Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II detoxification enzymes involved in glycosylation of lipophilic endobiotics and xenobiotics, including phytoalexins. Nicotine, one of the most abundant secondary plant metabolites in tobacco, is highly toxic to herbivorous insects. Plant-herbivore competition is the major impetus for the evolution of large superfamilies of UGTs and other detoxification enzymes. However, UGT functions in green peach aphid (Myzus persicae) adaptation are unknown. In this study, we show that UGT inhibitors (sulfinpyrazone and 5-nitrouracil) significantly increased nicotine toxicity in M. persicae nicotianae, suggesting that UGTs may be involved in nicotine tolerance. In total, 101 UGT transcripts identified in the M. persicae genome/transcriptome were renamed according to the UGT Nomenclature Committee guidelines and grouped into 11 families, UGT329, UGT330, UGT339, UGT341–UGT345, and UGT348–UGT350, with UGT344 containing the most (57). Ten UGTs (UGT330A3, UGT339A2, UGT341A6, UGT342B3, UGT343C3, UGT344D5, UGT344D8, UGT348A3, UGT349A3, and UGT350A3) were highly expressed in M. persicae nicotianae compared to M. persicae sensu stricto. Knockdown of four UGTs (UGT330A3, UGT344D5, UGT348A3, and UGT349A3) significantly increased M. persicae nicotianae sensitivity to nicotine, suggesting that UGT expression in this subspecies may be associated with nicotine tolerance and thus host adaptation. This study reveals possible UGTs relevant to nicotine adaptation in tobacco-consuming M. persicae nicotianae, and the findings will facilitate further validation of the roles of these UGTs in nicotine tolerance.

Nicotine is one of the most toxic secondary plant metabolites in nature and it is highly toxic to herbivorous insects. The overexpression of CYP6CY3 and its homologous isozyme CYP6CY4 in Myzus persicae nicotianae is correlated with nicotine tolerance. The expanded (AC)_n repeat in promoter is the cis element for CYP6CY3 transcription. These repeat sequences are conserved in the CYP6CY3 gene from Aphis gossypii and the homologous P450 genes in Acyrthosiphon pisum. The potential transcriptional factors that may regulate CYP6CY3 were isolated by DNA pulldown and sequenced in order to investigate the underlying transcriptional regulation mechanism of CYP6CY3. These identified transcriptional factors, AhR and ARNT, whose abundance was highly correlated with an abundance of the CYP6CY3 gene, were validated. RNAi and co-transfection results further confirm that AhR and ARNT play a major role in the transcriptional regulation of the CYP6CY3 gene. When the CYP6CY3 transcript is destabilized by AhR/ARNT RNAi, the transcription of the CYP6CY4 is dramatically up-regulated, indicating a compensatory mechanism between the CYP6CY3 and CYP6CY4 genes. Our present study sheds light on the CYP6CY3 and CYP6CY4 mediated nicotine adaption of M. persicae nicotianae to tobacco. The current studies shed light on the molecular mechanisms that underlie the genotypic and phenotypic changes that are involved in insect host shifts and we conclude that AhR/ARNT regulate the expression of CYP6CY3 and CYP6CY4 cooperatively, conferring the nicotine adaption of M. persicae nicotianae to tobacco

Chemosensory proteins (CSPs) are a class of transporters in arthropods. Deeper research on CSPs showed that CSPs may be involved in some physiological processes beyond chemoreception, such as insect resistance to pesticides. We identified two upregulated CSPs in two resistant strains of Aphis gossypii Glover. To understand their role in the resistance of aphids to pesticides, we performed the functional verification of CSP1 and CSP4 in vivo and in vitro. Results showed that the sensitivity of the thiamethoxam-resistant strain to thiamethoxam increased significantly with the silencing of CSP1 and CSP4 by RNAi (RNA interference), and the sensitivity of the spirotetramat-resistant strain to spirotetramat increased significantly with the silencing of CSP4. Transgenic Drosophila melanogaster expressing CSPs exhibited stronger resistance to thiamethoxam, spirotetramat, and alpha-cypermethrin than the control did. In the bioassay of transgenic Drosophila, CSPs showed different tolerance mechanisms for different pesticides, and the overexpressed CSPs may play a role in processes other than resistance to pesticides. In brief, the present results prove that CSPs are related to the resistance of cotton aphids to insecticides.

Nuclear factor erythroid 2 related factor 2 (Nrf2) belongs to the cap ‘n’ collar basic region leucine zipper (CNC-bZIP) transcription factor family, and is activated by diverse oxidants, pro-oxidants, antioxidants, and chemo-preventive agents. Transcriptional regulation of a battery of detoxifying and antioxidant genes by Nrf2 has been shown to be important for protection against oxidative stress or chemically-induced cellular damages. In our research, we cloned the full length CncC gene from the Spodoptera frugiperda, named as SfCncC. The cDNA of the SfCncC consists of 2652 nucleotides that include a 2196-nucleotide open reading frame (ORF), encoding 731 amino acid residues, and 239- and 217-bp non-coding regions flanking at the 5’- and 3’-ends of the cDNA, respectively. Sequence analysis indicated SfCncC has the conserved domain (CNC-bZIP domain and a tetrapeptide motif, ETGE) character of Nrf2 and showed high identity compared with the CncC/Nrf2 from other insect and vertebrate species. Over-expression of SfCncC can up-regulate the transcription and activity of the SOD gene in Sf9 cells, and the RNAi of SfCncC in Sf9 cells and larvae of S. frugiperda can dramatically reduce the transcriptional level and activity of the SOD gene, as determined by real-time quantitative PCRs. So the SfCncC is involved in the Keap1-Nrf2-ARE pathway, acting the same as the transcriptional factor Nrf2 in vertebrate, and plays a role for host cell defense. The functional characterization of SfCncC provides the fundamental basis for us to further understand the regulatory mechanism of anti-oxidants and anti-xenobiotics in S. frugiperda.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II detoxification enzymes involved in glycosylation of lipophilic endobiotics and xenobiotics, including phytoalexins. Nicotine, one of the most abundant secondary plant metabolites, is highly toxic to herbivorous insects. Plant-herbivore competition has been the major impetus for evolution of large superfamilies of UGTs and other detoxification enzymes. However, UGT functions in Myzus persicae adaptation have remained unknown. In this study, UGT inhibitors (sulfinpyrazone and 5-nitrouracil) significantly increased nicotine toxicity in M. persicae nicotianae, suggesting UGTs may be involved in nicotine tolerance. One hundred one UGT transcripts identified from the M. persicae genome/transcriptome were renamed according to UGT Nomenclature Committee guidelines and clustered into 11 families: UGT329, UGT330, UGT339, UGT341-UGT345, and UGT348-UGT350. UGT344 contained the most UGTs (fifty-seven). Ten UGTs (UGT330A3, UGT339A2, UGT341A6, UGT342B3, UGT343C3, UGT344D5, UGT344D8, UGT348A3, UGT349A3 and UGT350A3) were overexpressed in M. persicae nicotianae compared with M. persicae sensu stricto. UGT330A3/UGT344D5/UGT348A3/UGT349A3 knockdown significantly increased M. persicae nicotianae nicotine sensitivity, suggesting UGT upregulation in this subspecies may be associated with nicotine tolerance and thus host adaptation. This study reveals possible UGTs relevant to nicotine adaptation in tobacco-consuming M. persicae nicotianae and might facilitate further validation of the roles of these UGTs in nicotine tolerance.