Molecular cloning and effects of Tm14-3-3ζ-silencing on larval survivability against E. Coli and C. albicans in tenebrio molitor

Abstract

The 14-3-3 family of proteins performs key regulatory functions in phosphorylationdependent signaling pathways including cell survival and proliferation, apoptosis, regulation of chromatin structure and autophagy. In this study, the zeta isoform of 14-3-3 proteins (designated as Tm14-3-3ζ) was identified from the expressed sequence tags (ESTs) and RNA sequencing (RNASeq) database of the coleopteran pest, Tenebrio molitor. Tm14-3-3ζ messenger RNA (mRNA) is expressed at higher levels in the immune organs of the larval and adult stages of the insect and exhibit almost five-fold induction within 3 h post-infection of the larvae with Escherichia coli and Candida albicans. To investigate the biological function of Tm14-3-3ζ, a peptide-based Tm14-3-3ζ polyclonal antibody was generated in rabbit and the specificity was confirmed using Western blot analysis. Immunostaining and confocal microscopic analyses indicate that Tm14-3-3ζ is mainly expressed in the membranes of midgut epithelial cells, the nuclei of fat body and the cytosol of hemocytes. Gene silencing of Tm14-3-3ζ increases mortality of the larvae at 7 days post-infection with E. coli and C. albicans. Our findings demonstrate that 14-3-3ζ in T. molitor is essential in the host defense mechanisms against bacteria and fungi.