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Title: Both UDP N-acetylglucosamine pyrophosphorylases of Tribolium castaneum are critical for molting, survival, and fecundity

Author
item ARAKANE, YASUYUKI - Kansas State University
item BAGUINON, MARILYN - Kutztown University
item JASRAPURIA, SINU - Kansas State University
item CHAUDHARI, SUJATA - Kansas State University
item DOYUNGAN, ALISON - Kansas State University
item KRAMER, KARL - Collaborator
item MUTHUKRISHNAN, SUBBARATNAM - Kansas State University
item Beeman, Richard

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/27/2010
Publication Date: 1/15/2011
Citation: Arakane, Y., Baguinon, M., Jasrapuria, S., 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 Biochemistry and Molecular Biology. 41(1):42-50. doi: http://dx.doi.org/10.1016/j.ibmb.2010.09.011.

Interpretive Summary: Chitinous structures on insects such as the exoskeleton and digestive sac are vital for insect survival and could be exploited by appropriately-targeted biopesticides. We identified two new “UAP” genes in the red flour beetle that are required to generate the basic building blocks not only of chitin, but also other vital insect sugar polymers and sugar-proteins. All other invertebrates examined have only one UAP gene. One of the two beetle genes is similar to the corresponding one in other invertebrates, while the other seems to have unique functions in nutrition and growth. If the second gene is eliminated, the insects die, apparently of starvation. Our ongoing gene discovery efforts in this pest insect continue to reveal new weaknesses that may lead to new methods of insect control.

Technical Abstract: A bioinformatics search of the genome of the red flour beetle, Tribolium castaneum, resulted in the identification of two genes encoding proteins closely related to UDP-N-acetylglucosamine pyrophosphorylases (UAP), which provide the activated precursor, UDP-N-acetylglucosamine, for the synthesis of chitin, glycoproteins, glycosylphosphoinositide (GPI) anchors of some membrane proteins, and other substrates. This is in contrast to other arthropods whose genomes have been completely sequenced, all of which have only a single copy of this gene. The two T. castaneum UAP genes, TcUAP1 and TcUAP2, share both nucleotide and amino acid sequence identities of about 60%. RT-PCR analysis revealed that the two genes differ in their developmental and tissue-specific patterns of expression. RNA interference (RNAi) indicated roles for TcUAP1 and TcUAP2 at the molt and intermolt stages, respectively: RNAi for TcUAP1 resulted in specific arrest at the larval-larval, larval-pupal, or pupal-adult molts, depending on time of injection of double-stranded RNAs, whereas RNAi for TcUAP2 prevented larval growth or resulted in pupal paralysis. Analysis of elytral cuticle indicated loss of structural integrity and chitin staining after RNAi for TcUAP1, but not after RNAi for TcUAP2. Loss of peritrophic matrix (PM)-associated chitin was also observed following RNAi for TcUAP1, but not after RNAi for TcUAP2. Down-regulation of transcripts for either TcUAP gene at the mature adult stage resulted in cessation of oviposition in females, as well as fat body depletion and eventual death in both sexes. These results indicate that both TcUAP genes are critical for beetle development and survival. While only TcUAP1 is clearly associated with synthesis of cuticular or PM chitin, both of these genes appear to have additional critical role(s) unrelated to chitin synthesis, presumably in the glycosylation of proteins and/or secondary metabolites.