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Hawthorn spider mite,  Amphitetranychus viennensis  Zacher, is one of the most devastating pests of deciduous fruit trees. The overall goal of this research is to develop a standardized protocol for real-time quantitative reverse transcription PCR (RT-qPCR) analysis in  A. viennensis  following the MIQE (minimum information for publication of Quantitative real time PCR experiments) guidelines. Based on the previous knowledge, we hypothesized that internal references for RT-qPCR analysis reside in housekeeping genes (HKGs). To test this hypothesis, we examined the stability of nine HKGs from  A. viennensis , including  18S ribosomal RNA  ( 18S ),  28S ribosomal RNA  ( 28S ),  Elongation factor 1-alpha  ( EF1A ),  Actin3 ,  V-ATP vacuolar-type H + -ATPase  ( V-ATPase ), α -tubulin  (α -tubulin ),  Ribosomal protein L13  ( RPL13 ),  40S ribosomal protein S9  ( RPS9 ), and  Glyceraldehyde 3-phosphate dehydrogenase  ( GAPDH ). The expression profile of these candidates under intrinsic conditions was evaluated by a panel of computational programs, including geNorm, Normfinder, BestKeeper, and ΔCt method. Based on RefFinder, a comprehensive software integrating all four above-mentioned algorithms,  V-ATPase ,  Actin3 , and  GAPDH  are the top three reference genes, which are stably expressed across all the intrinsic conditions, including developmental stage, sex, and diapause. In addition, we compared reference genes recommended for different developmental stages among the nine cell-content feeding arthropods, including four spider mites,  A. viennensis, Tetranychus urticae, Tetranychus cinnabarinus , and  Panonychus citri , and five hemipterans,  Myzus persicae, Aphis gossypii, Toxoptera citricida, Lipaphis erysimi , and  Sogatella furcifera . Not surprisingly, rRNAs and ribosomal proteins, the most abundant RNA species, is the top choice, and follows by  EF1A, Actin, GAPDH , and  tubulin.  Information present here lays the foundation for the genomic and functional genomic research in cell-content feeding arthropods in general and  A. viennensis  in particular.

frontiers in physiology

Selection of Reference Genes for RT-qPCR Analysis Under Intrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis (Acarina: Tetranychidae)