Tribolium castaneum Larval Gut Transcriptome and Proteome: A Resource for the Study of the Coleopteran Gut

Abstract

Tribolium castaneum is an important agricultural pest and an advanced genetic model for coleopteran insects. We have taken advantage of the recently acquired T. castaneum genome to identify T. castaneum genes and proteins in one of the more critical environmental interfaces of the insect, the larval alimentary tract. Genetic transcripts isolated from the T. castaneum larval gut were labeled and hybridized to a custom array containing oligonucleotides from predicted genes in the T. castaneum genome. Through a ranking procedure based on relative labeling intensity, we found that approximately 17.6% of the genes represented in the array were predicted to be highly expressed in gut tissue. Several genes were selected to compare relative expression levels in larval gut, head, or carcass tissues using quantitative real-time PCR, and expression levels were, with few exceptions, consistent with the gut rankings. In parallel with the microarrays, proteins extracted from the T. castaneum larval gut were subjected to proteomic analysis. Two-dimensional electrophoretic analysis combined with MALDI-TOF resulted in the identification of 37 of 88 selected protein samples. As an alternative strategy, one-dimensional electrophoretic separation of T. castaneum larval gut proteins followed by two-dimensional nano-HPLC and ESI-MS/MS resulted in the identification of 98 proteins. A comparison of the proteomic studies indicated that 16 proteins were commonly identified in both, whereas 80 proteins from the proteomic analyses corresponded to genes with gut rankings indicative of high expression in the microarray analysis. These data serve as a resource of T. castaneum transcripts and proteins in the larval gut and provide the basis for comparative transcriptomic and proteomic studies related to the gut of coleopteran insects.

Sodium Solute Symporter and Cadherin Proteins Act as Bacillus thuringiensis Cry3Ba Toxin Functional Receptors in Tribolium castaneum

“…The coleopteran model insect Tribolium castaneum (Tc) is a major global pest of stored products for human consumption for which many genetic and genomics tools have been developed (22), so it constitutes an ideal subject for the identification of new biopesticide targets based in Bt. In this work, we carried out receptor binding and ligand blot experiments in Tc larvae with the coleopteran-specific toxin Cry3Ba, previously shown to be active against this insect pest (23).…”

Comparative gut transcriptome analysis reveals differences between virulent and avirulent Russian wheat aphids, Diuraphis noxia

“…The gut transcriptomes of both hematophagous and phytophagous insects have been investigated extensively (Morris et al 2009, Wang et al 2011, Zhang et al 2010, Boissière et al 2012 However, we found it surprising to note the presence of greater sequence diversity in the RWA2 gut library, which shared maximum homology with that of several insect species.…”

Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference

“…To evaluate the RT-PCR based TcCTLP expression data, we have compared our results with microarray and proteomic data recently reported for the larval gut of Tribolium (Morris et al, 2009). In that study, ranking scores between 1 and 100 were assigned to the transcripts of gut-expressed genes, which reflect the extent of gene expression in the larval gut based on a custom array hybridization experiment covering about 12,000 GLEAN sequences of the T. castaneum genome.…”

“…In addition to their well-established function in digestion, they function in molting and metamorphosis, and act as highly specific regulators of innate immune responses, including melanization and the expression of antimicrobial peptides (Kanost et al, 2004;Law et al, 1977;Neurath, 1984;Srinivasan et al, 2006). The latter functions are tightly controlled by serpins, specific serine proteinase inhibitors that irreversibly inhibit their target enzymes by a suicide mechanism involving major conformational changes (Gettins, 2002).…”

“…Several conserved amino acid residues in the S1 pocket have been recognized as important determinants of substrate specificity, but these may be not the sole decisive factors for specifying the cleavage site (Hedstrom, 2002). Most S1A peptidases are secreted into the extracellular space as inactive, zymogenic precursor proteins, which become activated by proteolytic cleavage at conserved sites (Bode et al, 1978;Neurath and Dixon, 1957;Page and Di Cera, 2008). Zymogen activation reflects a powerful regulatory mechanism that is frequently embedded in a cascade of successive proteolytic steps that together initiate physiological processes in due time at the right place.…”