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Title: Characterization and expression of the beta-N-acetylglucosaminidase gene family of Tribolium castaneum

Author
item HOGENKAMP, DAVID - KANSAS STATE UNIV
item ARAKANE, YASUYUKI - KANSAS STATE UNIV
item KRAMER, KARL - 5430-05-30 RETIRED
item MUTHUKRISHNAN, SUBBARATNAM - KANSAS STATE UNIV
item Beeman, Richard

Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/1/2007
Publication Date: 3/1/2008
Citation: 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 Tribolium castaneum. Journal of Insect Biochemistry and Molecular Biology 38: 478-489.

Interpretive Summary: The chitin-containing insect exoskeleton (“skin”) represents both a unique physiological adaptation and a promising target for biopesticide disruption. Each time an insect sheds its old skin, the chitin is digested into smaller components, and these components are recycled to manufacture the next skin. We have identified four insect genes called “NAGs” that are essential for the final stage of chitin degradation into the smallest component parts. Each of the four NAG genes has a slightly different function in chitin degradation, presenting 4 new targets for pest control intervention. Continued identification of the individual genes required for processes unique to insects will expand our options for selective biopesticide design.

Technical Abstract: Enzymes belonging to the Beta-N-acetylglucosaminidase (NAG) family cleave chitin oligosaccharides produced by the action of chitinases on chitin into the constituent N-acetylglucosamine monomer. Four genes encoding putative NAGs in the red flour beetle, Tribolium castaneum, namely TcNAG1, TcFDL, TcNAG2, and TcNAG3, and three other related hexosaminidases were identified by searching the recently completed genome (Tribolium Genome Sequencing Consortium, submitted). Full-length cDNAs for all four NAGs were cloned and sequenced, and the exon-intron organization of the corresponding genes was determined. Analyses of their developmental expression patterns indicated that, although all four of the NAGs are transcribed during most developmental stages, each gene had a distinct spatial and temporal expression pattern. TcNAG1 transcripts are the most abundant, particularly at the late pupal stage, while TcNAG3 transcripts are least abundant, even at their peak levels in the late larval stages. The function of each NAG during different developmental stages was assessed by observation of lethal phenotypes after gene-specific dsRNA-mediated transcript depletion (RNAi) as verified by real-time PCR. TcNAG1 RNAi was most effective in interrupting all three types of molts, larval-larval, larval-pupal, and pupal-adult. Treated insects died after failing to completely shed their old cuticles. Knockdown of transcripts for the other three NAG genes resulted in phenotypes similar to those of TcNAG1 dsRNA-treated insects, but the effects were somewhat variable and less severe. Sequence comparisons with other insect homologs suggested that TcFDL, unlike the other NAGs, may have a role in N-glycan processing in addition to its apparent role in cuticular chitin turnover. These results support the hypothesis that TcNAGs participate in chitin turnover and/or N-glycan processing during insect development and that each NAG fulfills an essential and distinct function.