Detection of Chitin Synthase Mutations in Lufenuron-Resistant Spodoptera frugiperda in China.

Spodoptera frugiperda (J. E. Smith), is commonly known as fall armyworm, native to tropical and subtropical regions of America, is an important migratory agricultural pest. It is important to understand the resistance and internal mechanism of action of S. frugiperda against lufenuron in China. Lufenuron is one of the main insecticides recommended for field use in China and has a broad prospect in the future. We conducted a bioassay using the diet-overlay method and found that the current S. frugiperda in China are still at a low level of resistance to lufenuron. Secondly, we examined whether the mutation I1040M (I1042M in Plutella xylostella), associated with lufenuron resistance, was produced in the field. And then we tested the expression of chitin synthase SfCHSA and SfCHSB in different tissues, and the changes of these two genes after lufenuron induction. The results showed that there is still no mutation generation in China and there is a significant change in the expression of SfCHSA under the effect of lufenuron. In conclusion, our study suggests that field S. frugiperda populations in 2019 and 2020 were less resistant to lufenuron. In fall armyworm, chitin synthases included SfCHSA and SfCHSB genes, and after induction treatment with lufenuron, the expression of the SfCHSA gene was significantly increased. In SfCHSA, no mutation has been detected in the site associated with lufenuron resistance. Secondly, in S. frugiperda larvae, the SfCHSA gene was the highest in the head of the larvae, followed by the integument; while the SfCHSB gene was mainly concentrated in the midgut. Therefore, we believe that the SfCHSA gene plays a greater role in the resistance of S. frugiperda to lufenuron than the SfCHSB gene. It is worth noting that understanding the level of resistance to lufenuron in China, the main mechanism of action of lufenuron on larvae, and the mechanism of resistance to lufenuron in S. frugiperda will help in crop protection as well as in extending the life span of this insecticide.

Plant volatile compound methyl benzoate is highly effective against Spodoptera frugiperda and safe to non-target organisms as an eco-friendly botanical-insecticide.

Recent studies have indicated that the plant volatile methyl benzoate (MB) exhibits significant insecticidal bioactivity against several common insects. However, the potential environmental hazards of MB and its safety to non-target organisms is poorly understood. In the present study, these characteristics were investigated through laboratory experiments and field investigations. The results revealed that MB was highly toxic to the agricultural pest, fall armyworm Spodoptera frugiperda. Compared with the commercial pesticide lambda-cyhalothrin, the toxicities of MB against S. frugiperda larvae and adults were comparable and 3.41 times higher, respectively. Behavioral bioassays showed that the percentage repellency of MB to S. frugiperda larvae was 56.72 %, and MB induced 69.40 % oviposition deterrence rate in S. frugiperda female adults. Furthermore, in terms of median lethal concentration (LC50) and median lethal doses (LD50), MB exhibited non-toxic effects on non-target animals with 3-d LC50 of > 1 % to natural predators (Coccinella septempunctata and Harmonia axyridis), 3-d LD50 of 467.86 µg/bee to the bumblebee Bombus terrestris, 14-d LC50 of 971.09 mg/kg to the earthworm Eisenia fetida, and 4-d LC50 of 47.30 mg/L to the zebrafish Brachydanio rerio. The accumulation of MB in the soil and earthworms was found to be extremely limited. Our comparative study clearly demonstrated that MB is effective as a selective botanical pesticide against S. frugiperda and it is safe to use in the tested environment, with no toxic effects on non-target animals and natural predators.

MicroRNA-263b confers imidacloprid resistance in Sitobion miscanthi (Takahashi) by regulating the expression of the nAChRß1 subunit.

The Chinese wheat aphid Sitobion miscanthi (CWA) is an important harmful pest in wheat fields. Imidacloprid plays a critical role in controlling pests with sucking mouthparts. However, imidacloprid-resistant pests have been observed after insecticide overuse. Point mutations and low expression levels of the nicotinic acetylcholine receptor ß1 (nAchRß1) subunit are the main imidacloprid-resistant mechanisms. However, the regulatory mechanism underlying nAChRß1 subunit expression is poorly understood. In this study, a target of miR-263b was isolated from the 5'UTR of the nAchRß1 subunit in the CWA. Low expression levels were found in the imidacloprid-resistant strain CWA. Luciferase reporter assays showed that miR-263b could combine with the 5'UTR of the nAChRß1 subunit and suppress its expression by binding to a site in the CWA. Aphids treated with the miR-263b agomir exhibited a significantly reduced abundance of the nAchRß1 subunit and increased imidacloprid resistance. In contrast, aphids treated with the miR-263b antagomir exhibited significantly increased nAchRß1 subunit abundance and decreased imidacloprid resistance. These results provide a basis for an improved understanding of the posttranscriptional regulatory mechanism of the nAChRß1 subunit and further elucidate the function of miRNAs in regulating susceptibility to imidacloprid in the CWA. These results provide a better understanding of the mechanisms of posttranscriptional regulation of nAChRß1 and will be helpful for further studies on the role of miRNAs in the regulation of nAChRß1 subunit resistance in homopteran pests.

Overexpression of PxαE14 Contributing to Detoxification of Multiple Insecticides in Plutella xylostella (L.).

The diamondback moth, Plutella xylostella (L.), has evolved with varying degrees of resistance to almost all major classes of insecticides and has become the most resistant pest worldwide. The multiresistance to different types of insecticides has been frequently reported in P. xylostella, but little is known about the mechanism. In this study, a carboxylesterase (CarE) gene, PxαE14, was found significantly overexpressed in a field-evolved multiresistant P. xylostella population and can be dramatically induced by eight of nine tested insecticides. Results of the real-time quantitative polymerase chain reaction (RT-qPCR) showed that PxαE14 was predominantly expressed in the midgut and malpighian tubule of larvae. Knockdown of PxαE14 dramatically increased the susceptibility of the larvae to ß-cypermethrin, bifenthrin, chlorpyrifos, fenvalerate, malathion, and phoxim, while overexpression of PxαE14 in Drosophila melanogaster increased the tolerance of the fruit flies to these insecticides obviously. More importantly, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay showed that the recombinant PxαE14 expressed in Escherichia coli exhibited metabolic activity against the six insecticides. The homology modeling, molecular docking, and molecular dynamics simulation analyses showed that these six insecticides could stably bind to PxαE14. Taken together, these results demonstrate that constitutive and inductive overexpression of PxαE14 contributes to detoxification of multiple insecticides involved in multiresistance in P. xylostella. Our findings provide evidence for understanding the molecular mechanisms underlying the multiresistance in insect pests.

Silencing of CYP6AS160 in Solenopsis invicta Buren by RNA interference enhances worker susceptibility to fipronil.

Cytochrome P450 monooxygenases play a key role in pest resistance to insecticides by detoxification. Four new P450 genes, CYP6AS160, CYP6AS161, CYP4AB73 and CYP4G232 were identified from Solenopsis invicta. CYP6AS160 was highly expressed in the abdomen and its expression could be induced significantly with exposure to fipronil, whereas CYP4AB73 was not highly expressed in the abdomen and its expression could not be significantly induced following exposure to fipronil. Expression levels of CYP6AS160 and CYP4AB73 in workers were significantly higher than that in queens. RNA interference-mediated gene silencing by feeding on double-stranded RNA (dsRNA) found that the levels of this transcript decreased (by maximum to 64.6%) when they fed on CYP6AS160-specific dsRNA. Workers fed dsCYP6AS160 had significantly higher mortality after 24 h of exposure to fipronil compared to controls. Workers fed dsCYP6AS160 had reduced total P450 activity of microsomal preparations toward model substrates p-nitroanisole. However, the knockdown of a non-overexpressed P450 gene, CYP4AB73 did not lead to an increase of mortality or a decrease of total P450 activity. The knockdown effects of CYP6AS160 on worker susceptibility to fipronil, combined with our other findings, indicate that CYP6AS160 is responsible for detoxification of fipronil. Feeding insects dsRNA may be a general strategy to trigger RNA interference and may find applications in entomological research and in the control of insect pests in the field.

Combined Transcriptomic and Proteomic Analysis of Myzus persicae, the Green Peach Aphid, Infected with Cucumber Mosaic Virus.

Aphids transmit CMV (cucumber mosaic virus) in a non-persistent manner. However, little is known about the mechanism of CMV transmission. In this study, an integrated analysis of the mRNA and protein was performed to identify important putative regulators involved in the transmission of CMV by aphids. At the level of transcription, a total of 20,550 genes (≥2-fold expression difference) were identified as being differentially expressed genes (DEGs) 24 h after healthy aphid transfer to infected tobacco plants using the RNA-seq approach. At the protein level, 744 proteins were classified as being differentially abundant between virus-treated and control M. persicae using iTRAQ (isobaric tags for relative and absolute quantitation) analysis. The combined mRNA and protein analysis enabled the identification of some viral putative regulators, such as cuticle proteins, ribosomal proteins, and cytochrome P450 enzymes. The results show that most of the key putative regulators were highly accumulated at the protein level. Based on those findings, we can speculate that the process by which aphids spread CMV is mainly related to post-translational regulation rather than transcription.

Coordinative mediation of the response to alarm pheromones by three odorant binding proteins in the green peach aphid Myzus persicae.

Odorant binding proteins (OBPs) play an essential role for insect chemosensation in insect peripheral nervous systems of antennae. Each antennal sensilla contains more than one OBP at high concentrations but the interactions and cooperation between co-localized OBPs are rarely reported. In present study, we cloned, expressed and purified eight OBPs of the green peach aphid Myzus persicae. The effects of knocking down the expression of these OBP genes by RNAi on the electrophysiological and behavioural responses of M. persicae to the aphid alarm pheromone, (E)-ß-farnesene (EßF) were investigated. The results showed that the aphids could still be repelled by EßF when the expression of each of three OBP genes was individually knocked down. However, the simultaneous knockdown of MperOBP3/7/9 expression significantly reduced the electrophysiological response and the repellent behaviours of M. persicae to EßF than the single OBP gene knockdown (P < 0.05). Rather than a normal saturation binding curve of individual OBP, the binding curve of MperOBP3/7/9 is bell-shaped with a higher affinity for the fluorescent probe N-phenyl-1-naphthylamine (1-NPN). The competitive binding assays confirmed that MperOBP3, MperOBP7, MperOBP9 and MperOBP3/7/9 mixture exhibited a stronger binding affinity for EßF, than for sex pheromones and plant volatiles with a dissociation constant of 2.5 µM, 1.1 µM, 3.9 µM and 1.0 µM, respectively. The competitive binding curve of MperOBP3/7/9 mixture to EßF is shallow without bottom plateau, suggesting a conformational change and a rapid dissociation after the displacement of all 1-NPN (in vivo after the saturation binding of all OBPs by EßF). The interaction between OBPs and formation of a heterogeneous unit may facilitate the delivery EßF to the OR at electrophysiological and behavioural levels during insect odorant signal transduction thus mediate M. persicae response to the alarm pheromone EßF.

Detection of ryanodine receptor target-site mutations in diamide insecticide-resistant Spodoptera frugiperda in China.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a widely distributed pest of corn. Since it invaded China in 2018, it has caused serious damage to local corn production. Chlorantraniliprole, an anthranilic diamide insecticide, has been widely used to control lepidopteran pests. Tetrachloropyramid is a new allosteric modulator insecticide developed based on chlorantraniliprole, so it has a similar mechanism and insecticidal effect. In this study, we investigated resistance levels to chlorantraniliprole and tetrachloropyramid in S. frugiperda from 13 populations in China. Among the populations tested, the relative highest resistance to chlorantraniliprole occurred in the Guangzhou population, and the most susceptible to chlorantraniliprole was found in the Wuhan population. The lethal dosage LD50 value of the Guangzhou population against chlorantraniliprole was 27.8-fold higher than that of the Wuhan population. Minimal differences were observed among S. frugiperda populations in terms of sensitivity to tetrachloropyramid. Heterozygous mutations at the I4734 site of the ryanodine receptor (RyR) were found, while no mutations were found in the G4891 site. The mutations were detected in only two of the 786 individuals analyzed, one from the Qinzhou population and other from the Anshun population (frequency below 2% in both cases). There were no significant differences in the expression levels of RyR between Guangzhou and Wuhan populations. In summary, our results indicate that: (i) S. frugiperda has low resistance levels to diamide insecticides in China; and (ii) the differences in relative resistance among the 13 populations analyzed are not caused by the mutations in RyR or the expression of RyR.

Characterization and target gene analysis of microRNAs in the antennae of the parasitoid wasp Microplitis mediator.

MicroRNAs (miRNAs), a class of non-coding single-strand RNA molecules encoded by endogenous genes, are about 21-24 nucleotides long and are involved in the post-transcriptional regulation of gene expression in plants and animals. Generally, the types and quantities of miRNAs in the different tissues of an organism are diverse, and these divergences may be related to their specific functions. Here we have identified 296 known miRNAs and 46 novel miRNAs in the antennae of the parasitoid wasp Microplitis mediator by high-throughput sequencing. Thirty-three miRNAs were predicted to target olfactory-associated genes, including odorant binding proteins (OBPs), chemosensory proteins, odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors. Among these, 17 miRNAs were significantly highly expressed in the antennae, four miRNAs were highly expressed both in the antennae and head or wings, while the remaining 12 miRNAs were mainly expressed in the head, thorax, abdomen, legs and wings. Notably, miR-9a-5p and miR-2525-3p were highly expressed in male antennae, whereas miR-1000-5p and novel-miR-13 were enriched in female antennae. The 17 miRNAs highly expressed in antennae are likely to be associated with olfaction, and were predicted to target one OBP (targeted by miR-3751-3p), one IR (targeted by miR-7-5p) and 14 ORs (targeted by 15 miRNAs including miR-6-3p, miR-9a-5p, miR-9b-5p, miR-29-5p, miR-71-5p, miR-275-3p, miR-1000-5p, miR-1000-3p, miR-2525-3p, miR-6012-3p, miR-9719-3p, novel-miR-10, novel-miR-13, novel-miR-14 and novel-miR-28). These candidate olfactory-associated miRNAs are all likely to be involved in chemoreception through the regulation of chemosensory gene expression in the antennae of M. mediator.

Molecular characterization and expression of sensory neuron membrane proteins in the parasitoid Microplitis mediator (Hymenoptera: Braconidae).

Sensory neuron membrane proteins (SNMPs), homologs of the human fatty acid transport protein CD36 family, are observed to play a significant role in chemoreception, especially in detecting sex pheromone in Drosophila and some lepidopteran species. In the current study, two full-length SNMP transcripts, MmedSNMP1 and MmedSNMP2, were identified in the parasitoid Microplitis mediator (Hymenoptera: Braconidae). Quantitative real-time polymerase chain reaction analysis showed that the expression of MmedSNMP1 was significantly higher in antennae than in other tissues of both sexes. In addition, the MmedSNMP1 transcript was increased dramatically in newly emerged adults and there were no significant differences between adults with or without mating and parasitic experiences. However, compared with MmedSNMP1, the expression of MmedSNMP2 was widely found in various tissues, significantly increased at half-pigmented pupae stage and remained at a relatively constant level during the following developmental stages. It was found that MmedSNMP1 contained eight exons and seven introns, which was highly conserved compared with other insect species. In situ hybridization assay demonstrated that MmedSNMP1 transcript was distributed widely in antennal flagella. Among selected chemosensory genes (odorant binding protein, odorant receptor, and ionotropic receptor genes), MmedSNMP1 only partially overlapped with MmedORco in olfactory sensory neurons of antennae. Subsequent immunolocalization results further indicated that MmedSNMP1 was mainly expressed in sensilla placodea of antennae and possibly involved in perceiving plant volatiles and sex pheromones. These findings lay a foundation for further investigating the roles of SNMPs in the chemosensation of parasitoids.

Overexpression of UDP-glycosyltransferase potentially involved in insecticide resistance in Aphis gossypii Glover collected from Bt cotton fields in China.

BACKGROUND: The cotton aphid Aphis gossypii Glover is one of the most destructive insect pests. It has evolved resistance to numerous insecticides around the world due to the application of insecticides. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) have been reported to potentially facilitate the detoxification process of imidacloprid and thiamethoxam in A. gossypii. RESULTS: In this study, the field populations of A. gossypii developed different levels of resistance to multiple insecticides. A UGT inhibitor, 5-nitrouracil, dramatically increased the toxicity of acetamiprid in resistant populations, moderately increased the toxicity of sulfoxaflor in the imidacloprid susceptible (IMI_S) population, and populations from Yuncheng in Shanxi Province (SXYC) and Jingzhou in Hubei Province (HBJZ), and increased the toxicity of bifenthrin in the IMI_S and HBJZ populations, but there was no synergism on omethoate or carbosulfan. Quantitative real-time PCR analysis revealed that UGT344B4 and UGT344C7 were overexpressed in all field populations, and UGT344N4 was overexpressed in the SDBZ and HBZJ populations. Furthermore, the suppression of UGT344B4 or UGT344C7 by RNA interference significantly increased the susceptibility to bifenthrin in the IMI_S population and the susceptibility to sulfoxaflor in the SXYC population. CONCLUSION: These results suggested that UGTs are potentially involved in the detoxification of neonicotinoid, sulfoximine, and pyrethroid insecticides in A. gossypii. Furthermore, the overexpression of UGTs could be associated with insecticide resistance in field populations of A. gossypii. The results might be helpful for the management of insecticide resistance in field populations of A. gossypii. © 2019 Society of Chemical Industry.

Antennal ionotropic receptors IR64a1 and IR64a2 of the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidate) collaboratively perceive habitat and host cues.

Ionotropic receptors (IRs), as a member of the conserved chemoreceptor families in the peripheral nervous system, play a critical role in the chemoreception of Drosophila. However, little is known about IRs in Hymenoptera insects. Here, we comprehensively characterized the gene structure, topological map and chemosensory roles of antennal IRs (MmedIRs) in the hymenopteran parasitoid wasp Microplitis mediator. We found that the IRs were conserved across various insect species. In the in situ hybridization assays, most IRs showed female antennae biased features, and there was no co-expression of the IRs and the olfactory receptor co-receptor (ORco). Moreover, three IR co-expressed complexes, IR75u-IR8a, IR64a1-IR8a and IR64a2-IR8a, were detected. Two genes with high similarity, IR64a1 and IR64a2, were located in distinct neurons but projected to the same sensillum. In two-electrode voltage-clamp recordings, IR64a1 was widely tuned to the chemicals from habitat cues released from host plants over long distances, whereas IR64a2 responded to a narrow range host cues and plant odors with low-volatility. Notably, IR64a2 was able to perceive Z9-14: Ald, a vital sex pheromone component that is released from Helicoverpa armigera, which is the preferred host of M. mediator. Furthermore, most ligands of IR64a1 and IR64a2 can trigger electrophysiological responses in female wasps. We propose that IR64a1 and IR64a2 collaboratively perceive habitat and host cues to assist parasitoids in efficiently seeking hosts.

Differential expression of genes in greenbug (Schizaphis graminum Rondani) treated by imidacloprid and RNA interference.

BACKGROUND: Insecticides act as toxins, inhibitors of digestion and deterrents, and affect the expression of many genes in insects. To assess key genes associated with the detoxification or regulation of imidacloprid in greenbug, Schizaphis graminum (Rondani), the transcriptome and digital gene expression (DGE) profile were analyzed using Illumina sequencing. RESULTS: In total, 48 763 494 clean reads were obtained by sequencing. Expression profile analysis showed that 2782 unigenes were differently expressed between the imidacloprid treatment and control groups. After exposure to imidacloprid, the expression levels of 1846 unigenes were upregulated and 936 were downregulated in comparison with controls. Expression patterns of the top 20 highly expressed genes show that they could be involved in the detoxification of imidacloprid. Silencing of multidrug resistance-associated gene (MRA), GATA-binding gene (GAT) and takeout-like precursor gene (TLP) resulted in increasing susceptibility to imidacloprid. CONCLUSIONS: The differentially expressed genes in S. graminum have potential regulatory or detoxification roles in response to imidacloprid. These results should be useful in understanding the molecular mechanisms of greenbug adaption to imidacloprid. © 2018 Society of Chemical Industry.

Toxicity and Sublethal Effects of Flupyradifurone, a Novel Butenolide Insecticide, on the Development and Fecundity of Aphis gossypii (Hemiptera: Aphididae).

The cosmopolitan pest Aphis gossypii (Glover) causes considerable economic losses on various crops by its feeding damage and transmitting diseases around the world. Flupyradifurone is a novel butenolide pesticide; its toxicity on A. gossypii parent generation (F0) was estimated following treatment with LC25 concentration for 48 h. The adult longevity and fecundity of the F0 individuals treated by flupyradifurone showed no significant decrease in comparison with the control. Life table method was used to evaluate the sublethal effects on progeny population (F1). Results showed that the development time of the fourth instar and the preadult as well as the total pre-reproductive period were significantly prolonged, while their fecundity was significantly decreased compared with the control. Additionally, the intrinsic rate of increase (r), the finite rate of increase (λ), and the net reproductive rate (R0) of F1 were all significantly lower in the group treated by LC25 than in the control group. These results reveal that the sublethal concentration of flupyradifurone could suppress the population growth of A. gossypii and indicate that this novel insecticide may be as a useful tool in pest management.

Identification and characterization of chemosensory genes in the antennal transcriptome of Spodoptera exigua.

Chemical senses are crucial for insect behaviors such as host preference, mate choice and oviposition site selection. Various protein families are involved in these processes, including odorant receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), chemosensory proteins (CSPs), odorant binding proteins (OBPs) and sensory neuron membrane proteins (SNMPs). To better understand the olfactory mechanism in Spodoptera exigua, we conducted transcriptome analysis of adult antennae and identified a total of 157 candidate chemosensory genes encoding 51 ORs, 20 IRs, 7 GRs, 32 CSPs, 45 OBPs and 2 SNMPs. Quantitative real time PCR (qPCR) analysis of the tissue- and sex-specific expression profiles of ORs, GRs and IRs revealed that these genes could be detected in at least one tissue tested. SexiOR6, 11, 13 and 16, which were predicted to be pheromone receptors based on phylogenetic analysis, exhibited male-specific antennae expression. SexiOR18, 26, 28, 30, 34, 39, and 40 exhibited female-biased expression. SexiGR1, SexiGR2 and SexiGR3, are predicted carbon dioxide receptors, and the former was expressed specifically in antennae, and the latter two were expressed both in antennae and labial palps. SexiIRs had diverse expression profiles. SexiIR8a and SexiIR25a were quite conserved and expressed at high levels. This work will greatly facilitate the understanding of olfactory system in S. exigua and provides valuable information for further functional studies of the chemoreception mechanism in Lepidopteran moths.

Key site residues of pheromone-binding protein 1 involved in interacting with sex pheromone components of Helicoverpa armigera.

Pheromone binding proteins (PBPs) are widely distributed in insect antennae, and play important roles in the perception of sex pheromones. However, the detail mechanism of interaction between PBPs and odorants remains in a black box. Here, a predicted 3D structure of PBP1 of the serious agricultural pest, Helicoverpa armigera (HarmPBP1) was constructed, and the key residues that contribute to binding with the major sex pheromone components of this pest, (Z)-11- hexadecenal (Z11-16:Ald) and (Z)-9- hexadecenal (Z9-16:Ald), were predicted by molecular docking. The results of molecular simulation suggest that hydrophobic interactions are the main linkage between HarmPBP1 and the two aldehydes, and four residues in the binding pocket (Phe12, Phe36, Trp37, and Phe119) may participate in binding with these two ligands. Then site-directed mutagenesis and fluorescence binding assays were performed, and significant decrease of the binding ability to both Z11-16:Ald and Z9-16:Ald was observed in three mutants of HarmPBP1 (F12A, W37A, and F119A). These results revealed that Phe12, Trp37, and Phe119 are the key residues of HarmPBP1 in binding with the Z11-16:Ald and Z9-16:Ald. This study provides new insights into the interactions between pheromone and PBP, and may serve as a foundation for better understanding of the pheromone recognition in moths.

Monitoring insecticide resistance and diagnostics of resistance mechanisms in the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) in China.

Myzus persicae (Sulzer) is one of the most serious agricultural pests in China, and management strategies mainly rely on insecticidal treatment. To evaluate the resistance of field populations of M. persicae to seven insecticides, we assessed the susceptibility of 11 field populations collected from eight provinces in China using leaf-dip bioassays. Toxicity assays showed that M. persicae field populations have developed several levels of resistance to each tested insecticide. For pyrethroids, the field populations have developed a high level of resistance to ß-cypermethrin and cypermethrin, while the resistance to bifenthrin is still low. The resistance ratios of field populations to imidacloprid ranged from 1.48 to 52.36, and eight populations have developed moderate to high resistance. Resistance to acetamiprid is low, and only two populations have a moderate level of resistance. Most of the field populations of M. persicae developed moderate to high resistance to methomyl and omethoate. To investigate potential resistance mechanisms, we analyzed the enzyme activity of carboxylesterases, the type of amplified esterase genes, as well as the kdr (L1014F) mutation. All of the field populations exhibited a higher esterase activity compared to the laboratory susceptible strain. An amplified FE4, as well as the L1014F mutation, were also found in all of our experimental field populations. These results provide valuable insight into the current status of insecticide resistance and will prove to be a valuable resource in designing appropriate resistance management strategies for M. persicae in China.

The Cuticle Protein Gene MPCP4 of Myzus persicae (Homoptera: Aphididae) Plays a Critical Role in Cucumber Mosaic Virus Acquisition.

Myzus persicae (Sulzer) (Homoptera: Aphididae) is one of the most important agricultural pests worldwide. In addition to sucking phloem sap, M. persicae also transmits Cucumber mosaic virus (CMV) as a vector in a nonpersistent manner. At present, the infection mechanism remains unclear, especially the process of aphid virus acquisition. In this study, we isolated four M. persicae cuticle protein genes (MPCP1, MPCP2, MPCP4, and MPCP5) from M. persicae. The relative amount of the gene encoding Cucumber mosaic virus capsid protein (CMV CP) and the transcript levels of these four cuticle protein genes were investigated in aphid virus acquisition by feeding the tobacco preinfested by CMV. The relative expression of MPCP1, MPCP2, and MPCP4 were significantly higher than MPCP5 at 24 h after aphids feeding on virus-infested tobacco. Yeast two-hybrid assays demonstrated that the protein encoded by MPCP4 gene was closely associated with the CMV CP through the direct interaction. Moreover, the ability of M. persicae to acquire CMV was suppressed by RNA interference of MPCP4. All these lines of evidence indicate that MPCP4, as a viral putative receptor in the stylet of aphid, plays an important role in aphid acquisition of CMV.

Transcriptional responses of detoxification genes to four plant allelochemicals in Aphis gossypii.

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.

Identification of microRNAs and their response to the stress of plant allelochemicals in Aphis gossypii (Hemiptera: Aphididae).

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.