To obtain accurate and reliable results from quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, it is necessary to select suitable reference genes as standards for normalizing target gene expression data. QRT-PCR is a popular analytical methodology for studying gene expression and it has been used widely in studies of Aphis gossypii Glover in recent years. However, there is absence of study on the stability of the expression of reference genes in A. gossypii. In this study, eight commonly used candidate reference genes, including 18S, 28S, β-ACT, GAPDH, EF1α, RPL7, α-TUB, and TBP, were evaluated under various experimental conditions to assess their suitability for use in the normalization of qRT-PCR data. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated by performing normalizations of expression data for the HSP70 gene. The results showed the most suitable combinations of reference genes for the different experimental conditions. For experiments based on divergent developmental stages, EF1α, β-ACT, and RPL7 are the optimal reference gene combination, both EF1α and β-ACT are the optimal combination used in the experiments of different geographical populations, whereas for experiments of the temperature changes, the combination of GAPDH and RPL7 is optimal, both 18S and β-ACT are an optimal combination for feeding assay experiments. These research results should be useful for the selection of the suitable reference genes to obtain reliable qRT-PCR data in the gene expression study of A. gossypii.

MicroRNAs (miRNAs) are a group of short non-coding RNAs involved in the inhibition of protein translation or in mRNA degradation. Although the regulatory roles of miRNAs in various biological processes have been investigated, there is as yet an absence of studies about the regulatory roles of miRNAs involved in the metabolism of plant allelochemicals in insects. We constructed five small RNA libraries from apterous Aphis gossypii adults that had fed on an artificial diet containing various allelochemicals. Using Illumina sequencing, a total of 73.27 million clean reads was obtained, and 292 miRNAs were identified from A. gossypii. Comparative analysis of read counts indicated that both conserved and novel miRNAs were differently expressed among the five libraries, and the differential expression was validated via qRT-PCR. We found that the transcript levels of several miRNAs were increased or decreased in all of the allelochemical treatment libraries compared to the control. The putative target genes of the miRNAs were predicted using in silico tools, and the target genes of several miRNAs were presumed to be involved in the metabolism of xenobiotic compounds. Furthermore, the target prediction results were confirmed using dual luciferase reporter assay, and Ago-miR-656a-3p was demonstrated to regulate the expression of CYP6J1 post-transcriptionally through binding to the 3′ UTR of CYP6J1. Our research results indicate that miRNAs may be involved in the metabolism of plant allelochemicals in A. gossypii, and these results also represent an important new small RNA genomics resource for further studies on this topic.

Aphis gossypii Glover (Hemiptera: Aphididae) can damage a variety of agricultural crops, so it is very important for cotton aphids to evolve adaptive mechanisms to various allelochemicals from host plants. Our results aim to provide a fundamental and rich resource for exploring aphid functional genes in A. gossypii. A transcriptome data set and five expression profile data sets of A. gossypii samples were analyzed by Illumina sequencing platform. In total, 53,763,866 reads were assembled into 1,963,516 contigs and 28,555 unigenes. Compared with the control, 619 genes were significantly up- or downregulated in the treatment group by 2-tridecanone. There were 516, 509, and 717 of differential expression genes in tannic acid, quercetin, and gossypol treatment groups, respectively. Furthermore, there were 4 of 54 putative cytochrome P450 genes and 1 of 7 putative carboxylesterases downregulated in all treatment groups by four plant allelochemicals. When aphids fed on 2-tridecanone, tannic acid, and quercetin, only one P450 gene was upregulated. These results show that plant allelochemical stress can induce differential gene expression in A. gossypii. The differential response information of gene expression based on a large-scale sequence would be useful to reveal molecular mechanisms of adaptation for A. gossypii to plant allelochemicals.

Background: Most aphids exhibit wing polyphenism in which wingless and winged morphs produce depending on the population density and host plant quality. Although the influence of environmental factors on wing polyphenism of aphids have been extensively investigated, molecular mechanisms underlining morph differentiation (i.e. wing development /degeneration), one downstream aspect of the wing polyphenism, has been poorly understood.Results: We examined the expression levels of the twenty genes involved in wing development network, and onlyvestigial (vg) showed significantly different expression levels in both whole-body and wall-body of third instar nymphs, with 5.4- and 16.14- fold higher expression in winged lines compared to wingless lines, respectively inRhopalosiphum padi.vgexpression was higher in winged lines compared to wingless lines in third, fourth instar nymphs and adults. Larger difference expression was observed in third (21.38-fold) and fourth (20.91-fold) instar nymphs relative to adults (3.12-fold). Suppression ofvgusing RNAi repressed the wing development of third winged morphs. Furthermore, dual luciferase reporter assay revealed that the miR-147 can target thevgmRNA. Modulation of miR-147b levels by microinjection of its agomir (mimic) decreasedvgexpression levels and repressed wing development.Conclusions: Our findings suggest thatvgis essential for wing development inR. padiand that miR-147b modulates its expression.

The fall armyworm, Spodoptera frugiperda is a major agricultural pest in China, and has migrated from its continuous breeding area to other parts of China. In our study, the biological behaviors of S. frugiperda fed on maize, wheat, barley, faba beans, and soya beans were evaluated in a growth chamber. Results indicated that maize-fed S. frugiperda larvae performed well, as evidenced by shorter larva-adult periods, adult pre-oviposition period (APOP), total pre-oviposition period (TPOP), and generation time (T), and a higher survival rate, intrinsic (r) and finite (λ) rate of increase, and net reproductive rate (R_o), However, S. frugiperda larvae performed weakly when fed barley and faba bean plants, as indicated by lower survival rates, r, and λ, and longer pre-adult period, TPOP, and T. A heavier pupal weight of both sexes was recorded on faba beans (0.202 g) and a lighter weight on barley (0.169 g). Fecundity was higher when fed faba beans and maize, and lower when fed wheat and barley. Thus, maize was the most optimal and barley was the least optimal host plant, followed by faba beans, for S. frugiperda larvae growth and development. This study enhances our knowledge of S. frugiperda in these host plants and can help in the design of management approaches.