Agrawal Sinu, Kelkenberg Marco, Begum Khurshida, Steinfeld Lea, Williams Clay E, Kramer Karl J, Beeman Richard W, Park Yoonseong, Muthukrishnan Subbaratnam, Merzendorfer Hans
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, USA.
Department of Biology, Chemistry, University of Osnabrück, Osnabrück 49069, Germany.
Insect Biochem Mol Biol. 2014 Jun;49:24-34. doi: 10.1016/j.ibmb.2014.03.009. Epub 2014 Mar 26.
The peritrophic matrix (PM) in the midgut of insects consists primarily of chitin and proteins and is thought to support digestion and provide protection from abrasive food particles and enteric pathogens. We examined the physiological roles of 11 putative peritrophic matrix protein (PMP) genes of the red flour beetle, Tribolium castaneum (TcPMPs). TcPMP genes are differentially expressed along the length of the midgut epithelium of feeding larvae. RNAi of individual PMP genes revealed no abnormal developmental phenotypes for 9 of the 11 TcPMPs. However, RNAi for two PMP genes, TcPMP3 and TcPMP5-B, resulted in depletion of the fat body, growth arrest, molting defects and mortality. In situ permeability assays after oral administration of different-sized FITC-dextran beads demonstrated that the exclusion size of the larval peritrophic matrix (PM) decreases progressively from >2 MDa to <4 kDa from the anterior to the most posterior regions of the midgut. In the median midguts of control larvae, 2 MDa dextrans were completely retained within the PM lumen, whereas after RNAi for TcPMP3 and TcPMP5-B, these dextrans penetrated the epithelium of the median midgut, indicating loss of structural integrity and barrier function of the larval PM. In contrast, RNAi for TcPMP5-B, but not RNAi for TcPMP3, resulted in breakdown of impermeability to 4 and 40 kDa dextrans in the PM of the posterior midgut. These results suggest that specific PMPs are involved in the regulation of PM permeability, and that a gradient of barrier function is essential for survival and fat body maintenance.
昆虫中肠的围食膜（PM）主要由几丁质和蛋白质组成，被认为有助于消化，并能保护昆虫免受粗糙食物颗粒和肠道病原体的侵害。我们研究了赤拟谷盗11个假定的围食膜蛋白（PMP）基因（TcPMPs）的生理作用。TcPMP基因在取食幼虫中肠上皮的不同部位呈现差异表达。对单个PMP基因进行RNA干扰后发现，11个TcPMP中有9个没有出现异常发育表型。然而，对两个PMP基因TcPMP3和TcPMP5 - B进行RNA干扰后，导致脂肪体消耗、生长停滞、蜕皮缺陷和死亡。口服不同大小的异硫氰酸荧光素（FITC） - 葡聚糖珠后进行的原位通透性分析表明，幼虫围食膜（PM）的截留尺寸从中肠最前端到最后端逐渐减小，从大于2 MDa降至小于4 kDa。在对照幼虫的中肠中部，2 MDa的葡聚糖完全保留在围食膜腔内，而对TcPMP3和TcPMP5 - B进行RNA干扰后，这些葡聚糖穿透了中肠中部的上皮细胞，表明幼虫围食膜的结构完整性和屏障功能丧失。相比之下，对TcPMP5 - B进行RNA干扰而非对TcPMP3进行RNA干扰，导致后肠围食膜对4 kDa和40 kDa葡聚糖的不透性破坏。这些结果表明，特定的PMP参与围食膜通透性的调节，并且屏障功能梯度对于生存和脂肪体维持至关重要。
