Ultrastructural changes caused by Snf7 RNAi in larval enterocytes of western corn rootworm (Diabrotica virgifera virgifera Le Conte)

Abstract

The high sensitivity to oral RNA interference (RNAi) of western corn rootworm (WCR, Diabrotica virgifera virgifera Le Conte) provides a novel tool for pest control. Previous studies have shown that RNAi of DvSnf7, an essential cellular component of endosomal sorting complex required for transport (ESCRT), caused deficiencies in protein de-ubiquitination and autophagy, leading to WCR death. Here we investigated the detailed mechanism leading to larval death by analyzing the ultrastructural changes in midgut enterocytes of WCR treated with double-stranded RNA (ds-DvSnf7). The progressive phases of pathological symptoms caused by DvSnf7-RNAi in enterocytes include: 1) the appearance of irregularly shaped macroautophagic complexes consisting of relatively large lysosomes and multi-lamellar bodies, indicative of failure in autolysosome formation; 2) cell sloughing and loss of apical microvilli, and eventually, 3) massive loss of cellular contents indicating loss of membrane integrity. These data suggest that the critical functions of Snf7 in insect midgut cells demonstrated by the ultrastructural changes in DvSnf7 larval enterocytes underlies the conserved essential function of the ESCRT pathway in autophagy and membrane stability in other organisms.

Figure 1. Midgut ultrastructural profiles of untreated, dsGFP-treated and dsDvSnf7-treated western corn rootworm (WCR) second instar larvae fed for 5 days.

The letter L with orange glowing background indicates the region of gut lumen, white arrows indicate endosomes, magenta arrows indicate lysosomes, black arrows indicate autolysosomes and MLB is multi-lamellar bodies. C′ is for the magnified view of macroautophagic complex in the midgut of dsDvSnf7-treated individuals. Scale bars  = 4 µm (a, b, c), 1 µm (a′, b′) and 500 nm (c′). D refers to macroautophagic complexes counted in enterocytes of untreated, dsGFP-treated and dsDvSnf7-treated larval midgut. Error bars represent mean ± S.D. of three individuals.

Figure 2. Progression of pathology in the midgut of dsDvSnf7-treated WCR larvae.

Observed symptoms were summarized in four phases (1 = lightest to 4 = most severe). Highlighted are macroautophagic complexes (blue ellipses) (diagram, A′), gut lumen (L with orange glowing background) filled with cellular debris and loss of microvilli (white arrowheads) (diagrams, B, B″), large vacuoles (diagrams, B′, B″, C′) (green arrows), luminal membrane disintegration (black arrowheads) (diagram, C″) and infolding of the basal membrane (transparent black arrowheads) (diagrams, C″, D′, D″), progressive cell sloughing (asterisks) (diagrams, A″, B, C). Panels E-E″ show midgut of dsGFP-treated control larvae. Highlighted are endosomes (white arrows), lysosomes (magenta arrows) and autolysosomes (black arrow). refers to grey area denoting sectional artifact (cracks in the tissue section). Scale bars  = 4 µm (A-E) and 2 µm (A′-E′ and A″-E″).