A protocol for assessment of direct effects of RNAi to earthworms

TY - ABST

T1 - A protocol for assessment of direct effects of RNAi to earthworms

AU - de Pinto, Roberta

AU - Valera López, Helena

AU - Strandberg, Morten Tune

AU - Kostov, Kaloyan

AU - Krogh, Paul Henning

N1 - Conference code: XI

PY - 2018/7/2

Y1 - 2018/7/2

N2 - Studies of non-target effects of RNAi require adjustment and modification of existing procedures such as the ISO and OECD standards as these procedures are mainly adopted to the soil exposure route. Earthworms, Lumbricidae, are an important part of the soil ecosystems and thus commonly included in the risk assessment of the new plant breeding techniques (NBT). However, the mechanisms and fate of ingested small interfering RNA molecules in this phylum are not well studied. We present a protocol for artificial exposure of RNAi molecules to earthworms aiming to study if dsRNA can cross the gut barrier, enter into the internal organs, and exert its silencing effect on the COI, cytochrome oxidase 1, mRNA, a crucial part of the respiratory energy producing system. The elements of the protocol include 1) reverse transcription of mRNA to cDNA, 2) dsRNA synthesis by PCR, 3) preparing chitosan/dsRNA nanoparticles acting as a carrier for food exposure of dsRNA (Zhang et al. 2015. J. Vis. Exp. (2015) DOI 10.3791/52523) 4) introduce dsRNA into the internal organs of earthworms and induce silencing and finally, 5) perform qPCR of the targeted mRNA to confirm the silencing effect. Thus, the protocol describes both how to study fate and effects of the RNAi molecules. If COI or another gene apt for silencing will be successful in the earthworm L. terrestris, this could become a suggested positive control agent for future non-target studies of RNAi. The testing of environmental effects of NBT require such positive control RNAi’s being comparable to the mode-of-action of new untested RNAi compounds. If the mode of action is of another nature than the RNAi being tested, the positive control ultimately cannot validate the test conditions because they are not addressed by the positive control agent.

AB - Studies of non-target effects of RNAi require adjustment and modification of existing procedures such as the ISO and OECD standards as these procedures are mainly adopted to the soil exposure route. Earthworms, Lumbricidae, are an important part of the soil ecosystems and thus commonly included in the risk assessment of the new plant breeding techniques (NBT). However, the mechanisms and fate of ingested small interfering RNA molecules in this phylum are not well studied. We present a protocol for artificial exposure of RNAi molecules to earthworms aiming to study if dsRNA can cross the gut barrier, enter into the internal organs, and exert its silencing effect on the COI, cytochrome oxidase 1, mRNA, a crucial part of the respiratory energy producing system. The elements of the protocol include 1) reverse transcription of mRNA to cDNA, 2) dsRNA synthesis by PCR, 3) preparing chitosan/dsRNA nanoparticles acting as a carrier for food exposure of dsRNA (Zhang et al. 2015. J. Vis. Exp. (2015) DOI 10.3791/52523) 4) introduce dsRNA into the internal organs of earthworms and induce silencing and finally, 5) perform qPCR of the targeted mRNA to confirm the silencing effect. Thus, the protocol describes both how to study fate and effects of the RNAi molecules. If COI or another gene apt for silencing will be successful in the earthworm L. terrestris, this could become a suggested positive control agent for future non-target studies of RNAi. The testing of environmental effects of NBT require such positive control RNAi’s being comparable to the mode-of-action of new untested RNAi compounds. If the mode of action is of another nature than the RNAi being tested, the positive control ultimately cannot validate the test conditions because they are not addressed by the positive control agent.

M3 - Conference abstract for conference

T2 - iPLANTA workshop on “Biosafety issues associated with RNAi” at the XI European Congress of Entomology in Naples (Italy), July 2-3, 2018

Y2 - 2 July 2018 through 3 July 2018

ER -