Chitvan Khajuria

Validation of Reference Housekeeping Genes for Gene Expression Studies in Western Corn Rootworm ( Diabrotica virgifera virgifera )

Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.

Parental RNA interference of genes involved in embryonic development of the western corn rootworm, Diabrotica virgifera virgifera LeConte

RNA interference (RNAi) is being developed as a potential tool for insect pest management and one of the most likely target pest species for transgenic plants that express double stranded RNA (dsRNA) is the western corn rootworm. Thus far, most genes proposed as targets for RNAi in rootworm cause lethality in the larval stage. In this study, we describe RNAi-mediated knockdown of two developmental genes, hunchback (hb) and brahma (brm), in the western corn rootworm delivered via dsRNA fed to adult females. dsRNA feeding caused a significant decrease in hb and brm transcripts in the adult females. Although total oviposition was not significantly affected, there was almost complete absence of hatching in the eggs collected from females exposed to dsRNA for either gene. These results confirm that RNAi is systemic in nature for western corn rootworms. These results also indicate that hunchback and brahma play important roles in rootworm embryonic development and could provide useful RNAi targets in adult rootworms to prevent crop injury by impacting the population of larval progeny of exposed adults. The ability to deliver dsRNA in a trans-generational manner by feeding to adult rootworms may offer an additional approach to utilizing RNAi for rootworm pest management. The potential to develop parental RNAi technology targeting progeny of adult rootworms in combination with Bt proteins or dsRNA lethal to larvae may increase opportunities to develop sustainable approaches to rootworm management involving RNAi technologies for rootworm control.

Long dsRNA but not siRNA initiates RNAi in western corn rootworm larvae and adults

Transgenic maize plants expressing dsRNA targeting western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) v‐ATPase subunit C mRNA for RNAi provided significant root protection from WCR larval feeding damage in greenhouse assays compared to negative controls. Transcribed hairpin dsRNA in WCR‐resistant maize plants was present as both intact hairpin‐derived dsRNA and plant‐processed siRNA. Therefore, the ability of dsRNA and siRNA targeting Dv v‐ATPase CmRNA to cause an RNAi response was studied in both WCR larvae and adults. In 9‐day diet‐based feeding assays, dsRNA of at least 60 bp in length resulted in high levels of larval mortality. In contrast, 15‐, 25‐ or 27‐bp dsRNAs or pooled 21‐bp siRNAs did not cause mortality of exposed larvae. When larvae were fed with diet overlaid with siRNAs, Dv v‐ATPase C transcript levels did not change. Conversely, when WCR larvae were fed with diet overlaid with 184‐bp dsRNA, the mRNA level was reduced by >20‐fold relative to yfp dsRNA negative control. Similarly, 184‐bp dsRNA caused 100% mortality of WCR adults, whereas the mortality of adults fed on diet treated with siRNAs was similar to the negative control. Feeding adults with siRNAs on diet did not affect the level of Dv v‐ATPase CmRNA transcripts, whereas adults fed with the 184‐bp dsRNA showed approximately 35‐fold reduction in the target mRNA level. Similar results were obtained with the WCR adults injected with 184‐bp dsRNA or 21‐bp siRNA. These results suggest that only long dsRNA or hairpin‐derived dsRNA is effective in causing lethal knock‐down of Dv v‐ATPase CmRNA. These results have implications for efficacious plant‐delivered dsRNA for the protection of transgenic maize from WCR feeding damage and for the risk assessment of transgenic maize expressing insecticidal dsRNA.

Knockdown of RNA Interference Pathway Genes in Western Corn Rootworms (Diabrotica virgifera virgifera Le Conte) Demonstrates a Possible Mechanism of Resistance to Lethal dsRNA

RNA interference (RNAi) is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2), an endonuclease responsible for formation of small interfering RNA’s and Argonaute 2 (Ago2), an essential catalytic component of the RNA-induced silencing complex (RISC) have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2) did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA’s. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance.

Systemic RNAi in western corn rootworm, Diabrotica virgifera virgifera, does not involve transitive pathways

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double‐stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA‐dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi‐mediated knockdown of Dv v‐ATPase C mRNA throughout the WCR gut and other tissues using high‐sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v‐ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.

Carbon dioxide receptor genes and their expression profile in Diabrotica virgifera virgifera

AbstractBackgroundDiabrotica virgifera virgifera, western corn rootworm, is one of the most devastating species in North America. D. v. virgifera neonates crawl through the soil to locate the roots on which they feed. Carbon dioxide (CO2) is one of the important volatile cues that attract D. v. virgifera larvae to roots. ResultsIn this study, we identified three putative D. v. virgifera gustatory receptor genes (Dvv_Gr1, Dvv_Gr2, and Dvv_Gr3). Phylogenetic analyses confirmed their orthologous relationships with known insect CO2 receptor genes from Drosophila, mosquitoes, and Tribolium. The phylogenetic reconstruction of insect CO2 receptor proteins and the gene expression profiles were analyzed. Quantitative analysis of gene expression indicated that the patterns of expression of these three candidate genes vary among larval tissues (i.e., head, integument, fat body, and midgut) and different development stages (i.e., egg, three larval stages, adult male and female).Conclusion The Dvv_Gr2 gene exhibited highest expression in heads and neonates, suggesting its importance in allowing neonate larvae to orient to its host plant. Similar expression patterns across tissues and developmental stages for Dvv_Gr1 and Dvv_Gr3 suggest a potentially different role. Findings from this study will allow further exploration of the functional role of specific CO2 receptor proteins in D. v. virgifera.

Patterns of Gene Expression in Western Corn Rootworm (Diabrotica virgifera virgifera) Neonates, Challenged with Cry34Ab1, Cry35Ab1 and Cry34/35Ab1, Based on Next-Generation Sequencing

With Next Generation Sequencing technologies, high-throughput RNA sequencing (RNAseq) was conducted to examine gene expression in neonates of Diabrotica virgifera virgifera (LeConte) (Western Corn Rootworm, WCR) challenged with individual proteins of the binary Bacillus thuringiensis insecticidal proteins, Cry34Ab1 and Cry35Ab1, and the combination of Cry34/Cry35Ab1, which together are active against rootworm larvae. Integrated results of three different statistical comparisons identified 114 and 1300 differentially expressed transcripts (DETs) in the Cry34Ab1 and Cry34/35Ab1 treatment, respectively, as compared to the control. No DETs were identified in the Cry35Ab1 treatment. Putative Bt binding receptors previously identified in other insect species were not identified in DETs in this study. The majority of DETs (75% with Cry34Ab1 and 68.3% with Cry34/35Ab1 treatments) had no significant hits in the NCBI nr database. In addition, 92 DETs were shared between Cry34Ab1 and Cry34/35Ab1 treatments. Further analysis revealed that the most abundant DETs in both Cry34Ab1 and Cry34/35Ab1 treatments were associated with binding and catalytic activity. Results from this study confirmed the nature of these binary toxins against WCR larvae and provide a fundamental profile of expression pattern of genes in response to challenge of the Cry34/35Ab1 toxin, which may provide insight into potential resistance mechanisms.