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Despite the agricultural importance of species in the Grapholitini (Lepidoptera: Tortricidae), and the value of gene expression analysis for improved population management, few gene expression studies based on quantitative real-time PCR (qPCR) have been conducted for this tribe. Part of the reason for this lack of information is that suitable reference genes, which are fundamental for accurate normalization of qPCR studies, have not been identified for the tribe. Thus, the expression stability of six potential reference genes (ACT, AK, COI, EF1, ENO and TUB) was assessed in three different tissues (whole body, midgut and cuticle) of Cryptophlebia peltastica (Meyrick), Cydia pomonella (L.) and Thaumatotibia leucotreta (Meyrick). Additionally, these reference genes were tested using T. leucotreta at different temperatures (15°C, 25°C and 35°C) with and without baculovirus infection. Suitable reference genes were identified for the whole body and midgut tissue of all three species, and for cuticle tissue of Cy. pomonella and T. leucotreta. When T. leucotreta was infected with the virus at all temperature conditions ACT, AK and EF1 were found to be the most suitable reference genes for experimental normalization. In general, for all tissue types, species and stress conditions, AK and EF1 were the best-performing reference genes. However, even though the three species analysed were closely related and within the same tribe, each species required varying gene combinations for suitable normalization. This study provides the first reference gene evaluation for the Tortricidae, and paves the way for future qPCR analysis in Tortricidae.

Microsatellite (simple sequence repeats, SSR) and mitochondrial DNA markers were used to assess the structure of European codling moth populations showing different levels of susceptibility towards one of the most important biocontrol agents used in apple production, the Cydia pomonella granulovirus CpGV-M. In 638 C. pomonella individuals from 33 different populations a total of 92 different alleles were scored using six SSR loci. The global estimate of genetic differentiation for all 33 populations was not significantly different from zero, thus indicating a lack of genetic differentiation. AMOVA analysis revealed a very weak but significant variance among C. pomonella populations from different geographic regions, however, no significant variation was evident between CpGV-M resistant or susceptible C. pomonella populations. Sequence analysis of a fragment of the cytochrome oxidase subunit 1 in eight C. pomonella populations resulted in 27 haplotypes, which were grouped in two distinct clusters. Again, no genetic differentiation between CpGV-M resistant and susceptible codling moth populations was detectable. In addition, Structure analysis using microsatellites and association tests with mtDNA haplotypes found neither population-level nor individual correlations associated with CpGV-M resistance. Accordingly, this lack of population structure does not allow discriminating between one or several, separate origins of CpGV-M resistance.