Kelkenberg Marco, Odman-Naresh Jothini, Muthukrishnan Subbaratnam, Merzendorfer Hans
Department of Biology, Chemistry, University of Osnabrück, Osnabrück 49069, Germany.
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, USA.
Insect Biochem Mol Biol. 2015 Jan;56:21-8. doi: 10.1016/j.ibmb.2014.11.005. Epub 2014 Nov 20.
In most insects, the peritrophic matrix (PM) partitions the midgut into different digestive compartments, and functions as a protective barrier against abrasive particles and microbial infections. In a previous study we demonstrated that certain PM proteins are essential in maintaining the PM's barrier function and establishing a gradient of PM permeability from the anterior to the posterior part of the midgut which facilitates digestion (Agrawal et al., 2014). In this study, we focused on the effects of a reduction in chitin content on PM permeability in larvae of the red flour beetle, Tribolium castaneum. Oral administration of the chitin synthesis inhibitor diflubenzuron (DFB) only partially reduced chitin content of the larval PM even at high concentrations. We observed no nutritional effects, as larval growth was unaffected and neutral lipids were not depleted from the fat body. However, the metamorphic molt was disrupted and the insects died at the pharate pupal stage, presumably due to DFB's effect on cuticle formation. RNAi to knock-down expression of the gene encoding chitin synthase 2 in T. castaneum (TcCHS-2) caused a complete loss of chitin in the PM. Larval growth was significantly reduced, and the fat body was depleted of neutral lipids. In situ PM permeability assays monitoring the distribution of FITC dextrans after DFB exposure or RNAi for TcCHS-2 revealed that PM permeability was increased in both cases. RNAi for TcCHS-2, however, led to a higher permeation of the PM by FITC dextrans than DFB treatment even at high doses. Similar effects were observed when the chitin content was reduced by feeding DFB to adult yellow fever mosquitos, Aedes aegypti. We demonstrate that the presence of chitin is necessary for maintaining the PM's barrier function in insects. It seems that the insecticidal effects of DFB are mediated by the disruption of cuticle synthesis during the metamorphic molt rather than by interfering with larval nutrition. However, as DFB clearly affects PM permeability, it may be suitable to increase the efficiency of pesticides targeting the midgut.
在大多数昆虫中，围食膜（PM）将中肠分隔成不同的消化区室，并作为一种保护屏障，抵御磨损颗粒和微生物感染。在之前的一项研究中，我们证明某些围食膜蛋白对于维持围食膜的屏障功能以及从中肠前部到后部建立围食膜通透性梯度以促进消化至关重要（Agrawal等人，2014年）。在本研究中，我们重点关注几丁质含量降低对赤拟谷盗（Tribolium castaneum）幼虫围食膜通透性的影响。即使在高浓度下，口服几丁质合成抑制剂氟虫脲（DFB）也只能部分降低幼虫围食膜的几丁质含量。我们未观察到营养方面的影响，因为幼虫生长未受影响，脂肪体中的中性脂质也未耗尽。然而，变态蜕皮受到干扰，昆虫在预蛹期死亡，推测是由于DFB对表皮形成的影响。通过RNA干扰敲除赤拟谷盗中编码几丁质合酶2的基因（TcCHS - 2）的表达，导致围食膜中几丁质完全缺失。幼虫生长显著减缓，脂肪体中的中性脂质耗尽。在DFB暴露或针对TcCHS - 2进行RNA干扰后，通过原位围食膜通透性测定监测异硫氰酸荧光素标记的葡聚糖的分布，结果表明在这两种情况下围食膜通透性均增加。然而，即使在高剂量下，针对TcCHS - 2的RNA干扰导致异硫氰酸荧光素标记的葡聚糖对围食膜的渗透高于DFB处理。当通过向成年埃及伊蚊喂食DFB来降低几丁质含量时，也观察到了类似的效果。我们证明几丁质的存在对于维持昆虫围食膜的屏障功能是必要的。似乎DFB的杀虫作用是通过在变态蜕皮期间破坏表皮合成介导的，而不是通过干扰幼虫营养。然而，由于DFB明显影响围食膜通透性，它可能适合提高针对中肠的杀虫剂的效率。
