Physiological and cellular responses caused by RNAi- mediated suppression of Snf7 orthologue in western corn rootworm (Diabrotica virgifera virgifera) larvae.

Ingestion of double stranded RNA (dsRNA) has been previously demonstrated to be effective in triggering RNA interference (RNAi) in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), providing potential novel opportunities for insect pest control. The putative Snf7 homolog of WCR (DvSnf7) has previously been shown to be an effective RNAi target for insect control, as DvSnf7 RNAi leads to lethality of WCR larvae. Snf7 functions as a part of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway which plays a crucial role in cellular housekeeping by internalization, transport, sorting and lysosomal degradation of transmembrane proteins. To understand the effects that lead to death of WCR larvae by DvSnf7 RNAi, we examined some of the distinct cellular processes associated with ESCRT functions such as de-ubiquitination of proteins and autophagy. Our data indicate that ubiquitinated proteins accumulate in DvSnf7 dsRNA-fed larval tissues and that the autophagy process seems to be impaired. These findings suggest that the malfunctioning of these cellular processes in both midgut and fat body tissues triggered by DvSnf7 RNAi were the main effects leading to the death of WCR. This study also illustrates that Snf7 is an essential gene in WCR and its functions are consistent with biological functions described for other eukaryotes

Cry3Bb1-Resistant Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte) Does Not Exhibit Cross-Resistance to DvSnf7 dsRNA.

There is a continuing need to express new insect control compounds in transgenic maize against western corn rootworm, Diabrotica virgifera virgifera (LeConte) (WCR). In this study three experiments were conducted to determine cross-resistance between the new insecticidal DvSnf7 dsRNA, and Bacillus thuringiensis (Bt) Cry3Bb1; used to control WCR since 2003, with field-evolved resistance being reported. Laboratory susceptible and Cry3Bb1-resistant WCR were evaluated against DvSnf7 dsRNA in larval diet-incorporation bioassays. Additionally, the susceptibility of seven field and one field-derived WCR populations to DvSnf7 (and Cry3Bb1) was assessed in larval diet-overlay bioassays. Finally, beetle emergence of laboratory susceptible and Cry3Bb1-resistant WCR was evaluated with maize plants in the greenhouse expressing Cry3Bb1, Cry34Ab1/Cry35Ab1, or DvSnf7 dsRNA singly, or in combination.The Cry3Bb1-resistant colony had slight but significantly (2.7-fold; P<0.05) decreased susceptibility to DvSnf7 compared to the susceptible colony, but when repeated using a field-derived WCR population selected for reduced Cry3Bb1 susceptibility, there was no significant difference (P<0.05) in DvSnf7 susceptibility compared to that same susceptible population. Additionally, this 2.7-fold difference in susceptibility falls within the range of DvSnf7 susceptibility among the seven field populations tested. Additionally, there was no correlation between susceptibility to DvSnf7 and Cry3Bb1 for all populations evaluated. In greenhouse studies, there were no significant differences (P<0.05) between beetle emergence of susceptible and Cry3Bb1-resistant colonies on DvSnf7 and Cry34Ab1/Cry35Ab1, and between DvSnf7 and MON 87411 (DvSnf7 + Cry3Bb1) for the Cry3Bb1-resistant colony. These results demonstrate no cross-resistance between DvSnf7 and Cry3Bb1 against WCR. Therefore, pyramiding DvSnf7 with Bt proteins such as Cry3Bb1 and Cry34Ab1/Cry35Ab1 will provide a valuable IRM tool against WCR that will increase the durability of these Bt proteins. These results also illustrate the importance of using appropriate bioassay methods when characterizing field-evolved resistant WCR populations

Binary Toxins from Bacillus thuringiensis Active against the Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest

Immuno staining of WCR tissues with Anti-ubiquitin.

<p>DsRNA for DvSnf7 (dsDvSnf7) and GFP (dsGFP) were overlaid on diet at 1 µg/ml of diet. Second instar WCR larvae were exposed to dsRNAs for five days. Whole mounts of midgut and fat bodies were used for immuno-staining. Panels A, B, C, D show nuclear DNA (DAPI) staining of midgut and fat bodies. Panels A’, B’, C’, D’ show Alexa-546 detecting ubiquitin. Panels A”, B”, C”, D” show merged images of DAPI and Alexa-546. Panels A*, B*, C*, D* show monochrome images of respective treated tissues at higher magnification. Red arrowheads mark the ubiquitin puncta. Scale Bar: White –50 µm, Red –10 µm.</p

Model depicting endosomal-autophagy pathway involved in intracellular sorting and degradation of receptors along with other macromolecules in a normal cell (left) and a DvSnf7 deficient cell (right).

<p>In the normal cell, internalization and ubiquitination of cargo (1), de-ubiquitination of cargo (2), biogenesis of MVB (multi-vesicular bodies) (3), formation of autophagosomes engulfing macromolecules (4), and fusion of late endosomes, autophagosome and lysosomes into autolysosmes for degradation of cargo and macromolecules (5) are depicted. In the DvSnf7 deficient cell, the impairment of de-ubiquitination, accumulation of autophagosomes, and failure of fusion of endosomes, autophagosomes and lysosomes and autolysosmal activity are highlighted.</p

Suppression of DvSnf7 in WCR tissues as determined by real-time PCR.

<p>DsRNA for DvSnf7 and GFP (control) were overlaid on diet at 1 µg/ml of diet. Second instar WCR larvae were exposed to dsRNAs for five days. Midgut and fat bodies were dissected from WCR for total RNA extraction. Tissues from WCR larvae sampled prior to dsRNA exposure were used as the control. Mean ± S.E. of three biological replicates are shown. Stars represent values significantly different from controls (p = 0.050; t-test; SAS 9.2).</p

Avidin staining in WCR tissues.

<p>DsRNA for DvSnf7 (dsDvSnf7) and GFP (dsGFP) were overlaid on diet at 1 µg/ml of diet. Second instar WCR larvae were exposed to dsRNAs for five days. Midgut and fat bodies were dissected from WCR for staining. Panels A, B, C, D show nuclear DNA staining (DAPI) in midgut and fat bodies. Panels A’, B’, C’, D’ show Texas-Red conjugated avidin staining. Panels A”, B”, C”, D” show merged images of DAPI and Texas-Red. Scale Bar: A” & B”- 50 µm, C” & D” – 20 µm.</p

Lysotracker staining of WCR tissues.

<p>DsRNA for DvSnf7 (dsDvSnf7) and GFP (dsGFP) were overlaid on diet at 1 µg/ml of diet. One set- of second instar WCR larvae were exposed to dsRNAs for four days and then starved for 24 h. Another set of larvae were exposed to dsRNAs continuously for five days. Midgut and fat bodies were dissected from both sets of larvae and used for Lysotracker staining. Panels A–D show Lysotracker and nuclear staining (DAPI) merged images of starved samples. Panels E–H show Lysotracker and nuclear staining (DAPI) merged images of continuously fed samples. Green punctate marks denote Lysotracker staining that detects active acidic lysosomes undergoing autophagy (panels C &D). Scale Bar: 50 µm.</p

Mean Percent Beetle Emergence¹ for Susceptible (Gass-S) and Cry3Bb1-Resistant (Gass-R) Western Corn Rootworm Feeding on <i>Bt</i> and RNAi-traited Maize.

<p>Mean Percent Beetle Emergence<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169175#t005fn001" target="_blank">¹</a> for Susceptible (Gass-S) and Cry3Bb1-Resistant (Gass-R) Western Corn Rootworm Feeding on <i>Bt</i> and RNAi-traited Maize.</p