PEST CONTROL BY TARGETING INSECT CHITIN METABOLISM

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0213739
• Project Start Date: Jan 1, 2008
• Project End Date: Dec 31, 2012
• Program Code: [(N/A)]- (N/A)

Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506

Performing Department
BIOCHEMISTRY

Non Technical Summary
Chitin is an essential component of cuticle, midgut peritrophic matrix (also known as peritrophic membrane or PM), and several other anatomical structures of insects. Precise regulation of chitin metabolism is a complex and intricate process that is critical for insect growth, metamorphosis, organogenesis, and survival. Chitin content of the insect is influenced directly by several enzymes, including chitin synthase (CHS), chitinase (CHT) and N-acetyl-glucosaminidase (NAG). Modulation of the physical properties of chitin-containing structures of insects is accomplished, in part, by the deacetylation of chitin by chitin deacetylase (CDA). Partially deacetylated chitin has different protein binding and structural properties compared to chitin. The presence of a multiplicity of isozymes of CHS, CHT, NAG, and CDA in insects is suggestive of evolutionary diversification and functional specialization within each class of enzymes during development or in different tissues. In addition to these enzymes, other non-enzymatic proteins such as "retroactive" (RTV) and Knickkopf (KNK) have been postulated to bind to chitin and shown to be required in the assembly of chitin microfibrils. Our recent results have indicated that down-regulation of the synthesis of several proteins directly involved in chitin metabolism, as well as other regulatory proteins of chitin metabolism, causes lethality at distinct stages of development of Tribolium castaneum, the red flour beetle which is a major stored product insect of agricultural importance. Stored product insects such as the red flour beetle, lesser grain borer and other beetles cause millions of dollars in damage annually to raw grain or to processed foods such as baked goods. Chitin represents a vulnerable and insect-selective target which can be exploited for insect control. We will identify and obtain detailed knowledge of the target genes in insects involved in chitin metabolism hich will help develop rational approaches to control pests selectively without deleterious effects on other useful insects. We will study the mode of action of genes of chitin metabolism as well as chitin insecticides such as benzoylphenylureas are effective also against lepidopteran pests such as the fall army worm and the tobacco hornworm as well as cockroaches. A thorough knowledge of the effects of chitin-modifying enzymes on cuticle may also help develop biomimetic materials for a variety of industrial and pharmaceutical applications.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
216	3110	1000	50%
216	3110	1060	50%

Knowledge Area
216 - Integrated Pest Management Systems;

Subject Of Investigation
3110 - Insects;

Field Of Science
1060 - Biology (whole systems); 1000 - Biochemistry and biophysics;

Keywords

insect growth regulators

chitin metabolism

tribolium

drosophila

Goals / Objectives
Objectives A. Determine tissue specificities and developmental patterns of expression of candidate genes in chitin metabolism. B. Analyze the functions of genes involved in chitin metabolism and the effects of RNAi-mediated transcript depletion on insect growth and development. C. Identify in vivo target sites/steps affected by insect growth regulators (IGRs) and other inhibitors of chitin metabolism. D. Purify and characterize the key enzymes of chitin metabolism, and analyze the effects of inhibitors of chitin metabolism on these enzymes in vitro. We anticipate that there will be selective expression of genes encoding specific isozymes or proteins at different developmental stages and/or in specific tissues. Complementary patterns of gene expression are expected between members in the chitin biosynthetic and chitin degradative branches in the epidermal and gut cells. Specific genes may be expressed in epidermis, gut and trachea and at different developmental stages (e.g. tracheal tube morphogenesis). We anticipate that different genes involved in chitin metabolism in Tribolium will affect molting/development at different stages during cuticle remodeling (embryonic development, larval-larval, larval-pupal, pupal-adult molt, adult maturation, and egg production) and in selective tissues (epidermis, gut, tracheae, etc). Some of the genes (CHS, CHT, CDA, and cystic) may influence chitin content as well as physical properties of cuticle associated with different organs or anatomical structures (e.g. head capsule, body skeleton, elytra, wing, trachea, mouthparts, spiracles, gin traps, urogomphi, and PM). Other genes (e.g. rtv, knk, SUR, KIR and Syntaxin) may be involved in chitin assembly and turnover. RNAi studies at different developmental stages may reveal the target tissue and stage of development when the expression of a specific gene is crucial. Dietary Calcofluor and WGA bind to chitin chains and may interfere with chitin polymerization and/or binding of peritrophins during PM assembly, thereby affecting PM integrity. These experiments will determine whether sulfonyl ureas (SU) or diazoxide interferes with transport of CHS to the plasma membrane and/or assembly of chitin chains into microfibrils. Our results will reveal for the first time whether SUs affect both cuticular and PM-associated chitin synthesis. Both cuticle and PM chitin synthesis may be affected by regulating vesicular transport of CHS enzymes. If difficulties are encountered in expressing these chitinases in Hi-5 cells, we will express them in yeast. This system has the advantage of being rapid and is capable of yielding milligram amounts of the secreted proteins from a liter of culture medium. The one possible drawback of the system is that the yeast-expressed proteins will exhibit fungal glycosylation patterns. If glycosylation does not interfere with the activity or properties of the chitinases, this system will be a valuable fall back option.

Project Methods
1.) We must determine the developmental pattern of expression and tissue-specificity of each gene to determine optimal timing for injection of dsRNAs and the tissues in which changes are expected. Expression patterns of Tribolium genes predicted to be involved in chitin metabolism during development and tissue-specificity of expression will be determined by real time PCR, in situ hybridization, western blotting, immunohistochemistry and confocal microscopy, and using transgenic promoter/reporter constructs. 2.) We will identify regions that are unique by DNA sequence comparisons. Using an in vitro transcription kit, dsRNA corresponding to unique regions of each target gene will be synthesized and purified. Administration of dsRNA into the dorsal abdomens during growth stages will be carried out. Observations will be made on a daily basis using light microscopy and video-recording to follow effects on development of embryos, egg hatching, 3 stages of molting, and fecundity. We will determine the developmental stage at time of death, chitin content of cuticle, PM and tracheae, protein levels by western blotting and integrity of cuticle, PM, trachea and other chitin-containing structures by light microscopy. 3.) We will test the hypothesis that IGRs target distinct proteins of chitin metabolism by comparing the effects of IGRs with those of RNAi experiments. We will follow the effects of the inhibitors administered at LD25 levels to identify their primary effects. We will analyze the surviving animals for growth abnormalities, fecundity, egg hatch, abnormal body structures including cuticle, gut and reproductive organs, chitin content of cuticle and PM, and other visual phenotypes such as inability to shed the larval or pupal cuticle, size shrinkage and loss of weight. The transcript levels of genes involved in chitin metabolism will be determined in control and inhibitor-treated animals using real time PCR. Alteration in levels of PM-associated chitin will be determined by chemical analysis and FITC-CBD staining. Changes in the assembly of procuticular and tracheal chitin lamellae and PM integrity will be visualized by confocal microscopy after staining. We will determine whether SUs target transport of vesicles from the ER to the Golgi and then to the plasma membrane. The intracellular locations of CHSs (and other integral membrane proteins and organelle-specific marker proteins) will be determined by immunolocalization with specific primary antibodies. We will fractionate epidermal and gut tissue homogenates to obtain membrane fractions expected to contain transport vesicles from control and treated animals. We will look for similarities between effects of dietary inhibitors and those of RNAi experiments with specific dsRNAs for proteins involved in chitin metabolism. These experiments may identify target proteins for that inhibitor. 4.) Using the AcMNPV vector in Hi-5 cells, we will express other CHT, NAG and CDA genes from Tribolium. The biochemical properties of the enzymes will be studied. We will test combinations of purified chitinases and NAGs to determine the degree of synergism in degradation of high molecular weight forms of chitin.

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

Outputs
OUTPUTS: By screening the newly completed red flour beetle genome database we have identified about fifty genes encoding proteins with chitin binding domains including several chitinase or chitinase-related proteins, peritrophins and cuticular proteins analogous to peritrophins. Using RNA interference techniques, we have established the functions of several of these proteins. We have also functionally characterized two chitin organizing proteins namely Knickkopf and Retroactive. PARTICIPANTS: S. Muthukrishnan PI - Designed project and analyzed data and edited publications; R. Beeman PI - Designed project and analyzed data and edited publications; Y. Arakane - Performed the experiments, analyzed data and wrote the publications; Sinu Jasrapuria- Performed the experiments, analyzed data and wrote publications; Sujata Chaudhari - Performed the experiments, analyzed data and wrote publications; Meera Kumari - Performed the experiments, analyzed data TARGET AUDIENCES: Farmers, agricultural chemical industry, graduate students, visiting scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have identified several critical proteins involved in chitin metabolism that are essential for insect survival, molting and fecundity and organization of chitin into laminae. Many of these proteins are essential for insect molting and the proper laminar organization of the insect exoskeleton. Some of these may prove to be potential targets for insecticides.

Publications

• Muthukrishnan, S., Merzendorfer, H., Arakane, Y., Kramer, K.J. (2012). Chitin metabolism in insects. In: Insect Biochemistry and Molecular Biology. Gilbert, L. I. (ed.), Elsevier Inc., San Diego, ISBN:-978-0-12-384747-8. pp.193-235.
• Merzendorfer, H., Kim, H.S., Chaudhari, S.S., Kumari, M., Specht, C.A., Butcher, S., Brown, S.J., Manak, J.R., Beeman R.W. Kramer K.J., and Muthukrishnan, S. (2012) Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species, Tribolium castaneum. Insect Biochem. Mol. Biol., 42: 264-276.
• Arakane, Y, Lomakin, J., Gehrke, S.H., Hiromasa, Y., Tomich, J.M., Muthukrishnan, S., Beeman, R.W., Kramer, K.J. and Kanost, M.R. (2012) Formation of rigid, non-flight forewings (elytra) of a beetle requires two major cuticular proteins. PLoS Genetics 8(4): e1002682. doi:10.1371/journal.pgen.1002682
• Jasrapuria, S., Specht, C.A., Kramer, K.J., Beeman, R.W., Muthukrishnan, S. (2012) Gene Families of Cuticular Proteins Analogous to Peritrophins (CPAPs) in Tribolium castaneum Have Diverse Functions. PLoS ONE 7(11): e49844. doi:10.1371/journal.pone.0049844

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We have previously identified several genes encoding proteins of chitin metabolism and assembly in the genome of the red flour beetle, Tribolium castaneum. During the past year using RNA interference, we have demonstrated that many of these genes are important for molting, survival and proper flexibility of leg joints. We also found that the chitin content of the cuticle is affected by the following genes, chitin synthases, chitin deacetylases and two chitin organizing proteins, Knickkopf (Knk) and retroactive (Rtv). The effects of KNK on chitin levels are indirect because simultaneous down-regulation of both chitinases and Knk result in restoration of chitin levels. These results indicate a role of Knk in protecting chitin from degradation from chitinases Transmission electron microscopic studies have revealed that the laminar organization of chitin is lost upon RNAi for Knk and chitinases suggesting a second role of Knk on laminar organization of chitin. PARTICIPANTS: S. Muthukrishnan, PI - Designed project and analyzed data and edited publications; R. Beeman, PI - Designed project and analyzed data and edited publications; Y. Arakane - Performed the experiments, analyzed data and wrote the publications; Sinu Jasrapuria- Performed the experiments, analyzed data; Sujata Chaudhari - Performed the experiments, analyzed data; Meera Kumari - Performed the experiments, analyzed data. TARGET AUDIENCES: Farmers, agricultural chemical industry, graduate students, visiting scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our results have established a new paradigm for the stability of chitin in the newly synthesized cuticle during the period of molting when the chitin in the old cuticle is being digested. Our experiments have established that chitinases are not excluded from the newly synthesized cuticle, but rather Knk protects chitin from chitinases by binding and/or organizing chitin into bundles and laminae. Thus the old model that the envelope layer is the basis of the protection of the new cuticle from chitinase must be modified.

Publications

• Arakane, Y., Baguinon, M., Jasrapuria, J., Chaudhari, S., Doyungan, A., Kramer, K.J., Muthukrishnan, S., Beeman, R. W. (2011) Both UDP N-acetylglucosamine pyrophosphorylases of Tribolium castaneum are critical for molting, survival and fecundity. Insect Biochem. Mol. Biol. 41: 42-50.
• Zhang J, Zhang X, Arakane Y, Muthukrishnan S, Kramer KJ, Ma E, Zhu KY. (2011) Comparative genomic analysis of chitinase and chitinase-like genes in the African malaria mosquito (Anopheles gambiae). PLoS One. 6(5):e19899. Epub 2011 May 18.
• Zhang J, Zhang X, Arakane Y, Muthukrishnan S, Kramer KJ, Ma E, Zhu KY. (2011) Identification and characterization of a novel chitinase-like gene cluster (AgCht5) possibly derived from tandem duplications in the African malaria mosquito, Anopheles gambiae. Insect Biochem Mol Biol. 2011: 41:521-528.
• Chaudhari, S. S., Arakane, Y., Specht, C. A., Moussian, B., Boyle, D. L., Park, Y., Kramer, K. J., Beeman, R. W. , Muthukrishnan, S. (2011) The Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton. Proc Natl Acad Sci USA. 108:17028-17033.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: By screening the newly completed red flour beetle genome database we have identified about fifty genes encoding proteins with chitin binding domains. These include several chitinases, chitinase-related proteins, peritrophins, and cuticular proteins analogous to peritrophins. Using RNA interference techniques, we have established the functions of several of these proteins. In particular, we have determined that several proteins with chitin-binding domains and two membrane-bound proteins are essential for normal molting and maintaining the structural integrity of the insect cuticle. PARTICIPANTS: S. Muthukrishnan PI - Designed project and analyzed data and edited publications; R. Beeman PI - Designed project and analyzed data and edited publications; Y. Arakane - Performed the experiments, analyzed data and wrote the publications; Sinu Jasrapuria- Performed the experiments, analyzed data; Sujata Chaudhari - Performed the experiments, analyzed data; Meera Kumari - Performed the experiments, analyzed data. TARGET AUDIENCES: Farmers, agricultural chemical industry, graduate students, visiting scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have determined that there is functional specialization among the more than 50 proteins with chitin-binding domains that are involved in chitin metabolism. Insect survival, molting, fecundity, and chitin content are affected in different ways when the activities of individual genes of chitin metabolism are down-regulated. We have also identified some genes whose protein products are affected by the insecticide diflubenzuron. Some of these genes/proteins may prove to be potential targets for insecticides.

Publications

• Arakane, Y., Muthukrishnan, S. (2010). Insect chitinases and chitinase-like proteins. Cellular Molecular Life Sciences, 67:201-216.
• Jasrapuria, S., Arakane, Y., Osman, G., Kramer, K.J., Beeman, R.W., Muthukrishnan, S. (2010). Insect Biochem Mol. Biol. Genes encoding proteins with peritrophin A-type chitin binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function. Insect Biochem Mol. Biol. 40:214-227.
• Arakane, Y., Dittmer, N., Kramer, K.J., Muthukrishnan, S., Beeman, R.W., and Kanost, M. (2010). Identification, mRNA expression and functional analyses of some yellow family genes in Tribolium castaneum. Insect Biochem Mol. Biol. 40:259-266.
• Broehan, G., Arakane, Y., Beeman, R.W., Kramer, K.J., Muthukrishnan, S., and Merzendorfer, H. (2010). Chymotrypsin-like peptidases from Tribolium castaneum: RNA interference reveals physiological functions in molting. Insect Biochem. Mol. Bio. 40:274-283.
• Mansur, J.F., Figueira-Mansur, J., Santos, A.S., Santos-Junior, H., Ramos, I.B., Neves de Medeiros, M., Machado, E.A., Kaiser, C.R., Muthukrishnan, S., Masuda, H., Vasconcellos, A.M.H., Melo, A.C.A., Moreira, M.F. (2010). The effect of lufenuron, a chitin synthesis inhibitor, on oogenesis of Rhodnius prolixus. Pesticide Biochemistry and Physiology 98:59-67.
• Chitvan, K., Buschman, L.L., Chen, M.S., Muthukrishnan, S., and Zhu, K.Y. (2010). A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae. Insect Biochem. Mol. Biol., 40:621-629.
• Arakane, Y., Baguinon, M., Jasrapuria, J., Chaudhari, S., Doyungan, A., Kramer, K.J., Muthukrishnan, S., Beeman, R.W. (2010). Two Uridine-diphosphatate N-acetylglucosamine pyrophosphorylases are critical for Tribolium castaneum molting and survival. Insect Biochem. Mol. Biol. doi:10.1016/j.ibmb.2010.09.011

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: By screening the newly completed red flour beetle genome database we have identified about fifty gene encoding proteins with chitin binding domains including several chitinase or chitinase-related proteins, peritrophins and cuticular proteins analogous to peritrohins. Using RNA interference techniques, we have established the functions of several of these proteins. PARTICIPANTS: S. Muthukrishnan PI - Designed project and analyzed data and edited publications, R. Beeman PI - Designed project and analyzed data and edited publications, Y. Arakane - Performed the experiments, analyzed data and wrote the publications, Sinu Jasrapuria- Performed the experiments, analyzed data, Sujata Chaudhari - Performed the experiments, analyzed data TARGET AUDIENCES: Farmers, agricultural chemical industry, graduate students, visiting scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have identified several critical proteins involved in chitin metabolism that are essential for insect survival, molting and fecundity. Some of these may prove to be potential targets for insecticides

Publications

• Arakane, Y., Begum, K., Dixit, R., Park, Y., Specht, C. A., Kramer, K. J., Beeman, R. W. (2009). Analysis of functions of the chitin deacetylase gene family in Tribolium castaneum. Insect Biochem Molec Biol. . 38: 959-962.
• Arakane, Y., Muthukrishnan, S. (2009) Insect chitinases and chitinase-like proteins. Cellular Molecular Life Sciences, DOI:10.1007/s00018-009-0161-9

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

Outputs
OUTPUTS: We have developed RNA interference techniques for analyzing the functions of genes involved in chitin metabolism. The results from our research effort have been presentations at Second Arthropod Genomics Conferences in Kansas City and in Departmental seminars on campus. We have trained scientists from other laboratories in US to use these techniques. We have also trained graduate and undergraduate students. PARTICIPANTS: S. Muthukrishnan: Project Director- planned and supervised research; edited publications; presented seminars; Y. Arakane: research Assistant Professor - planned and conducted research; supervised research; wrote research publications; presented seminars; Sinu Jasrapuria: graduate student - conducted research, presented seminars; Sujata Chaudhari: graduate student - conducted research, presented seminars. Partner Organizations - University of Massachusetts medical school: U. Mass medical school cooperated with us in the analysis of insect samples for chitin and chitosan content. University of Osnabrueck, Germany cooperated with us on studies on diflubenzuron. Collaborators and Contacts - Dr. Charles A Spect of U. Mass cooperated with us in the analysis of insect samples for chitin and chitosan content. Dr. Hans Merzendorfer of the University of Osnabrueck, Germany cooperated with us on studies on diflubenzuron, an insecticide. Training or Professional development - Two graduate students Sinu Jasapuria and Sujata Chaudhari received doctoral training in conducting research. Dr. Hans Merzendorfer and his student Gunnar Broehan from the University of Osnabrueck, Germany received training in microinjection of insects. TARGET AUDIENCES: Students and other scientists in our field. We presented our work in the form of seminars and taught students in class and in the laboratory. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have been able to demonstrate that the isozymes of chitin metabolism have distinct functions in terms of being expressed at different developmental stages or in different tissues. We have shown that different isozymes have distinct functions in some cases.

Publications

• Zhu, Q., Arakane, Y., Beeman, R.W., Kramer, K.J., Muthukrishnan, S. (2008) Functional specialization among insect chitinase family genes revealed by RNA interference.Proc NatlAcadSci USA. 105: 6650-6655.
• Arakane, Y., Specht, C.A., Kramer, K.J., Muthukrishnan, S., Beeman, R.W. (2008) Chitin synthases are required for survival, fecundity, embryonic hatching in the red flour beetle, Triboliumcastaneum. Insect Biochem. Mol. Biol. 38: 959-962.
• Zhu, Q., Arakane, Y., Banerjee, D., Beeman, R.W., Kramer, K.J., Muthukrishnan, S.(2008) Domain organization and phylogenetic analysis of chitinase family of proteins in three species of insects. Insect Biochem Mol Biol 38: 452-466
• Zhu, Q., Arakane, Y., Beeman, R.W., Kramer, K.J., Muthukrishnan, S. (2008) Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Triboliumcastaneum. Insect Biochem. Molec. Biol. 38: 467-477
• Hogenkamp, D.G., Arakane, Y., Kramer, K.J., Muthukrishnan, S., Beeman, R.W. (2008) Characterization and expression of the beta-N-acetylglucosaminidase gene family of Triboliumcastaneum. Insect Biochem. Molec. Biol. 38: 478-489
• Dixit, R., Arakane. Y., Specht, C.A., Richard, C., Kramer, K.J., Beeman, R.W., Muthukrishnan, S. (2008) Domain organization and phylogenetic analysis of proteins from the chitin deacetylase gene family of Triboliumcastaneum and three other species of insects. Insect Biochem Mol Biol.38:440-51.