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Background: Afidopyropen is a novel insecticide with high selectivity between sucking insects such as the peach aphids Myzus persicae and natural enemies like the seven-spotted lady beetle Coccinella septempunctata. However, the mechanisms of selective action for afidopyropen remain unknown., Results: The LC 50 values of afidopyropen to the 1st-4th instar larvae and adult C. septempunctata were 372- to more than 7267-fold higher than that to adult M. persicae. Though the activity of cytochrome P450s in M. persicae was 6.1- to 7.5-fold higher than that in C. septempunctata, the latter has much higher activities of carboxylesterase (CarEs) and glutathione S-transferases (GSTs), and the crude enzyme of C. septempunctata and M. persicae showed similar metabolism efficiency to afidopyropen. Molecular docking results demonstrated that afdopyropen showed higher binding affinity to the vanilloid-type transient receptor potential (TRPV) channel of M. persicae (-9.1 kcal/mol) than to that of C. septempunctata (-8.2 kcal/mol). And the EC 50 value of afdopyropen to the TRPV channel of C. septempunctata (41 360 nM) was 19 885-fold higher than that in M. persicae (2.08 nM)., Conclusions: Our results demonstrated that the significantly different sensitivity of M. persicae and C. septempunctata TRPV channel to afidopyropen play a key role in the high selectivity of afidopyropen. These findings provide new insights into the selective mechanisms of afidopyropen against insect pests and natural enemies as well as the theory support for coordinated application of chemical control and biological control. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

The cotton aphid, Aphis gossypii , is a polyphagous pest that stunts host plant growth via direct feeding or transmitting plant virus. Due to the long-term application of insecticides, A. gossypii has developed different levels of resistance to numerous insecticides. We found that five field populations had evolved multiple resistances to neonicotinoids. To explore the resistance mechanism mediated by uridine diphosphate glycosyltransferases (UGTs), two upregulated UGT genes in these five strains, UGT350C3 and UGT344L7 , were selected for functional analysis of their roles in neonicotinoid detoxification. Transgenic Drosophila bioassay results indicated that compared with the control lines, the UGT350C3 and UGT344L7 overexpression lines were more tolerant to thiamethoxam, imidacloprid, and dinotefuran. Knockdown of UGT350C3 and UGT344L7 significantly increased A. gossypii sensitivity to thiamethoxam, imidacloprid, and dinotefuran. Molecular docking analysis demonstrated that these neonicotinoids could bind to the active pockets of UGT350C3 and UGT344L7. This study provides functional evidence of neonicotinoid detoxification mediated by UGTs and will facilitate further work to identify strategies for preventing the development of neonicotinoid resistance in insects.

Myrosinase (Myr) catalyzes the hydrolysis of glucosinolates, yielding biologically active metabolites. In this study, glucoraphanin (GRA) extracted from broccoli seeds was effectively hydrolyzed using a Myr-obtained cabbage aphid ( Brevicoryne brassicae ) ( Bb Myr) to produce (R)-sulforaphane (SFN). The gene encoding Bb Myr was successfully heterologously expressed in Escherichia coli , resulting in the production of 1.6 g/L (R)-SFN, with a remarkable yield of 20.8 mg/g broccoli seeds , achieved using recombination E. coli whole-cell catalysis under optimal conditions (pH 4.5, 45 °C). Subsequently, Bb Myr underwent combinatorial simulation-driven mutagenesis, yielding a mutant, DE9 (N321D/Y426S), showing a remarkable 2.91-fold increase in the catalytic efficiency ( k cat / K M ) compared with the original enzyme. Molecular dynamics simulations demonstrated that the N321D mutation in loopA of mutant DE9 enhanced loopA stability by inducing favorable alterations in hydrogen bonds, while the Y426S mutation in loopB decreased spatial resistance. This research lays a foundation for the environmentally sustainable enzymatic (R)-SFN synthesis.

Insect trehalases have been identified as promising new targets for pest control. These key enzymes are involved in trehalose hydrolysis and plays an important role in insect growth and development. In this contribution, plant and microbial compounds, namely validamycin A, amygdalin, and phloridzin, were evaluated for their effect, through trehalase inhibition, on Acyrthosiphon pisum aphid. The latter is part of the Aphididae family, main pests as phytovirus vectors and being very harmful for crops. Validamycin A was confirmed as an excellent trehalase inhibitor with an half maximal inhibitory concentration and inhibitor constant of 2.2 × 10 -7 and 5 × 10 -8  M, respectively, with a mortality rate of ~80% on a A. pisum population. Unlike validamycin A, the insect lethal efficacy of amygdalin and phloridzin did not correspond to their trehalase inhibition, probably due to their hydrolysis by insect β-glucosidases. Our docking studies showed that none of the three compounds can bind to the trehalase active site, unlike their hydrolyzed counterparts, that is, validoxylamine A, phloretin, and prunasin. Validoxylamine A would be by far the best trehalase binder, followed by phloretin and prunasin., (© 2024 Wiley Periodicals LLC.)

Chemoenzymatic synthesis of non-natural terpenes using the promiscuous activity of terpene synthases allows for the expansion of the chemical space of terpenoids with potentially new bioactivities. In this report, we describe protocols for the preparation of a novel aphid attractant, (S)-14,15-dimethylgermacrene D, by exploiting the promiscuity of (S)-germacrene D synthase from Solidago canadensis and using an engineered biocatalytic route to convert prenols to terpenoids. The method uses a combination of five enzymes to carry out the preparation of terpenoid semiochemicals in two steps: (1) diphosphorylation of five or six carbon precursors (prenol, isoprenol and methyl-isoprenol) catalyzed by Plasmodium falciparum choline kinase and Methanocaldococcus jannaschii isopentenyl phosphate kinase to form DMADP, IDP and methyl-IDP, and (2) chain elongation and cyclization catalyzed by Geobacillus stearothermophilus (2E,6E)-farnesyl diphosphate synthase and S. canadensis (S)-germacrene D synthase to produce (S)-germacrene D and (S)-14,15-dimethylgermacrene D. Using this method, new non-natural terpenoids are readily accessible and the approach can be adopted to produce different terpene analogs and terpenoid derivatives with potential novel applications., (Copyright © 2024. Published by Elsevier Inc.)

Insect trehalases are glycoside hydrolases essential for trehalose metabolism and stress resistance. We here report the extraction and purification of Acyrthosiphon pisum soluble trehalase (ApTreh-1), its biochemical and structural characterization, as well as the determination of its kinetic properties. The protein has been purified by ammonium sulphate precipitation, first followed by an anion-exchange and then by an affinity chromatography. The SDS-PAGE shows a main band at 70 kDa containing two isoforms of ApTreh-1 (X1 and X2), identified by mass spectrometry and slightly contrasting in the C-terminal region. A phylogenetic tree, a multiple sequence alignment, as well as a modelled 3D-structure were constructed and they all reveal the ApTreh-1 similarity to other insect trehalases, i.e. the two signature motifs 179 PGGRFRELYYWDTY 192 and 479 QWDFPNAWPP 489 , a glycine-rich region 549 GGGGEY 554 , and the catalytic residues Asp336 and Glu538. The optimum enzyme activity occurs at 45 °C and pH 5.0, with K m and V max values of ~ 71 mM and ~ 126 µmol/min/mg, respectively. The present structural and functional characterization of soluble A. pisum trehalase enters the development of new strategies to control the aphids pest without significant risk for non-target organisms and human health., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

During evolution, enzymes can undergo shifts in preferred substrates or in catalytic activities. An intriguing question is how enzyme function changes following horizontal gene transfer, especially for bacterial genes that have moved to animal genomes. Some insects have acquired genes that encode enzymes for the biosynthesis of bacterial cell wall components and that appear to function to support or control their obligate endosymbiotic bacteria. In aphids, the bacterial endosymbiont Buchnera aphidicola provides essential amino acids for aphid hosts but lacks most genes for remodeling of the bacterial cell wall. The aphid genome has acquired seven genes with putative functions in cell wall metabolism that are primarily expressed in the aphid cells harboring Buchnera . In analyses of aphid homogenates, we detected peptidoglycan (PGN) muropeptides indicative of the reactions of PGN hydrolases encoded by horizontally acquired aphid genes but not by Buchnera genes. We produced one such host enzyme, Ap LdcA, and characterized its activity with both cell wall derived and synthetic PGN. Both Ap LdcA and the homologous enzyme in Escherichia coli, which functions as an l,d-carboxypeptidase in the cytoplasmic PGN recycling pathway, exhibit turnover of PGN substrates containing stem pentapeptides and cross-linkages via l,d-endopeptidase activity, consistent with a potential role in cell wall remodeling. Our results suggest that Ap LdcA derives its functions from the promiscuous activities of an ancestral LdcA enzyme, whose acquisition by the aphid genome may have enabled hosts to influence Buchnera cell wall metabolism as a means to control symbiont growth and division. IMPORTANCE Most enzymes are capable of performing biologically irrelevant side reactions. During evolution, promiscuous enzyme activities may acquire new biological roles, especially after horizontal gene transfer to new organisms. Pea aphids harbor obligate bacterial symbionts called Buchnera and encode horizontally acquired bacterial genes with putative roles in cell wall metabolism. Though Buchnera lacks cell wall endopeptidase genes, we found evidence of endopeptidase activity among peptidoglycan muropeptides purified from aphids. We characterized a multifunctional, aphid-encoded enzyme, Ap LdcA, which displays l,d-endopeptidase activities considered promiscuous for the Escherichia coli homolog, for which these activities do not contribute to its native role in peptidoglycan recycling. These results exemplify the roles of enzyme promiscuity and horizontal gene transfer in enzyme evolution and demonstrate how aphids influence symbiont cell wall metabolism.

The pea aphid Acyrthosiphon pisum hosts different facultative symbionts (FS) which provide it with various benefits, such as tolerance to heat or protection against natural enemies (e.g., fungi, parasitoid wasps). Here, we investigated whether and how the presence of certain FS could affect phenoloxidase (PO) activity, a key component of insect innate immunity, under normal and stressed conditions. For this, we used clones of A. pisum of different genetic backgrounds (LL01, YR2 and T3-8V1) lacking FS or harboring one or two (Regiella insecticola, Hamiltonella defensa, Serratia symbiotica + Rickettsiella viridis). Gene expression and proteomics analyses of the aphid hemolymph indicated that the two A. pisum POs, PPO1 and PPO2, are expressed and translated into proteins. The level of PPO genes expression as well as the amount of PPO proteins and phenoloxidase activity in the hemolymph depended on both the aphid genotype and FS species. In particular, H. defensa and R. insecticola, but not S. symbiotica + R. viridis, caused a sharp decrease in PO activity by interfering with both transcription and translation. The microinjection of different types of stressors (yeast, Escherichia coli, latex beads) in the YR2 lines hosting different symbionts affected the survival rate of aphids and, in most cases, also decreased the expression of PPO genes after 24 h. The amount and activity of PPO proteins varied according to the type of FS and stressor, without clear corresponding changes in gene expression. These data demonstrate that the presence of certain FS influences an important component of pea aphid immunity., (© 2020 Institute of Zoology, Chinese Academy of Sciences.)

The black bean aphid, Aphis fabae (Homoptera: Aphididae), is a widespread pest that has more than 200 hosts in the world. Insecticide resistance (IR) due to frequent applications is the major limitation in integrated pest management programs. Biochemical resistance is a common type of IR in which the insecticide is detoxified by one or more enzymes of the pest before reaching its target site. In this study, the IR of A. fabae populations to chlorpyrifos was evaluated in two single sprayed fields (fields A and C) and one replicated spraying field (field B) in comparison with a susceptible population (field H) during 2015. After treatments, total protein content and the activity of two detoxifying enzymes, esterases (ESTs) and glutathione S-transferases (GSTs), and acetylcholinesterase (AChE) in the populations were determined. Results clearly showed higher total protein content for the field populations compared to the susceptible population. The total protein content in field B population was significantly more than other populations. The total protein contents in Field A, B and C were 2.81, 2.89 and 1.06-fold more than susceptible strain, respectively. Higher actives of enzymes were observed in fields A, B, and C populations compared to the susceptible population (field H). The highest activity of GSTs and ESTs was observed in the field B population. Taken together, the present study demonstrated a significant IR to chlorpyrifos in the sprayed populations of A. fabae that can be attributed to the higher activity of their detoxification enzymes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cry toxins produced by Bacillus thuringiensis are well known for their high insecticidal activities against Lepidoptera, Diptera, and Coleoptera; however, their activities against Aphididae are very low. Recently, it has been reported that a Cry41-related toxin exhibited moderate activity against the aphid Myzus persicae , and thus, it is highly desirable to uncover its unique mechanism. In this paper, we report that Cathepsin B, calcium-transporting ATPase, and symbiotic bacterial-associated protein ATP-dependent-6-phosphofructokinase were pulled down from the homogenate of M. persicae as unique proteins that possibly bound to Cry41-related toxin. Cathepsin B has been reported to cleave and inactivate antiapoptotic proteins and plays a role in caspase-initiated apoptotic cascades. In this study, Cathepsin B was expressed in Escherichia coli and purified, and in vitro interaction between recombinant Cathepsin B and Cry41-related toxin was demonstrated. Interestingly, we found that addition of Cry41-related toxin obviously enhanced Cathepsin B activity. We propose a model for the mechanism of Cry41-related toxin as follows: Cry41-related toxin enters the aphid cells and enhances Cathepsin B activity, resulting in acceleration of apoptosis of aphid cells.

Histone acetylation is an evolutionarily conserved epigenetic mechanism of eukaryotic gene regulation which is tightly controlled by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In insects, life-history traits such as longevity and fecundity are severely affected by the suppression of HAT/HDAC activity, which can be achieved by RNA-mediated gene silencing or the application of chemical inhibitors. We used both experimental approaches to investigate the effect of HAT/HDAC inhibition in the pea aphid (Acyrthosiphon pisum) a model insect often used to study complex life-history traits. The silencing of HAT genes (kat6b, kat7, and kat14) promoted survival or increased the number of offspring, whereas targeting rpd3 (HDAC) reduced the number of viviparous offspring but increased the number of premature nymphs, suggesting a role in embryogenesis and eclosion. Specific chemical inhibitors of HATs/HDACs showed a remarkably severe impact on life-history traits, reducing survival, delaying development, and limiting the number of offspring. The selective inhibition of HATs and HDACs also had opposing effects on aphid body weight. The suppression of HAT/HDAC activity in aphids by RNA interference or chemical inhibition revealed similarities and differences compared to the reported role of these enzymes in other insects. Our data suggest that gene expression in A. pisum is regulated by multiple HATs/HDACs, as indicated by the fitness costs triggered by inhibitors that suppress several of these enzymes simultaneously. Targeting multiple HATs or HDACs with combined effects on gene regulation could, therefore, be a promising approach to discover novel targets for the management of aphid pests., (© 2019 Wiley Periodicals, Inc.)

To develop new potential pesticides, a series of matrine-cholesterol derivatives were prepared by modifications of two non-food bioactive products matrine and cholesterol. Two N-phenylsulfonylmatrinic esters (5i and 5j) showed the most potent insecticidal activity against Mythimna separata Walker. Two N-benzylmatrinic esters (5e and 5g) exhibited the most promising aphicidal activity against Aphis citricola Van der Goot. Especially compound 5e showed good control effects in the greenhouse against A. citricola. Some interesting results of their structure-activity relationships were also observed. By reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR) analysis of HMG-CoA reductase in apterous adults of A. citricola, it demonstrated that matrine and cholesterol may be the HMG-CoA reductase inhibitors, and the hydroxyl of cholesterol or the lactam ring of matrine may be important for acting with HMG-CoA reductase in A. citricola., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Buprofezin, a chitin synthesis inhibitor that can be used for the control of hemipteran pests, especially melon aphid, Aphis gossypii. The impact of low lethal concentrations of buprofezin on the biological parameters and expression profile of CHS1 gene were estimated for two successive generations of A. gossypii. The present result shows that the LC 15 and LC 30 of buprofezin significantly decreased the fecundity and longevity of both generations. Exposure of F 0 individuals to both concentrations delay the developmental period in F 1 . Furthermore, the survival rate, intrinsic rate of increase (r), finite rate of increase (λ), and net reproductive rate (R 0 ) were reduced significantly in progeny generation at both concentrations. However, the reduction in gross reproductive rate (GRR) was observed only at LC 30 . Although, the mean generation time (T) prolonged substantially at LC 30 . Additionally, expression of the CHS1 gene was significantly increased in F 0 adults. Significant increase in the relative abundance of CHS1 mRNA transcript was also observed at the juvenile and adult stages of F 1 generation following exposure to LC 15 and LC 30 . Therefore, our results show that buprofezin could affect the biological traits by diminishing the chitin contents owing to the inhibition of chitin synthase activity in the succeeding generation of melon aphid.

Myzus persicae is a major pest of many crops including canola and Brassica vegetables, partly because it vectors plant viruses. Previously it has been reported that double-stranded RNA delivered to aphids by injection, artificial diet or transgenic plants has knocked down target genes and caused phenotypic effects. While these studies suggest that RNA interference (RNAi) might be used to suppress aphid populations, none have shown effects sufficient for field control. The current study analyses the efficacy of dsRNA directed against previously reported gene-targets on Green peach aphid (Myzus persicae) strains. No silencing effect was observed when dsRNA was delivered in artificial diet with or without transfection reagents. dsRNA produced in planta also failed to induce significant RNAi in M. persicae. Transcriptome analyses of the midgut suggested other potential targets including the Ferritin heavy chain transcripts, but they also could not be knocked down with dsRNA. Here we show that dsRNA is rapidly degraded by midgut secretions of Myzus persicae. Analysis of the transcriptome of the M. persicae midgut revealed that an ortholog of RNases from other insects was abundant.

The alarm behavior plays a key role in the ecology of aphids, but the site and molecular mechanism for the biosynthesis of aphid alarm pheromone are largely unknown. Farnesyl diphosphate synthase (FPPS) catalyzes the synthesis of FPP, providing the precursor for the alarm pheromone (E)-β-farnesene (EβF), and we speculate that FPPS is closely associated with the biosynthetic pathway of EβF. We firstly analyzed the spatiotemporal expression of FPPS genes by using quantitative reverse transcription-polymerase chain reaction, showing that they were expressed uninterruptedly from the embryonic stage to adult stage, with an obvious increasing trend from embryo to 4th-instar in the green peach aphid Myzus persicae, but FPPS1 had an overall significantly higher expression level than FPPS2; both FPPS1 and FPPS2 exhibited the highest expression in the cornicle area. This expression pattern was verified in Acyrthosiphon pisum, suggesting that FPPS1 may play a more important role in aphids and the cornicle area is most likely the site for EβF biosynthesis. We thus conducted a quantitative measurement of EβF in M. persicae by gas chromatography-mass spectrometry. The data obtained were used to perform an association analysis with the expression data, revealing that the content of EβF per aphid was significantly correlated with the mean weight per aphid (r = 0.8534, P = 0.0307) and the expression level of FPPS1 (r = 0.9134, P = 0.0109), but not with that of FPPS2 (r = 0.4113, P = 0.4179); the concentration of EβF per milligram of aphid was not correlated with the mean weight per aphid or the expression level of FPPS genes. These data suggest that FPPS1 may play a key role in the biosynthesis of aphid alarm pheromone.

The pea aphid, Acyrthosiphon pisum, has an incomplete immune system compared to those of other insect species; some conserved components and pathways in other species are missing in its genome. As a core component of the insect immune system, prophenoloxidase (PPO) genes are retained in the pea aphid. Early studies have also shown the presence of phenoloxidase activity in specific tissues or cells in the pea aphid and suggested its involvement in response to immune challenges. In this study, we knocked down the expression of PPO genes in the pea aphid using double-stranded RNA-based interference, and quantitative PCR analysis and an enzyme activity assay confirmed our success in the PPO gene knockdown. In bacterial and fungal infection experiments, we observed that the knockdown of PPO resulted in more live bacterial cells and fungal spores in the body of the aphids and higher mortality of the aphids after infection. Our study provides evidence supporting a critical role of PPO in the defence of the pea aphid., (© 2018 The Royal Entomological Society.)

Farnesyl diphosphate synthase (FPPS) catalyzes the formation of FPP, providing the precursor for the biosynthesis of (E)-β-farnesene (EβF) in plants, but it is unknown if FPPS supplies the precursor for the biosynthesis of EβF, the major component of aphid alarm pheromone, though our previous studies support the hypothesis that EβF is synthesized by the aphid itself. Here, we used two cohorts of the green peach aphid Myzus persicae separately, reared on pepper plant and artificial diet to test the correlations among droplet emission, EβF quantity, and FPPS gene expression. It was found that the proportion of aphids emitting cornicle droplets and the quantity of EβF per milligram of aphid were both significantly different between the two cohorts, which were positively correlated with the expression of the two FPPS genes ( MpFPPS1/ 2) in M. persicae. These results were further confirmed by RNAi-mediated knockdown of MpFPPS1/ 2. Specifically, knockdown of MpFPPS1/ 2 imposed no significant cost on the survival of aphid but remarkably increased the number of offspring per aphid; most importantly, knockdown of MpFPPS1/ 2 significantly reduced the proportion of aphids emitting droplets and the quantity of EβF calculated as per the weight of aphid. Our results suggest that both FPPS genes are involved in the production of EβF in M. persicae and cornicle droplet emission is closely associated with the EβF release in the aphid., (© 2018 Wiley Periodicals, Inc.)

The bird cherry-oat aphid, Rhopalosiphum padi (L.), is an insect pest that persistently attacks wheat crops worldwide. Glutathione S-transferases (GSTs) are important detoxification enzymes that play roles in insecticide resistance. In this study, we identified two GST genes (RpGSTS1 and RpGSTS2) from R. padi. Phylogenetic analysis indicated that the genes are associated with the sigma class of insect GSTs. The RpGSTS1 and RpGSTS2 contain nine α-helices and five β-sheets connected by loops, and had 60 and 50% homology with the 3D structure of the Blattella germanica GST5. We tested the toxicity of chlorpyrifos, imidacloprid, isoprocarb, sulfoxaflor, and λ-cyhalothrin to R. padi, and found that the toxicity of five insecticides to the aphid varied. The detoxification activity of GSTs and the expression patterns of RpGSTS1 and RpGSTS2 after insecticide treatments were also analyzed. Compared to the control, the GST activity was increased by 23, 18.5, 13, and 11.5% in aphids treated by LC50 concentrations of chlorpyrifos, isoprocarb, imidacloprid, and sulfoxaflor, respectively. Exposure to different chemical insecticides showed different effects on the expression of RpGSTS1 and RpGSTS2. These results indicate that RpGSTS1 and RpGSTS2 have unique biochemical characteristics and may play roles in resistance to insecticides in R. padi.

In recent decades, man-made electric fields have greatly increased the intensity of electrostatic fields that are pervasively present in the environment. To better understand the physiological alterations exhibited by herbivorous insects in response to changing electric environments, we determined the activities of anti-oxidative enzymes and the metabolic rate of Sitobion avenae Fabricius (Hemiptera: Aphididae) over multiple generations in response to direct and host-seed exposure to a high-voltage electrostatic field (HVEF) of varying strength for different durations. Under controlled greenhouse conditions, 20-min direct exposure of S. avenae and wheat seeds to a 2- or 4-kV/cm HVEF resulted in significantly increased superoxide dismutase (SOD) activity in the sixth, 11th, 16th, and 21st generations relative to the control activities, whereas significantly decreased SOD activity was detected in the second generation. In addition, the activities of catalase (CAT) and peroxidase (POD) in S. avenae showed significant decreases over multiple generations. We also examined the suppressive effects of the duration of 4-kV/cm treatment on aphid physiology. The results showed that exposure to the 4-kV/cm HVEF for 20 min exerted adverse effects on CAT and POD activities and significantly decreased the metabolic rates of S. avenae, as demonstrated through evaluations of CO 2 production rate, and these parameters were not significantly affected by higher HVEF durations. Overall, these findings increase our understanding of plant-pest interactions under novel HVEF environments and provide information that can improve integrated management strategies for S. avenae. Bioelectromagnetics. 40:52-61, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

The bird cherry-oat aphid, Rhopalosiphum padi (L.), is a major insect pest of cereal crops in many countries. Imidacloprid has been widely used for controlling piercing-sucking insect pests worldwide, but its sublethal effects on R. padi have not been well addressed. In this study, we investigated the sublethal effects of imidacloprid on biological parameters and five enzyme activities of R. padi. The LC10, LC20, and LC25 of imidacloprid to adult aphids were 0.0053, 0.0329 and 0.0659 mg L-1, respectively. These concentrations significantly decreased pre-adult survival rate, but prolonged the development duration of 1st instar nymphs, pre-oviposition period, and adult longevity. Adult oviposition period was also extended by LC20. The intrinsic rate of increase (r), net reproductive rate (R0), and finite rate (λ) decreased at all three concentrations, whereas mean generation time (T) increased. Moreover, LC20 and LC25 significantly inhibited superoxide dismutase (SOD) activity, but increased catalase (CAT) activity. Acetylcholinesterase (AChE) activity also increased at LC20. However, cytochrome P450 enzyme and peroxidase (POD) activity did not differ between imidacloprid treatments and the control. In conclusion, the imidacloprid concentrations tested here have negative impacts on the performance of R. padi by reducing its nymphal survival, extending the development duration of some stages, decreasing the rate of population growth, and altering enzyme activities., Competing Interests: The authors have declared that no competing interests exist.

Cytochrome P450 monooxygenases represent a key detoxification mechanism in neonicotinoids resistance in Aphis gossypii Glover. Synergism analysis has indicates that P450s are involved in thiamethoxam resistance. In this study, expression changes in the transcripts of P450 genes were determined in thiamethoxam-susceptible and thiamethoxam-resistant strains. Nine P450 genes in CYP3 clade were significantly overexpressed in the resistant strain (especially CYP6CY14, which was increased 17.67-fold) compared with the susceptible strain. Transcripts of ecdysone synthesis-related P450 genes, including CYP302A1, CYP306A1, CYP307A1 and CYP315A1, were up-regulated in the resistant strain, which may accelerate molting hormone production. The ecdysone response genes (ecdysone receptor (EcR), ultra-spiracle (USP) and Broad-complex protein (Br-C)) were overexpressed in the resistant strain. RNA interference (RNAi) targeting CYP6CY14 significantly increased the sensitivity of the resistant aphid to thiamethoxam. The results of the present study indicate the possible involvement of these P450 genes in thiamethoxam resistance. Our findings may facilitate further work to validate the roles of these P450s in thiamethoxam resistance based on heterologous expression, and show that screening the expression changes in P450 genes can reveal the impact of thiamethoxam on ecdysone synthesis-related P450 genes. These results are useful for understanding the mechanism of thiamethoxam resistance and will contribute to the management of insecticide-resistant cotton aphids in China., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cytochrome P450 monooxygenases play a key role in detoxification mechanism for spirotetramat resistance in Aphis gossypii Glover. However, only one P450 genes (CYP6DA2), among thirty-five P450 genes identified from Aphis gossypii transcriptome database, has been reported to play important role in spirotetramat resistance in previous resistance level until now. In this study, after the confirmation of the rise of resistance level and important roles of P450s in spirotetramat resistance by the synergism analysis, the gene expression changes were determined for P450 genes in spirotetramat susceptible and resistant strains. Compared with the susceptible strain, CYP6CY4, CYP6CY14, CYP6CY18 and CYP6DC1 in CYP3 Clade were up-regulated in resistant nymphs, with the CYP6CY14, CYP6CY4, CYP6DC1, and CYP6CY18 increased to 2.54-, 1.51-, 1.31- and 1.29-fold, respectively. Eight genes in CYP3 Clade, three genes in CYP4 Clade and one gene in Mito Clade were down-regulated. In resistant adult aphids, CYP380C6 in CYP4 Clade, CYP353B1 in CYP2 Clade, and CYP307A1 in Mito Clade were up-regulated under spirotetramat stress, with the CYP380C6, CYP353B1 and CYP307A1 increased to 2.89-, 1.91-, and 1.38-fold, respectively. In contrast, the other P450 genes were almost down-regulated, especially these P450 genes in CYP3 Clade, CYP4 Clade and Mito Clade. RNA interference of CYP380C6 significantly increased the sensitivity of the resistant adults and nymphs to spirotetramat, while suppression of CYP6CY14 could not increase the toxicity of spirotetramat. These results indicate the possible involvement of the CYP380C6 genes in spirotetramat resistance at present very high resistance levels. Screening the expression changes of P450 genes under different spirotetramat resistance levels in the genome-scale will provide an overall view on the possible metabolic factors in the resistance development. The results may facilitate further work to validate the roles of P450 in spirotetramat resistance with heterologous expression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Laccase 1 (Lac1), a polyphenol oxidase, has been proposed to be involved in insect iron metabolism and immunity responses. However, little information is available on the roles of Lac 1 in insect-plant interactions. The grain aphid Sitobion avenae is one of the most destructive pests of cereal, directly drawing phloem sap and transmitting viruses. In the present study, we first cloned the open reading frame (ORF) of Lac 1 from S. avenae, and the putative protein sequence was predicted to have a carboxyl-terminal transmembrane domain. We found that SaLac1 had higher expression levels in the fourth and adult stages using reverse transcription real-time quantitative PCR (RT-qPCR). SaLac 1 was highly expressed in the salivary gland and midgut and also in wingless compared with winged morphs. After feeding on aphid-resistant wheat with a high total phenol content, the expression level of SaLac 1 increased significantly. RNA interference (RNAi) by oral feeding successfully inhibited the transcript levels of SaLac 1, and the knockdown of Lac 1 significantly decreased the survival rate of S. avenae on aphid-resistant wheat. Our study demonstrated that S. avenae Lac1 was involved in the detoxification of phenolic compounds in wheat and was essential for the aphid to adapt to resistant plants., (© 2018 Wiley Periodicals, Inc.)

Carboxylesterases (CarEs) play an important role in detoxifying insecticides in insects. Over-expression and structural modification of CarEs have been implicated in the development of organophosphate (OP) insecticide resistance in insects. A previous study identified four nonsynonymous mutations (resulting in four amino acid residue substitutions) in the open reading frame of the carboxylesterase gene of resistant cotton aphids compared to the omethoate susceptible strain, which has possibly influenced the development of resistance to omethoate (a systemic OP insecticide). The current study further characterized the function of these mutations, both alone and in combination, in the hydrolysis of OP insecticides. The metabolism results suggest that the combination of four mutations, mainly existing in the laboratory-selected OP-resistant cotton aphid population, increased the OP hydrolase activity (approximately twofold) at the cost of detectable carboxylesterase activity. The functional studies of single or multiple mutations suggest the positive effect of H104R, A128V and T333P on the acquisition of OP hydrolase activity, especially the combination of H104R with A128V or T333P. K484R substitution decreased both the OP hydrolase activity and the CarE activity, indicating that this mutation primarily drives the negative effect on the acquisition of OP hydrolase activity amongst these four mutations in the resistant strain. The modelling and docking results are basically consistent with the metabolic results, which strongly suggest that the structural gene modification is the molecular basis for the OP resistance in this laboratory-selected cotton aphid strain., (© 2017 The Royal Entomological Society.)

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 β-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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The cotton aphid, Aphis gossypii (Glover), is a destructive pest that affects numerous crops throughout the world. Pyrethroid resistance has become endemic in A. gossypii populations in the cotton growing areas of China due to wide- spread application of insecticides. To assess the extent of pyrethroid resistance, bioassays were conducted on field populations collected from several cotton cultivation areas from 2010 to 2015. The frequency of a known resistance-associated sodium channel mutation (M918 L) in A. gossypii was evaluated and the bioassay of bifenthrin with or without the synergist was performed to illuminate the mechanisms underlying resistance to pyrethroids., Results: The field populations exhibited very high levels of resistance to both beta-cypermethrin and deltamethrin. Pretreatment with synergists, DEF and PBO, significantly increased the toxicity of bifenthrin to cotton aphid populations collected from Bt cotton fields in China. Further, 96.8-100% of individuals with the M918 L mutation (including both RR and RS individuals) were observed in various populations, and only 2.8-3.2% of individuals with wild-type homozygotes (SS) were detected., Conclusion: The mutation M918 L in the voltage-gated sodium channel along with detoxifying metabolism was contributed to the pyrethroid resistance in the field populations of Aphis gossypii from cotton growing regions of China. And insecticides with different modes of action should be recommended for the control of A. gossypii in the future. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Acetyl-coenzyme A carboxylase (ACC) catalyses the carboxylation of acetyl-coenzyme A (acetyl-CoA) to produce malonyl-CoA during the de novo synthesis of fatty acids. Spirotetramat, an inhibitor of ACC, is widely used to control a range of sucking insects, including the Aphis gossypii. In the present study, Reverse transcription quantitative real-time PCR (RT-qPCR) results demonstrated that ACC was significantly overexpressed in a laboratory-selected spirotetramat-resistant strain compared with the susceptible strain. ACC RNA interference significantly suppressed fecundity and led to cuticle formation deficiencies in resistant adults and nymphs compared with the control. The full-length ACC gene was sequenced from both resistant and susceptible cotton aphids, and a strong association was found between spirotetramat resistance and 14 amino acid substitutions in the biotin carboxylase domain and carboxyl transferase domain of the ACC gene. Furthermore, ACC activity was higher in resistant aphids than in the susceptible strain, and ACC in the resistant aphids exhibited significant insensitivity to spirotetramat and spirotetramat-enol. The results indicate that the overexpressed insensitive (mutated) ACC target played an important role in the high levels of spirotetramat resistance observed here. This association of amino acid substitution with resistance is the first report of a potential target site mechanism affecting spirotetramat in the cotton aphid., (© 2017 The Royal Entomological Society.)

Acetylcholinesterase (AChE) is the primary target of organophosphate- and carbamate-based insecticides. We sequenced the full-length cDNAs of two AChE genes from the brown citrus aphid Aphis (Toxoptera) citricidus (Kirkaldy). These two genes, Tcace1 and Tcace2, which encode TcAChE1 and TcAChE2, respectively, had a shared amino acid identity of 29% and were highly similar to other insect ace1 and ace2 genes, respectively, having specific functional motifs. Potential differences in enzymatic function were characterized by the heterologous expression of the two genes using a baculovirus system in Sf9 insect cells. Both of the recombinant AChEs had high specific activities for three typical substrates, acetylthiocholine iodide, butyrylthiocholine iodide, and propinylthiocholine iodide. TcAChE1 had a lower Michaelis-Menten constant value and a higher maximal reaction velocity than recombinant TcAChE2, indicating a higher affinity for substrates and greater catalytic efficiency, respectively. Bioassays showed a greater sensitivity of recombinant TcAChE1 to the 10 tested insecticides. Silencing of Tcace1 and Tcace2 by RNA interference significantly increased the susceptibility of A. citricidus to malathion and carbaryl; however, silencing Tcace1 resulted in a higher mortality rate than silencing Tcace2. Additionally, the specific enzyme activity decreased more after silencing Tcace1 than after silencing Tcace2. Thus, TcAChE1 plays a major role in postsynaptic neurotransmission in A. citricidus., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The green peach aphid, Myzus persicae , is a vector of the Potato leafroll virus (PLRV, Luteoviridae), transmitted exclusively by aphids in a circulative manner. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip as compared with the weed physalis, and this was a transient effect caused by a host-switch response. A trend of higher PLRV titer in physalis-reared aphids as compared with turnip-reared aphids was observed at 24 h and 72 h after virus acquisition. The major difference in the proteomes of these aphids was the up-regulation of predicted lysosomal enzymes, in particular the cysteine protease cathepsin B (cathB), in aphids reared on turnip. The aphid midgut is the site of PLRV acquisition, and cathB and PLRV localization were starkly different in midguts of the aphids reared on the two host plants. In viruliferous aphids that were reared on turnip, there was near complete colocalization of cathB and PLRV at the cell membranes, which was not observed in physalis-reared aphids. Chemical inhibition of cathB restored the ability of aphids reared on turnip to transmit PLRV in a dose-dependent manner, showing that the increased activity of cathB and other cysteine proteases at the cell membrane indirectly decreased virus transmission by aphids. Understanding how the host plant influences virus transmission by aphids is critical for growers to manage the spread of virus among field crops., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Piperonyl butoxide (PBO) is a well-known insecticide synergist capable of interacting with phase 1 metabolic enzymes, specifically esterases and cytochrome P450s. In this study, structure-activity relationship analyses were used to characterise the interaction of around 30 analogues of PBO with the esterase FE4 and the P450 CYP6CY3 from insecticide-resistant Myzus persicae (Sulzer), in order to predict the synthesis of more potent inhibitors., Results: Enzyme inhibition studies were performed against esterase and oxidase activities and, together with in silico modelling, key activity determinants of the analogues were identified and optimised. Novel analogues were then designed and synthesised, some of which showed greater inhibition against both enzymatic systems: specifically, dihydrobenzofuran moieties containing an alkynyl side chain and a butyl side chain against FE4, and benzodioxole derivatives with a propyl/butyl side chain and an alkynyl ether moiety for CYP6CY3., Conclusions: In vitro assays identified potential candidate synergists with high inhibitory potency. The in vivo confirmation of such results will allow consideration for a possible use in agriculture. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Chemical investigation of the aerial parts of Atraphaxis frutescens resulted in the isolation of five 7-methoxyflavonols with pyrogallol B-ring moieties (1-5), a fisetinidol glucoside (13), and a benzyl glycoside (18), together with 26 known compounds including flavonoids, phenylpropanoid amides, anthraquinone glycosides, lignans, and a benzyl derivative. The principal chemical structural feature of the isolated compounds was either a pyrogallol or catechol B-ring moiety, and they showed potent 1,1-diphenyl-2-picrylhydrazyl radical scavenging activities. To assess the effects of these antioxidants on biological enzymes, their inhibitory effects against an insect phenoloxidase and a mushroom tyrosinase were evaluated. This study indicated that insect phenoloxidase was inhibited by phenylpropanoid amides and that mushroom tyrosinase was inhibited by the characteristic 7-methoxyflavonol 3-O-rhamnopyranosides.

Peroxidases (POXs) make up a large superfamily of enzymes that act in a wide range of biological mechanisms, including maintaining appropriate redox balances within cells, among other actions. In this study, we cloned a sequence that encodes a POX protein, SaPOX, from wheat aphids, Sitobion avenae. Amino acid sequence alignment showed the SaPOX sequence was conserved with POXs from other insect species. SaPOX mRNA accumulations were present in all nymphal and adult stages, at higher levels during the first and second instar, and lower during later stages in the life cycle. Ingestion of dsRNA specific to POX led to reduced SaPOX mRNA accumulation. Sitobion avenae nymphs continuously exposed to dietary dsPOX via an artificial diet led to reduced survival rate and ecdysis index. We infer that POX is important to maintain the growth and development of S. avenae., (© 2016 Wiley Periodicals, Inc.)

Myzus persicae (Sulzer) is one of the most important agricultural pests in China, which caused serious losses every year. For resistance monitoring, twelve populations of this pest were collected from tobacco field in Chongqing, China, and their sensitivities to 4 insecticides were tested. Results showed that only WL (RR=6.51) and FJ (RR=6.03) populations have developed minor resistance to imidacloprid, and the others still remained susceptible. One population (NC) has reached a high resistance level to cyhalothrin (RR=41.28), five populations showed medium level (10.36≤RR≤20.45), and the other six remained susceptible (0.39≤RR≤3.53). As regards carbosulfan, three populations have developed medium resistance, four populations showed only minor resistance, and the other five (0.81≤RR≤3.97) were still susceptible. Population SZ developed a medium level (RR=14.83) to phoxim, the other 11 were susceptible (0.29≤RR≤2.41). To analysis the potential resistance mechanism, inhibition effects of synergists and detoxifying enzyme activities were detected. The results indicated that the MFO was the most important detoxifying enzyme conferring imidacloprid resistance, and CarE was most important to cyhalothrin, carbosulfan and phoxim. Our study provided a comprehensive survey of insecticide resistance of M. persicae in Chongqing, and suggested that different counties should take corresponding management to delay the insecticide resistance development and prolong the usefulness of insecticides., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Terrestrial insects deposit a layer of hydrocarbons (HCs) as waterproofing agents on their epicuticle. The insect-specific CYP4G genes, subfamily members of P450, have been found in all insects with sequenced genomes to date. They are critical for HC biosynthesis in Drosophila; however, their functional roles in other insects including the piercing-sucking hemipterous aphids remain unknown. In this study, we presented the molecular characterization and a functional study of the CYP4G51 gene in the pea aphid, Acyrthosiphon pisum (Harris). CYP4G51 transcript was detectable across the whole life cycle of A. pisum, and was prominently expressed in the aphid head and abdominal cuticle. Up-regulation of CYP4G51 under desiccation stress was more significant in the third instar nymphs compared with the adults. Also, up-regulation of CYP4G51 was observed when the aphids fed on an artificial diet compared with those fed on the broad bean plant, and was positively correlated with a high level of cuticular HCs (CHCs). RNAi knockdown of CYP4G51 significantly reduced its expression and caused reductions in both internal and external HCs. A deficiency in CHCs resulted in aphids being more susceptible to desiccation, with increased mortality under desiccation stress. The current results confirm that CYP4G51 modulates HC biosynthesis to protect aphids from desiccation. Moreover, our data also indicate that saturated and straight-chain HCs play a major role in cuticular waterproofing in the pea aphid. A. pisum CYP4G51 could be considered as a novel RNAi target in the field of insect pest management., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Chitin synthase (CHS) is a crucial enzyme involved in the final step of the insect chitin biosynthetic pathway. In this study, we cloned the full-length cDNA sequence of a chitin synthase gene (TCiCHS) from the brown citrus aphid, Toxoptera citricida, an important citrus pest and the main vector of citrus tristeza virus worldwide. TCiCHS was expressed during the entire lifecycle and in all insect tissues examined. Expression was highest in first-second-instar nymphs, nymph-adult transitions and in the abdomen (6.7-fold higher than head). Embryos had a higher expression level than the integument. Fourth-instar nymphs were exposed to 5 and 500 mg/l concentrations of the chitin synthesis inhibitor diflubenzuron (DFB) for 48 h and had the highest mortality at the 500 mg/l concentration. The mRNA expression levels of TCiCHS were significantly enhanced upon the exposure of nymphs to both low and high DFB concentrations. Silencing of TCiCHS occurred through plant-mediated double-stranded RNA (dsRNA) feeding. Most dsRNA-fed nymphs were unable to moult to the next stage, and the expression of TCiCHS decreased 48% compared with controls. These results demonstrate that TCiCHS plays an important role in nymph to adult development, is possibly help identify molecular targets for To. citricida control., (© 2016 The Royal Entomological Society.)

The daily susceptibility rhythm of the cotton aphid, Aphis gossypii Glover, to diazinon and the corresponding changes in the activity of three xenobiotic detoxifying enzymes-cytochrome P450 monooxygenases (P450), glutathione S-transferases (GSTs), and esterases-were investigated. Bioassays were conducted to estimate the median lethal doses (LD50) of diazinon at six different zeitgeber times (ZT0, 4, 8, 12, 16, and 20) under constant light (LL) and lighting conditions of 16 h of light and 8 h of darkness (LD). The results showed that the highest susceptibility occurred at the onset of night (ZT16) and 4 h before this time point (ZT12) under the LD condition. The endogenous rhythmicity of susceptibility was ensured, as the highest susceptibility occurred at the same time under the LL condition. The circadian changes in susceptibility to diazinon were almost coincident with changes in esterase and GSTs activity, but not in P450 activity. We also found rhythmic changes in energy components of whole-body aphids, with similar patterns of circadian changes of proteins, lipids, and soluble carbohydrates, but not glycogen, under LL and LD conditions. These photoperiod conditions (LD and LL) showed different fluctuation in trends of energy resources and of course, different quantities. Our study represents the first report of circadian control of insecticide susceptibility in aphids and provides insights into more efficient control of these pests by unveiling the times of day during which aphids are more susceptible to insecticides with attention to endogenous physiological phenomena., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The aphid species Sitobion avenae and Rhopalosiphum padi are the most important pests in wheat growing regions of many countries. In this study, we investigated the sublethal effects of imidacloprid on fecundity, longevity, and enzyme activity in both aphid species by comparing 3-h exposure for one or three generations. Our results indicated that 3-h exposure to sublethal doses of imidacloprid for one generation had no discernible effect on the survival, fecundity, longevity, or enzyme activity levels of aphids. However, when pulse exposures to imidacloprid were sustained over three generations, both fecundity and longevity were significantly decreased in both S. avenae and R. padi. Interestingly, the fecundity of R. padi had almost recovered by the F5 generation, but its longevity was still deleteriously affected. These results indicated that R. padi laid eggs in shorter time lags and has a more fast resilience. The change in reproduction behavior may be a phenomenon of R. padi to compensate its early death. If this is stable for the next generation, it means that the next generation is more competitive than unexposed populations, which could be the reason underlying population outbreaks that occur after longer-term exposure to an insecticide. This laboratory-based study highlights the sublethal effects of imidacloprid on the longevity and fecundity of descendants and provides an empirical basis from which to consider management decisions for chemical control in the field.

NADPH-cytochrome P450 reductase (CPR) plays an important role in the cytochrome P450 (CYP)-mediated metabolism of endogenous and exogenous substrates. CPR has been found to be associated with insecticide metabolism and resistance in many insects. However, information regarding CPR in the bird cherry-oat aphid, Rhopalosiphum padi, is unavailable. In the current study, a full-length cDNA (2,476 bp) of CPR (RpCPR) encoding 681 amino acids was cloned from R. padi. Nucleotide sequence and deduced amino acid sequence analysis showed that RpCPR exhibits characteristics of classical CPRs and shares high identities with those of other insects, especially with the pea aphid, Acyrthosiphon pisum. The mRNA of RpCPR was expressed at all developmental stages, with the highest expression level found in the second instar and the lowest in adult. Expression levels of RpCPR in isoprocarb-resistant and imidacloprid-resistant strains were 3.74- and 3.53-fold higher, respectively, than that of a susceptible strain. RpCPR expression could also be induced by low concentrations (LC30) of isoprocarb and imidacloprid. Moreover, we sequenced the open reading frame (ORF) of RpCPR from 167 field samples collected in 11 geographical populations. Three hundred and thirty-four SNPs were detected, of which, 65 were found in more than two individuals. One hundred and ninety-four missense mutations were present in the amino acid sequence, of which, the P484S mutant had an allele frequency of 35.1%. The present results suggest that RpCPR may play an important role in the P450-mediated insecticide resistance of R. padi to isoprocarb and imidacloprid and possibly other insecticides. Meanwhile, RpCPRmaintains high genetic diversity in natural individuals, which provides the possibility of studying potential correlations between variants and certain special physiological characters.

Background: Myzus persicae nicotianae is an important pest in Greece, controlled mainly by neonicotinoids. Monitoring of the aphid populations for resistance mechanisms is essential for effective control., Results: Two new RFLP-based diagnostics for the detection of the M918T (super-kdr pyrethroid resistance) and nAChR R81T (neonicotinoid resistance) mutations were applied, along with other established assays, on 131 nicotianae multilocus genotypes (MLGs) collected from tobacco and peach in Greece in 2012-2013. Furthermore, we present resistance data from aphid clones (>500, mainly nicotianae) collected in 2006-2007. About half of the clones tested with a diagnostic dose of imidacloprid were tolerant. The R81T mutation was not found in the 131 MLGs and 152 clones examined. Over half (58.6%) of a subset of 29 clones showed a 9-36-fold overexpression of CYP6CY3. M918T was found at low to moderate frequencies. The kdr and MACE mechanisms and carboxylesterase-based resistance were found at high frequency in all years., Conclusion: The aphid retains costly resistance mechanisms even in the absence of pressure from certain insecticides, which could be attributed to factors related to climate and genetic properties of the populations. The indication of build-up of resistance/tolerance to neonicotinoids, related to CYP6CY3 overexpression, is a matter of concern. © 2015 Society of Chemical Industry., (© 2015 Society of Chemical Industry.)

The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is an important cosmopolitan and extremely polyphagous species capable of causing direct and indirect damage to various crops. Insecticide resistance in melon aphids is of particular concern. To determine the basis of resistance, organophosphate (OP)-resistant strains of A. gossypii were obtained by continuous selection with dimethoate in the laboratory, and resistance mechanisms were investigated along with susceptible strains. Three resistant strains LKR-1, LKR-2, and LKR-3 exhibiting 270-, 243-, and 210-fold resistance obtained after 30 generations of selection with dimethoate, respectively, were utilized in this study. The role of acetylcholinesterase (AChE), a target enzyme for OPs and carbamates (CMs), was investigated. AChE enzyme assay revealed that there was no significant change in the activities of AChE in resistant and susceptible strains. However, AChE inhibitory assay showed that 50% of the enzyme activity in resistant strains was inhibited at significantly higher concentration of dimethoate (131.87, 158.65, and 99.29 µmolL(−1)) as compared with susceptible strains (1.75 and 2.01 µmolL(−1)), indicating AChE insensitivity owing to altered AChE. Molecular diagnostic tool polymerase chain reaction-restriction fragment length polymorphism revealed the existence of two consistent non-synonymous point mutations, single-nucleotide polymorphism, viz., A302S (equivalent to A201 in Torpedo californica Ayres) and S431F (equivalent to F331 in T. californica), in the AChE gene Ace2 of resistant strains. Further, cloning and sequencing of a partial fragment of Ace2 (897 bp) gene from susceptible and resistant strains revealed an additional novel mutation G221A in resistant strains, LKR-1 and LKR-2. Susceptible Ace2 genes shared 99.6 and 98.9% identity at the nucleic acid and amino acid levels with resistant ones, respectively. Functional analysis of these point mutations was assessed by in silico docking studies using the modeled wild-type and naturally mutated AChE2. Computational analysis showed that the conformational changes in AChE2 active site due to structural gene substitutions (A302S, S431F, and G221A) significantly reduced the level of ligand (OP-dimethoate, omethoate, and CM-pirimicarb) binding, suggesting that they are potentially associated with resistance development. These results unambiguously suggested that multiple mutations located in the enzyme active site are responsible for AChE insensitivity to dimethoate and are likely the molecular basis for dimethoate resistance in these selected field populations of A. gossypii.

Endosymbionts can fundamentally alter host physiology. Whether such changes are beneficial or detrimental to one or both partners may depend on the dynamics of the symbiotic relationship. Here we investigate the relationship between facultative symbionts and host immune responses. The pea aphid, Acyrthosiphon pisum, maintains an obligate primary symbiont, but may also harbour one or more facultative, secondary symbionts. Given their more transient nature and relatively recent adoption of a symbiotic lifestyle compared to primary symbionts, secondary symbionts may present a challenge for the host immune system. We assessed the response of several key components of the cellular immune system (phenoloxidase activity, encapsulation, immune cell counts) in the presence of alternative secondary symbionts, investigating the role of host and secondary symbiont genotype in specific responses. There was no effect of secondary symbiont presence on the phenoloxidase response, but we found variation in the encapsulation response and in immune cell counts based largely on the secondary symbiont. Host genotype was less influential in determining immunity outcomes. Our results highlight the importance of secondary symbionts in shaping host immunity. Understanding the complex physiological responses that can be propagated by host-symbiont associations has important consequences for host ecology, including symbiont and pathogen transmission dynamics., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study aims to explore the aphicidal activity and underlying mechanism of Illicium verum Hook. f. that is used as both food and medicine. The contact toxicity of the extracts from I. verum fruit with methyl alcohol (MA), ethyl acetate (EA), and petroleum ether (PE) against Myzus persicae (Sulzer), and the activities of acetylcholinesterase (AChE) and glutathione S-transferases (GSTs) of M. persicae after contact treatment were tested. The results showed that MA, EA, and PE extracts of 1.000 mg/l caused, respectively, M. persicae mortalities of 68.93%, 89.95% and 74.46%, and the LC50 of MA, EA, and PE extracts were 0.31, 0.14 and 0.27 mg/l at 72 h after treatment, respectively; the activities of AChE and GSTs in M. persicae were obviously inhibited by the three extracts, as compared with the control, with strong dose and time-dependent effects, the inhibition rates on the whole reached more than 50.00% at the concentration of 1.000 mg/l at 72 h after treatment. The inhibition of the extracts on AChE and GSTs activities (EA extract > PE extract > MA extract) were correlated with theirs contact toxic effects, so it is inferred that the decline of the metabolic enzymes activities may be one of important reasons of M. persicae death. The study results suggested that I. verum extracts have potential as a eco-friendly biopesticide in integrated pest management against M. persicae., (© The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America.)

A laboratory-selected spirotetramat-resistant strain (SR) of cotton aphid developed 579-fold and 15-fold resistance to spirotetramat in adult aphids and 3rd instar nymphs, respectively, compared with a susceptible strain (SS) [26]. The SR strain developed high-level cross-resistance to alpha-cypermethrin and bifenthrin and very low or no cross-resistance to the other tested insecticides. Synergist piperonyl butoxide (PBO) dramatically increased the toxicity of spirotetramat and alpha-cypermethrin in the resistant strain. RT-qPCR results demonstrated that the transcriptional levels of CYP6A2 increased significantly in the SR strain compared with the SS strain, which was consistent with the transcriptome results [30]. The depletion of CYP6A2 transcripts by RNAi also significantly increased the sensitivity of the resistant aphid to spirotetramat and alpha-cypermethrin. These results indicate the possible involvement of CYP6A2 in spirotetramat resistance and alpha-cypermethrin cross-resistance in the cotton aphid. These together with other cross-resistance results have implications for the successful implementation of resistance management strategies for Aphis gossypii., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Carboxylesterases are mainly involved in the mediation of metabolic resistance of many insects to organophosphate (OP) insecticides. Carboxylesterases underwent two divergent evolutionary events: (1) quantitative mechanism characterized by the overproduction of carboxylesterase protein; and (2) qualitative mechanism caused by changes in enzymatic properties because of mutation from glycine/alanine to aspartate at the 151 site (G/A151D) or from tryptophan to leucine at the 271 site (W271L), following the numbering of Drosophila melanogaster AChE. Qualitative mechanism has been observed in few species. However, whether this carboxylesterase mutation mechanism is prevalent in insects remains unclear. In this study, wild-type, G/A151D and W271L mutant carboxylesterases from Culex pipiens and Aphis gossypii were subjected to germline transformation and then transferred to D. melanogaster. These germlines were ubiquitously expressed as induced by tub-Gal4. In carboxylesterase activity assay, the introduced mutant carboxylesterase did not enhance the overall carboxylesterase activity of flies. This result indicated that G/A151D or W271L mutation disrupted the original activities of the enzyme. Less than 1.5-fold OP resistance was only observed in flies expressing A. gossypii mutant carboxylesterases compared with those expressing A. gossypii wild-type carboxylesterase. However, transgenic flies universally showed low resistance to OP insecticides compared with non-transgenic flies. The flies expressing A. gossypii W271L mutant esterase exhibited 1.5-fold resistance to deltamethrin, a pyrethroid insecticide compared with non-transgenic flies. The present transgenic Drosophila system potentially showed that a quantitative increase in carboxylesterases induced broader resistance of insects to insecticides than a qualitative change., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A resistant strain of the Aphis glycines Matsumura (CRR) has developed 76.67-fold resistance to lambda-cyhalothrin compared with the susceptible (CSS) strain. Synergists piperonyl butoxide (PBO), S,S,S-Tributyltrithiophosphate (DEF) and triphenyl phosphate (TPP) dramatically increased the toxicity of lambda-cyhalothrin to the resistant strain. Bioassay results indicated that the CRR strain had developed high levels of cross-resistance to chlorpyrifos (11.66-fold), acephate (8.20-fold), cypermethrin (53.24-fold), esfenvalerate (13.83-fold), cyfluthrin (9.64-fold), carbofuran (14.60-fold), methomyl (9.32-fold) and bifenthrin (4.81-fold), but did not have cross-resistance to chlorfenapyr, imidacloprid, diafenthiuron, abamectin. The transcriptional levels of CYP6A2-like, CYP6A14-like and cytochrome b-c1 complex subunit 9-like increased significantly in the resistant strain than that in the susceptible. Similar trend were observed in the transcripts and DNA copy number of CarE and E4 esterase. Overall, these results demonstrate that increased esterase hydrolysis activity, combined with elevated cytochrome P450 monooxygenase detoxicatication, plays an important role in the high levels of lambda-cyhalothrin resistance and can cause cross-resistance to other insecticides in the CRR strain., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Sieve tubes serve as transport conduits for photo-assimilates and other resources in angiosperms and are profitable targets for piercing-sucking insects such as aphids. Sieve-tube sap also contains significant amounts of proteins with diverse functions, for example in signalling, metabolism, and defence. The identification of salivary proteases in Acyrthosiphon pisum led to the hypothesis that aphids might be able to digest these proteins and by doing so suppress plant defence and access additional nitrogen sources. Here, the scarce knowledge of proteases in aphid saliva is briefly reviewed. In order to provide a better platform for discussion, we conducted a few tests on in vitro protease activity and degradation of sieve-tube sap proteins of Cucurbita maxima by watery saliva. Inhibition of protein degradation by EDTA indicates the presence of different types of proteases (e.g. metalloproteses) in saliva of A. pisum. Proteases in the watery saliva from Macrosiphum euphorbiae and A. pisum were able to degrade the most abundant phloem protein, which is phloem protein 1. Our results provide support for the breakdown of sieve-element proteins by aphid saliva in order to suppress/neutralize the defence responses of the plant and to make proteins of sieve-tube sap accessible as a nitrogen source, as is discussed in detail. Finally, we discuss whether glycosylation of sieve-element proteins and the presence of protease inhibitors may confer partial protection against the proteolytic activity of aphid saliva., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Acetyl-CoA carboxylase (ACC) catalyzes the committed and rate-limiting step in fatty acid biosynthesis. The two partial reactions, carboxylation of biotin followed by carboxyl transfer to the acceptor acetyl-CoA, are performed by two separate domains in animal ACCs. The cyclic keto-enol insecticides and acaricides have been proposed to inhibit insect ACCs. In this communication, we show that the enol derivative of the cylic keto-enol insecticide spirotetramat inhibited ACCs partially purified from the insect species Myzus persicae and Spodoptera frugiperda, as well as the spider mite (Tetranychus urticae) ACC which was expressed in insect cells using a recombinant baculovirus. Steady-state kinetic analysis revealed competitive inhibition with respect to the carboxyl acceptor, acetyl-CoA, indicating that spirotetramat-enol bound to the carboxyltransferase domain of ACC. Interestingly, inhibition with respect to the biotin carboxylase substrate ATP was uncompetitive. Amino acid residues in the carboxyltransferase domains of plant ACCs are important for binding of established herbicidal inhibitors. Mutating the spider mite ACC at the homologous positions, for example L1736 to either isoleucine or alanine, and A1739 to either valine or serine, did not affect the inhibition of the spider mite ACC by spirotetramat-enol. These results indicated different binding modes of the keto-enols and the herbicidal chemical families., (Copyright © 2014 Elsevier Ltd. All rights reserved.)