Rhopalosiphum padi (L.) (Hemiptera: Aphididae) is an important cosmopolitan pest in cereal crops. Reference genes can significantly affect qRT-PCR results. Therefore, selecting appropriate reference genes is a key prerequisite for qRT-PCR analyses. This study was conducted to identify suitable qRT-PCR reference genes in R. padi. We systematically analyzed the expression profiles of 11 commonly used reference genes. The ΔCt method, the BestKeeper, NormFinder, geNorm algorithms, and the RefFinder online tool were used to evaluate the suitability of these genes under diverse experimental conditions. The data indicated that the most appropriate sets of reference genes were β-actin and GAPDH (for developmental stages), AK and TATA (for populations), RPS18 and RPL13 (for tissues), TATA and GAPDH (for wing dimorphism), EF-1α and RPS6 (for antibiotic treatments), GAPDH and β-actin (for insecticide treatments), GAPDH, TATA, RPS18 (for starvation-induced stress), TATA, RPS6, and AK (for temperatures), and TATA and GAPDH (for all conditions). Our study findings, which revealed the reference genes suitable for various experimental conditions, will facilitate the standardization of qRT-PCR programs, while also improving the accuracy of qRT-PCR analyses, with implications for future research on R. padi gene functions.

Insect pests are primarily controlled by insecticides. However, the sensitivity decreases and insecticide resistance is problematic for the effective management of agriculturally important insects, including Metopolophium dirhodum, which is an aphid that commonly feeds on cereals. The insecticide sensitivity status and potential resistance of M. dirhodum field populations remain relatively unknown. In this study, the susceptibility of 19 M. dirhodum populations from seven provinces in Northern China to neonicotinoids, pyrethroids, organophosphates, and a macrolide (abamectin) was determined in 2017–2019. The results indicated that two populations were highly resistant to thiamethoxam, with a relative resistance ratio (RLR) of 134.03 and 103.03, whereas one population was highly resistant to beta-cypermethrin (RLR of 121.42). On the basis of the RLR, the tested M. dirhodum populations ranging from susceptible to showing moderate levels of resistance to imidacloprid (RLR of 1.50 to 57.29), omethoate (RLR of 1.07 to 18.73), and abamectin (RLR of 1.10 to 25.89), but they were ranging from susceptible to showing tolerance or low levels of resistance to bifenthrin (RLR of 1.14 to 6.02) and chlorpyrifos (RLR of 1.11 to 7.59). Furthermore, a pair-wise correlation analysis revealed a significant correlation between the median lethal concentrations (LC₅₀) for beta-cypermethrin and thiamethoxam, reflecting the cross-resistance between these two insecticides. The data obtained in our study provide timely information about aphid insecticide sensitivity, which may be used to delay the evolution of M. dirhodum insecticide resistance in Northern China.

Sulfoxaflor belongs to a new class of insecticides which are effective against many sap-feeding pests. Sitobion miscanthi, Rhopalosiphum padi, and Metopolophium dirhodum are the predominant pests coexisting on wheat plants. It is unknown whether these aphid species have developed resistance to sulfoxaflor. Here, the susceptibilities of three wheat aphid species from different regions of China to sulfoxaflor were evaluated. The results showed that two S. miscanthi, one R. padi, and two M. dirhodum field populations were highly resistant to sulfoxaflor. Additionally, 13 S. miscanthi, 9 R. padi, and 4 M. dirhodum field populations were moderately resistant to sulfoxaflor. Analysis of differences in toxicity showed that the susceptibility levels of R. padi in 9 of 20 regions, M. dirhodum in 5 of 9 regions, and M. dirhodum in 3 of 9 regions to sulfoxaflor were greater than those of S. miscanthi, S. miscanthi, and R. padi in the same regions, respectively. Thus, each wheat aphid species has field populations that are highly sulfoxaflor resistant. The R. padi and M. dirhodum populations were more susceptible to sulfoxaflor than those of S. miscanthi. These findings provide new insights into insecticide resistance development and rational sulfoxaflor use.

Sitobion miscanthi and Schizaphis graminum (Rondani) are the 2 main aphid species that occur simultaneously, causing significant loss to wheat production. Acetamiprid has been used to control a variety of pests, including aphids. In this study, the sublethal effect of acetamiprid on S. miscanthi and S. graminum was evaluated using life-table analyses. The results showed that acetamiprid has a high toxicity to S. miscanthi and S. graminum with a LC₅₀ of 1.90 and 3.58 mg/L at 24 h, respectively. The adult longevity and fecundity of S. miscanthi and S. graminum F₀ generation were significantly reduced after being exposed to a sublethal concentration of acetamiprid. Additionally, the sublethal concentration of acetamiprid had negative transgenerational effects on S. miscanthi and S. graminum, which showed a significant decrease in fecundity and population life-table parameters involving age-stage-specific survival rate (s_xj), age-specific survival rate (l_x), and intrinsic rate of increase (r). Furthermore, the population projections showed that the total population size of S. miscanthi and S. graminum was significantly lower in the aphid group exposed to sublethal concentration of acetamiprid compared to the control group. These results suggest that sublethal concentration of acetamiprid suppresses the population growth of S. miscanthi and S. graminum. This finding is beneficial to the control of wheat aphids, and is important to fully understand the role of acetamiprid in integrated pest management.

Sulfoxaflor belongs to a new class of insecticides that is effective against many sap-feeding pests. In this study on Sitobion miscanthi (Takahashi) (i.e., the predominant wheat pest), a highly sulfoxaflor-resistant (SulR) population was obtained from a field. Its resistance to the other seven insecticides and its biological fitness were analyzed using a leaf-dip method and a two-sex life table approach, respectively. Compared with the relatively susceptible (SS) population, the SulR population was highly resistant to sulfoxaflor, with a relative insecticide resistance ratio (RR) of 199.8 and was moderately resistant to beta-cypermethrin (RR = 14.5) and bifenthrin (RR = 42.1) but exhibited low resistance to chlorpyrifos (RR = 5.7). Additionally, the SulR population had a relative fitness of 0.73, with a significantly prolonged developmental period as well as a lower survival rate and poorer reproductive performance than the SS population. In conclusion, our results suggest that S. miscanthi populations that are highly resistant to sulfoxaflor exist in the field. The possibility that insects may develop multi-resistance between sulfoxaflor and pyrethroids is a concern. Furthermore, the high sulfoxaflor resistance of S. miscanthi was accompanied by a considerable fitness cost. The study data may be useful for improving the rational use of insecticides and for exploring novel insecticide resistance mechanisms.

Beta-cypermethrin is one of the most widely used pyrethroid insecticides. However, its continuous and extensive use developed pests’ resistance to beta-cypermethrin. To identify candidate genes for potential resistance phenotypes and facilitate more targeted studies, we screened out a wide range of resistance-related genes by comparing multiple transcripts before and after the induction of multiple agents. In this study, transcriptomes were compared to elucidate the mechanisms and genetic basis of potential resistance between Rhopalosiphum padi (R. padi) sensitive (SS) and resistant (Beta-R) strains to beta-cypermethrin (resistance ratio: 4588.48). These two strains of aphids were treated with a spray solution of lethal beta-cypermethrin concentration (i.e., LC₅₀). To obtain diverse transcripts, we obtained 17,985,440–25,478,353 clean data from different transcript groups, of which 17,183 genes were annotated. Subsequently, these transcripts were divided into multiple groups for comparison purposes to obtain more comprehensive genes related to resistance. There were 178 to 2856 differentially expressed genes (DEGs) in these transcript groups. The DEGs, including the enriched ones, were classified according to the GO and KEGG Pathway databases. Besides, some drug-resistant DEGs were related to cuticle proteins and detoxification metabolic processes. Among them, 17 genes related to cuticle protein were upregulated and 20 were downregulated, 11 genes related to P450 were upregulated and 25 were downregulated, 7 genes related to UGT were upregulated and 15 were downregulated, 2 genes related to ABC transporter were upregulated and 4 were downregulated, 2 genes related to trypsin were upregulated and 1 were downregulated. Finally, qRT-PCR by DEGs confirmed the observed trend in the RNA sequencing expression profile, and most of the results were consistent between qRT-PCR and RNA sequencing (RNA-seq). The results of this study are highly significant in understanding the resistance phenomenon in R. padi and other similar wheat aphids, establishing the valuable basis for further research in the complex mechanism of R. padi resistance to beta-cypermethrin.