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Helicoverpa armigera and Aphis gossypii are two important insect species that feed on cotton plants. These insects have distinct abilities to induce plant resistance and tolerate plant toxins, which results in interspecific competition imbalance that may be fatal to the low-tolerance A. gossypii and force these insects to develop avoidance behaviors and subsequently separate from their niche. We implemented ecological experiments to test the effects of H. armigera-induced plant resistance and behavioral avoidance in A. gossypii, and employed transcriptomics and metabolomics analyses to reveal changes in resistance genes and metabolites in plants.Our results demonstrate that cotton plants induced by H. armigera cause significant inhibitory and avoidance effects on A. gossypii insect populations. Electrical penetration graph (EPG) analysis showed changes in plant resistance induced by H. armigera leading to a decreased feeding efficiency of A. gossypii. In addition, genes associated with jasmonic acid and ethylene signaling pathways were significantly up-regulated in cotton plants after H. armigera induction, which led to a significant up-regulation of metabolites inducing plant resistance. These observations were corroborated by bioactivity analysis on metabolites, which showed that jasmonic acid, gossypol and tannins have significant inhibitory effects on A. gossypii populations. In contrast, methylparaben is associated with avoidance behaviors on A. gossypii populations.Our study suggests that the differences in the ability to induce plant resistance and tolerance between two non-predatory insects were lethal to low-tolerance A. gossypii insects, which might be a major factor determining their niche differentiation. This was further demonstrated by screening anti-insect and bio-hormonal metabolites. Our study provides a reference for investigating the evolutionary relationship between non-predatory insects and insights to implement effective insect biocontrol. © 2022 Society of Chemical Industry.

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.

Agricultural soils and microplastics (MPs) are hotspots for antibiotic resistance genes (ARGs). Plastic mulch is the most important source of MPs in agricultural soil. ARGs, mobile genetic elements (MGEs), and their host profiles in long-term mulch MP-exposed soils remain unclear. In the present study, metagenomics was used to investigate the distribution patterns of ARGs and MGEs in eight Chinese provinces with a long history of plastic mulch use. A total of 204 subtypes of ARGs and thousands of MGEs (14 integrons, 28 insertions, and 2993 plasmids) were identified. A similar diversity of ARGs was found among MPs film-contaminated sites. The types of ARGs with a high abundance were more concentrated, and multidrug resistance genes were the dominant ARGs. Soils from regions with a longer history of plastic film use (such as Xinjiang province) had a higher abundance of ARGs and MGEs. The distribution of ARGs and MGEs exhibited a modular network distribution pattern. A total of 27 ARG subtypes and 29 MGEs showed co-occurrence network relationships. More than 10 common hosts of ARGs and MGEs, such as Pseudomonas, were found, and their abundances were highest in three provinces, including Xinjiang. This study may help elucidate the impact mechanism of long-term MP residues on the occurrence and spread of ARGs in soil.

Insect endosymbionts are widespread in nature and known to play key roles in regulating host biology. As a secondary endosymbiont, bacteria in the genus Hamiltonella help cotton aphids (Aphis gossypii) defend against parasitism by parasitoid wasps, however, the potential negative impacts of these bacteria on cotton aphid biology remain largely unclear.This study aims to evaluate the potential impacts of Hamiltonella on the growth and development of cotton aphids based on life table parameters and RNA sequencing. The results showed that infection with Hamiltonella resulted in smaller body type and lower body weight in aphids. Compared to the control group, there were significant differences in the finite and intrinsic rates of increase and mean generation time. Furthermore, the RNA sequencing data revealed that the genes related to energy synthesis and nutrient metabolism pathways were significantly downregulated and genes related to molting and nervous system pathways were significantly upregulated in the Hamiltonella population.Our results confirm that Hamiltonella retarded the growth and development of cotton aphids accompanied by the downregulation of genes related to energy synthesis and nutrient metabolism, which provides new insights into aphid-symbiont interactions and may support the development of improved aphid management strategies. © 2022 Society of Chemical Industry.

The lady beetle Propylea japonica is a dominant natural predator of insect pests in farmland ecosystems and an important non-target indicator insect for the environmental safety assessment of GM crops. The commercial cultivation of GM crops may cause P. japonica to frequently be exposed to the Bt protein environment. In this study, the biological characteristics, enzyme activity, and expression levels of detoxification and metabolism in P. japonica were studied after Cry1B protein treatment. No significant differences were observed in developmental duration, emergence rate, or body weight at different ages after feeding larvae 0.5 mg/mL of Cry1B protein compared with the control. Furthermore, there were no significant differences in the activities of glutathione S-transferase (GST), catalase (CAT), and peroxidase (POD) after feeding 0.25 mg/mL and 0.5 mg/mL Cry1B protein. However, when the concentration of Cry1B protein increased to 1.0 mg/mL, the activities of the GST, CAT, and POD increased significantly. Compared with the control group, there were no significant differences in the expression levels of most detoxification metabolism related genes; only a few genes had changed expression levels at the individual concentrations (CYP345B1, CYP4Q2, CYP9F2, GST, and microsomal GST). Overall, these results suggest that Cry1B protein has little or no effect on the biological characteristics of P. japonica. Genes related to enzyme activity and detoxification are differentially expressed at high concentration stimulation. Therefore, this research suggests that the potential risks of Cry1B for the predator P. japonica are negligible.

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.

With the aging population worldwide, osteoporosis, as an age-related bone metabolic disease, is becoming a hot issue in public health. However, it is still a great challenge to realize osteoporotic bone healing due to the alteration of the bone microenvironment in osteoporosis patients. In this study, a nano-structured akermanite (nAK) coating was in situ constructed on Ti–6Al–4V implants to improve osteoporotic bone repair. In vitro studies indicated that both the surface nano-topography and bioactive ions released from the nAK coatings promoted the proliferation, osteogenesis, angiogenesis and inhibited osteoclastogenesis of ovariectomy rabbit-derived bone marrow mesenchymal stem cells (OVX-rBMSCs). Furthermore, the nAK-coated Ti–6Al–4V implants improved new bone formation and osseointegration in an osteoporosis rabbit model in vivo. These results indicated that the AK coating with a nano-structured surface on the Ti–6Al–4V implant could synergistically promote bone formation and osseointegration for osteoporosis patients. This may be a promising strategy to improve the bone regeneration and osseointegration capability of orthopedic implants under osteoporosis conditions.

Osteoporosis (OP) is a major systemic bone disease leading to an imbalance in bone homeostasis which remains a challenge in the current treatment of bone defects. Our previous studies on strontium (Sr) doping apparently stimulated osteogenesis of bioceramics, which suggested a promising strategy for the treatment of bone defects. However, the potential effects and the underlying mechanisms of Sr-doping on osteoporotic bone defects still remain unclear. Autophagy is a conventional self-degradation process of cells involved in bone homeostasis and regeneration under physiological and pathological conditions. Therefore, it is essential to design appropriate biomaterials and investigate the associated osteogenic mechanisms via autophagy. Based on this hypothesis, Sr-doped 45S5 bioglass (Sr/45S5) was fabricated, and ovariectomy bone marrow-derived mesenchymal stem cells (OVX-BMSCs) were applied as the in vitro cell culture model. First, the optimal Sr-doping concentration of 10 mol% was screened by cell proliferation, ALP staining, alizarin red S staining and the real-time PCR assay. Then, the results of western blot (WB) analysis showed that Sr-induced osteogenic differentiation of OVX-BMSCs was associated with time-dependent modulation of autophagy and related to the AKT/mTOR signaling pathway. Meanwhile, the autophagy in Sr-induced osteogenic differentiation of OVX-BMSCs was detected by WB, immunofluorescence staining and transmission electron microscopy. Furthermore, the effect of osteogenic differentiation of OVX-BMSCs has been significantly inhibited by the administration of autophagy inhibitors and the AKT/mTOR pathway inhibitors, respectively, in the early and late periods of osteogenic differentiation. Finally, the results of the model of femoral condyle defects in OVX-rats indicated that Sr10/45S5 granules remarkably enhanced bone regeneration which provided the evidences in vivo. Our research indicates that Sr-doping provides a promising strategy to promote osteogenic differentiation of OVX-BMSCs and bone regeneration in osteoporotic bone defects via early improvement of autophagy and late activation of the Akt/mTOR signaling pathway.

Angiogenesis is closely coupled with osteogenesis and has equal importance. Thus, promoting angiogenesis during the bone repair process is vital for ideal bone regeneration. As important mediators of cell-cell communication and biological homeostasis, mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been proved to be highly involved in bone and vascular regeneration. Because hypoxia microenvironment promotes the proangiogenic activity of MSCs, in the present study, we investigate the effect and underlying molecular mechanisms of sEVs from hypoxia-preconditioned MSCs (hypo-sEVs) on angiogenesis and develop an effective strategy to promote vascularized bone regeneration. Compared to sEVs from normoxia MSCs (nor-sEVs), hypo-sEVs promoted the proliferation, migration, and angiogenesis of HUVECs and ultimately enhanced bone regeneration and new blood vessel reconstruction in a critical-size calvarial bone defect model. miRNA sequence and the verified results showed that miR-210-3p in hypo-sEVs was increased via HIF-1α under hypoxia. The upregulated miR-210-3p in hypo-sEVs promoted angiogenesis by downregulating EFNA3 expression and enhancing the phosphorylation of the PI3K/AKT pathway. Thus, this study suggests a successful strategy to enhance vascularized bone regeneration by utilizing hypo-sEVs via the miR-210-3p/EFNA3/PI3K/AKT pathway. STATEMENT OF SIGNIFICANCE: Considering the significance of vascularization in ideal bone regeneration, strategies to promote angiogenesis during bone repair are required. Hypoxia microenvironment can promote the proangiogenic potential of mesenchymal stem cells (MSCs). Nonetheless, the therapeutic effect of small extracellular vesicles (sEVs) from hypoxia-preconditioned MSCs on cranio-maxillofacial bone defect remains unknown, and the underlying mechanism is poorly understood. This study shows that hypo-sEVs significantly enhance the proliferation, migration, and angiogenesis of HUVECs as well as promote vascularized bone formation. Moreover, this work indicates that HIF-1α can induce overexpression of miR-210-3p under hypoxia, and miR-210-3p can hinder EFNA3 expression and subsequently activate the PI3K/AKT pathway. The application of hypo-sEVs provides a facile and promising strategy to promote vascularized bone regeneration in a critical-size bone defect model.

As microplastics became the focus of global attention, the hazards of plastic plasticizers (PAEs) have gradually attracted people's attention. Agricultural soil is one of its hardest hit areas. However, current research of its impact on soil ecology only stops at the microorganism itself, and there is still lack of conclusion on the impact of soil metabolism. To this end, three most common PAEs (Dimethyl phthalate: DMP, Dibutyl phthalate: DBP and Bis (2-ethylhexyl) phthalate: DEHP) were selected and based on high-throughput sequencing and metabolomics platforms, the influence of PAEs residues on soil metabolic functions were revealed for the first time. PAEs did not significantly changed the alpha diversity of soil bacteria in the short term, but changed their community structure and interfered with the complexity of community symbiosis network. Metabolomics indicated that exposure to DBP can significantly change the soil metabolite profile. A total of 172 differential metabolites were found, of which 100 were up-regulated and 72 were down-regulated. DBP treatment interfered with 43 metabolic pathways including basic metabolic processes. In particular, it interfered with the metabolism of residual steroids and promoted the metabolism of various plasticizers. In addition, through differential labeling and collinear analysis, some bacteria with the degradation potential of PAEs, such as Gordonia, were excavated.

In insects, cytochrome P450 monooxygenases (P450s or CYPs) play an important role in the detoxification and metabolism of exogenous plant allelochemicals. In this study, a P450 gene CYP6AB12 was identified and characterized from Spodoptera litura. The cDNA contains an open reading frame (ORF) encoding 511 amino acid residues. CYP6AB12 was expressed at different ages of S. litura, with the highest levels found in the third and fourth instar larvae. Its highest expression was found in the midgut and fat body of fourth instar larvae fed with gossypol. Moreover, these expression levels were substantially increased compared with those from larvae fed with control diet. Gene silencing was then conducted by smearing dsRNA mixed with nanomaterials onto the cuticle. CYP6AB12 expression was significantly decreased in the midgut and fat body, and the net weight increase was substantially lower than that of the control group, indicating that the treatment group had more sensitivity to gossypol than the control. These results reveal that CYP6AB12 plays an important role in the detoxification and metabolism of gossypol, thus further confirming that P450s have a broad ability to detoxify and metabolize plant allelochemicals. It provides an important molecular basis for the exploration of detoxification metabolism and pest control of S. litura.

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.

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.

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.

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.

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.

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.