Goji fruit fly, Neoceratitis asiatica, is a major pest on the well-known medicinal plant Lycium barbarum. Dissecting molecular mechanisms of infestation and host selection of N. asiatica will contribute to the determination of best management practices for pest fly control. Gene expression normalization by Real-time quantitative PCR (qPCR) requires the selection and validation of appropriate reference genes (RGs). Hence, 15 candidate RGs were selected from transcriptome data of N. asiatica. Their expression stability was evaluated with five algorithms (∆Ct, Normfinder, GeNorm, BestKeeper, and RefFinder) for sample types differing in the developmental stage, sex, tissue type, and in response to five different abiotic stresses. Our results indicated that the RGs β-Actin + GST for sex, RPL32 + EF1α for tissue type, RPS13+ EF1α for developmental stages along with odor stimulation, color induction, and starvation-refeeding stresses, EF1α + GAPDH under insecticide stress, RPS13 + RPS18 under temperature stress, respectively, were selected as the most suitable RGs for qPCR normalization. Overall, RPS18 and EF1α were the two most stable RGs in all conditions, while RPS15 and EF1β were the least stable RGs. The corresponding suitable RGs and one unstable RG were used to normalize a target odorant-binding protein OBP56a gene in male and female antennae, different tissues, and under odor stimulation. The results of OBP56a expression were consistent with transcriptome data. Our study is the first research on the most stable RGs selection in N. asiatica, which will facilitate further studies on the mechanisms of host selection and insecticide resistance in N. asiatica.

Background: Insect olfactory proteins can transmit chemical signals in the environment that serve as the basis for foraging, mate searching, predator avoidance and oviposition selection. Semanotus bifasciatus is an important destructive borer pest, but its olfactory mechanism is not clear. We identified the chemosensory genes of S. bifasciatus in China, then we conducted a phylogenetic analysis of the olfactory genes of S. bifasciatus and other species. And the expression profiles of odorant binding proteins (OBPs) genes in different tissues and different genders of S. bifasciatus were determined by quantitative real-time PCR for the first time. Results: A total of 32 OBPs, 8 chemosensory proteins (CSPs), 71 odorant receptors (ORs), 34 gustatory receptors (GRs), 18 ionotropic receptors (IRs), and 3 sensory neuron membrane proteins (SNMPs) were identified. In the tissue expression analysis of OBP genes, 7 OBPs were higher expressed in antennae, among them, SbifOBP2, SbifOBP3, SbifOBP6, SbifOBP7 and SbifOBP20 were female-biased expression, while SbifOBP1 was male-biased expression and SbifOBP22 was no-biased expression in antennae. In addition, the expressed levels of SbifOBP4, SbifOBP12, SbifOBP15, SbifOBP27 and SbifOBP29 were very poor in the antennae, and SbifOBP4 and SbifOBP29 was abundant in the head or legs, and both of them were male-biased expression. While SbifOBP15 was highly expressed only at the end of the abdomen with its expression level in females three times than males. Other OBPs were expressed not only in antennae but also in various tissues. Conclusion: We identified 166 olfactory genes from S. bifasciatus, and classified these genes into groups and predicted their functions by phylogenetic analysis. The majority of OBPs were antenna-biased expressed, which are involved in odor recognition, sex pheromone detection, and/or host plant volatile detection. However, also some OBPs were detected biased expression in the head, legs or end of the abdomen, indicating that they may function in the different physiological processes in S. bifasciatus.

Carposina coreana is an important pest of Cornus officinalis, distributed in China, Korea, and Japan. In recent years, its damage to C. officinalis has become increasingly serious, causing enormous economic losses in China. This study and prediction of current and future suitable habitats for C. coreana in China can provide an important reference for the monitoring, early warning, prevention, and control of the pest. In this study, the potential distributions of C. coreana in China under current climate and future climate models were predicted using the maximum entropy (MaxEnt) model with ArcGIS software. The distribution point data of C. coreana were screened using the buffer screening method. Nineteen environmental variables were screened using the knife-cut method and variable correlation analysis. The parameters of the MaxEnt model were optimized using the kuenm package in R software. The MaxEnt model, combined with key environmental variables, was used to predict the distribution range of the suitable area for C. coreana under the current (1971–2000) and four future scenarios. The buffer screening method screened data from 41 distribution points that could be used for modeling. The main factors affecting the distribution of C. coreana were precipitation in the driest month (Bio14), precipitation in the warmest quarter (Bio18), precipitation in the coldest quarter (Bio19), the standard deviation of seasonal variation of temperature (Bio4), minimum temperature in the coldest month (Bio6), and average temperature in the coldest quarter (Bio11). The feature class (FC) after the kuenm package optimization was a Q-quadratic T-threshold combination, and the regularization multiplier (RM) was 0.8. The suitable areas for C. coreana under the current climate model were mainly distributed in central China, and the highly suitable areas were distributed in southern Shaanxi, southwestern Henan, and northwestern Hubei. The lowest temperature in the coldest month (Bio6), the average temperature in the coldest quarter (Bio11), and the precipitation in the warmest quarter (Bio18) all had good predictive ability. In future climate scenarios, the boundary of the suitable area for C. coreana in China is expected to shift northward, and thus, most of the future climate scenarios would shift northward.

Insecticide resistance has long been a problem in crop pest control. Bactericera gobica is a major pest on the well-known medicinal plants Lycium barbarum L. Investigating insecticide resistance mechanisms of B. gobica will help to identify pesticide reduction strategies to control the pest. Gene expression normalization by RT-qPCR requires the selection and validation of appropriate reference genes (RGs). Here, 15 candidate RGs were selected from transcriptome data of B. gobica. Their expression stability was evaluated with five algorithms (Delta Ct, GeNorm, Normfinder, BestKeeper and RefFinder) for sample types differing in response to five insecticide stresses and in four other experimental conditions. Our results indicated that the RGs RPL10 + RPS15 for Imidacloprid and Abamectin; RPL10 + AK for Thiamethoxam; RPL32 + RPL10 for λ-cyhalothrin; RPL10 + RPL8 for Matrine; and EF2 + RPL32 under different insecticide stresses were the most suitable RGs for RT-qPCR normalization. EF1α + RPL8, EF1α + β-actin, β-actin + EF2 and β-actin + RPS15 were the optimal combination of RGs under odor stimulation, temperature, developmental stages and both sexes, respectively. Overall, EF2 and RPL8 were the two most stable RGs in all conditions, while α-TUB and RPL32 were the least stable RGs. The corresponding suitable RGs and one unstable RG were used to normalize a target cytochrome P450 CYP6a1 gene between adult and nymph stages and under imidacloprid stress. The results of CYP6a1 expression were consistent with transcriptome data. This study is the first research on the most stable RG selection in B. gobica nymphs exposed to different insecticides, which will contribute to further research on insecticide resistance mechanisms in B. gobica.