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Cytochrome P450 monooxygenase (P450 or CYP) plays an important role in the metabolism of insecticides and plant allelochemicals by insects. CYP321B1, a novel Spodoptera litura P450 gene, was identified and characterized. CYP321B1 contains a 1488 bp open reading frame (ORF) that encodes a 495 amino acid protein. In fourth instar larvae, the highest CYP321B1 expression levels were found in the midgut and fat body. In the tannin feeding test, tannin can significantly induce the expression of CYP321B1 in the midgut and fat body of 4th instar larvae. To verify the function of CYP321B1, RNA interference and metabolome analysis were performed. The results showed that silencing CYP321B1 significantly reduced the rate of weight gain under tannin induction. Metabolome analysis showed silencing affected 47 different metabolites, mainly involved in secondary metabolite biosynthesis and amino acid metabolism, including amino acids, lipid fatty acids, organic acids and their derivatives. Henoxyacetic acid and cysteamine are the most highly regulated metabolites, respectively. These findings demonstrate that CYP321B1 plays an important role in tannin detoxification and metabolism. Functional knowledge about metabolite detoxification genes in this major herbivorous insect pest can provide new insights into this biological process and provide new targets for agricultural pest control.

The cotton-melon aphid Aphis gossypii is a sap-sucking insect that is considered a serious global pest. The species is distributed over a large geographical range and uses a wide variety of hosts, with some populations being specialized to attack different plant species. Here, we provide de novo chromosome-level genome assemblies of a cotton specialist population (Hap1) and a cucurbit specialist population (Hap3). We achieved this by using a combination of third-generation sequencing platforms, namely Illumina and Hi-C sequencing technologies. We were able to anchor a total of 334.89 Mb (scaffold N50 of 89.13 Mb) and 359.95 Mb (scaffold N50 of 68.88 Mb) to four chromosomes for Hap1 and Hap3, respectively. Moreover, our results showed that the X-chromosome of Hap3 (113.01 Mb) was significantly longer than that of Hap1 (100.26 Mb), with a high level of sequence conservation between the aphid species. We also report variation in the number of protein-coding genes and repeat sequences between Hap1 and Hap3. In particular, olfactory and gustatory receptor genes underwent a high level of gene duplication and expansion events in A. gossypii, including between Hap1 and Hap3. Moreover, we identified two glutathione S-transferase genes which underwent single gene duplications in Hap3, and tandem duplication and inversion events affecting the cytochrome P450 monooxygenase between Hap1 and Hap3, all of which include the CYP3 family. Our results illustrate the variance in the genomic composition of two specialized A. gossypii populations and provide a helpful resource for the study of aphid population evolution, host adaption and insecticide resistance.

Propylea japonica has been regarded as one of the most remarkable natural enemies against aphid in China. However, the mechanism of juvenile hormone (JH) regulation of reproduction in P. japonica is still unclear. In this study, we investigated the JH titers of P. japonica and the development of the ovaries. We selected the six different developmental stages of ladybeetle females for transcriptome sequencing. We identified 583 genes involved in insect reproduction regulation, including 107 insect hormone synthesis signaling pathway-related genes and 476 nutrition-sensing signaling pathway-related genes. Transcriptome analysis indicated that a large number JH synthesis- and metabolism-related enzyme genes and some potential nutrient signal sensing- and transduction-related genes were significantly differentially expressed during P. japonica development. We investigated the effects of Met gene silencing on the reproduction of female adults and found that the ovarian maturation, vitellogenesis, and follicular epithelium development in the dsMet treatment group were significantly inhibited.

Background Aphis gossypii, a polyphagous and recurrent pest induced by pesticides, causes tremendous loss in the yield of crops each year. Previous studies of pesticide-induced sublethal effects mechanism mainly focus on gene level. The symbiotic bacteria are also important participants of this mechanism, but their roles in hormesis are still unclear. Results In this study, life table parameters and 16S rRNA sequencing were applied to evaluate the sublethal and transgenerational effects of sulfoxaflor on adult A. gossypii after 24-h LC20 (6.96 mg L-1 ) concentration exposure. The results indicated that the LC20 of sulfoxaflor significantly reduced the finite rate of increase (λ) and net reproductive rate (R0 ) of parent generation (G0), and significantly increased mean generation time (T) of G1 and G2, but not of G3 and G4. Both reproductive period and fecundity of G1 and G2 were significantly higher than those of the control. Furthermore, our sequencing data revealed that more than 95% bacterial communities were dominated by the phylum Proteobacteria, in which the maximum proportion genus was the primary symbiont Buchnera and the facultative symbiont Arsenophonus. Compared to those of the control, the abundance and composition of symbiotic bacteria of A. gossypii for three successive generations (G0-G2) were changed after G0 A. gossypii was exposed to sulfoxaflor: the diversity of the bacterial community was decreased, but the abundance of Buchnera was increased (G0), while the abundance of Arsenophonus was decreased. Contrary to G0, G1 and G2 cotton aphid exhibited an increased relative abundance of Arsenophonus in sublethal treatment group. Conclusion Taken together, our results provide an insight into the interactions among pesticide resistance, aphids, and symbionts, which will eventually help to better manage the resurgence of A. gossypii. This article is protected by copyright. All rights reserved.

The complete mitochondrial genome of Aphis gossypii Glover cucumber biotype was sequenced using traditional PCR amplification coupled with Sanger sequencing. The genome is 15,870 bp long, with 83.7...

Research on microplastic pollution of terrestrial soils is catching up with the aquatic environment, especially agricultural soil systems. Plastic residues have caused various environmental problems in mulch film extensively used agricultural areas. However, studies focusing specifically on the potential influence of mulch film residues on the metabolic cycle of soil systems have yet to be conducted. Here, high-throughput sequencing combined with metabolomics were first used to study the effects of residual mulch on soil microbial communities and related metabolic functions. Plastic film treatment did not significantly affect soil physicochemical properties including pH, organic matter and nitrogen, etc in short term. However, it did significantly changed overall community structure of soil bacteria, and interfered with complexity of soil bacterial symbiosis networks; exposure time and concentration of residues were particularly important factors affecting community structure. Furthermore, metabolomics analysis showed that film residue significantly changed soil metabolite spectrum, and interfered with basic carbon and lipid metabolism, and also affected basic cellular processes such as membrane transport and, in particular, interfered with the biosynthesis of secondary metabolites, as well as, biodegradation and metabolism of xenobiotics. Additionally, through linear discriminant and collinear analysis, some new potential microplastic degrading bacteria including Nitrospira, Nocardioidaceae and Pseudonocardiaceae have been excavated.

The ladybird beetle Propylea japonica is an important natural enemy in agro-ecological systems. Studies on the strong tolerance of P. japonica to high temperatures and insecticides, and its population and phenotype diversity have recently increased. However, abundant genome resources for obtaining insights into stress-resistance mechanisms and genetic intra-species diversity for P. japonica are lacking. Here, we constructed the P. japonica genome maps using Pacific Bioscience (PacBio) and Illumina sequencing technologies. The genome size was 850.90 Mb with a contig N50 of 813.13 kb. The Hi-C sequence data were used to upgrade draft genome assemblies; 4,777 contigs were assembled to 10 chromosomes; and the final draft genome assembly was 803.93 Mb with a contig N50 of 813.98 kb and a scaffold N50 of 100.34 Mb. Approximately 495.38 Mb of repeated sequences was annotated. The 18,018 protein-coding genes were predicted, of which 95.78% were functionally annotated, and 1,407 genes were species-specific. The phylogenetic analysis showed that P. japonica diverged from the ancestor of Anoplophora glabripennis and Tribolium castaneum ~ 236.21 million years ago. We detected that some important gene families involved in detoxification of pesticides and tolerance to heat stress were expanded in P. japonica, especially cytochrome P450 and Hsp70 genes. Overall, the high-quality draft genome sequence of P. japonica will provide invaluable resource for understanding the molecular mechanisms of stress resistance and will facilitate the research on population genetics, evolution and phylogeny of Coccinellidae. This genome will also provide new avenues for conserving the diversity of predator insects.

Parasitism usually causes considerable changes in lipids and fatty acids by redirecting the development of the host. In this study, changes in weight and in free fatty acid content of cotton aphids were recorded after aphids had been parasitized. Results showed that the weight of parasitized Aphis gossypii was increased compared to nonparasitized aphids, and significantly increased weights were detected at 1, 2, and 3 instars after parasitization by Lysiphlebia japonica. Free fatty acid test kits and GC-MS showed that the fatty acid content increased in the early stage of parasitization but decreased after 3 days of parasitization. Seven genes related to the fatty acid synthesis pathway were significantly upregulated in the parasitized aphids, where they were 1.96-10.97 times greater. Our data described the change that occurs in the fatty acid content of parasitized A. gossypii.

Bacteria and insects have a mutually beneficial symbiotic relationship. Bacteria participate in several physiological processes such as reproduction, metabolism, and detoxification of the host. Adelphocoris suturalis is considered a pest by the agricultural industry and is now a major pest in cotton, posing a serious threat to agricultural production. As with many insects, various microbes live inside A. suturalis. However, the microbial composition and diversity of its life cycle have not been well-studied. To identify the species and community structure of symbiotic bacteria in A. suturalis, we used the HiSeq platform to perform high-throughput sequencing of the V3–V4 region in the 16S rRNA of symbiotic bacteria found in A. suturalis throughout its life stages. Our results demonstrated that younger nymphs (1st and 2nd instar nymphs) have higher species richness. Proteobacteria (87.06%) and Firmicutes (9.43%) were the dominant phyla of A. suturalis. At the genus level, Erwinia (28.98%), Staphylococcus (5.69%), and Acinetobacter (4.54%) were the dominant bacteria. We found that the relative abundance of Erwinia was very stable during the whole developmental stage. On the contrary, the relative abundance of Staphylococcus, Acinetobacter, Pseudomonas, and Corynebacterium showed significant dynamic changes at different developmental stages. Functional prediction of symbiotic bacteria mainly focuses on metabolic pathways. Our findings document symbiotic bacteria across the life cycle of A. suturalis, as well as differences in both the composition and richness in nymph and adult symbiotic bacteria. Our analysis of the bacteria in A. suturalis provides important information for the development of novel biological control strategies.

The predatory ladybird beetle, Harmonia axyridis, is a predominant natural enemy of pest insects in cotton fields. Commercialization of genetically modified crops has promoted the increased use of the herbicide glyphosate. In this study, to assess potential negative effects of glyphosate on beneficial non-target organisms in cotton fields, we first examined how glyphosate exposure affected the development and endosymbiotic bacterial community of H. axyridis. The results showed that the survival rate, development duration, pupation rate and emergence rate of H. axyridis under low and high concentrations of glyphosate exposure were not significantly changed, but glyphosate did significantly reduce the body weight of H. axyridis. Based on 16S rRNA sequencing, there were no significant differences in the diversity or richness of the endosymbiotic bacteria of H. axyridis before and after glyphosate exposure. The dominant bacterial phyla Firmicutes and Proteobacteria and genera Staphylococcus and Enterobacter remained the same regardless of treatment with glyphosate, however the abundance and copy number of these bacteria were altered. Glyphosate treatment significantly reduced the abundance and gene copy number of Staphylococcus and increased the abundance and gene copy number of Enterobacter. This is the first report demonstrating that glyphosate can reduce the body weight H. axyridis and alter the bacterial endosymbiont community by affecting the abundance and gene copy number of dominant bacteria.

Background The worldwide pest Aphis gossypii has three-winged morphs in its life cycle, namely, winged parthenogenetic female (WPF), winged gynopara (GP), and winged male, which are all produced by a wingless parthenogenetic female (WLPF). Most studies on A. gossypii have focused on WPF, while few have investigated GP and male. The shared molecular mechanism underlying the wing differentiation in the three wing morphs of A. gossypii remains unknown. The wing differentiation of WPF was explored in a previous study. Herein, GP and male were induced indoors. The characters of the body, internal genitals, wing veins, and fecundity of GP and male were compared with those of WPF or WLPF. Compared with WLPF, the shared and separate differentially expressed genes (DEGs) were identified in these three-wing morphs. Results Newly-born nymphs reared in short photoperiod condition (8 L:16D, 18 °C) exclusively produced gynoparae (GPe) and males in adulthood successively, in which the sex ratio was GP biased. A total of 14 GPe and 9 males were produced by one mother aphid. Compared with WLPF, the three-wing morphs exhibited similar morphology and wing vein patterns but were obviously discriminated in the length of fore- and underwings, reproductive system, and fecundity. A total of 37 090 annotated unigenes were obtained from libraries constructed using the four morphs via RNA sequencing (RNA-Seq). In addition, 10 867 and 19 334 DEGs were identified in the pairwise comparison of GP versus WLPF and male versus WLPF, respectively. Compared with WLPF, the winged morphs demonstrated 2 335 shared DEGs (1 658 upregulated and 677 downregulated). The 1 658 shared upregulated DEGs were enriched in multiple signaling pathways, including insulin, FoxO, MAPK, starch and sucrose metabolism, fatty acid biosynthesis, and degradation, suggesting their key roles in the regulation of wing plasticity in the cotton aphid. Forty-four genes that spanned the range of differential expression were chosen to validate statistical analysis based on RNA-Seq through the reverse transcription quantitative real time polymerase chain reaction (RT-qPCR). The comparison concurred with the expression pattern (either up- or downregulated) and supported the accuracy and reliability of RNA-Seq. Finally, the potential roles of DEGs related to the insulin signaling pathway in wing dimorphism were discussed in the cotton aphid. Conclusions The present study established an efficiently standardized protocol for GP and male induction in cotton aphid by transferring newly-born nymphs to short photoperiod conditions (8 L:16D, 18 °C). The external morphological characters, especially wing vein patterns, were similar among WPFs, GPe, and males. However, their reproductive organs were strikingly different. Compared with WLPF, shared (2 335) and exclusively (1 470 in WLPF, 2 419 in GP, 10 774 male) expressed genes were identified in the three-wing morphs through RNA-Seq, and several signaling pathways that are potentially involved in their wing differentiation were obtained, including insulin signaling, starch and sucrose metabolism.

Lysozymes are used in sterilisation, antisepsis, dairy additives, inflammation, and cancer. One transgenic goat line expressing high levels of human lysozyme (hLZ) in goat milk has been developed in China. Herein, we established an event-specific real-time polymerase chain reaction (real-time PCR) method to detect the transgenic hLZ goat line. The developed method has high specificity, sensitivity and accuracy, and a wide quantitative dynamic range. The limit of detection and limit of quantification was 5 and 10 copies per reaction, respectively. The practical sample analysis results showed that the method could identify and quantify transgenic lysozyme content in trace samples in routine lab analyses. Furthermore, the potential applicability in risk assessment, such as molecular characterisation and gene horizontal transfer, was confirmed. We believe that this method is suitable for the detection of transgenic hLZ goat line and its derivate.

Spodoptera litura is a major insect with a cosmopolitan distribution and strong resistance to multiple insecticides. Determining the molecular basis and key candidate genes of the insecticide resistance of S. litura may help in managing this insect. In this study, fifth-instar S. litura larvae were subjected to transcriptome analysis at 6, 12, 24, 48, and 72 h after feeding on an LC20 dose of avermectin. The result showed that genes responding to avermectin changed dynamically with different gene counts and resistance mechanisms at the fifth instar based on a metabolic pathway map. These responses included degrading the insecticide by a series of P450 and glutathione-S-transferase enzymes starting at the 12 h time point, with subsequent increases in the number of genes involved and shifts to TOLL and immune deficiency (IMD) pathways at 48 h after feeding the insecticide. Weighted correlation network analysis (WGCNA) determined a co-expression module related to the avermectin response at 12 and 24 h (r = 0.403, p = 0.0371; r = 0.436, p = 0.023), in which a hub gene (LOC111358940) related to metalloproteinase activity was identified. In addition, Analysis of the genes in the co-expression module further revealed that eight genes encoding UDP-glucuronosyltransferases were directly associated with insecticide response in S. litura. These results provide better understanding of the avermectin response mechanism of S. litura and may be useful in developing improved control strategies for this species. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s13205-021-02651-9) contains supplementary material, which is available to authorized users.

Background Endosymbiotic bacteria have been reported to mediate interactions between parasitoids and their insect hosts. How parasitic wasps influence changes in host microbial communities and the relationship between them are of great importance to the study of host-parasitoid co-evolutionary and ecological interactions. However, these interactions remain largely unreported for interactions between in Aphis gossypii and Lysiphlebia japonica. Results In this study, we characterize the bacterial microbiota of L. japonica wasps at different developmental stages and monitor changes over time in the bacterial microbiota of their parasitized and non-parasitized aphid hosts, using metagenomic analysis of 16S rDNA sequencing data. Proteobacteria, Firmicutes, and Actinobacteria were the three most abundant bacterial phyla identified in L. japonica. We found that parasitism was associated with an increased abundance of Buchnera nutritional endosymbionts, but decreased abundance of Acinetobacter, Arsenophonus, Candidatus_Hamiltonella, and Pseudomonas facultative symbionts in aphid hosts. Functional analysis of enriched pathways of parasitized aphids showed significant differences in the 'transport and metabolism of carbohydrates' and 'amino acid, lipid, and coenzyme biosynthesis' pathways. Notably, the composition of symbiotic bacteria in wasp larvae was highly similar to that of their aphid hosts, especially the high abundance of Buchnera. Conclusion The results provide a conceptual framework for L. japonica interactions with A. gossypii in which the exchange of symbiotic microbes provides a means by which microbiota can potentially serve as evolutionary drivers of complex, multilevel interactions underlying the ecology and co-evolution of these hosts and parasites. This article is protected by copyright. All rights reserved.

Simple Summary The Aphis gossypii is a global problem for its pesticide resistance with substantial economic and ecological cost and a wide host range, including cotton and cucurbits. The development of insecticide resistance is rapid and widespread and threatens crop productivity. Biopesticides have emerged as a better alternative for pest control. Cucurbitacin B (CucB) and epigallocatechin gallate (EGCG) are the major secondary metabolites of host plants cucurbits and cotton. In this study, we used cotton- and cucurbit-specialized aphids (CO and CU) as a study system to better understand the effects of CucB and EGCG on cotton aphid. Our study showed that CucB and EGCG can significantly reduce the population-level fitness of A. gossypii, affect their ability to adapt to nonhost plants and alter the levels of some detoxifying enzymes, which showed a potential to be developed into new biopesticides against the notorious aphids. Abstract Aphis gossypii (Glover) is distributed worldwide and causes substantial economic and ecological problems owing to its rapid reproduction and high pesticide resistance. Plant-derived cucurbitacin B (CucB) and epigallocatechin gallate (EGCG) are known to have insecticidal and repellent activities. However, their insecticidal activity on cotton- and cucurbit-specialized aphids (CO and CU), the two important host biotypes of A. gossypii, remains to be investigated. In the present study, we characterized, for the first time, the effects of these two plant extracts on the two host biotypes of A. gossypii. CucB and EGCG significantly reduced the A. gossypii population-level fitness and affected their ability to adapt to nonhost plants. Activities of important detoxification enzymes were also altered, indicating that pesticide resistance is weakened in the tested aphids. Our results suggest that CucB and EGCG have unique properties and may be developed as potential biopesticides for aphid control in agriculture.

Given the ever-increasing commercial planting of transgenic plants across the world, an evaluation of their impacts on non-target organisms is as an important part of the risk assessment process. Propylea japonica is a dominant non-target predator and pollen feeder insect that is prevalent in Bt cotton fields, and it is thus in direct contact with Bt proteins. However, the effect of Bt proteins on P. japonica has not received much attention. In this study, the effects of Cry1Ac and/or Cry2Ab proteins on P. japonica were investigated from three aspects. First, no significant differences in the diversity of the microbiota nor change in species composition and community structure were observed among Cry protein treatments. Firmicutes are the most abundant bacterial phylum present in P. japonica, followed by Proteobacteria and Actinobacteria. The most abundant genus was Staphylococcus. Second, the expression levels of the detoxification and digestion-related genes did not change significantly in any Cry protein treatment. Third, none of the Cry proteins affected the population fitness of P. japonica. These results indicated that P. japonica was not sensitive to Bt proteins, suggesting that growing Bt cotton expressing Cry1Ac and/or Cry2Ab will pose negligible risks to P. japonica.

The widespread commercialization of genetically modified (GM) cotton makes it important to assess the potential impact of this recombinant crop on non-target organisms. As important natural enemies of cotton field predators, green lacewing Chrysoperla sinica larvae are exposed to Bt insecticidal proteins expressed by GM cotton by feeding on herbivorous pests, and adults are directly exposed to Bt proteins by cotton pollen consumption. However, potential impacts of transgenic Bt cotton on C. sinica remain unclear. In this study, we evaluated the effects of two transgenic cotton varieties, CCRI41 and CCRI45, which express Cry1Ac (Bt toxin) and CpTI (Cowpea Trypsin Inhibitor), on C. sinica larvae and adults. After being fed with cotton aphids Aphis gossypii reared on transgenic cotton, the survival rate, developmental duration, pupation rate, and emergence rate of larvae were not adversely affected. After being fed two types of transgenic cotton pollen, the 7-day weight of adults and the preoviposition period and the cumulative oviposition of females were not significantly different from control specimen. Taken together, these results indicate that the potential risks of the two tested GM cotton varieties for the predator C. sinica are negligible. Capsule: Our study indicated that GM cotton varieties CCRI41 and CCRI45 have no adverse effects on insect predator C. sinica.

Background: Three wing morphs exists in the life cycle of the worldwide pest Aphis gossypii, i.e., wing parthenogenetic female (WPF), gynopara (GP) and male, which were produced mostly by crowding and host quality, photoperiod, loss of X chromosome, respectively. However, the shared molecular mechanism underlying their wing differentiation remains an enigma. Here we firstly induced gynoparae and males indoors and compared the characters of these wing morphs in body, internal genitals and fecundity. Then we identified the shared and separate differentially expressed genes (DEGs) and signaling pathways potentially involved in the wing morphs regulation in WPF, GP and male compared to wingless parthenogenetic female (WLPF). Results: Newly-born nymphs reared in short photoperiod condition exclusively produce gynoparae and males in adulthood successively, in which the sex ratio is gynoparae biased. Compared with WLPF, three wing morphs have similar morphology in bodies but is obviously discriminated in the reproductive system and fecundity. Built upon our previous study, 37 090 annotated unigenes were obtained from libraries constructed by the four morphs above through RNA-sequencing, in which 10 867, 19 334 DEGs were identified in pairwise comparison of GP vs. WLPF, Male vs. WLPF, respectively. Furthermore, 2 335 shared DEGs including 1 658 up- and 677 downregulated were obtained in these wing morphs compared to WLPF. The 1 658 shared up-regulated DEGs were enriched in multiple signaling pathways including insulin, FoxO, MAPK, strarch and sucrose metabolism, fatty acid biosynthesis and degradation which hint their key roles in the regulation of wing plasticity in cotton aphid. Gene expression levels were validated by using Pearson’s correlation (r) and potential roles of 15 DEGs related to the insulin signaling pathway in cotton wing dimorphism were discussed. Conclusions: The results of this study establish a solid foundation for deciphering molecular mechanisms underlying the switch between wingless and wing morphs in the cotton aphid and provide valuable resources for future research on the host-alternating aphid species.

Elevated CO2 concentrations can change the multi-level nutritional relationship of the ecosystem through the cascading effect of the food chain. To date, few studies have investigated the effects of elevated CO2 concentration on the Araneae species through the tritrophic system. Hylyphantes graminicola (Araneae: Linyphiidae) is distributed widely in Asia and is a dominant predator in cotton fields. This study investigated chemical components in the food chain of cotton (Gossypium hirsutum)—cotton aphid (Aphis gossypii)—predator (H. graminicola) and compared the development, reproduction, and predation of H. graminicola under ambient (400 ppm) and elevated concentration of CO2 (800 ppm). The results showed that the elevated CO2 concentration increased the chemicals of cotton and cotton aphid, but it did not affect the nutrients, development, reproduction, and predation of the spider. However, the survival rate of the spider was significantly decreased in elevated CO2. The results will further our understanding of the role of natural enemies in an environment with elevated CO2 concentration.

Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L. japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L. japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L. japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT, 18S rRNA, and RPL13 during different stages; AK, RPL13, and TBP among sexes; EF1A, PPI, and RPL27 in different tissues, and EF1A, RPL13, and PPI in adults fed on different diets. Moreover, the expression profile of a target gene (odorant receptor 1, OR1) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L. japonica and will benefit subsequent genomics and functional genomics research on this natural enemy.

Risk assessments of insecticidal Cry proteins from Bacillus thuringiensis (Bt) cotton in soils have been mainly based on microcosm and short-term experiments. There is a lack of longer-term field results, especially when Bt-cotton residues are incorporated into the soil. In this study, we used a six-year field trial to evaluate how cultivation duration and Cry1Ac-inputted modes of Bt-cotton affect the persistence of Cry1Ac proteins and soil microbe-mediated enzymatic properties. The results showed that the persistence of Cry1Ac proteins increased with cultivation duration and periodic residue incorporation of the transgenic Bt-cotton variety ZM41. Moreover, temporal residue incorporation had a relatively larger contribution to the persistence of Cry1Ac proteins in the soil than their release in the growth period. Regardless of Bt-cotton cultivation or residue incorporation, soil microbial biomass was significantly suppressed. However, the dehydrogenase activity was significantly stimulated in Bt-cotton cultivation but suppressed in residue incorporation. The activities of β -glucosidase, nitrate reductase, phosphomonoesterase and arylsulfatase were significantly stimulated in soils with Bt-cotton residue incorporation. Based on a structural equation model analysis, the change in enzymatic activity of these four enzymes was attributed to both a direct effect from Cry1Ac proteins and an indirect effect via dehydrogenase.

Chrysoperla sinica (Tjeder) is widely recognized as an important holometabolous natural enemy of various insect pests in different cropping systems and as a non-target surrogate in environmental risk assessment of Bt rice (i.e., genetically modified rice to express a toxin gene from Bacillus thuringiensis). Like other complex organisms, abundant microbes live inside C. sinica; however, to date, microbiome composition and diversity of the whole life cycle in C. sinica has not yet been well characterized. In the current study, we analyze the composition and biodiversity of microbiota across the whole life cycle of C. sinica by using high-throughput Illumina sequencing of the 16S ribosomal RNA gene. Collectively, Proteobacteria and Firmicutes dominated the microenvironment at all stages, but their relative abundances fluctuated by host developmental stage. Interestingly, eggs, neonates, and adults shared similar microbes, including an abundance of Rickettsia and Wolbachia. After larva feeding, Staphylococcus, Enterobacteriaceae, and Serratia were enriched in larvae and pupa, suggesting that food may serve as a major factor contributing to altered microbial community divergence at different developmental stages. Our findings demonstrated that C. sinica harbor a variety of bacteria, and that dynamic changes in community composition and relative abundances of members of its microbiome occur during different life cycle stages. Evaluating the role of these bacterial symbionts in this natural enemy may assist in developing environmental risk assessments and novel biological control strategies.

Background Aphis gossypii is a worldwide sap-sucking pest with a variety of hosts and a vector of more than 50 plant viruses. The strategy of wing polyphenism, mostly resulting from population density increasing, contributes to the evolutionary success of this pest. However, the related molecular basis remains unclear. Here, we identified the effects of postnatal crowding on wing morph determination in cotton aphid, and examined the transcriptomic differences between wingless and wing morphs. Results Effect of postnatal crowding on wing determination in A. gossypii was evaluated firstly. Under the density of 5 nymphs·cm− 2, no wing aphids appeared. Proportion of wing morphs rised with the increase of density in a certain extent, and peaked to 56.1% at the density of 20 nymphs·cm− 2, and reduced afterwards. Then, transcriptomes of wingless and wing morphs were assembled and annotated separately to identify potentially exclusively or differentially expressed transcripts between these two morphs, in which 3 126 and 3 392 unigenes annotated in Nr (Non-redundant protein sequence) database were found in wingless or wing morphs exclusively. Moreover, 3 187 up- and 1 880 down-regulated genes were identified in wing versus wingless aphid. Pathways analysis suggested the involvement of differentially expressed genes in multiple cellular signaling pathways involved in wing morphs determination, including lipid catabolic and metabolism, insulin, ecdysone and juvenile hormone biosynthesis. The expression levels of related genes were validated by the reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) soon afterwards. Conclusions The present study identified the effects of postnatal crowding on wing morphs induction and demonstrated that the critical population density for wing morphs formation in A. gossypii was 20 nymphs·cm− 2. Comparative transcriptome analysis provides transcripts potentially expressed exclusively in wingless or wing morph, respectively. Differentially expressed genes between wingless and wing morphs were identified and several signaling pathways potentially involved in cotton aphid wing differentiation were obtained.

Gut microbiota has been suggested as an important component of host defense. However, microbiota in other parts of the body have rarely been characterized. In our present work, we give a detailed view of the body of Spodoptera exigua larvae, the midgut with intestinal contents (MG), midgut excluding intestinal contents (PM), intestinal contents (IC) and remaining parts excluding the MG (RM), with the objective of revealing the complexity of microorganisms and comparing the biodiversity of the S. exigua larvae-associated microbiota. Our results provide the first evidence that third and fifth larvae samples of S. exigua were dominated by members of the phyla Firmicutes and Proteobacteria. In addition, there were no obvious differences in relative abundances of microbiota among MG, PM and RM at the phylum level. MG3 and MG5 (nearly RM5) harbored the richest microbial community, with much higher abundance of Halomonas, Pseudomonas and Methylobacterium, respectively. Intestinal contents contained the lowest diversity and highest abundance of Enterococcus. Knowledge of the bacteria in a major herbivorous insect, such as S. exigua, can facilitate the acquisition of special biological resources for agricultural pest control.

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Common varieties of genetically modified (GM) cotton increasingly display insect-resistant properties via expression of bacterial-derived toxins from Bacillus thuringiensis (Bt). This necessitates a deeper understanding of the possible effects of these crops on non-target insects. The mirid bug Apolygus lucorum is a major pest in cotton production in China, however, the effect of GM cotton on this non-target species is currently virtually unknown. This insect is exposed to these transgenic plants by consuming genetically modified (GM) leaves. In this study, laboratory experiments were conducted to assess the toxicity of CCRI41 and CCRI45, (genetically modified cotton varieties which express the toxins Cry1Ac and CpTI (Cowpea Trypsin Inhibitor)) on nymphs and adults of A. lucorum. There was no detectable increase in mortality after A. lucorum fed on GM cotton leaves for 20 days. While we detected trace amounts of Cry1Ac proteins in both A. lucorum nymphs and adults (10 ng/g fresh weight), the expression of genes related to detoxification did not detectably differ from those feeding on non-GM cotton. Our binding assays did not show Cry1Ac binding to receptors on the midgut brush border membrane from either A. lucorum nymphs or adults. Our findings collectively indicate that feeding on leaves of the GM cotton varieties CCRI41 and CCRI45 have few toxic effects on A. lucorum.

Insulin signalling in insects, as in mammals, regulates various physiological functions, such as reproduction. However, the molecular mechanism by which insulin signals orchestrate ovarian stem cell proliferation, vitellogenesis, and oviposition remains elusive. Here, we investigate the functions of the phosphoinositide 3-kinase (PI3K)-serine/threonine kinase (Akt) pathway, GTPase Ras/mitogen-activated protein kinase (MAPK) pathway, and their downstream messengers in a natural predator, Chrysopa pallens, by the RNAi method. When C. pallens vitellogenin gene 1 (CpVg1) expression was knocked down, the follicle maturation was arrested and total fecundity was reduced. Silencing C. pallens insulin receptor 1 (CpInR1) suppressed Vg transcription and reduced egg mass and hatching rate. Depletion of C. pallens insulin receptor 2 (CpInR2) transcripts lowered Vg transcript level, hampered ovarian development and decreased reproductive output. Knockdown of C. pallens Akt (CpAkt) and C. pallens extracellular-signal-regulated kinase (Cperk) caused phenotypes similar to those caused by knockdown of CpInR2. Disruption of C. pallens transcription factor forkhead box O (CpFoxO) expression caused no significant effects on ovarian development, but sharply impaired total fecundity. Interference with the expression of C. pallens target of rapamycin (CpTor) gene and C. pallens cAMP-response element binding protein (CpCreb) gene led to a down-regulation of Vg transcription, blocking of ovariole growth, and decrease in egg quality. These results suggested the two CpInRs orchestrate oogenesis and oviposition via two signalling pathways to guarantee natural reproduction in the green lacewing, C. pallens.

Molecular characterization of exogenous DNA integrations in host genome is a key aspect in risk assessment of bioengineered crops. However, gaining a clear understanding of the molecular characters of a bioengineered crop using conventional techniques remains a challenging task. Herein, we report the full molecular characterization of one new transgenic rice event G6H1 via a paired-end sequencing approach and bioinformatics analysis pipelines. Also, the molecular characterization reported was validated using conventional PCR, Sanger sequencing, and digital PCR. The results showed there is only one copy of the exogenous DNA inserted, which is located within chromosome 7 of the G6H1 genome. There is no other unintended integration of sequences from the transformation plasmid. These results indicated that the paired-end sequencing approach, combined with bioinformatics pipeline developed, is well suited to elucidate the molecular characteristics of bioengineered crops, and is efficient, low cost, and comprehensive.

DNA Phosphorothioate (PT), replacing a non-bridging phosphate oxygen atom with a sulfur atom, is one kind of common DNA modification in bacteria. Whole genome scale description of the location and frequency of PT modification is the key to understand its biological function. Herein we developed a novel method, named with iodine-induced cleavage quantitative real-time PCR (IC-qPCR), to evaluate the frequency of PT modification at a given site in bacterial DNA. The efficiency, dynamic range, sensitivity, reproducibility and accuracy of IC-qPCR were well tested and verified employing an E. coli B7A strain as example. The amplification efficiency of IC-qPCR assay ranged from 91% to 99% with a high correlation coefficient ≥0.99. The limit of quantification was determined as low as 10 copies per reaction for the 607710 and 1818096 sites, and 5 copies for the 302695 and 4120753 sites. Based on the developed IC-qPCR method, the modification frequency of four PTs in E. coli B7A was determined with high accuracy, and the results showed that the PT modification was partial and that the modification frequency varied among investigated PT sites. All these results showed that IC-qPCR was suitable for evaluating the PT modification, which would be helpful to further understand the biological function of PT modification.

The cotton‐melon aphid, Aphis gossypii Glover, is a worldwide‐spreading species, and pesticide‐resistant populations are increasing rapidly. In this study, investigations were performed based on Illumina HiSeq sequencing of the 16S rDNA V4 region for the bacterial communities embodied as intracellular symbionts under natural and in pesticide‐treated populations of A. gossypii. The results revealed that more than 82% of bacterial communities belonged to the phylum Proteobacteria in which the maximum proportion (53.24%) was of the genus Arsenophonus; Hamiltonella composed 22.31; and 1.37% was of the genus Acinetobacter. The relative abundance of Hamiltonella was obvious, vertically transmitted, divided into two groups, and its infection influenced the bacterial communities in A. gossypii. Symbiont density and composition were changed in samples tested on different days. Azadirachtin and phoxim influenced on the composition of bacterial communities. Different biomarkers were used for pesticide‐treated samples with LEfSe results. These findings will increase awareness regarding bacterial communities in naturally occurring populations of A. gossypii and pave the way to study the relationship between symbionts and pesticide resistance.

Propylea japonica is a very important predator in agricultural ecosystems, which could be exposed to Bt protein. In this study, the bacterial community of P. japonica fed with normal food and food containing Cry2Ab protein was characterized for the first time using qPCR and high-throughput sequencing approaches. Results showed no effect of Cry2Ab on P. japonica development and reproduction. The most abundant bacterial phylum was Firmicutes, and the most abundant genus was Staphylococcus. The total bacteria copy number was not significantly different across four larval stages. Bacteria species composition was gathered more closely in feed on sucrose solution (sucrose-fed) than in larvae only fed on pea aphid (aphid-fed), the diversity indices of some operational taxonomic unit (OTU) were significantly different between sucrose-fed and aphid-fed samples. Different instar larval stages of P. japonica fed with sucrose solution containing Cry2Ab Bt protein and found no effect on microbial community composition and total bacteria copy numbers. However, effects on relative abundance of microbes, copy numbers of Corynebacterium 1 and Glutamicibacter arilaitensis were observed significantly lower in Bt-fed first and fourth larval stages. Low and high concentrations of Cry2Ab protein altered the microbial abundance relative to sucrose-fed P. japonica and copy numbers of G. arilaitensis and Staphylococcus xylosus were significantly lower in Bt-fed samples than control sucrose-fed. Our results are the first report showing that feeding on Cry2Ab protein does not alter microbial species composition in P. japonica, but effects gene copy number of some dominant bacteria. Further investigations are needed to assess the effect of copy number change on P. japonica.

Transgenic insect-resistant cotton expressing the Cry1Ac and/or CpTI protein has been released into the environment for commercial cultivation for over a decade in China. In this consecutive 3-year field study (2007–2009), we characterized the microbial populations in two different transgenic cotton fields and one non-transgenic cotton field to assess the environmental impact of long-term cultivation of transgenic insect-resistant plants in credible field conditions. We obtained samples at four different growth stages of cotton (seedling, budding, boll forming and boll opening). The results showed significant seasonal variation in the number of bacteria, fungi, azotobacter, denitrifying bacteria and ammonia-oxidizing bacteria and in diversity indices of microorganisms, but no significant difference in the number of each microbial population or diversity indices attributable to long-term cultivation of transgenic cotton.

With the large-scale release of genetically modified (GM) crops, there are ecological concerns on transgene movement from GM crops to non-GM counterparts and wild relatives. In this research, we conducted greenhouse experiments to measure pollen-mediated gene flow (PGF) in the absence and presence of pollinators (Bombus ignitus, Apis mellifera and Pieris rapae) in one GM cotton (resistant to the insect Helicoverpa armigera and the herbicide glyphosate) and two non-GM lines (Shiyuan321 and Hai7124) during 2012 and 2013. Our results revealed that: (1) PGF varied depending on the pollinator species and was highest with B. ignitus (10.83%) and lowest with P. rapae (2.71%); (2) PGF with B. ignitus depended on the distance between GM and non-GM cottons; (3) total PGF to Shiyuan321 (8.61%) was higher than to Hai7124 (4.10%). To confirm gene flow, we tested hybrids carrying transgenes for their resistance to glyphosate and H. armigera and most hybrids showed strong resistance to the herbicide and insect. Our research confirmed that PGF depended on pollinator species, distance between plants and the receptor plant.

Gossypium hirsutum has proven difficult to sequence owing to its complex allotetraploid (AtDt) genome. Here we produce a draft genome using 181-fold paired-end sequences assisted by fivefold BAC-to-BAC sequences and a high-resolution genetic map. In our assembly 88.5% of the 2,173-Mb scaffolds, which cover 89.6%∼96.7% of the AtDt genome, are anchored and oriented to 26 pseudochromosomes. Comparison of this G. hirsutum AtDt genome with the already sequenced diploid Gossypium arboreum (AA) and Gossypium raimondii (DD) genomes revealed conserved gene order. Repeated sequences account for 67.2% of the AtDt genome, and transposable elements (TEs) originating from Dt seem more active than from At. Reduction in the AtDt genome size occurred after allopolyploidization. The A or At genome may have undergone positive selection for fiber traits. Concerted evolution of different regulatory mechanisms for Cellulose synthase (CesA) and 1-Aminocyclopropane-1-carboxylic acid oxidase1 and 3 (ACO1,3) may be important for enhanced fiber production in G. hirsutum.

A new microbial α-amylase inhibitor gene was cloned and characterized. The encoded, recombinant, α-amylase inhibitor protein was induced and expressed by isopropyl β-d-1-thiogalactopyranoside (IPTG) in Escherichia coli M15 cells. The effects of the α-amylase inhibitor protein on Helicoverpa armigera larvae were studied. Compared to the control, the weight of H. armigera larvae fed the diet with recombinant α-amylase inhibitor protein added at a concentration of 20 μg/g was reduced by 49.8%. The total soluble protein of H. armigera larvae fed the diet with the α-amylase inhibitor protein added was also reduced by 36.8% compared to the control. The recombinant α-amylase inhibitor protein showed inhibition activity against α-amylase of H. armigera. These results suggested that this α-amylase inhibitor protein may be a promising bioinsecticide candidate for controlling H. armigera.

Evolution of pest resistance reduces the efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays or in transgenic crops. Although several pests have evolved resistance to Bt crops in the field, information about the genetic basis of field-evolved resistance to Bt crops has been limited. In particular, laboratory-selected resistance to Bt toxin Cry1Ac based on recessive mutations in a gene encoding a toxin-binding cadherin protein has been identified in three major cotton pests, but previous work has not determined if such mutations are associated with field-selected resistance to Bt cotton. Here we show that the most common resistance alleles in field populations of cotton bollworm, Helicoverpa armigera , selected with Bt cotton in northern China, had recessive cadherin mutations, including the deletion mutation identified via laboratory selection. However, unlike all previously studied cadherin resistance alleles, one field-selected cadherin resistance allele conferred nonrecessive resistance. We also detected nonrecessive resistance that was not genetically linked with the cadherin locus. In field-selected populations, recessive cadherin alleles accounted for 75–84% of resistance alleles detected. However, most resistance alleles occurred in heterozygotes and 59–94% of resistant individuals carried at least one nonrecessive resistance allele. The results suggest that resistance management strategies must account for diverse resistance alleles in field-selected populations, including nonrecessive alleles.

The attenuation of disease resistance in transgenic insect-resistant cotton has become one of the important factors restricting cotton production in China. Two transgenic insect-resistant cotton lines and their parental conventional cotton lines were used as the testing materials. The effects of root exudates of these cotton lines on the spore germination and mycelial growth of Fusarium oxysporum were studied and the components, contents of amino acids and sugars were determined. The results showed that the resistance of the two insect-resistant cotton lines to F. oxysporum was inferior to the parental lines, and that their root exudates promoted fungal spore germination and mycelial growth. Considerable differences in the components and contents of both, amino acids and sugars were found between the root exudates of transgenic cotton lines and their parental lines, where the disease indices were highly correlated with the total amount of sugars in the root exudates.

With the expansion of the planted area of transgenic Bt+CpTI cotton, the effects of this crop on non-target organisms in soil, including earthworms, are becoming the most important aspect of their ecological risk assessment. Laboratory toxicity studies were conducted to determine the effects of transgenic Bt+CpTI cotton leaves, containing high concentrations of the Bt toxin and cowpea trypsin inhibitor, on the earthworm Eisenia foetida. In comparison with the non-transgenic cotton line Zhong23, transgenic Bt+CpTI cotton Zhong41 had no significant acute toxicity on E. foetida. Moreover, the leaves of transgenic Bt+CpTI cotton were more suitable than the non-transgenic cotton Zhong23 for E. foetida growth and reproduction (time of reproduction, the number of cocoons and newly incubated offspring).