Tank-Mix Adjuvants Enhance Pesticide Efficacy by Improving Physicochemical Properties and Spraying Characteristics for Application to Cotton with Unmanned Aerial Vehicles.

Tank-mix adjuvants have been used to reduce spray drift and facilitate the efficacy of pesticides applied with unmanned aerial vehicles (UAVs). However, the effects of specific adjuvants on pesticide characteristics and the mechanism of action remain unclear. Herein, we analyzed the effects of three different types of tank-mix adjuvants (plant oil; mineral oil; and mixture of alcohol and ester) on the surface tension (ST), contact angle, wetting, permeation, evaporation, spray performance, and aphid-control effects of two types of pesticides. The mineral oil adjuvant Weichi (WCH) was highly effective in reducing the pesticide solution ST, improving the wetting and penetration ability, increasing droplet size, and promoting droplet deposition. The mixed alcohol and ester adjuvant Quanrun (QR) showed excellent wetting and antievaporation properties and promoted droplet deposition. A plant oil adjuvant (Beidatong) moderately improved wetting and penetration ability and reduced droplet drift. Field tests showed that the control efficiencies (CEs) of two pesticides were increased after the addition of adjuvants, even with 20% reductions in pesticide application. When the UAV was operated at 1.5 m, the CEs of two pesticides were increased from 65.39 and 66.63% to 73.11-76.52% and 77.91-88.31%, respectively. When operated at 2.5 m, the CEs were increased from 51.24 and 68.60% to 65.06-75.70% and 77.57-92.59%, respectively. Especially, the CEs of pesticides with WCH and QR increased obviously. Importantly, neither WCH nor QR inhibited hatching of the critical insect natural enemy ladybird beetle at concentrations used in the field. This study provides a framework for assessment of tank-mix adjuvants in aerial sprays and directly demonstrates the value of specific adjuvants in improving pesticide bioavailability and minimizing associated environmental pollution.

Exploring the potential of biochar for the remediation of microbial communities and element cycling in microplastic-contaminated soil.

The detrimental effects of microplastics (MPs) on soil microbial and elemental raise significant environmental concerns. The potential of remediation with biochar to mitigate these negative impacts remains an open question. The remediation effects of biochar derived from corn and cotton straw on MPs concerning soil microorganisms and element cycling were investigated. Specifically, biochar induced substantial remediations in microbial community structure following MP exposure, restoring and fortifying the symbiotic network while exerting dominance over microbial community changes. A combined treatment of biochar and MPs exhibited a noteworthy increase in the abundance of NH4+, NO3-, and available phosphorous by 0.46-2.1 times, reversing the declining trend of dissolved organic carbon, showing a remarkable increase by 0.36 times. This combined treatment also led to a reduction in the abundance of the nitrogen fixation gene nifH by 0.46 times, while significantly increasing the expression of nitrification genes (amoA and amoB) and denitrification genes (nirS and nirK) by 22.5 times and 1.7 times, respectively. Additionally, the carbon cycle cbbLG gene showed a 2.3-fold increase, and the phosphorus cycle gene phoD increased by 0.1-fold. The mixed treatment enriched element-cycling microorganisms by 4.8-9.6 times. In summary, the addition of biochar repaired the negative effects of MPs in terms of microbial community dynamics, element content, gene expression, and functional microbiota. These findings underscore the crucial role of biochar in alleviating the adverse effects of MPs on microbial communities and elemental cycling, providing valuable insights into sustainable environmental remediation strategies.

Combined effects of mulch film-derived microplastics and pesticides on soil microbial communities and element cycling.

Pesticides and microplastics (MPs) derived from mulch film in agricultural soil can independently impact soil ecology, yet the consequences of their combined exposure remain unclear. Therefore, the effects of simultaneous exposure to commonly used pesticides (imidacloprid and flumioxazin) and aged mulch film-derived MPs on soil microorganisms and element cycles in cotton fields were investigated. The combined exposure influenced soil microorganisms, alongside processes related to carbon, nitrogen, and phosphorus cycles, exhibiting effects that were either neutralized or enhanced compared to individual exposures. The impact of pesticides in combined exposure was notably more significant and played a dominant role than that of MPs. Specifically, combined exposure intensified changes in soil bacterial community and symbiotic networks. The combined exposure neutralized NH4+, NO3-, DOC, and A-P contents, shifting from 0.33 % and 40.23 % increase in MPs and pesticides individually to a 40.24 % increase. Moreover, combined exposure resulted in the neutralization or amplification of the nitrogen-fixing gene nifH, nitrifying genes (amoA and amoB), and denitrifying genes (nirS and nirK), the carbon cycle gene cbbLG and the phosphorus cycle gene phoD from 0.48 and 2.57-fold increase to a 2.99-fold increase. The combined exposure also led to the neutralization or enhancement of carbon and nitrogen cycle functional microorganisms, shifting from a 1.53-fold inhibition and 10.52-fold increase to a 6.39-fold increase. These findings provide additional insights into the potential risks associated with combined pesticide exposure and MPs, particularly concerning soil microbial communities and elemental cycling processes.

Impact of a glyphosate-based herbicide on the longevity, fertility, and transgenerational effects on Chrysopa pallens (Rambur) (Neuroptera: Chrysopidae).

Glyphosate-based herbicides (GBHs) are common herbicide formulations used in the field and are increasingly used worldwide with the widespread cultivation of herbicide-tolerant genetically modified crops. As a result, the risk of arthropod exposure to GBH is increasing rapidly. Chrysopa pallens (Rambur) (Neuroptera: Chrysopidae) is a common predatory natural enemy in agroecosystems, which is exposed to GBH (Roundup®) while preying on pests. To identify and characterize the potential effects of GBH on C. pallens, the life tables of C. pallens larvae and adults fed with GBH were constructed. Moreover, the effects of GBH treatment on the expression of genes involved in insulin signalling in adults were analyzed using qRT-PCR. The results showed that GBH treatment altered the pupal period and preadult stage of C. pallens larvae. However, it did no effect on longevity, fecundity, and population parameters and two insulin receptor genes (InR1, InR2), a serine/threonine kinase (Akt), an extracellular-signal-regulated kinase (erk), and vitellogenin (Vg1) expression of C. pallens. Adults feeding on GBH significantly altered development, longevity, and differences in the mean generation time of the F0 generation. However, GBH feeding only minimally influenced the growth and population parameters of the F1 generation. In addition, InR1, InR2, erk, and Vg1 expression in the F0 generation were downregulated on the fifth day of feeding on GBH. Furthermore, the expression levels of InR1, InR2, Akt, erk, and Vg1 in C. pallens decreased with the increase of GBH concentration, although the expression levels returned to control levels on the tenth day. Overall, the consumption of the GBH by larvae and adults of C. pallens had minimal effect on the growth and population parameters of C. pallens. The findings of this study can provide a reference for elucidating the environmental risks of GBH, guiding the optimal use of glyphosate in agricultural practices in the future.

Fitness effect and transcription profile reveal sublethal effect of nitenpyram on the predator Chrysopa pallens (Neuroptera: Chrysopidae).

Although neonicotinoids are widely used and important insecticide, there are growing concerns about their effect on nontarget insects and other organisms. Moreover, the effects of nitenpyram (NIT), a second generation of neonicotinoid insecticides, on Chrysopa pallens are still unclear. Therefore, this study purposed to investigate the acute toxicity of NIT to C. pallens using the spotting method. To examine the potential effects of a sublethal dose of NIT (LD30 , 1.85 ng of active ingredient per insect) on C. pallens, we constructed the life tables and analyzed the transcriptome data. The life table results showed that the period of second instar larvae, adult pre-oviposition period and total pre-oviposition period were significantly prolonged after exposure to sublethal dose of NIT, but had no significant effects on the other instars, longevity, oviposition days, and fecundity. The population parameters, including the preadult survival rate, gross reproduction rate, net reproductive rate, the intrinsic rate of increase, and finite rate of increase, were not significantly affected, and only the mean generation time was significantly prolonged by NIT. Transcriptome analysis showed that there were 68 differentially expressed genes (DEGs), including 50 upregulated genes and 18 downregulated genes. Moreover, 13 DEGs related to heat shock protein, nose resistant to fluoxetine protein 6, and prophenoloxidas were upregulated. This study showed the potential effects of sublethal doses of NIT on C. pallens and provided a theoretical reference for the comprehensive application of chemical and biological control in integrated pest management.

Effects of long-term microplastic pollution on soil heavy metals and metal resistance genes: Distribution patterns and synergistic effects.

Heavy metals (HMs) and microplastics (MPs) are two emerging factors threatening global food security. Whether long-term MPs pollution will affect the distribution of HMs and their resistance genes (MRGs) in soil is unknown. Here, metagenomic approach was used to decipher the fate of MRGs in cropland soils with long-term film MPs residues. Similar distribution pattern of MRGs was formed in long-term film MPs contaminated soil. A total of 202 MRG subtypes were detected, with resistance genes for Multimetal, Cu, and As being the most prevalent type of MRGs. MRGs formed a modular distribution of five clusters centered on MRGs including ruvB in long-term film MPs contaminated soil. MRGs also formed tight co-occurrence networks with mobile genetic elements (MGEs: integrons, insertions and plasmids). Redundancy analysis showed that HMs together with microbial communities and MGEs affected the distribution of MRGs in soil. Thirteen genera including Pseudomonas were identified as potential hosts for MRGs and MGEs. The research provides preliminary progress on the synergistic effect of HMs and MPs in affecting soil ecological security. The synergistic effect of MPs and HMs needs to be considered in the remediation of contaminated soils.

Knockdown of CYP9A9 increases the susceptibility to lufenuron, methoxyfenozide and a mixture of both in Spodoptera exigua.

Lufenuron (LUF) and Methoxyfenozide (MET) as Insect Growth Regulators (IGRs) contribute to the current control of the catastrophic crop pest, Spodoptera exigua (Lepidoptera, Noctuidae). Yet S. exigua has evolved resistance to LUF and MET, which is possibly mediated by cytochrome P450 monooxygenases (P450s), particularly from the CYP3 clade family, as it plays a key role in the detoxification of insecticides. However, a mixture of LUF and MET (MML) (optimal ratio: 6:4) remains highly insecticidal. Here, we analysed the response of S. exigua to sublethal concentrations of LUF, MET, and MML via transcriptomics. Twelve differentially expressed genes (DEGs) encoding CYP3 clade members were observed in transcriptomes and CYP9A9 was significantly upregulated after treatment with LUF, MET, and MML. Further, CYP9A9 was most highly expressed in the midgut of L4 S. exigua larvae. RNAi-mediated knockdown of CYP9A9 reduced the activity of CYP450 and increased the susceptibility of S. exigua larvae to LUF, MET, and MML. Thus, CYP9A9 plays a key role in the detoxification of LUF, MET, and MML in S. exigua. These findings provide new insights into insecticidal actions of IGRs, which can be applied to the establishment of novel pest management strategies.

Tracking antibiotic resistance genes in microplastic-contaminated soil.

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.

Microplastics mulch film affects the environmental behavior of adsorption and degradation of pesticide residues in soil.

Plastic pollution in the soil ecosystem is currently receiving worldwide attention. However, little is known whether the presence of microplastics (MPs) in soil will affect the environmental behavior of pesticide residues in soil. Here, the effect of the addition of new mulch MPs (New-MPs), aged mulch MPs (Aged-MPs) and biodegradable mulch MPs (BioD-MPs) on the adsorption and degradation behaviors of two pesticides (imidacloprid and flumioxazin) in soil was investigated. Three MPs slowed down rapid adsorption stage of pesticides in soil and delayed the time to reach adsorption equilibrium. Adsorption rates: Soil > Soil + New-MPs > Soil + Aged-MPs > Soil + BioD-MPs. Three MPs enhanced the adsorption strength of the soil system for the two pesticides, and the aging treatment of the MPs enhanced this effect. Three MPs affected the degradation process of the two pesticides. New-MPs promoted the degradation of two pesticides imidacloprid and flumioxazin, and the degradation half-lives were shortened to 0.93 and 0.85 times, respectively; while Aged-Mps and BioD-MPs delayed the degradation process of two pesticides, and the degradation half-lives were extended to 1.64 times and 1.21 times, respectively. The effect was more significant with the increase of MPs and pesticides concentration. Pesticide polarity, surface structure and functional groups of MPs are potentially important reasons for the differences in adsorption and degradation of MPs-soil systems. Our findings provide a deep insight into understanding the mechanism of interaction between MPs and pesticide residues in soil environment.

Knockdown of UDP-N-acetylglucosamine pyrophosphorylase and chitin synthase A increases the insecticidal efficiency of Lufenuron to Spodoptera exigua.

Spodoptera exigua (Lepidoptera, Noctuidae) has been responsible for causing considerable and widespread agricultural losses worldwide. Owing to strong selective pressure, S. exigua showed increased resistance to Lufenuron (LUF). Consequently, RNA interference (RNAi)-based insecticides had more benefits than chemical insecticides. Therefore, to enhance the insecticidal activity of LUF to S. exigua, in the present study, we aimed to elucidate the impact of double-stranded RNAs (dsRNAs) on S. exigua larval susceptibility to LUF. First, the transcriptome of S. exigua was sequenced following the treatment with LUF. By comparing the upregulated and downregulated GO enrichment, chitin binding and chitin metabolic processes were the significantly enriched pathways. According to transcriptome sequencing, 8 genes associated with chitin biosynthesis, 8 chitin degradation genes, and 17 cuticle protein genes were obtained. UDP-N-acetylglucosamine pyrophosphorylase (UAP) and Chitin synthase A (CHSA) showed significantly downregulated expression after treatment with different sublethal doses of LUF. Downregulation of UAP increased mortality from 31.97% to 47.91% when the larvae were exposed to LUF. A significant increase in the mortality of S. exigua from 30.63% to 50.19% was observed following LUF administration after dsCHSA. In addition, the expression analysis of genes associated with chitin biosynthesis was significantly changed after LUF treatment, dsRNAs-RNAi, and their combination (LUF-dsRNAs). Significant differences were observed in the chitin content between the control group at 72 h after treatments. Results of the present study can help further elucidate the understanding of the combined effects of RNAi and LUF on S. exigua. Additionally, this research provides a suitable foundation for future studies with the aim to develop an efficient method of delivery for large-scale pest control in the fields.

Evaluation of the Toxicity and Sublethal Effects of Acetamiprid and Dinotefuran on the Predator Chrysopa pallens (Rambur) (Neuroptera: Chrysopidae).

Neonicotinoid insecticides affect the physiology or behavior of insects, posing risks to non-target organisms. In this study, the effects of sublethal doses of two neonicotinoid insecticides, acetamiprid and dinotefuran, against Chrysopa pallens (Rambur) (Neuroptera: Chrysopidae) were determined and compared. The results showed that acetamiprid and dinotefuran at LD10 (8.18 ng a.i. per insect and 9.36 ng a.i. per insect, respectively) and LD30 (16.84 ng a.i. per insect and 15.01 ng a.i. per insect, respectively) significantly prolonged the larval stages and pupal stages (except acetamiprid LD10), compared to control. In addition, acetamiprid and dinotefuran at LD30 significantly prolonged the adult preoviposition period (APOP) and total preoviposition period (TPOP). In contrast, the two insecticides at LD10 and LD30 had no significant effect on the longevity, fecundity, reproductive days, preadult survival rate (%), intrinsic rate of increase (r), net reproductive rate (R0), and finite rate of increase (λ). These results provide a theoretical basis for the rational use of these two insecticides and the utilization and protection of C. pallens.

Microbiology combined with metabonomics revealing the response of soil microorganisms and their metabolic functions exposed to phthalic acid esters.

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.

Integrated microbiology and metabolomics analysis reveal plastic mulch film residue affects soil microorganisms and their metabolic functions.

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.

Combined Transcriptomic Analysis and RNA Interference Reveal the Effects of Methoxyfenozide on Ecdysone Signaling Pathway of Spodoptera exigua.

Spodoptera exigua is a worldwide pest afflicting edible vegetables and has developed varying levels of resistance to insecticides. Methoxyfenozide (MET), an ecdysteroid agonist, is effective against lepidopteran pests such as S. exigua. However, the mechanism of MET to S. exigua remains unclear. In this study, we analyzed the expression patterns of genes related to the ecdysone signaling pathway in transcriptome data treated with sublethal doses of MET and analyzed how expression levels of key genes affect the toxicity of MET on S. exigua. Our results demonstrated that 2639 genes were up-regulated and 2512 genes were down-regulated in S. exigua treated with LC30 of MET. Of these, 15 genes were involved in the ecdysone signaling pathway. qPCR results demonstrated that ecdysone receptor A (EcRA) expression levels significantly increased in S. exigua when treated with different doses of MET, and that the RNAi-mediated silencing of EcRA significantly increased mortality to 55.43% at 72 h when L3 S. exigua larvae were exposed to MET at the LC30 dose. Additionally, knocking down EcRA suppressed the most genes expressed in the ecdysone signaling pathway. The combination of MET and dsEcRA affected the expression of E74 and enhanced the expression of TREA. These results demonstrate that the adverse effects of sublethal MET disturb the ecdysone signaling pathway in S. exigua, and EcRA is closely related to MET toxic effect. This study increases our collective understanding of the mechanisms of MET in insect pests.

Midgut de novo transcriptome analysis and gene expression profiling of Spodoptera exigua larvae exposed with sublethal concentrations of Cry1Ca protein.

Spodoptera exigua (Hübner) is a polyphagous pest on agricultural crops, whose control is based mainly on the application of chemical insecticides. Bacillus thuringiensis (Bt) is one of the most important biological agents that have been successfully applied as a biological control, and Cry1Ca protein is considered to be active against S. exigua. Therefore, to understand the response of S. exigua to Cry1Ca protein, high-throughput sequencing was used to analyse the S. exigua larval midgut after treatment with sublethal concentrations of Cry1Ca protein. Transcriptome data showed that a total of 98,571 unigenes with an N50 value of 1135 bp and a mean length of 653 bp were obtained. Furthermore, 2962 differentially expressed genes (DEGs) were identified after Cry1Ca challenge, including 1508 up-regulated and 1454 down-regulated unigenes. Among these DEGs, detoxification (CYP, CarE, and GST) and Bt resistance (ALP, APN, and ABC transporter)-related genes were differentially expressed in the midgut of S. exigua after Cry1Ca treatment. However, most DEGs of protective enzymes were down-regulated, while most DEGs related with serine protease and REPAT were up-regulated. Furthermore, almost all DEGs related to the immune signaling pathway, antimicrobial protein, and lysozyme were up-regulated by Cry1Ca treatment. These results indicated that the detoxification enzyme, protective enzymes, Bt resistance-related genes, serine protease, REPAT, and the immune response might have been involved in the response of S. exigua to Cry1Ca protein. In summary, analysis of the transcriptomal expression of genes involved in Cry1Ca protein against S. exigua provided potential clues for elucidating the host response processes and defensive mechanisms underlying Cry1Ca toxicity.

Three GPI-anchored alkaline phosphatases are involved in the intoxication of Cry1Ca toxin to Spodoptera exigua larvae.

Glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (ALP) isoforms are crucial for the intoxication of crystal proteins (Cry) to several insect species. We cloned five SeALPs from the larval midgut of Spodoptera exigua, a major pest of many crops. All five SeALPs contain a signal peptide at the N-terminus, a phosphatase domain, and a GPI-anchor site at the C-terminus. Additionally, the sequences encode two or three potential N-glycosylation sites. The five SeALPs were highly expressed at the larval stage, especially in the larval gut or Malpighian tubules. Ingestion over four consecutive days of double-stranded RNAs (dsRNAs) targeting SeALP1, SeALP2, SeALP3, SeALP4, and SeALP5 significantly reduced the corresponding mRNA levels by 60.0%, 40.0%, 65.6%, 48.1%, and 69.1% respectively, compared with the levels in control larvae that fed on non-specific dsRNA (dsEGFP). When larvae that previously ingested phosphate buffered saline (PBS)-, dsEGFP-, or five dsSeALPs-overlaid diets were then exposed to a diet containing Cry1Ca, the larval mortalities after six days were 70.0%, 71.8%, 49.1%, 54.9%, 65.3%, 52.5%, and 77.4%, respectively. ANOVA analysis revealed that the larvae that previously fed on the dsSeALP1-, dsSeALP2-, and dsSeALP4-overlaid diets had significantly lower mortalities than those that previously ingested the PBS-, dsEGFP-, dsSeALP3- and dsSeALP5-overlaid diets. The results suggest that SeALP1, SeALP2 and SeALP4 are involved in the intoxication of Cry1Ca to S. exigua larvae.

The Spodoptera exigua (Lepidoptera: Noctuidae) ABCC2 Mediates Cry1Ac Cytotoxicity and, in Conjunction with Cadherin, Contributes to Enhance Cry1Ca Toxicity in Sf9 Cells.

In insects, the mode of Cry1A toxins action has been studied in detail and many receptors that participate in the process are known. Recent evidence has revealed that an ABC transporter (ABCC2) is involved in conferring resistance to Cry1A toxins and that ABCC2 could be a receptor of Cry1A. However, it is not known whether Cry1Ca interacts with the same receptor proteins as Cry1A. In this study, we report the cloning of an ABC transporter gene, SeABCC2b, from the midgut of Spodoptera exigua (Hübner) larvae, and its expression in Sf9 cells for a functional analysis. The addition of Cry1Ca and Cry1Ac to Sf9 cell culture caused swelling in 28.5% and 93.9% of the SeABCC2-expressing cells, respectively. In contrast, only 7.4% and 1.3% of the controls cells swelled in the presence of Cry1Ca and Cry1Ac. Thus, SeABCC2b-expressing Sf9 cells had increased susceptibility to Cry1Ca and Cry1Ac. Similarly, S. exigua cadherin (SeCad1b) expressed in Sf9 cells caused 47.1% and 1.8% of the SeCad1b-expressing cells to swell to Cry1Ca and Cry1Ac exposure. Therefore, Sf9 cells expressing SeCad1b were more sensitive to Cry1Ca than Cry1Ac. Together, our data suggest that SeABCC2b from S. exigua mediates Cry1Ac cytotoxicity and, in conjunction with SeCad1b, contributes to enhance Cry1Ca toxicity in Sf9 cells.

A novel molecule Me6TREN promotes angiogenesis via enhancing endothelial progenitor cell mobilization and recruitment.

Critical limb ischaemia is the most severe clinical manifestation of peripheral arterial disease. The circulating endothelial progenitor cells (EPCs) play important roles in angiogenesis and ischemic tissue repair. The increase of circulating EPC numbers by using mobilization agents is critical for obtaining a better therapeutic outcome in patients with ischemic disease. Here, we firstly report a novel small molecule, Me6TREN (Me6), can efficiently mobilize EPCs into the blood circulation. Single injection of Me6 induced a long-lasting increase in circulating Flk-1(+) Sca-1(+) EPC numbers. In a mouse hind limb ischemia (HLI) model, local intramuscular transplantation of these Me6-mobilized cells accelerated the blood flow restoration in the ischemic muscles. More importantly, systemic administration of Me6 notably increased the capillary density, arteriole density and regenerative muscle weight in the ischemic tissue of HLI. Mechanistically, we found Me6 reduced stromal cell-derived factor-1α level in bone marrow by up-regulation of matrix metallopeptidase-9 expression, which allowed the dissemination of EPCs into peripheral blood. These data indicate that Me6 may represent a potentially useful therapy for ischemic disease via enhancing autologous EPC recruitment and promote angiogenesis.

RNA interference-mediated knockdown of three putative aminopeptidases N affects susceptibility of Spodoptera exigua larvae to Bacillus thuringiensis Cry1Ca.

Aminopeptidase N (APN) isoforms in insects have been documented to be involved in the mode of action of insecticidal crystal proteins (Cry) from Bacillus thuringiensis. Here we cloned two novel Seapns from the larval midgut of Spodoptera exigua, a major pest of many crops of economic importance in China. According to a phylogenetic analysis, these two novel SeAPNs, along with the four SeAPN isoforms already described, belong to six different clades. All the six SeAPNs share similar structural features. From N- to C-terminus a signal peptide, a gluzincin aminopeptidase motif, a zinc binding/gluzincin motif, and a glycosylphosphatidylinositol-anchor sequence are located. The six Seapn genes were highly expressed at the larval stage, especially in the larval gut. Ingestion during four consecutive days of double-stranded RNAs (dsRNAs) targeting Seapn1, Seapn2, Seapn3, Seapn4, Seapn5 and Seapn6 significantly reduced corresponding mRNA levels by 55.6%, 45.5%, 43.2%, 56.8%, 45.4%, and 46.0% respectively, compared with those recorded in control larvae fed on non-specific dsRNA (dsegfp). When the larvae that previously ingested phosphate buffered saline (PBS)-, dsegfp-, or six dsSeapns-overlaid diets were then exposed to a diet containing Cry1Ca, the larval mortalities were 71.2%, 69.3%, 52.0%, 77.2%, 43.3%, 62.0%, 65.4% and 53.8% respectively recorded after 6days. ANOVA analysis revealed that the larvae previously fed on dsSeapn1-, dsSeapn3-, and dsSeapn6-overlaid diets had significantly lower mortalities than those previously ingested PBS-, dsegfp-, dsSeapn2-, dsSeapn4- and dsSeapn5-overlaid diets. Thus, these results suggest that SeAPN1, SeAPN3 and SeAPN6 may be candidate receptors for Cry1Ca in S. exigua.

A cadherin-like protein from the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) is a putative Cry1Ac receptor.

In S. exigua, ingestion of Cry1Ac reduces larval growth, shortens lifespan, and decreases copulation and oviposition of the adults. Cadherin-like protein SeCad1b in S. exigua has recently been published. Here, we tested whether SeCad1b mediates the negative effects of Cry1Ac. We identified three potential Cry toxin binding regions in SeCad1b, i.e., (879) EIAIQITDTNN(889) , (1357) SLLTVTI(1363) , and (1436) GVISLNFQ(1443) . We expressed and purified a truncated cadherin, rSeCad1bp, and its interspecific homologue, rHaBtRp, from H. armigera that contain the putative toxin binding regions. Using a toxin overlay assay, we found that rSeCad1bp specifically binds to biotinylated Cry1Ac in a dose-dependent manner. We also discovered that an addition of rSeCad1bp and rHaBtRp enhances the suppression of larval growth by Cry1Ac, although rSeCad1bp is less suppressive than rHaBtRp. Finally, RNA interference-mediated knockdown of SeCad1b reduced approximately 80% of the target gene and significantly alleviated the negative effect of CrylAc on larval growth. We infer that the S. exigua SeCad1b is a functional receptor of Cry1Ac.