Effects of Nitrogen Supply on Induced Defense in Maize (Zea mays) against Fall Armyworm (Spodoptera frugiperda).

How nitrogen (N) supply affects the induced defense of plants remains poorly understood. Here, we investigated the impacts of N supply on the defense induced in maize (Zea mays) against the fall armyworm (Spodoptera frugiperda). In the absence of herbivore attack or exogenous jasmonic acid (JA) application, N supply increased plant biomass and enhanced maize nutrient (soluble sugar and amino acid) contents and leaf area fed by S. frugiperda (the feeding leaf area of S. frugiperda larvae in maize supplemented with 52.2 and 156.6 mg/kg of N was 4.08 and 3.83 times that of the control, respectively). When coupled with herbivore attack or JA application, maize supplemented with 52.2 mg/kg of N showed an increased susceptibility to pests, while the maize supplemented with 156.6 mg/kg of N showed an improved defense against pests. The changes in the levels of nutrients, and the emissions of volatile organic compounds (VOCs) caused by N supply could explain the above opposite induced defense in maize. Compared with herbivore attack treatment, JA application enhanced the insect resistance in maize supplemented with 156.6 mg/kg of N more intensely, mainly reflecting a smaller feeding leaf area, which was due to indole emission and two upregulated defensive genes, MPI (maize proteinase inhibitor) and PAL (phenylalanine ammonia-lyase). Hence, the optimal N level and appropriate JA application can enhance plant-induced defense against pests.

Effects of Salicylic Acid Concentration and Post-Treatment Time on the Direct and Systemic Chemical Defense Responses in Maize (Zea mays L.) Following Exogenous Foliar Application.

Salicylic acid (SA) plays a critical role in allergic reactions of plants to pathogens and acquired systemic resistance. Thus far, although some research has been conducted on the direct effects of different concentrations of SA on the chemical defense response of treated plant parts (leaves) after at multiple post-treatments times, few research has reported on the systematic effects of non-treated parts (roots). Therefore, we examined direct and systemic effects of SA concentration and time following foliar application on chemical defense responses in maize variety 5422 with two fully expanded leaves. In the experiments, maize leaves were treated with different SA concentrations of 0.1, 0.5, 1.0, 2.5, 5.0 mM, and then, the presence of defense chemicals and enzymes in treated leaves and non-treated roots was measured at different time points of 3, 12, 24, 48, 72 h following SA foliar application. The results showed that direct and systemic effects of SA treatment to the leaf on chemical defense responses were related to SA concentration and time of measurement after spraying SA. In treated leaves, total phenolics content increased directly by 28.65% at the time point of 12 h following foliar application of 0.5 mM SA. DIMBOA (2,4-dihydroxy-7-methoxy-2H, 1, 4-benzoxazin-3 (4H)-one) content was directly enhanced by 80.56~551.05% after 3~72 h following 0.5~5.0 mM SA treatments. Polyphenol oxidase and superoxide dismutase activities were directly enhanced after 12~72 h following 0.5~5.0 mM SA treatments, whereas peroxidase and catalase activities were increased after 3~24 h following application of 1.0~5.0 mM SA. In non-treated roots, DIMBOA content and polyphenol oxidase activity were enhanced systematically after 3~48 h following 1.0~5.0 mM SA foliar treatments. Superoxide dismutase activities were enhanced after 3~24 h following 0.5~2.5 mM SA applications, but total phenolics content, peroxidase and catalase activity decreased in some particular concentrations or at the different times of measurement in the SA treatment. It can be concluded that SA foliar application at 1.0 and 2.5 mM produces strong chemical defense responses in maize, with the optimal induction time being 24 h following the foliar application.

Lead stress affects the reproduction of Spodoptera litura but not by regulating the vitellogenin gene promoter.

Lead (Pb) stress affects hormone-mediated responses (e.g., reproduction) in insects. In this study, the effects of Pb stress (12.5-50 mg Pb/kg in larval artificial diets) on the reproduction of the common cutworm Spodoptera litura (Lepidoptera: Noctuidae) were investigated after 7 generations. The results showed that Pb stress did not reduce the longevity of adult females, but 50 mg Pb/kg significantly reduced the longevity of adult males, regardless of the generation. After 50 mg Pb/kg stress for one or 7 generations, the peak time of egg-laying was delayed, and egg production and hatchability were decreased significantly. The vitellin content in eggs was significantly inhibited by Pb stress. The S. litura vitellogenin (Vg) gene promoter was cloned and analyzed. Multiple putative transcription factors were predicted for the 2321 bp Vg promoter region, including the TATA box, GATA, basic helix-loop-helix (bHLH) transcription factor, Broad-Complex (BR-C) binding sites, etc. The fragment from -2222 to -211 bp of the Vg promoter was the activation domain for Vg, whereas the region from -211 to -55 bp repressed the activity of the Vg promoter. The construct promoter (-782/+76) in Trichoplusia ni (Hi5) cells significantly improved Vg expression, which was not affected by Pb stress (1 or 10 mg/ml). Therefore, Pb stress significantly inhibited the reproduction of S. litura but not by regulating the Vg promoter.

Cabbage cultivars influence transfer and toxicity of cadmium in soil-Chinese flowering cabbage Brassica campestris-cutworm Spodoptera litura larvae.

We executed a pot experiment to examine the differences of absorption, chemical forms, subcellular distribution, and toxicity of Cd between two cultivars of Chinese flowering cabbage Brassica campestris [Lvbao701 (low-Cd cultivar) and Chicaixin No.4 (high-Cd cultivar)]. Compared to Chicaixin No.4, the presence of Lvbao701 enhanced the proportion of insoluble Cd forms in soil, Lvbao701 roots and leaves had higher proportion of Cd converted into insoluble phosphate precipitates and pectate-or protein-bound forms and lower proportion of inorganic Cd, which result in low accumulation and toxicity of Cd to Lvbao701 and cutworm Spodoptera litura fed on Lvbao701 leaves. Instead of total Cd, Cd transfer and toxicity in B. campestris-S. litura system depend on chemical Cd forms in soil and cabbages and subcellular Cd distributions in cabbages and insects, and the proportions of them were not the highest among all chemical forms and subcellular distributions of Cd. Although exchangeable Cd was major Cd chemical form in cabbage planted soil, Cd bound to iron and manganese oxides and to organic matter were significantly correlated with growth indices and photosynthesis parameters of cabbages. Despite major part of Cd was precipitated in cell wall of roots, Cd in organelle fraction was closely associated with the fitness of cabbages. Metal-rich granules, not cytosolic fraction (the major subcellular Cd distribution), affected the food utilization of S. litura. Therefore, cabbage cultivars significantly affected Cd transfer and toxicity in B. campestris-S. litura system, and the use of Lvbao701 in Cd polluted soil could reduce potential risks for Cd entering food chains.

Accumulation and excretion of zinc and their effects on growth and food utilization of Spodoptera litura (Lepidoptera: Noctuidae).

By exposing larvae of the holometabolous insect Spodoptera litura to the artificial diets supplemented with a range of Zinc (Zn) contents, we investigated Zn ingestion, excretion and accumulation in the insect throughout its life cycle. The effects of Zn stress on the survival, growth and food utilization of S. litura were also determined. Zn concentrations in the body (larvae, pupae, and adults), faeces, exuviates, puparium, eggs increased with the increasing Zn concentrations in the diets, while Zn excretion and accumulation by S. litura in 750 mg/kg Zn treatments was lower than the 600 mg/kg Zn treatment. In the 450 mg/kg Zn treatment, the Zn accumulation in S. litura at different developmental stages differed as follows: larvae > pupa > adult. S. litura ingested Zn via feeding and could excrete most of the Zn via faeces (compared with Zn excretion via exuviates) to reduce its internal Zn accumulation (compared with Zn ingestion). Survival and weight were significantly inhibited, and the prolonged period of development (larvae, pupae) and shortened longevity of adults were found in S. litura exposed to Zn stress greater than 450 mg Zn/kg. In the 150-450 mg/kg Zn treatments, the 6th instar larvae increased their relative consumption rate (RCR) and approximate digestibility (AD) (namely, food eaten) to gain weight, which resulted in greater Zn accumulation in the body. Therefore, below the threshold level (being close to 450 mg/kg Zn), S. litura seemed to have a strong homeostatic adjustment ability (increase the amount of food eaten, thereby increasing AD, RCR and Zn excretion via faeces and exuviates) to sustain their weight, and Zn was beneficial and harmless. Although larvae treated with 750 mg/kg Zn had a similar RCR and AD as the control, a reduced weight gain and prolonged larval period resulted in significantly lower relative growth rate (RGR), which indicated surviving insects may allocate more energy from foods for detoxification than for growth.

Effects of Cry1Ab-expressing Bt rice straw return on juvenile and adult Eisenia fetida.

A 90 day experiment was conducted in the laboratory to investigate the potential effects of transgenic Cry1Ab-expressing rice (Bacillus thuringiensis (Bt) rice: T775 and its F1 hybrid) straw return on earthworm Eisenia fetida, compared to non-Bt rice (TYHZ) straw. Juvenile E. fetida could survive, grow up, mature and reproduce offspring well in a Bt rice treated test during the whole experiment. The significantly higher relative growth rate (RGR) was found in earthworms from Bt rice treatment than from non-Bt rice treatment on the 7th day. The period of sexual maturity for earthworms from Bt rice treatments was shortened significantly, compared to non-Bt rice treatments. Adult E. fetida survived with weight loss under Bt rice treatments. On the 7th and 15th day, earthworm RGR decreased and glutathione peroxidase (GSH-PX) activity increased under Bt rice straw treatments. Significantly fewer offspring were produced by earthworms from Bt rice than non-Bt rice treatments on the 60th and 75th day. Enzyme-linked immunosorbent assay (ELISA) determined a sharp decrease of Cry1Ab in straw mixed soil along with the experimental time, regardless of juvenile or adult earthworm treatments. Cry1Ab concentration in the earthworms from the juvenile group was significantly higher than those from the adult group. Bt rice straw return had significant effects on soil nutrients, especially on the content of total and available phosphorus. In view of two bioassays, Bt rice (T775 and its F1 hybrid) straw return presented different effects on E. fetida from the juvenile (no deleterious effect) and adult (a little negative effect) groups, that were not directly related to Cry1Ab presence and nutrient differences among the three rice variety treatments.

Stacked Bt maize and arthropod predators: exposure to insecticidal Cry proteins and potential hazards.

Genetically engineered (GE) crops with stacked insecticidal traits expose arthropods to multiple Cry proteins from Bacillus thuringiensis (Bt). One concern is that the different Cry proteins may interact and lead to unexpected adverse effects on non-target species. Bi- and tri-trophic experiments with SmartStax maize, herbivorous spider mites (Tetranychus urticae), aphids (Rhopalosiphum padi), predatory spiders (Phylloneta impressa), ladybeetles (Harmonia axyridis) and lacewings (Chrysoperla carnea) were conducted. Cry1A.105, Cry1F, Cry3Bb1 and Cry34Ab1 moved in a similar pattern through the arthropod food chain. By contrast, Cry2Ab2 had highest concentrations in maize leaves, but lowest in pollen, and lowest acquisition rates by herbivores and predators. While spider mites contained Cry protein concentrations exceeding the values in leaves (except Cry2Ab2), aphids contained only traces of some Cry protein. Predators contained lower concentrations than their food. Among the different predators, ladybeetle larvae showed higher concentrations than lacewing larvae and juvenile spiders. Acute effects of SmartStax maize on predator survival, development and weight were not observed. The study thus provides evidence that the different Cry proteins do not interact in a way that poses a risk to the investigated non-target species under controlled laboratory conditions.

Biomagnification of persistent organic pollutants (POPs) in detritivorous, phytophagous, and predatory invertebrates: How POPs enter terrestrial food web?

Invertebrates are primary contributors to fluxes of nutrients, energy, and contaminants in terrestrial food webs, but the trophodynamic of contaminants in invertebrate food chains is not fully understood. In this study, occurrence and biomagnification of persistent organic pollutants (POPs) were assessed in detritivorous, phytophagous, and predatory invertebrate food chains. Detritivorous species (earthworm and dung beetle) have higher concentrations of POPs than other species. Different composition patterns and biomagnification factors (BMFs) of POPs were observed for invertebrate species. Negative correlations were found between BMFs and log KOW of POPs for detritivorous and most phytophagous species. In contrast, parabolic relationships between BMFs and log KOW were observed in snails and predatory species, possibly attributed to the efficient digestion and absorption of diet and POPs for them. Bioenergetic characteristics are indicative of the biomagnification potential of POPs in terrestrial wildlife, as suggested by the significant and positive correlation between basal metabolic rates (BMRs) and BMFs of BDE 153 for invertebrates, amphibians, reptiles, birds, and mammals. The estimations of dietary exposure suggest that the terrestrial predators, especially feeding on the underground invertebrates, could be exposed to high level POPs from invertebrates.

Defense of cabbages against herbivore cutworm Spodoptera litura under Cd stress and insect herbivory stress simultaneously.

Biotic (e.g., heavy metal) and abiotic stress (e.g., insect attack) can affect plant chemical defense, but little is known about the changes in plant defense when they occur concurrently. Herein, the impacts of heavy metal cadmium (Cd) stress and insect herbivory stress on the direct and indirect defense of two cultivar cabbages of Brassica campestris, the low-Cd cultivar Lvbao701 and the high-Cd cultivar Chicaixin No.4, against the herbivore cutworm Spodoptera litura were investigated. Although 10 mg kg-1 Cd stress alone inhibited leaf secondary metabolites (total phenolics, flavonoids), it reduced the feeding rate and odor selection of S. litura towards both cultivar cabbages, especially for Lvbao701, by increasing leaf Cd content and repellent volatile organic compounds (VOCs) (6-methyl-5-hepten-2-one, 7,9-di-tert-butyl-1-oxaspiro (4,5)deca-6,9-diene-2,8-dione), and reducing soluble sugar and attractive VOCs (3-methyl-3-pentanol, 2,5-hexanedione, tetradecanal). Under 2.5 mg kg-1 Cd and herbivory stress, although leaf total phenolics and flavonoids increased significantly, the feeding rate and odor selection of S. litura towards both cultivar cabbages increased, especially for Chicaixin No.4, indicating that the chemical defense of cabbages was depressed. Therefore, Cd stress alone improved the insect resistance of cabbages, whereas herbivory stress weakened the enhanced cabbages defence by Cd stress. The low-Cd cultivar Lvbao701 presented stronger insect resistance than Chicaixin No.4, suggesting that Lvbao701 application in Cd-polluted soil can not only decrease Cd transmission to higher levels in the food chain but also reduce pest occurrence.

Characterization of the complete chloroplast genome of Firmiana hainanensis (Malvaceae), an endemic and vulnerable tree species of China.

Firmiana hainanensis Kosterm. is a commercially valuable endemic tree species in China and has long been considered a globally vulnerable species. We assembled and characterized the complete chloroplast genome of this species by using Illumina pair-end sequencing data. The total chloroplast genome size was 161,559 bp, including two inverted repeats (IRs) of 25,612 bp, separated by a large single copy (LSC) and a small single copy (SSC) regions of 90,057 and 20,277 bp, respectively. A total of 130 genes were identified, including 85 protein-coding genes, 37 tRNA, and eight rRNA genes. Phylogenetic analysis showed that F. hainanensis was the most basal species in the genus Firmiana. The chloroplast genome of this species will provide a theoretical basis to understand the taxa's evolution further and is expected to contribute to its conservation efforts.

Effects of Exogenous Salicylic Acid Application to Aboveground Part on the Defense Responses in Bt (Bacillus thuringiensis) and Non-Bt Corn (Zea mays L.) Seedlings.

Bt (Bacillus thuringiensis) corn is one of the top three large-scale commercialized anti-insect transgenic crops around the world. In the present study, we tested the Bt protein content, defense chemicals contents, and defense enzyme activities in both the leaves and roots of Bt corn varieties 5422Bt1 and 5422CBCL, as well as their conventional corn 5422 seedlings, with two fully expanded leaves which had been treated with 2.5 mM exogenous salicylic acid (SA) to the aboveground part for 24 h. The result showed that the SA treatment to the aboveground part could significantly increase the polyphenol oxidase activity of conventional corn 5422, the Bt protein content, and peroxidase activities of Bt corn 5422Bt1, as well as the polyphenol oxidase and peroxidase activity of Bt corn 5422CBCL in the leaves. In the roots, the polyphenol oxidase and peroxidase activity of conventional corn 5422, the polyphenol oxidase and superoxide dismutase activities of Bt corn 5422Bt1, the DIMBOA (2,4-dihydroxy-7-methoxy-2H, 1, 4-benzoxazin-3 (4H)-one) content, and four defense enzymes activities of Bt corn 5422CBCL were systematically increased. These findings suggest that the direct effect of SA application to aboveground part on the leaf defense responses in Bt corn 5422CBCL is stronger than that in non-Bt corn. Meanwhile, the systemic effect of SA on the root defense responses in Bt corn 5422CBCL is stronger than that in conventional corn 5422 and Bt corn 5422Bt1. It can be concluded that the Bt gene introduction and endogenous chemical defense responses of corns act synergistically during the SA-induced defense processes to the aboveground part. Different transformation events affected the root defense response when the SA treatment was applied to the aboveground part.

Molecular characterization and expression patterns of Spodoptera litura heat shock protein 70/90, and their response to zinc stress.

Heat shock proteins (hsps) are highly evolutionarily conserved, whose expressions are altered with varying degrees in response to stress factors. Here, hsp70 and hsp90 cDNA were cloned from Spodoptera litura, and two hsps comprised corresponding family protein sequences, signature sequences and EEVD motif. They showed high homology to their counterparts in other species, and the phylogenetic analysis of hsp90 was consistent with the known insect classification. The hsp70/90 transcripts were present in S. litura midgut, cuticle and fat body, of which the highest levels of both were detected in fat body. The expression levels of both were different during four life stages, and the lowest and highest levels were detected in fifth instar larvae and adults, respectively. The hsp70 expression was induced significantly after the insects were exposed to 150 mg Zn/kg, but the down-regulation in other treatments showed significantly negative correlation with Zn accumulations in the body. The hsp90 expression was induced in all treated pupae and 150-450 mg Zn/kg treated larvae, while inhibited effects were observed in larvae exposed to 600-750 mg Zn/kg. Our results showed the induced response of S. litura hsp90 to Zn was more sensitive than hsp70, whereas inhibited response of hsp70 was much stronger than hsp90.

Integration of transcriptome and proteome reveals molecular mechanisms underlying stress responses of the cutworm, Spodoptera litura, exposed to different levels of lead (Pb).

Heavy metals are major environmental pollutants that affect organisms across different trophic levels. Herbivorous insects play an important role in the bioaccumulation, and eventually, biomagnification of these metals. Although effects of heavy metal stress on insects have been well-studied, the molecular mechanisms underlying their effects remain poorly understood. Here, we used the RNA-Seq profiling and isobaric tags for relative and absolute quantitation (iTRAQ) approaches to unravel these mechanisms in the polyphagous pest Spodoptera litura exposed to lead (Pb) at two different concentrations (12.5 and 100 mg Pb/kg; PbL and PbH, respectively). Altogether, 1392 and 1630 differentially expressed genes (DEGs) and 58, 114 differentially expressed proteins (DEPs) were identified in larvae exposed to PbL and PbH, respectively. After exposed to PbL, the main up-regulated genes clusters and proteins in S. litura larvae were associated with their metabolic processes, including carbohydrate, protein, and lipid metabolism, but the levels of cytochrome P450 associated with the pathway of xenobiotic biodegradation and metabolism were found to be decreased. In contrast, the main up-regulated genes clusters and proteins in larvae exposed to PbH were enriched in the metabolism of xenobiotic by cytochrome P450, drug metabolism-cytochrome P450, and other drug metabolism enzymes, while the down-regulated genes and proteins were found to be closely related to the lipid (lipase) and protein (serine protease, trypsin) metabolism and growth processes (cuticular protein). These findings indicate that S. litura larvae exposed to PbL could enhance food digestion and absorption to prioritize for growth rather than detoxification, whereas S. litura larvae exposed to PbH reduced food digestion and absorption and channelized the limited energy for detoxification rather than growth. These contrasting results explain the dose-dependent effects of heavy metal stress on insect life-history traits, wherein low levels of heavy metal stress induce stimulation, while high levels of heavy metal stress cause inhibition at the transcriptome and proteome levels.

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Heavy metal pollution is one of the thorniest issues in the world, which is a serious threat to ecosystems and food security. As an important link of food chain and food web in the ecosystem, herbivorous insects play important role in the transfer and accumulation of heavy metals. Consequently, more and more attentions have been paid on the potential effects of heavy metal pollution on herbivorous insects. Here, we reviewed the effects of heavy metal pollution on herbivorous insects with literature publshied during 2007 to 2018. Herbivorous insects were exposed to heavy metals pollution via four ways, including insect diets or food added with inorganic heavy metals, field exposure with heavy metals, "soil/substrate-plants-herbivorous insect" food chain and in vitro injection. Excessive accumulation of heavy metals in insect body results in decreased survival rate, reproductive capacity and population growth, the retarded growth and development. Physiological and biochemical toxicity of heavy metal pollution to herbivorous insects included cell ultra-structure destruction and DNA damage, decreased amount of energy materials, and changes in enzymes acti-vity and gene expression levels. However, herbivorous insects can resist heavy metal stress by producing metallothionein and enhancing activity of detoxification enzymes etc., which probably results in their adaptive evolution to heavy metal stress at low intensity or long-term and even improve their tolerance to other stresses (e.g., pesticides).

Sugarcane/soybean intercropping with reduced nitrogen input improves crop productivity and reduces carbon footprint in China.

Global climate change and decreases in available land are significant challenges that humans are currently facing. Alternative management approaches for sugarcane fields have great potential to help mitigate these problems in China. We hypothesized that soybean intercropping with reduced nitrogen input could increase crop productivity and reduce the carbon footprint (CF) of sugarcane fields in China. Therefore, a long-term field experiment from 2009 to 2017 in the Pearl River Delta of China was chosen to test this hypothesis. The results showed that the energy yields of sugarcane/soybean intercropping systems were 17.8%-39.4% higher than those of sugarcane monocropping systems. The energy yields of the same cropping systems using conventional and reduced N inputs (525 kg ha-1 and 300 kg ha-1) did not show a significant difference. Additionally, the CF values of the unit yield (CFY) for sugarcane/soybean intercropping were 3.2%-30.4% lower than those of the monocropping systems, showing the higher CF efficiency of the intercropping pattern, although the difference was not significant. The CF of the unit area (CFA) and the CFY of all the cropping patterns at the conventional N level were 19.5%-62.0% higher than that at the reduced N level, demonstrating that reducing the nitrogen input could significantly lower the CF of the sugarcane fields. In addition, the high N level cased negative effects in terms of increasing the crop productivity and reducing the CF of the soybean/sugarcane intercropping pattern. In conclusion, sugarcane/soybean intercropping with reduced N input improved crop productivity while lowering the CF of sugarcane fields in China. The sugarcane/soybean (1:2) intercropping with 300 kg N ha-1 system showed the best benefits in the Pearl River Delta of China. These advanced agricultural practices contributed to improved farmland use efficiency and clean production in an agricultural system.

Effects of zinc exposure on the reproduction of Spodoptera litura Fabricius (Lepidoptera: Noctuidae).

Reproductive toxicity of Zn to insects was investigated in this study. By exposing phytophagous insect Spodoptera litura Fabricius to Zn in artificial diets of larvae, we investigated the effects of Zn on reproduction at ecological and molecular levels. A significantly shorter period of laying eggs was observed in S. litura exposed to 300-750mg Zn/kg. The oviposition rate, fecundity and hatchability of female adults treated with 750mg Zn/kg were significantly lower than those of the controls (31.43%, 20.95% and 52%, respectively, compared to the control). The Zn accumulation and vitellin (Vn) content in eggs were tested by inductively coupled plasma-atomic emission spectrometry and Bradford combining Western-blot, respectively. The results showed that Zn accumulated in the eggs, which has affected the weight and Vn content of eggs with significant negative correlations. The down-regulated expression levels of vitellogenin (Vg) mRNA were detected by real-time polymerase chain reaction (RT-PCR): the relative quantity of Vg mRNA was less than half of the controls at higher than 450mg Zn/kg wet weight. These results indicated that excess Zn made expression of Vg gene down-regulated and caused poor accumulation of egg yolk, which led to a reduction in egg numbers and failure of eggs to hatch.

No Interactions of Stacked Bt Maize with the Non-target Aphid Rhopalosiphum padi and the Spider Mite Tetranychus urticae.

In the agroecosystem, genetically engineered plants producing insecticidal Cry proteins from Bacillus thuringiensis (Bt) interact with non-target herbivores and other elements of the food web. Stacked Bt crops expose herbivores to multiple Cry proteins simultaneously. In this study, the direct interactions between SmartStax® Bt maize producing six different Cry proteins and two herbivores with different feeding modes were investigated. Feeding on leaves of Bt maize had no effects on development time, fecundity, or longevity of the aphid Rhopalosiphum padi (Hemiptera: Aphididae), and no effects on the egg hatching time, development time, sex ratio, fecundity, and survival of the spider mite Tetranychus urticae (Acari: Tetranychidae). The results thus confirm the lack of effects on those species reported previously for some of the individual Cry proteins. In the Bt maize leaves, herbivore infestation did not result in a consistent change of Cry protein concentrations. However, occasional statistical differences between infested and non-infested leaves were observed for some Cry proteins and experimental repetitions. Overall, the study provides evidence that the Cry proteins in stacked Bt maize do not interact with two common non-target herbivores.

The Influence of Bt Maize Cultivation on Communities of Arbuscular Mycorrhizal Fungi Revealed by MiSeq Sequencing.

The cultivation of transgenic Bacillus thuringiensis (Bt) has received worldwide attention since Bt crops were first released. Its ecological risks on arbuscular mycorrhizal fungi (AMF) have been widely studied. In this study, after cultivation for five seasons, the AMF diversity and community composition of two Bt maize varieties, 5422Bt1 (event Bt11) and 5422CBCL (event MO10), which both express Cry1Ab protein, and their isoline non-Bt maize 5422, as well as Bt straw after cultivation had been returned to subsequent conventional maize variety, were analyzed using Illumina MiSeq sequencing. A total of 263 OTUs (operational taxonomic units) from 511,847 sequenced affiliated with the AMF which belonged to Mucoromycota phylum Glomeromycotina subphylum were obtained. No significant difference was detected in the AMF diversity and richness (Shannon, Simpson, ACE, and Chao 1 indices) and community composition in rhizosphere soils and roots between Bt and non-Bt treatment revealed by NMDS (non-metric multidimensional scaling) and NPMANOVA (non-parametric multivariate analysis). Moreover, Glomus was the most dominant genus in all samples. Although there was no significant difference in the AMF community in roots and rhizosphere soils between the Bt and non-Bt maize treatments, total phosphorus (TP), total nitrogen (TN), organic carbon (OC), and pH were driving factors affecting the AMF community, and their composition varied between rhizosphere soils and roots during the maturity period of the fifth season. Compared to our previous study, the results were identical. In conclusion, no significant difference was observed between the Bt and non-Bt treatments, and the Illumina MiSeq method had higher throughput and higher quality read cover, which gave us comprehensive insight into AMF communities in agro-ecosystems.

Effects of Cd accumulation on cutworm Spodoptera litura larvae via Cd treated Chinese flowering cabbage Brassica campestris and artificial diets.

By exposing herbivorous cutworm Spodoptera litura (Lepidoptera: Noctuidae) larvae to Cadmium (Cd) stress via Cd treated cabbages Brassica campestris and artificial diets, we investigated effects of Cd accumulation in larvae on their survival and food utilization. The results showed that Cd transferred from soils contaminated with different Cd concentrations through cabbages-larvae, and be mainly accumulated in larvae guts. There was a dose-response relationship between Cd accumulations in larvae cuticle, head, guts and Cd concentrations in artificial diets, and the highest one was found in the guts, regardless of generations. High Cd stress (10 mg kg-1 Cd in soil, 40.6, 81.2 mg kg-1 Cd in artificial diets) had inhibited effects on larvae growth and food utilization, whereas low Cd stress (Lvbao 701 planted in 2.5 mg kg-1 Cd soil, 4.06 mg kg-1 Cd in artificial diets) showed stimulated effect. Cd accumulations in the guts were significantly negative correlated with efficiency of conversion of ingested food (ECI) and relative growth rate (RGR) of larvae feeding on Cd treated diets or cabbages while were significantly positive correlated with relative consumption rate (RCR). Therefore, after S. litura larvae feed on Cd treated natural food or artificial diets, Cd could be transferred to different tissues, and mainly accumulated in the guts, which significantly affected growth and food utilization. Additionally, Cd stress via Cd treated artificial diets presented less detrimental effects on S. litura larvae than via Cd treated cabbages.

Effects of Cry1Ab Bt maize straw return on bacterial community of earthworm Eisenia fetida.

The eco-toxicological effects of Bacillus thuringiensis (Bt) maize on earthworm life-history traits were widely studied and the results were controversial, while their effects on earthworm bacterial community have been rarely studied. Here, effects of two hybrids of Bt maize [5422Bt1 (event Bt11) and 5422CBCL (MON810)] straw return on Eisenia fetida bacterial community were investigated by the terminal restriction fragment length polymorphism (T-RFLP) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) combing with DNA sequencing, compared to near-isogenic non-Bt maize (5422). Bt maize straw return had significant effects on soil nutrients, especially for available nitrogen (N). The significant differences were shown in soil bacterial community between Bt and non-Bt maize treatments on the 75th and 90th d, which was closely correlated with soil available N, P and K rather than Cry1Ab protein. There was no statistically significant difference in the bacterial community of earthworm gut contents between Bt and non-Bt maize treatments. The significant differences in the bacterial community of earthworm casts were found among three maize varieties treatments, which were closely correlated with Cry1Ab protein and N levels. The differentiated bacterial species in earthworm casts mainly belonged to Proteobacteria, including Brevundimonas, Caulobacter, Pseudomonas, Stenotrophomonas, Methylobacterium, Asticcacaulis and Achromobacter etc., which were associated with the mineralization, metabolic process and degradation of plants residues. Therefore, Bt maize straw return caused changes in the bacterial community of E. fetida casts, which was possibly caused by the direct (Cry1Ab protein) and non-expected effects (N levels) of Bt maize straw.