Selection of Reference Genes for RT-qPCR Analysis in Coccinella septempunctata to Assess Un-intended Effects of RNAi Transgenic Plants

Abstract

The development of genetically engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is reverse transcriptase-quantitative polymerase chain reaction, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata, a commonly used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper, the ΔCt method, and RefFinder, were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended, respectively, for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata, our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.

Responses of two ladybird beetle species (Coleoptera: Coccinellidae) to dietary RNAi

“…Transcript levels of A. bipunctata and C. septempunctata vATPase A were quantified using quantitative reverse transcription PCR (RT-PCR) and are presented relative to the levels determined for alpha tubulin (the sequence data were kindly provided by Heiko Vogel, Max-Planck-Institute for Chemical Ecology, Jena, Germany) which was selected as reference gene based on the results from previous studies to evaluate candidate reference genes in the same family. 54 In each of the three trials, additional ladybird beetle specimens (12 to 20, depending on the number of available neonates) were exposed to the same treatments. At days 1, 3, and 5 after the first provision of the test solution, up to four larvae per treatment were collected, snap-frozen in liquid nitrogen, and immediately transferred into RNAlater RNA Stabilization Reagent (Ambion, ThermoFisher Scientific) until further processing ( Fig.…”

Selection and evaluation of reference genes for expression analysis using quantitative real-time PCR in the Asian Ladybird Harmonia axyridis (Coleoptera: Coccinellidae)

“…Some recent studies have attempted to determine the stability of reference genes in arthropods [ 15 , 21 – 23 ]. Yang et al [ 24 , 25 ] and Pan et al [ 26 ] identified stable reference genes in three species of ladybeetles: Coleomegilla maculate , Coccinella septempunctata , and Hippodamia convergens . However, there are no available data on the most appropriate genes to use for H .…”

Validation of reference genes for quantitative real-time PCR in tiger beetles across sexes, body parts, sexual maturity and immune challenge

“…B-t is one of the major components of microtubules which function in many cellular processes. Previous works in insects have shown its suitability for developmental stages, tissues and dietary RNAi experiments in Coccinella septempunctata 26 , developmental stages in Liposcelis Bostrychophila 43 and across all the conditions tested in Drosophila melanogaster 18 , while it has also been tested in Tribolium castaneum 24 , Bemisia tabaci 17 , Dibrotica virgifera virgifera 27 , Apis mellifera 44 and two Bombus species 20 , not being ranked as the optimal gene in most of the cases. Even though Myo and Myo-Alc , related to ATP regulation and cell movement, were allocated as the best genes for sex comparison in C .…”

“… 17 verified its usefulness in Bemisia tabaci for abiotic factor, but it also presented good values for some biotic factor such as developmental stages. In addition, NADH was top-ranked as one of the most stables gene for developmental stages and tissue comparison in Coccinella septempunctata 26 and exhibited acceptable values in Coleomegilla maculata 25 .…”

“…That is the case of Tribolium castaneum 22 – 24 , for which different ribosomal proteins were found to be the suitable for broad-spectrum expression analyses. Two different species of Coccinellidae 25 , 26 , the western corn root worm Diabrotica virgifera virgifera 27 and the emerald ash borer Agrilus planipennis 28 have also been investigated.…”