RNAi of two chitin-binding protein genes disrupts peritrophic matrix barrier function in the larval midgut of Tribolium castaneum

The peritrophic matrix (PM) in the midgut of insects consisting primarily of the matrix polysaccharide chitin and proteins (PMPs) is thought to support digestion and protection from abrasive food particles and enteric pathogens. To investigate the physiological roles of PMPs, an RNAi study of all eleven PMP genes (TcPMPs) of the red flour beetle, Tribolium castaneum, was carried out. TcPMP genes are differentially expressed along the length of the midgut epithelium of feeding larvae.  RNAi of individual PMP genes reveals no developmental phenotypes for 9 of 11 TcPMPs, suggesting that many have redundant or non-essential functions. RNAi for TcPMP3 and TcPMP5-B, however, results in depletion of fat body, growth arrest, molting defects and mortality. In situ permeability assays after oral administration of FITC-conjugated dextrans of different molecular masses to larvae demonstrate that the exclusion size of the PM decreases progressively from the anterior to the posterior parts of the midgut from >2 MDa to < 4 kDa. In control larvae, 2 MDa dextrans are completely retained within the PM lumen in the median midgut, whereas after RNAi for TcPMP3 and TcPMP5-B, these dextrans penetrate the median midgut epithelium, indicating loss of structural integrity and barrier functions of larval PM. In adult females, while PMP3 RNAi affects fecundity and viability, only egg hatch is affected by PMP5-B RNAi.  These results indicate for the first time in any insect species that PMPs are essential for regulation of PM permeability, and that there is additional functional specialization even among individual PMPs.