Environmental RNAi pathways in the two-spotted spider mite

Abstract

Background RNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation and has applications that includes arthropod pest control. RNAi technologies are dependent on delivery method with the most convenient likely being feeding, which is effective in some animals while others are insensitive. The two-spotted spider mite, Tetranychus urticae, is prime candidate for developing RNAi approaches due to frequent occurrence of conventional pesticide resistance. Using a sequencing-based approach, the fate of ingested RNAs was explored to identify features and conditions that affect small RNA biogenesis from external sources to better inform RNAi design. Results Biochemical and sequencing approaches in conjunction with extensive computational assessment were used to evaluate metabolism of ingested RNAs in T. urticae. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. Processing of synthetic and plant host RNAs ingested during feeding were evaluated to identify active substrates for spider mite RNAi pathways. Through cataloging characteristics of biochemically purified RNA from these sources, trans-acting small RNAs could be distinguished from degradation fragments and their origins documented. Conclusions Using a strategy that delineates small RNA processing, we found many transcripts have the potential to enter spider mite RNAi pathways, however, trans-acting RNAs appear very unstable and rare. This suggests potential RNAi pathway substrates from ingested materials are mostly degraded and infrequently converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers for T.urticae.

Localized efficacy of environmental RNAi in Tetranychus urticae

“…2 and 4 ). This is consistent with levels of target gene downregulation obtained in other RNAi studies in T. urticae 39 – 41 , 48 – 53 . The partial reduction of target gene expression is frequently associated with the overall low effectiveness of RNAi 54 – 57 .…”

“…SID-like genes were not identified in the T. urticae genome, but similar to plants and the nematode C. elegans 38 , T. urticae contains multiple copies of RdRP encoding genes 34 . Consistent with the possibility that RdRP amplifies RNAi signal in T. urticae , transitive small RNAs (antisense transcripts mapping outside the target region within the target mRNA) were identified upon oral delivery of dsRNA 48 . The amplification of RNAi signal, the strength of the RNAi-induced phenotypes 37 , and the ability of maternally-injected RNAs to induce loss-of-function phenotype in the progeny 35 , suggest the highly efficient RNAi in mites.…”

Establishing RNAi for basic research and pest control and identification of the most efficient target genes for pest control: a brief guide

“…In contrast, many Lepidopterans were found to be quite resistant to environmental RNAi [ 86 , 107 ]. There are positive reports for several stinkbugs [ 52 , 108 – 110 ] while there are more mixed reports with respect to whiteflies [ 65 , 111 – 114 ], aphids [ 115 – 120 ] and spider mites [ 121 , 122 ]. Though RNAi was shown to work in several hemipteran pest species, its commercial use to target these species is currently out of scope due to the high concentrations of dsRNA necessary for a rather moderate response.…”

Characteristics of environmental RNAi in potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Psylloidea: Triozidae)

“…These 20–30 nucleotide (nt) long RNAs complementarily bind to the target transcript and trigger degradation of the transcript or blocking of protein synthesis using the transcript as template. Consequently, gene silencing, or RNAi, can be exploited to develop the next generation biopesticides, referred to as ‘environmental RNAi’ ( Ivashuta et al, 2015 ; Mondal et al, 2021 ). In RNAi technology, long double-stranded RNAs (dsRNA) or single-stranded RNAs (ssRNA) introduced to the target organism are scavenged to the RNAi machinery, usually immediately after ingestion, which is responsible for producing small RNAs that trigger destruction of the target transcript or the inhibition of translation ( Agrawal et al, 2003 ; Baum et al, 2007 ; Mondal et al, 2020 ).…”