Establishing an In Vivo Assay System to Identify Components Involved in Environmental RNA Interference in the Western Corn Rootworm

Abstract

The discovery of environmental RNA interference (RNAi), in which gene expression is suppressed via feeding with double-stranded RNA (dsRNA) molecules, opened the door to the practical application of RNAi-based techniques in crop pest management. The western corn rootworm (WCR, Diabrotica virgifera virgifera) is one of the most devastating corn pests in North America. Interestingly, WCR displays a robust environmental RNAi response, raising the possibility of applying an RNAi-based pest management strategy to this pest. Understanding the molecular mechanisms involved in the WCR environmental RNAi process will allow for determining the rate limiting steps involved with dsRNA toxicity and potential dsRNA resistance mechanisms in WCR. In this study, we have established a two-step in vivo assay system, which allows us to evaluate the involvement of genes in environmental RNAi in WCR. We show that laccase 2 and ebony, critical cuticle pigmentation/tanning genes, can be used as marker genes in our assay system, with ebony being a more stable marker to monitor RNAi activity. In addition, we optimized the dsRNA dose and length for the assay, and confirmed that this assay system is sensitive to detect well-known RNAi components such as Dicer-2 and Argonaute-2. We also evaluated two WCR sid1- like (sil) genes with this assay system. This system will be useful to quickly survey candidate systemic RNAi genes in WCR, and also will be adaptable for a genome-wide RNAi screening to give us an unbiased view of the environmental/systemic RNAi pathway in WCR.

Knockdown of nuclease activity in the gut enhances RNAi efficiency in the Colorado potato beetle, Leptinotarsa decemlineata , but not in the desert locust, Schistocerca gregaria

“…However, since S. gregaria has been shown to possess a very potent systemic RNAi response upon injection (Wynant et al, 2012), it seems less likely that problems with intracellular processing would be responsible for the refractoriness towards orally delivered dsRNA in this species.Several reports mention that competition of dsRNA fragments might occur when they are administered together (Miller et al, 2012;Miyata et al, 2014;Tomoyasu et al, 2008). Nevertheless, here, simultaneous delivery of multiple dsRNA fragments resulted in potent knockdowns of all targeted genes at once, indicating that the RNAi machinery was not saturated, even with up to four dsRNA fragments in S. gregaria.…”

“…Interestingly, a clear response could not be observed, suggesting that no potent environmental RNAi response exists in our lab strain of T. castaneum. It is noteworthy that this discrepancy was also mentioned by another recently published research paper (Miyata et al, 2014), while another research group more recently succeeded in delivering dsRNA through the diet of T. castaneum (Abd El Halim et al, 2016). This indicates that the ability to generate a potent environmental RNAi response in T.…”

“…However, dsRNA was applied via a leaf dip bioassay, using bacterial suspensions. In another example, in the Western corn rootworm, Diabrotica virgifera, a species that is regarded as highly responsive towards oral RNAi, a concentration of 100 ng of dsRNA topically applied on 200 µl of artificial diet was sufficient to generate a physiological response, while 10 ng appeared to be insufficient (Miyata et al, 2014). However, from both these examples, it is impossible to know exactly how much dsRNA was actually ingested by the animals.…”

Uptake and impact of natural diet-derived small RNA in invertebrates: Implications for ecology and agriculture

“…Again using the corn rootworm as an example, recent studies in this organism have established it as a valuable model for elucidating the molecular mechanisms of dietary sRNA uptake and function. [173][174][175] Parallel studies on mechanisms of sRNA packaging and stability of the dietary plants will also be paramount for generating a complete biological picture. The number and diversity of invertebrates is staggering, with 1.3 million currently recognized species, a number that is likely to grow by a factor of 5 in the coming years with new species discovery.…”

In silico identification of off-target pesticidal dsRNA binding in honey bees (Apis mellifera)

“…Off-target sequence homology increased significantly as the parent dsRNA increased in 138 length (linear regression: F 1,100 = 623, P < 0.001) (Figure 1a), with every increase of 100 nt in 139 the dsRNA resulting in 6 more predicted hits. This strong relationship between dsRNA length 140 and potential off-target binding can be further demonstrated using only the genes described in 141 Miyata et al (2014), in which the authors evaluated the effects of dsRNA length on RNAi 143 pesticidal specifically, and thus excluded from our overall analysis, the authors evaluated 144 silencing of the same gene targets (laccase 2 and ebony) using different sized dsRNAs to 145 evaluate efficacy. When we examined this suite of genes from a risk assessment perspective 146 using the same methodology as for the pesticidal RNAs, the longer dsRNAs returned 147 significantly more regions of off-target sequence homology in the honey bee genome (laccase 2:…”