Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae)

Abstract

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the sensitive method to quantify the expression levels of target genes on the basis of endogenous control. An appropriate reference gene set for normalization is essential for reliable results. The ghost moth, Thitarodes armoricanus, a host species of a medicinal fungus, Ophiocordyceps sinensis, is an economically important member of the Lepidoptera. Recent studies have focused on the mechanism of adaptation of this species to its high-altitude environment and host immune response to O. sinensis infection and RT-qPCR is commonly used in these studies to decipher the genetic basis of physiological functions. However, a thorough assessment of candidate reference genes in the genus Thitarodes is lacking. Here, the expression levels of eight candidate reference genes (ACT, EF, EIF4A, GAPDH, G6PDH, RPL13A, TUB and 18S) in T. armoricanus at different developmental stages and in different body parts of the seventh instar larvae were analyzed, along with larvae kept under low temperatures, larvae exposed to two fungal infections and larvae fed different diets. Three established software programs–Bestkeeper, geNorm and NormFinder–were employed to calculate variation among the treatments. The results revealed that the best-suited reference genes differed across the treatments, with EF, EIF4A and GAPDH found to be the best suited for the different developmental stages and larvae body parts; EF, EIF4A and RPL13A found to be the best suited for low-temperature challenge; and EF, EIF4A and TUB found to be the best suited for the fungal infections and dietary treatments. This study thus further contributes to the establishment of an accurate method for normalizing RT-qPCR results for T. armoricanus and serves as a reference for gene expression studies of related insect species.

Selection of housekeeping genes and demonstration of RNAi in cotton leafhopper, Amrasca biguttula biguttula (Ishida)

“…Various studies have already been accomplished by identifying HKGs in different species and tissues. Recently, numerous reference genes have been testified under different conditions in stinkbug (Bansal et al , 2016), western flower thrips [ 46 ], honey bee [ 47 ], fly [ 48 ], silkworm [ 49 ], moths [ 50 ] and beetles [ 51 ]. The analysis of reference genes in cotton leafhopper across different developmental stages showed variation in expression as reported in several other insects [ 52 ].…”

Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Larvae of Spodoptera litura Exposed to Azadirachtin Stress Conditions

“…It was reported that the expression of reference genes is not static in different species, tissues or experimental conditions (Lu et al, 2013 ), so the stability verifications of reference genes are necessary before evaluating target gene expression levels by RT-qPCR. Recently, more and more research considered this and the stability verifications were also performed in many Lepidoptera insects, for example: Plutella xylostella (Fu et al, 2013 ), Danaus plexippus (Pan et al, 2015 ), Sesamia inferens (Lu et al, 2015 ), Heliconius numata (Piron Prunier et al, 2016 ), Thitarodes armoricanus (Liu et al, 2016 ), Bombyx mori (Guo et al, 2016 ), Helicoverpa armigera (Chandra et al, 2017 ), Grapholita molesta (Wang et al, 2017 ), Chilo suppressalis (Xu et al, 2017 ), Spodoptera exigua (Płachetka-Bozek and Augustyniak, 2017 ), and grassland caterpillars (Zhang et al, 2017 ). As the important agricultural pest, the identification and validation of reference genes in S. litura with three biotic factors and four abiotic treatments were also investigated (Lu et al, 2013 ).…”

Automated identification of reference genes based on RNA-seq data

“…The first RGs were brought from Northerns and usually encoded proteins involved in structural functions and basic cell metabolism due to their theoretical expression invariability in most tissues. This initial election was revealed inappropriate [ 2 – 4 ] and the quest of more reliable RGs has been pursued in the literature [ 5 – 8 ].…”