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The cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is an important insect pest of cotton crops worldwide. The objectives of this study were to determine the acute toxicity of afidopyropen and whether there are any effects of sublethal concentrations of afidopyropen on the biological characteristics of A. gossypii. The results showed that afidopyropen possesses high acute toxicity to adult A. gossypii with a 72 h LC50 value of 1.062 mg L–1. A sublethal concentration (LC10) of afidopyropen significantly decreased adult longevity, fecundity and oviposition days of female adults in both F0 and F1 generations. The total pre-adult survival of F1 progeny was also significantly reduced by 30% at the LC10 of afidopyropen. In addition, the nymph developmental time, pre-adult period, adult pre-reproductive period (APRP), and total pre-reproductive period (TPRP) of the F1 progeny were significantly prolonged compared with the control. Several population parameters, including the net reproductive rate (R0), intrinsic rate of increase (r) and finite rate of increase (λ) of F1 progeny were significantly decreased by a sublethal afidopyropen concentration exposure. These results indicated that sublethal concentration of afidopyropen can significantly suppress A. gossypii population growth. It would be useful for assessing the overall effects of afidopyropen on A. gossypii.

Carboxylesterase (CarE) was considered as important phase-I detoxifying enzymes which participated in detoxification of different types of insecticides. Up-regulation of CarE genes has been proved playing a major role in insecticide resistance in many pest insects, but its involvement in resistance to insecticides in Plutella xylostella has been rarely reported. In this study, a CarE cDNA named PxαE8 was identified in P. xylostella, which has an open reading frame of 1 599 nucleotides and putatively encodes 532 amino acids. The investigation of spatial expression profiles of PxαE8 revealed that it was expressed in all developmental stages, especially in larvae and adults. The body part/tissue-specific expression profiles showed that the PxαE8 mainly expressed in fat body, malpighian tubule and hemolymph of larvae. Further, the relative expression of PxαE8 in two multi-resistant field populations, Hainan (HN) and Guangdong (GD) populations, was found 24.4- and 15.5-fold higher than that in susceptible population, respectively. Knockdown of PxαE8 by RNA interference dramatically increased the mortalities of larvae of HN population treated with LC50 of beta-cypermethrin and phoxim by 25.3 and 18.3%, respectively. These results suggested that up-regulation of PxαE8 was involved in resistance to both beta-cypermethrin and phoxim in P. xylostella, which shed light on further understanding of molecular mechanisms of multi-insecticide-resistance in P. xylostella and other pest insects.

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.

The fall armyworm (FAW), Spodoptera frugiperda, is a newly invasive, widespread agricultural pest in China. Understanding the suitability of the main field crops in Chinese agricultural system as host for this polyphagous herbivore is especially important for making control strategy. Here, after FAWs were fed three important oil-bearing crops (oilseed rape, soybean and sunflower) planted in China and resultant population parameters were compared using the age-stage, two-sex life table method, survival of larvae on soybean was significantly lower than that on oilseed rape and sunflower. Developmental duration of larvae on soybean was also the longest (23.3 days). The highest pupation rate was recorded on sunflower. The highest pupal mass (0.19 g) was attained on oilseed rape, significantly higher than on the other host plants; the lowest mass was on soybean (0.15 g). On soybean, oilseed rape and sunflower, respectively, the average generation period was 42.21, 39.10 and 40.44 d; the intrinsic rate of increase (r) was 0.0844, 0.1041 and 0.1134; the finite rate of increase (λ) was 1.0881, 1.1098 and 1.1202. While the most suitable host plant overall was sunflower, S. frugiperda completed development and increased its population on all three host plants. Thus, soybean, oilseed rape and sunflower were all suitable for FAW, and population monitoring and management of FAW in these crops should be increased.

Spodoptera frugiperda, the pest fall armyworm (FAW), is widespread in more than 100 countries. To date, planting insect-resistant transgenic crops is one of the main control methods in its native countries. In this study we evaluated Bt-transgenic maize (Bt maize) and non-transgenic (conventional) maize and six other host plants in greenhouse pot experiments and field trials for oviposition preference by the Chinese populations of FAW. In laboratory trials, female moths preferred to oviposit on maize with no significant preference between conventional and Bt maize. However, after conventional and transgenic maize were exposed to FAW larvae and damaged, oviposition was significantly higher on transgenic maize than on the conventional one. Field trials showed that for plants less damaged at an early stage (seedling stage), oviposition of FAW on transgenic and conventional maize was significantly higher than that on wheat, sorghum, foxtail millet, peanut and soybean while showing no significant difference between transgenic or conventional maize. FAW adults mainly laid eggs on Bt maize, while the larval density and leaf damage rating or percentage of damaged plants were significantly lower than on conventional maize. Larval density and its damage on conventional maize were significantly higher than that on Bt maize and the other five hosts. Thus, maize is a highly preferred and suitable host for S. frugiperda feeding and ovipositing, and Bt maize can be used as trap crop to protect other crops.

Floral resources, such as carbohydrate-rich nectar or pollen, can bolster fitness and raise reproductive output of adult lepidopterans. Here, we used laboratory experiments to assess how those plant-derived foods impact adult fecundity, reproductive physiology and flight performance of an invasive strain of the fall armyworm, Spodoptera frugiperda (FAW; Lepidoptera: Noctuidae) in China. More specifically, supplementary feeding on bee pollen and honey enhanced FAW flight duration, testis size, ovarian development, longevity and adult fecundity. FAW adults attained the longest pre-oviposition (10.8 days) and oviposition period (6.8 days) and longevity (19.2 days) on 5% acacia honey. Upon access to 2.5% acacia honey and 2.5‰ pine pollen, S. frugiperda attained the highest mating rate (79.7%), fecundity (644.9 eggs/female) and egg hatching rate (82.3%). Feeding on honey further delayed decay of male testes, while ovarian development was enhanced when female moths were allowed access to 2.5% honey and 2.5‰ pine pollen. Upon feeding on 5% honey solution, S. frugiperda engaged in flight over the longest duration (9.5 h), distance (29.9 km) and speed (3.1 km h−1). Honey had a comparatively greater effect on the above parameters than pollen. Our findings help decipher FAW invasion patterns and population dynamics, facilitate the development of nutritional attractants, and contribute to integrated pest management of this newly-invasive pest in eastern Asia.

Sitobion avenae (F.) and Rhopalosiphum padi (L.) are two important pests of wheat in China. They typically coexist in fields during the late period of wheat growth. Sulfoxaflor is a novel sulfoximine insecticide that demonstrates broad-spectrum efficacy, especially in targeting sap-feeding insects. This study was carried out to investigate the sublethal effects of sulfoxaflor on the development, longevity, and reproduction of two species of wheat aphids. Our results showed that sublethal concentrations of sulfoxaflor did not cause significant effects on the fecundity or the longevity of the parent generation (F0 generation) of either S. avenae or R. padi. However, it caused transgenerational sublethal effects. For S. avenae, adult longevity of F1 generation was significantly decreased. No significant differences were observed on the population parameters of S. avenae in the F1 generation. For R. padi, the adult preoviposition period (APOP) and the total preoviposition period (TPOP) of F1 generation were significantly reduced. The mean generation time (T) was significantly reduced in the R. padi F1 generation. What's more, the intrinsic rate of increase (rm) and the finite rate of increase (λ) were significantly increased in the R. padi F1 generation. Taken together, these results suggest that exposure to the LC25 of sulfoxafl had no effects on the parent generation of S. avenae or R. padi, but it reduced adult longevity of S. avenae as a negative effect and increased the rm and λ of R. padi in the first progeny generation, which may have an impact on the population dynamics of R. padi.

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.

In order to precisely assess gene expression level, a suitable internal reference gene must be chosen to quantify real-time reverse transcription polymerase chain reaction (RT-qPCR) data. For greenbug, Schizaphis graminum, a suitable reference gene for assessing the level of transcriptional expression of target genes has yet to be explored. In our study, eight reference genes, elongation fator 1 beta (Ef1β), TATA box binding protein (TBP), alpha-tubulin (α-TUB), 18S ribosomal (18S), 28S ribosomal (28S), glyceraldehyde-3-phosphate (GAPDH), actin (ACT), and ribosomal protein L18 (RPL18) were evaluated in S. graminum at different developmental stages, tissues, and insecticide treatments. To further explore whether these genes are suitable to serve as internal control, three software-based approaches (geNorm, BestKeeper, and NormFinder), ΔCt method, and one web-based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized gene expression data of three target genes, heat shock protein gene (HSP70), cytocrome P450 gene (SgraCYP18A1), and glutathione S-transferase (GST). We found that the most suitable reference genes varied considerably under different experimental conditions. For developmental stages, α-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACT were suitable reference genes; for insecticide treatments, 28S and α-TUB were suitable for normalizations of expression data. In addition, 28S and α-TUB were the suitable reference gene as they had the most stable expression among different developmental stages, tissues and insecticide treatments. This should be useful for the selection of the suitable reference genes to obtain reliable RT-qPCR data in the gene expression of S. graminum.

Insects have developed a good adaptive mechanism in response to environmental stresses in the long-term evolution. They have developed a helpful metabolism system to resist plant allelochemicals. Insects also harbor different kinds of symbiotic bacteria, which provide them a competitive advantage. Here, using cotton aphid as an example, we investigated the effects of four plant allelochemicals on the differential expression of symbiotic bacteria based on transcriptome data. We also studied the composition of symbiotic bacteria and function on pathway level in three kinds of aphids. We found that the bacteria have a significant role in resisting the plant allelochemicals stress and host plant selection by aphids. These results should be useful to investigate the environmental adaption mechanism of aphids in the view of symbiotic bacteria. These results would offer a new insight for improving strategy of aphids and developing new pest control systems.

Aphids are considered as one of the key pests for wheat production worldwide. Major aphid species that infest wheat in China include Sitobion avenae, Rhopalosiphum padi, Schizaphis graminum and Metopolophium dirhodum. However, during our wheat field survey in Wenshang County of Shangdong Province, China, we observed that Aphis gossypii can feed on wheat. The damage risk of A. gossypii on wheat was assessed using host shift method. A population of A. gossypii collected from a wheat field in 2015 and another population reared on cotton under laboratory conditions for a decade without exposure to insecticides were used in the study. The results of host shift demonstrated that the A. gossypii colony from wheat has not yet developed wheat specialization. Moreover, the assessment of A. gossypii fitness on wheat and cotton showed that fecundity and net reproductive rate of A. gossypii population fed on wheat was significantly higher comparing to the population fed on cotton, whether the initial host of A. gossypii population was wheat or cotton. This study raises a warning that the cotton aphid has potential to establish well on wheat and it may cause significant effects under specific circumstances. Therefore, future studies are required to evaluate the effects of A. gossypii on wheat production.

Carmine spider mites (Tetranychus cinnabarinus) and cotton aphids (Aphis gossypii) are both serious pests of cotton, and cause reductions in yields of this key agricultural crop. In order to gain insights into how plant defense responses induced by one herbivore species affect the behavior and performance of another, we examined how infestation with T. cinnabarinus influences the development of A. gossypii using cotton as a model. In this study, we measured the activities of several important biochemical markers and secondary metabolites in the leaves of cotton seedlings responding to infestation by T. cinnabarinus. Furthermore, the influences of T. cinnabarinus infestation on the development of A. gossypii in cotton were also examined. Our data showed that the activities of several key defense enzymes, including phenylalanine ammonia-lyase (PAL), peroxidase (POD), lipoxygenase (LOX), and polyphenol oxidase (PPO), were substantially increased in cotton seedlings responding to spider mite infestation. Further, the contents of gossypol and condensed tannins, key defensive compounds, were significantly enhanced in leaves of cotton seedlings following T. cinnabarinus infestation. Moreover, the T. cinnabarinus-induced production of defense enzymes and secondary metabolites was correlated with infestation density. The developmental periods of A. gossypii on cotton seedling leaves infested with T. cinnabarinus at densities of 10 and 15 individuals cm−2 were 1.16 and 1.18 times that of control, respectively. Meanwhile, the mean relative growth rates of A. gossypii on cotton leaves infested with T. cinnabarinus at densities of 8, 10 and 15 individuals cm−2 were significantly reduced. Therefore, these data suggested that the developmental periods of A. gossypii were significantly lengthened and the mean relative growth rates were markedly reduced when cotton aphids were reared on plants infested with high densities of spider mites. This research sheds light on the role that inducible defense responses played in plant-mediated interspecific interactions between T. cinnabarinus and A. gossypii.

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.

Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a full-length cDNA of carboxylesterase pxCCE016b was cloned and exogenously expressed in Escherichia coli at the first time, which contained a 1693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of the pxCCE016b was significantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore, pxCCE016b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1 293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfully with prokaryotic cell. Western blot analysis showed that it was coded by pxCCE016b. All the above findings provide important information for further functional study, although we are uncertainty whether the pxCCE016b gene is actually involved in chlorantraniliprole resistance.

The red imported fire ant (Solenopsis invicta) is a global major invasive pest, and has caused significant economic, social and environmental impacts since its invasion to mainland of China in 2004. To date, chemical control has been the most effective measure. However, the long-term use of chemicals would lead to an unexpected rebound. To understand the risks and explore the mechanisms of detoxification or induction to insecticides in S. invicta, the O-demethylase activity and expression of cytochrome P450 genes of workers and queens, and the effects of chlorpyrifos and fipronil exposure in workers were investigated. Biochemical assays showed the O-demethylase activity of cytochrome P450 was significantly higher in workers than in queens (1.66-fold), and was significantly induced in workers exposed to chlorpyrifos and fipronil, reaching a maximum (3.00- and 1.95-fold) at 48 h and then decreasing dramatically compared to controls (exposed to acetone counter-part). The relative expression levels of 12 cytochrome P450 expressed sequence tags (ESTs) in workers were significantly higher than in queens (from 2.3- to 36.4-fold). Multiple cytochrome P450 genes (except 9E4) were co-up-regulated (from 1.5- to 2.86-fold) in workers exposed to fipronil. These results indicated that the increased O-demethylase activity may result from the increased transcription levels of cytochrome P450 related to detoxification of insecticides in S. invicta. It appears that cytochrome P450 plays an important role in enhanced metabolic detoxification of insecticides. At the same time, it also provides the theoretical basis for resistance management and rational usage of insecticides to control S. invicta.

Differential effects of methamidophos and three pyrethroids on ATPase activity and membrane fluidity of mitochondria were investigated between the wolf spider (Pirata subpiraticus (Boes. et Str.)) and the rice stem borer (Chilo suppressalis (Walker)). Based on a comparison of LD50 values, the toxicities of the tested insecticides were higher to the wolf spider than to the rice stem borer. Cyhalothrin at 1×10−4 mmol L−1 caused inhibition of the mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities, and it's inhibitions on Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities were significantly higher in the wolf spider (44 and 28%) than in the rice stem borer (19 and 11%). Methamidophos at 1×10−4 mmol L−1 decreased Ca2+-Mg2+-ATPase activity by 16 and 27% in the wolf spider and the rice stem borer, respectively, but no significant effect on the specific activity of Na+-K+-ATPase was observed. The DPH (1,6-diphenyl-1,3,5-hexatriene) fluorescence polarization values of mitochondrial membranes were not significantly affected by methamidophos in either species. However, cyhalothrin and alpha-cypermethrin induced the values of DPH polarization of mitochondrial membrane increasing with the concentration of cyhalothrin and alpha-cypermethrin from 20 to 100 μmol L−1 in the rice stem borer and the wolf spider. Effect of ethofenprox on fluidity of the wolf spider and the rice stem borer was contrary. These results suggest that both inhibition of membrane ATPase and changes of membrane fluidity could be appended to the action mechanisms of pyrethroid insecticides.

Carbohydrate chains are the principal antigens by which Bacillus thuringiensis (Bt) identify receptor proteins. The interaction between the antigen and Bt causes a pore in the membrane of midgut epithelial cells of insects. Receptor proteins, such as aminopeptidase N and alkaline phosphatase, are glycoproteins. Cadherin is another cell surface receptor protein which has potential glycosylation sites. Glycosyltransferase is very important for the synthesis and modification of receptor proteins. It can indirectly influence the function of Bt. The 1 950 bp full-length cDNA encoding β-1,3-galactosyltransferase was cloned from the the midgut of Helicoverpa armigera by degenerative PCR combined with RACE techniques (GAL-Harm, GenBank accession no.: GQ904195.1) with two potential N-glycosylation sites (157NNTI160 and 272NKTL275). Protein sequence alignments revealed that H. armigera β-1,3-galactosyltransferase shared high identity with β-1,3-galactosyltransferase in other insect species. The expression level of the β-1,3-galactosyltransferase gene in Cry1Ac-resistant H. armigera larvae was 9.2-fold higher than that in susceptible strain. The function of β-1,3-galactosyltransferase was investigated using RNAi technique. The result showed Cry1Ac enhanced the toxicity against the siRNA-treated larvae compared with non-siRNA-treated ones, which indicated β-1,3-galactosyltransferase played an important role for the insecticidal toxicity of Cry1Ac in H. armigera.

The insecticide chlorantraniliprole exhibits good efficacy and plays an important role in controlling the diamondback moth, Plutella xylostella Linnaeus. However, resistance to chlorantraniliprole has been observed recently in some field populations. At present study, diamondback moths with resistance to chlorantraniliprole (resistant ratio (RR) was 82.18) for biochemical assays were selected. The assays were performed to determine potential resistance mechanisms. The results showed that the selected resistant moths (GDLZ-R) and susceptible moth could be synergized by known metabolic inhibitors such as piperonyl butoxide (PBO), triphenyl phosphate (TPP) and diethyl-maleate (DEM) at different levels (1.68–5.50-fold and 2.20–2.89-fold, respectively), and DEM showed the maximum synergism in both strains. In enzymes assays, a high level of glutathione-S-transferase (GST) was observed in the resistant moth, in contrast, moths that are susceptible to the insecticide had only 1/3 the GST activity of the resistant moths. The analysis of short-term exposure of chlorantraniliprole on biochemical response in the resistant strain also showed that GST activity was significantly elevated after exposure to a sub-lethal concentration of chlorantraniliprole (about 1/3 LC50, 12 mg L−1) 12 and 24 h, respectively. The results show that there is a strong correlation between the enzyme activity and resistance, and GST is likely the main detoxification mechanism responsible for resistance to chlorantraniliprole in P. xylostella L., cytochrome P450 monooxygenase (P450) and carboxy-lesterase (CarE) are involved in to some extent.

Cry toxins produced by Bacillus thuringiensis (Bt) are effective biological insecticides against certain insect species. However, there are potential risks of the evolved resistance of insects to Cry toxin owing to decreased binding of toxins to target sites in the brush border membranes of the larva midgut. The Cry toxins with different binding sites in the larval midgut have been considered to be a good combination to deploy in delaying resistance evolution. Bioassay results demonstrated that the toxicity of different Cry toxins ranked differently for each species. The toxicity ranking was Cry1Ac>Cry1Ab>Cry2Ab for Helicoverpa armigera, Cry1B>Cry1C>Cry2Ab for Spodoptera exigua, and Cry2Ab>Cry1B> Cry1C for S. litura. Only Cry2Ab was toxic to Agrotis ipsilon. Binding experiments were performed with 125I-Cry1Ab, 125I-Cry1Ac, 125I-Cry1B, 125I-Cry1C, 125I-Cry2Ab and the brush border membranes vesicles (BBMV) from H. armigera, S. exigua, S. litura and A. ipsilon. The binding of Cry1Ab and Cry1Ac was shown to be saturable by incubating with increasing concentrations of H. armigera BBMV (Kd=(45.00±2.01) nmol L−1 and (12.80±0.18) nmol L−1, respectively; Bmax=(54.95±1.79) ng and (55.44±0.91) ng, separately). The binding of Cry1B was shown to be saturable by incubating with increasing concentrations of S. exigua BBMV (Kd=(23.26±1.66) nmol L−1; Bmax=(65.37±1.87) ng). The binding of 125I-Cry toxins was shown to be non-saturable by incubating with increasing concentrations of S. litura and A. ipsilon BBMV. In contrast, Cry1B and Cry1C showed some combination with the BBMV of S. litura, and a certain amount of Cry2Ab could bind to the BBMV of A. ipsilon. These observations suggest that a future strategy could be devised for the focused combination of specific cry genes in transgenic crops to control target pests, widen the spectrum of insecticide effectiveness and postpone insect resistance evolution.

The 7-ethoxycoumarin O-deethylase (ECOD) activities of cytochrome P450s and differential expression of six cytochrome P450 genes induced by the volatiles from both damaged and undamaged maize plants were investigated in the cotton bollworm, Helicoverpa armigera (Hübner). The ECOD activity changed with time of exposure to maize volatiles. At 36 h after cotton bollworm larvae exposure to maize volatiles, the ECOD activities in cotton bollworm damaged and artificially damaged groups were 2.36 and 4.53 times higher than the control group respectively. The relative expression levels of CYP4S1, CYP6B2 and CYP6B7 in the cotton bollworm were significantly increased in artificially damaged plant group, which was 2.93, 5.09 and 10.66 times higher than that in the control group, respectively. The expression levels of CYP6B2, CYP6B6, CYP9A12, and CYP9A14 were much lower in the larvae exposure to volatiles from both healthy and pest damaged maize seedlings than in the control group at 12 h after larvae exposure to maize volatiles. For the cotton bollworm damaged maize group, the expression of CYP4S1 and CYP9A14 increased.

The gene expression and activity of (+)-δ-cadinene synthase during cotton development and in response to stress, as well as the spatial and temporal pattern of sesquiterpene biosynthesis, constitute one of chemical defense mechanisms in cotton plants. In order to explore the effects of omethoate on the cotton defense in relation to (+)-δ-cadinene synthase and gossypol, effects of omethoate treatments on activity of (+)-δ-cadinene synthase and gossypol content in cotton seedlings were investigated. Cotton seedlings treated with 400 mg L−1 omethoate exhibited a significant decrease in the specific activity of (+)-δ-cadinene synthase from 12 to 120 h after treating when compared to the untreated control; significantly lower (+)-δ-cadinene synthase activity was also observed in cotton seedlings treated with 200 mg L−1 omethoate from 72 to 120 h after treating; but for cotton seedlings treated with 100 mg L−1 omethoate, from 12 to 120 h after treating, no significant changes were observed for activity of (+)-δ-cadinene synthase. The gossypol content in cotton seedlings treated with 100, 200 or 400 mg L−1 omethoate for different time periods showed no significant changes compared to that of the control. These results indicated that the activity of (+)-δ-cadinene synthase in cotton seedlings in responses to exposure of omethoate at three concentrations for different time periods followed dose- and time-dependent responses to omethoate exposure. With omethoate as a chemical stress factor for cotton seedlings, the cotton defense in relation to the activity of (+)-δ-cadinene synthase in cotton seedlings may be affected by omethoate application.

Metaflumizone is a novel sodium channel blocker insecticide, which has been registered for controling the diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae) in China. It is unavoidable for DBMs to be exposed to sublethal dose of metaflumizone in brassicaceous vegetable field. Sublethal effects of metaflumizone at LC15 (1.06 mg L−1) and LC25 (2.03 mg L−1) on the biological characteristics were investigated. Pupation rate, pupal period and pupal weight were significantly declined comparing with the control (P=0.05) when third instar larvae were exposed to LC15 and LC25 of metaflumizone. Comparing the control group to LC15 and LC25 treatment groups, there were significant differences in the development periods of eggs, first instar larvae, pupae and total preoviposition period. The peak of age-specific fecundity in LC15 and LC25 treatment groups was lagged behind control group. Mean values of intrinsic rate of increase (rm), net reproductive rate (R0) and finite rate of increase (λ) observed from the LC15 and LC25 treatment groups were lower than those from the control group. But mean values of gross reproduction rate (GRR) was not different. The mean generation time (T) of the treatment groups was prolonged. Whether sublethal doses of metaflumizone could stimulate reproduction in the long term needs further investigation on continuous generations.

Cry toxins produced by Bacillus thuringiensis (Bt) are effective biological insecticides against certain insect species. In this study, bioassay results indicated that Cry1B and Cry1C were toxic to Spodoptera exigua. We also identified a cadherin-like gene in S. exigua that could enhance the toxicity of Cry1B and Cry1C. The cadherin-like gene identified from the larvae midgut tissue was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA of the gene consisted of 5 220 bp encoding 1 740 amino acid with a predicted molecular mass of 196 kD. BLAST search analysis showed that the predicted amino acid sequence had a high sequence identity to the published sequences of cadherin-like proteins from other Lepidoptera insects. Spatial expression of the cadherin-like gene detected by qRT-PCR analysis revealed that the cadherin-like gene was mainly present in the gut of 4th instar larvae and during different life stages. The results suggested that the commercial development of this synergist has the potential to enhance Cry1B and Cry1C toxicity against Lepidoptera insects.

The whitefly Bemisia tabaci are considered as a taxonomically complex that contained some destructive pests. Two of the most prevalent cryptic species are B. tabaci Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED). In an extensive field survey of the B. tabaci complex present throughout part of China from 2004 to 2007, we obtained 93 samples of B. tabaci from 22 provinces. We determined that these Chinese haplotypes included 2 invasive species (MEAM1 and MED), and 4 indigenous cryptic species (Asia II 1, Asia II 3, China 3 and Asia II 7) by sequencing mitochondrial cytochrome oxidose one gene (mtCOI). The diversity and genetic differentiation of a subset of 19 populations of B. tabaci were studied using cDNA amplified fragment length polymorphism (AFLP). Prior to 2007, MEAM1 was a dominant species in many provinces in China. By 2007, MED was dominant in 11 provinces. Both invasive and indigenous species were simultaneously found in some regions. Indigenous species of B. tabaci were found in six provinces in southern China. MED and MEAM1 have broad ranges of host plants, and indigenous species appeared to have much narrower host ranges. All Asia II 3 samples were found on cotton except one on aubergine. China 3 has more host plants than Asia II 3. Twelve samples of China 3 were collected from sweet potato, Japanese hop, squash and cotton. A total of 677 reproducible bands amplified with 5 AFLP primer combinations were obtained. The highest proportion of polymorphic bands was 98.7% and the lowest was 91.9%. Unweighted pair-group method analysis indicated that the clustering was independent of the different species. MED showed the lowest degree of similarity than the other species. The data indicate that both MEAM1and MED were rapidly established in China.

Top-down force is referred to arthropod pest management delivered by the organisms from higher trophic levels. In the context of prevalent adoption of transgenic Bt crops that produce insecticidal Cry proteins derived from Bacillus thuringiensis (Bt), it still remains elusive whether the top-down forces are affected by the insect-resistant traits that introduced into the Bt crops. We explored how Bt cotton affect the strength of top-down forces via arthropod natural enemies in regulating a non-target pest species, the cotton aphid Aphis gossypii Glover, using a comparative approach (i.e. Bt cotton vs. conventional cotton) under field conditions. To determine top-down forces, we manipulated predation/parasitism exposure of the aphid to their natural enemies using exclusion cages. We found that the aphid population growth was strongly suppressed by the dominant natural enemies including Coccinellids, spiders and Aphidiines parasitoids. Coccinellids, spiders and the assemblage of other arthropod natural enemies (mainly lacewings and Hemipteran bugs) are similarly abundant in both plots, but with the parasitoid mummies less abundant in Bt cotton plots compared to the conventional cotton plots. However, the lower abundance of parasitoids in Bt cotton plots alone did not translate into differential top-down control on A. gossypii populations compared to conventional ones. Overall, the top-down forces were equally strong in both plots. We conclude that transgenic Bt cotton does not disrupt the top-down forces regulating the cotton aphid in central China.

BackgroundRNA interference (RNAi) is an effective tool to examine the function of individual genes. Carboxylesterases (CarE, EC 3.1.1.1) are known to play significant roles in the metabolism of xenobiotic compounds in many insect species. Previous studies in our laboratory found that CarE expression was up-regulated in Aphis gossypii (Glover) (Hemiptera: Aphididae) adults of both omethoate and malathion resistant strains, indicating the potential involvement of CarE in organophosphorus (OP) insecticide resistance. Functional analysis (RNAi) is therefore warranted to investigate the role of CarE in A. gossypii to OPs resistance.ResultCarE expression in omethoate resistant individuals of Aphis gossypii was dramatically suppressed following ingestion of dsRNA-CarE. The highest knockdown efficiency (33%) was observed at 72 h after feeding when dsRNA-CarE concentration was 100 ng/µL. The CarE activities from the CarE knockdown aphids were consistent with the correspondingly significant reduction in CarE expression. The CarE activity in the individuals of control aphids was concentrated in the range of 650-900 mOD/per/min, while in the individuals of dsRNA-CarE-fed aphids, the CarE activity was concentrated in the range of 500-800 mOD/per/min. In vitro inhibition experiments also demonstrated that total CarE activity in the CarE knockdown aphids decreased significantly as compared to control aphids. Bioassay results of aphids fed dsRNA-CarE indicated that suppression of CarE expression increased susceptibility to omethoate in individuals of the resistant aphid strains.ConclusionThe results of this study not only suggest that ingestion of dsRNA through artificial diet could be exploited for functional genomic studies in cotton aphids, but also indicate that CarE can be considered as a major target of organophosphorus insecticide (OPs) resistance in A. gossypii. Further, our results suggest that the CarE would be a propitious target for OPs resistant aphid control, and insect-resistant transgenic plants may be obtained through plant RNAi-mediated silencing of insect CarE expression.

Sitobion avenae is a major agricultural pest of wheat in China. Using microsatellite markers, we studied the potential gene flow, genetic diversity, genetic differentiation, and genetic structure of seven S. avenae populations from different regions of China (Beijing, Hebei, Henan, Hubei, Jiangsu, Shandong, and Shanxi provinces). The populations from Henan, Shandong, and Jiangsu showed high levels of genic and genotypic diversity. By contrast, the genic diversity in the Beijing and Hebei populations was much lower. Despite this low genic diversity, the genotypic diversity of the Beijing population was higher than that of all of the other populations, except those from Jiangsu and Shandong. Overall, the genetic divergence among the seven S. avenae populations tested was high, though there was almost no differentiation between the Shandong and Henan populations. We observed significant negative correlation between the strength of gene flow and the geographic distances among populations. Based on genetic analysis, the seven S. avenae populations studied can be divided into four distinct clusters; (i) Hubei, (ii) Shanxi, (iii) Beijing and Hebei, and (iv) Shandong, Henan, and Jiangsu. The present results provide a basis for potentially optimizing integrated pest management (IPM) programs in China, through adapting control methods that target biological traits shared by various populations of the same genotype.

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 AgomiR- 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. [ABSTRACT FROM AUTHOR]

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