S., Campbell, J.H., Buchman, G.W., Boucias, D.G. 2010. Functional and translational analyses of a beta-glucosidase gene (glycosyl hydrolase family 1) isolated from the gut of the lower termite Reticulitermes flavipes. Insect Biochemistry and Molecular Biology. 40: 611-620.
  • Wheeler, M.M., X. Zhou, M.E. Scharf and F.M. Oi. 2007. Molecular and biochemical markers for monitoring dynamic shifts in cellulolytic protozoa in Reticulitermes flavipes. Insect Biochemistry and Molecular Biology 37 (12): 1366-1374.
  • Zhou, X., J.A. Smith, P.G. Koehler, F.M. Oi, G.W. Bennett and M.E. Scharf. 2007. Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite R. flavipes. Gene 395: 29-39.
  • Zhou X., M.R. Tarver and M.E. Scharf. 2007. Hexamerin-based regulation of juvenile hormone dependent gene expression underlies phenotypic plasticity in a social insect. Development 134: 601-610.
  • Zhou, X., Kovaleva, E.S., Wu-Scharf, D., Campbell, J.H., Buchman, G.W., Boucias, D.G., Scharf, M.E., 2010. Production and characterization of two recombinant beta-1,4-endoglucanases (GHF9) from the termite Reticulitermes flavipes. Archives of Insect Biochemistry and Physiology. 74: 147-162.
  • Sun, J.Z. and Scharf, M.E. 2010. Exploring and integrating cellulolytic systems of insects to advance biofuel technology. Insect Science 17: 163-165.
  • Scharf, M.E. and D.G. Boucias. 2010. Potential of termite-based pre-treatment strategies for use in bioethanol production. Insect Science 17: 1-9.
  • Tarver, M.R., Zhou, X., Scharf, M.E. 2010. Socio-environmental and endocrine influences on developmental and caste-regulatory gene expression in the eusocial termite Reticulitermes flavipes. BMC Molecular Biology 11: 28.
  • Wheeler MM, Tarver MR, Coy MR, Scharf ME. 2010. Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes. Archives of Insect Biochemistry and Physiology. 73: 30-48.
  • Tartar, A., M.M. Wheeler, X. Zhou, M.R. Coy, D.G. Boucias and M.E. Scharf. 2009. Parallel meta-transcriptome analyses of host and symbiont gene expression in the gut of the termite R. flavipes. Biotechnology for Biofuels. 2: 25.
  • Scharf, M.E., C.E. Buckspan, T.L. Grzymala and X. Zhou. 2007. Regulation of polyphenic caste differentiation in the termite Reticulitermes flavipes by interaction of intrinsic and extrinsic factors. Journal of Experimental Biology 210 (24): 4390-4398.


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Outputs from 2009 were again in both project areas of: (1) termite caste differentiation and (2) the termite gut. With respect to caste differentiation, (1a) novel semiochemicals have been purified and characterized, (1b) dozens of candidate developmental genes that respond to semiochemicals and hormones have been identified, (1c) critical responsive genes have been characterized using functional/ translational genomics and RNA interference, and (1d) steps to develop prototype termiticides have been initiated. With respect to the termite gut, (2a,2b) sequence verification from the termite gut and symbiont meta-transcriptomes has continued, and (2c) functional characterizations of gut genes using biochemistry approaches and RNA interference have continued. Results have been disseminated through peer-reviewed publications, extension materials / presentations, mass media interviews, and direct contacts with the pest control industry. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The target audiences of this research include the urban pest control community, the termite research community, pesticide research and development researchers and companies, bioenergy technology development and biorefinery industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Findings from 2009 contributed directly to the development of termite control technologies, and secondarily, to the bioenergy / renewable energy area. Control technologies include RNAi termiticides and enzymatic inhibitors. Bio/ renewable energy technologies include novel pre-treatment enzymes that contribute to biomass breakdown (ie, pre-treatment/ delignification) and release of fermentable sugars (ie, saccharification). Project evaluation reveals that progress is on-track and objectives are not in need of adjustment. Resources supported personnel, research costs and supplies/expenses directly associated with attainment of project objectives.

Publications

  • 6.Smith, J.A., M.E. Scharf, R.M. Pereira and P.G. Koehler. 2009. Comparison of gut carbohydrolase activity patterns in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers. Sociobiology 53(1): 13-22.
  • 4.Tarver M.R., E.A. Schmelz, J.R. Rocca and M.E. Scharf. 2009. Effects of soldier-derived terpenes on soldier caste differentiation in the termite Reticulitermes flavipes. Journal of Chemical Ecology 35: 256-264.
  • 5.Smith, J.A., M.E. Scharf, R.M. Pereira and P.G. Koehler. 2009. pH optimization of gut cellulase and xylanase activities from the Eastern subterranean termite, Reticulitermes flavipes. Sociobiology 54(1): 199-210.
  • 1.Scharf, M.E. and D.G. Boucias. 2010. Potential of termite-based pre-treatment strategies for use in bioethanol production. Insect Science. In Press.
  • 2.Wheeler, M.M., M.R.Tarver, M.R.Coy and M.E. Scharf. 2010. Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes. Archives of Insect Biochemistry and Physiology. 73: 30-48.
  • 3.Tartar, A., M.M. Wheeler, X. Zhou, M.R. Coy, D.G. Boucias and M.E. Scharf. 2009. Parallel meta-transcriptome analyses of host and symbiont gene expression in the gut of the termite R. flavipes. Biotechnology for Biofuels. 2: 25.


Progress 07/01/08 to 12/31/08

Outputs
OUTPUTS: Outputs from 2008 were in both project areas of: (1) termite caste differentiation, and (2) the termite gut. With respect to caste differentiation, (1a) novel semiochemicals have been identified, (1b) developmental genes that respond to semiochemicals and hormones have been identified, and (1c) some developmental genes have been preliminarily characterized using RNA interference. With respect to the termite gut, (2a,2b) comprehensive 1st-tier sequencing from the termite gut and symbiont meta-transcriptomes has been completed [~6,000 genes], and (2c) functional characterizations of gut genes using biochemistry approaches and RNA interference are underway. Results have been disseminated through peer-reviewed publications, extension materials / presentations, mass media interviews, and direct contacts with the pest control industry. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Findings from 2008 contributed directly to the development of termite control technologies, and secondarily, to the bioenergy / renewable energy area. Project evaluation reveals that progress is on-track and objectives are not in need of adjustment. Resources supported personnel, research costs and supplies/expenses directly associated with attainment of project objectives.

Publications

  • 2. Scharf, M.E., X. Zhou and M.A. Schwinghammer. 2008. Application of RNA interference in functional-genomics studies of a social insect. In Methods in Molecular Biology vol. 442: RNAi, design and applications (S. Barik, Ed.), pp. 205-229. Humana Press, Totowa, NJ.
  • 3.Tarver M.R., E.A. Schmelz and M.E. Scharf. 2008. Effects of soldier-derived terpenes on soldier caste differentiation in the termite Reticulitermes flavipes. J. Chem. Ecol. In Press.
  • 1. Scharf, M.E. and A. Tartar. 2008. Termite digestomes as sources for novel lignocellulases. Biofuels, Bioproducts and Biorefining. 2(6): 540-552.
  • 4. Smith, J.A., M.E. Scharf, R.M. Pereira and P.G. Koehler. 2008. Comparison of gut carbohydrolase activity patterns in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers. Sociobiology 53(1) In Press.
  • 5. Zhou, X., M.M. Wheeler, F.M. Oi and M.E. Scharf. 2008. RNA interference in the termite R. flavipes through ingestion of double-stranded RNA. Insect Biochem. Mol. Bio. 38: 805-815.
  • 6. Zhou, X., M.M. Wheeler, F.M. Oi and M.E. Scharf. 2008. Inhibition of termite cellulases by carbohydrate-based cellulase inhibitors: evidence from in vitro biochemistry and in vivo feeding studies. Pestic. Biochem. Physiol. 90(1): 31-41.
  • 7. Tarver, M.R and M.E. Scharf. 2008. Termite caste differentiation: a social affair. Pest Control Technology Magazine. 35(1): 82,84,86.