Background: Quantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used for quantification of mRNA as a way to determine key genes involved in different biological processes. For accurate gene quantification analysis, normalization of RT-qPCR data is absolutely essential. To date, normalization is most frequently achieved by the use of internal controls, often referred to as reference genes. However, several studies have shown that the reference genes used for the quantification of mRNA expression can be affected by the experimental set-up or cell type resulting in variation of the expression level of these key genes. Therefore, the evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of insects. For this purpose, ten candidate reference genes were investigated in three different tissues (midgut, Malpighian tubules, and fat body) of the oriental fruit fly, Bactrocera dorsalis (Hendel). Results: Two different programs, geNorm and Normfinder, were used to analyze the data. According to geNorm, α-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + α-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + α-TUB are the best choice for both males and females. However, α-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by Normfinder are quite the same as the results with geNorm; α-TUB is always one of the most stable genes in each sample validated by the two programs. Conclusions: In this study, we validated the suitable reference genes for gene expression profiling in different tissues of B. dorsalis. Moreover, appropriate reference genes were selected out for gene expression profiling of the same tissues taking the sexual differences into consideration. This work not only formed a solid basis for future gene expression study in B. dorsalis, but also will serve as a resource to screen reference genes for gene expression studies in any other insects.

The vitellogenin receptor (VgR) functions as an essential component in uptaking and transporting vitellogenin (Vg) in female adults, which is involved in ovary development and oviposition. This study aimed to clarify the molecular characteristics and function of VgR in the oriental fruit fly Bactrocera dorsalis (Hendel). Here, we identified the full-length of BdVgR (GenBank Accession No. JX469118), encoding a 1925 residue (aa) protein with a 214.72 kDa molecular mass and several typical motifs of low-density lipoprotein receptor superfamily (LDLR). Phylogenic analysis suggested that BdVgR was evolutionary conserved with other Dipteran VgRs. The expression of BdVgR was exclusively detected in the ovaries rather than head, thorax or other tissues. The developmental expression patterns showed that the signal of BdVgR was detectable in very beginning of adult stage, and positively correlated with the growth rate of ovaries and the expression levels of its ligands. In addition, we also demonstrated that the expression level of BdVgR, and ovary development were significantly suppressed after being injected with BdVgR-targeted dsRNA. Together, all of these results indicated that BdVgR was critical for yolk protein absorption and ovary maturation in B. dorsalis, playing a vital role in female reproduction.

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is a serious pest on a variety of economically important crops widely distributed in China, and its resistance to acaricides has quickly developed. In this study, we fully sequenced 13 GST genes of T. cinnabarinus (TcGSTs). The phylogenetic tree showed that five of them belonged to the delta class and the other eight belonged to the mu class. The alignment of gene sequences and comparison of gene expressions between a fenpropathrin-resistant strain (FR) and a susceptible strain (SS) showed that neither point mutation nor overexpression was detected in TcGSTs. However, when challenged by a sublethal dose of fenpropathrin, the mRNA levels of three GSTs from the mu class (TCGSTM2, TCGSTM3, and TCGSTM8) highly increased in FR, while in SS, the expression of these genes was still at the same level under the treatment. In conclusion, specific TcGSTs were identified that were inducible to stimulation by fenpropathrin, and proved that TcGSTs in FR were not constantly expressed at a high level, but could react much more quickly under the stress of fenpropathrin than SS.

The oriental fruit fly Bactrocera dorsalis (Hendel) causes devastating losses to agricultural crops world-wide and is considered to be an economically important pest. Little is known about the digestive enzymes such as serine proteases (SPs) in B. dorsalis, which are important both for energy supply and mitigation of fitness cost associated with insecticide tolerance. In this study, we identified five SP genes in the midgut of B. dorsalis, and the alignments of their deduced amino acid sequences revealed the presence of motifs conserved in the SP superfamily. Phylogenetic analyses with known SPs from other insect species suggested that three of them were trypsin-like proteases. Analyses of the expression profiles among the different developmental stages showed that all five genes were most abundant in larvae than in other stages. When larvae were continuously fed on diet containing 0.33 μg/g β-Cypermethrin, expression of all five genes were upregulated in the midgut but the larval development was delayed. Biochemical assays were consistent with the increased protease activity exhibited by SPs in the midgut after treatment with β-Cypermethrin. Taken together, these findings provide evidence for the hypothesis that enhanced SP activity may play an indirect role in relieving the toxicity stress of insecticide in B. dorsalis.