Genotypic variation in field-grown maize eliminates trade-offs between resistance, tolerance and growth in response to high pressure from the Asian corn borer.

Insect herbivory challenges plant survival, and coordination of the interactions between growth, herbivore resistance/tolerance is a key problem faced by plants. Based on field experiments into resistance to the Asian corn borer (ACB, Ostrinia furnacalis), we selected 10 inbred maize lines, of which five were resistant and five were susceptible to ACB. We conducted ACB larval bioassays, analysed defensive chemicals, phytohormones, and relative gene expression using RNA-seq and qPCR as well as agronomic traits, and found resistant lines had weaker inducibility, but were more resistant after ACB attack than susceptible lines. Resistance was related to high levels of major benzoxazinoids, but was not related to induced levels of JA or JA-Ile. Following combination analyses of transcriptome, metabolome and larval performance data, we discovered three benzoxazinoids biosynthesis-related transcription factors, NAC60, WRKY1 and WRKY46. Protoplast transformation analysis suggested that these may regulate maize defence-growth trade-offs by increasing levels of benzoxazinoids, JA and SA but decreasing IAA. Moreover, the resistance/tolerance-growth trade-offs were not observed in the 10 lines, and genotype-specific metabolic and genetic features probably eliminated the trade-offs. This study highlights the possibility of breeding maize varieties simultaneously with improved defences and higher yield under complex field conditions.

Roles of Ear Injury, Infestation, and Vector Activity by Ostrinia furnacalis (Asian Corn Borer) and Conogethes punctiferalis (Yellow Peach Moth) in Fusarium verticillioides Infection and Kernel Fumonisin Level.

Field experiments were conducted to evaluate the roles of two corn borers, Asian corn borer (ACB), Ostrinia furnacalis and yellow peach moth (YPM), Conogethes punctiferalis, in Fusarium verticillioides infection using green fluorescent protein (GFP) as a marker. Effects of insect injury, manual injury, and insecticide application on fumonisin production also were assessed. In this study, third instars of ACB and YPM significantly increased GFP-tagged F. verticillioides infection compared with the control, regardless of the fungal inoculation method. Besides acquiring F. verticillioides spores from leaf surfaces and transmitting them to ears, larvae of the ACB and YPM also injure maize ears, which allows F. verticillioides from leaves or silk to infect ears more easily. This suggests that ACB and YPM larvae are vectors of F. verticillioides, which can increase the occurrence of ear rot. Manual injuries significantly increased GFP-tagged F. verticillioides infection of ears, while effective insect control significantly reduced F. verticillioides infection of ears. Insecticide control of borers also significantly reduced fumonisin content in kernels. Larval infestations significantly increased fumonisins in kernels to levels higher than or very close to the European Union threshold (4,000 µg kg-1). Significant and high correlations among corn borer attack, F. verticillioides severity, and kernel fumonisin levels were discovered, confirming the important role of ACB and YPM activity in F. verticillioides infection and kernel fumonisin production.

Asian Corn Borer (Ostrinia furnacalis) Infestation Increases Fusarium verticillioides Infection and Fumonisin Contamination in Maize and Reduces the Yield.

Field trials based on manual infestation of the Asian corn borer (ACB) (Ostrinia furnacalis [Guenée]) and Fusarium verticillioides (Nirenberg) atomization were conducted on four maize hybrids to investigate the relationship between ACB infestation and F. verticillioides infection, yield loss, and fumonisin contamination in maize. Analysis of fumonisins B1 and B2 was carried out using an LC-MS/MS system. In this study, manual ACB infestation significantly promoted F. verticillioides infection (both symptomatic and symptomless) and grain fumonisin levels. Ear rot incidence and severity, symptomless kernel infection, and fumonisin contamination were significantly correlated to each other and to ACB damage severity. Manual ACB infestation increased fumonisin levels from 580 to 4,418 µg/kg in 2018; 6,059 to 10,681 µg/kg in 2019 spring-sown maize (2019A); and 2,042 to 5,060 µg/kg in 2019 summer-sown maize (2019B), with the threshold of the European Union (EU) being 4,000 µg/kg. The threshold was exceeded in spring of 2019 in untreated controls. Regarding yield, significant negative correlation between ACB damage and ear weight was observed in three seasons. These results indicated that ACB infestation can lead to severe quality degradation and yield loss of maize. Kernel fumonisin levels may exceed the concentration threshold of the EU in certain conditions, threatening the health of livestock and humans. Measures should be taken to reduce ACB infestation to ensure food and feed security.

Revealing the difference of α-amylase and CYP6AE76 gene between polyphagous Conogethes punctiferalis and oligophagous C. pinicolalis by multiple-omics and molecular biological technique.

BACKGROUND: Conogethes pinicolalis has been thought as a Pinaceae-feeding variant of the yellow peach moth, Conogethes punctiferalis. The divergence of C. pinicolalis from the fruit-feeding moth C. punctiferalis has been reported in terms of morphology, ecology, and genetics, however there is a lack of detailed molecular data. Therefore, in this study, we investigated the divergence of C. pinicolalis from C. punctiferalis from the aspects of transcriptomics, proteomics, metabolomics and bioinformatics. RESULTS: The expression of 74,611 mRNA in transcriptome, 142 proteins in proteome and 218 metabolites in metabolome presented significantly differences between the two species, while the KEGG results showed the data were mainly closely related to metabolism and redox. Moreover, based on integrating system-omics data, we found that the α-amylase and CYP6AE76 genes were mutated between the two species. Mutations in the α-amylase and CYP6AE76 genes may influence the efficiency of enzyme preference for a certain substrate, resulting in differences in metabolic or detoxifying ability in both species. The qPCR and enzyme activity test also confirmed the relevant gene expression. CONCLUSIONS: These findings of two related species and integrated networks provide beneficial information for further exploring the divergence in specific genes, metabolism, and redox mechanism. Most importantly, it will give novel insight on species adaptation to various diets, such as from monophagous to polyphagous.

Seed Myco-priming improves crop yield and herbivory induced defenses in maize by coordinating antioxidants and Jasmonic acid pathway.

BACKGROUND: Seed Myco-priming based on consortium of entomopathogenic fungi is very effective seed treatment against Ostrinia furnacalis herbivory. Maize regulates defense responses against herbivory by the production of defense-related enzymatic and non-enzymatic antioxidants, phytohormones, and their corresponding genes. Jasmonic acid (JA) plays a key role in plant-entomopathogenic fungi-herbivore interaction. RESULTS: To understand how a consortium of the entomopathogenic fungi Beauveria bassiana and Trichoderma asperellum induce changes in the response of maize to herbivory and increase the crop yield, 2-year field experiment, antioxidant enzymes, leaf transcriptome, and phytohormone were performed. Fungal inoculation enhanced the production of antioxidant enzymes and JA signaling pathway more than the normal herbivory. The comparison between single inoculated, consortium inoculated, and non-inoculated plants resulted in distinct transcriptome profiles representing a considerable difference in expression of antioxidant- and JA- responsive genes identified through Weighted gene co-expression network analysis (WGCNA) and expression analysis, respectively. Seed priming with a consortium of B. bassiana and T. asperellum significantly enhanced the expression of genes involved in antioxidants production and JA biosynthesis cascade, with the highest expression recorded at 24-h post O. furnacalis larval infestation. They reduced the larval nutritional indices and survival up to 87% and enhancing crop yield and gross return up to 82-96% over the year 2018 and 2019. CONCLUSION: From our results we suggest that a consortium of B. bassiana and T. asperellum can be used synergistically against O. furnacalis in maize under field condition and can mediate antioxidants- and JA- associated maize defense response by boosting up the expression of their responsive genes, thereby enhancing crop yield.

Toxicity of Cry1-Class, Cry2Aa, and Vip3Aa19 Bt proteins and their interactions against yellow peach Moth, Conogethes punctiferalis (Guenée) (Lepidoptera: Crambidae).

Transgenic plants expressing insecticidal proteins from the Bacillus thuringiensis (Bt) have provided an effective way to control target pests. However, the toxicity of Bt proteins against yellow peach moth (YPM), Conogethes punctiferalis (Guenée), one of the most serious maize pests in China, has not received much study. Therefore, we performed diet-overlay bioassays to evaluate the insecticidal activities of Cry1Ab, Cry1Ac, Cry1Fa, Cry1Ah, Cry1Ie, Cry2Aa, and Vip3Aa19, as well as the interaction between Cry1-Class, Cry2Aa, and Vip3Aa19 against YPM. Results showed that the LC50 values ranged from 1.08 to 178.12 ng/cm2 (protein/diet). Among these proteins, Cry1Ab and Cry1Ac had lower LC50 values and LC90 values. In YPM bioassays, the combinations of Cry2Aa with Cry1Ac, Cry1Ie, and Cry1Ab showed antagonism while a mixture of Cry2Aa with Cry1Fa and Cry1Ah exhibited synergism. When Vip3Aa19 was combined with Cry proteins, all combinations interacted positively, with variation in synergistic factors (SF). Three ratios 1:1, 1:2, and 2:1 of Cry1Ah and Vip3Aa19 protein combination showed SF values of 5.20, 5.63, and 8.98, respectively. These findings can be applied in the establishment of new pyramided transgenic crops with suitable candidates as well as in resistance management strategies.

Phenotypic responses and potential genetic mechanism of lepidopteran insects under exposure to graphene oxide.

Clarification of the interactions between engineered nanomaterials and multiple generations of insects is crucial to understanding the impact of nanotechnology on the environment and agriculture, particularly in toxicity management, pest management and genetic engineering. To date, there has been very limited information about nanoparticle-insect interactions at the genetic and proteomic levels. Here, we examined the phenotypic responses and potential mechanism of a lepidopteran insect Asian corn borer (ACB) to graphene oxide (GO). It was demonstrated that GO could significantly promote the growth of ACB. The transcriptomic and proteomic results consistently verified that GO might activate trypsin-like serine protease, glutathione S-transferase, heat shock protein and glycosyltransferase to further influence the development of ACB. RNA interference results indicated that the trypsin gene was one of the critical genes to accelerate the growth of ACB fed with GO diet. Moreover, physiological analysis showed potential alterations of the expression levels of genes and proteins, and more cholesterol (CE), triacylglycerides (TG) and lipids were accumulated in GO-exposed ACB. Our findings may help to reveal the phenotypic, physiological and genetic responses of insects under exposure to nanomaterials and to assess the environmental risks of other nanomaterials.

Effects of brief exposure to high temperatures on the survival and fecundity of Athetis lepigone (Lepidoptera: Noctuidae).

In Huang-Huai-Hai Summer Corn Region of China, brief periods of high summer temperatures have been reported with increasing frequency in recent years. Athetis lepigone is a cosmopolitan insect which causes severe damage on summer corn seedlings. To understand how high summer temperatures may affect the population dynamics of A. lepigone, we exposed different developmental stages (1, 2 and 4-day old eggs; 1, 6, 12 and 18-day old larvae; 1, 3 and 6-day old pupae; and 1 and 2-day old female and male adults) to 41 °C for periods of various length (0.5, 1, 2, 4 and 6 h): The rearing temperature (constant 26 °C) was used as control. After heat treatment, all individuals were transferred to a 26 °C climate chamber for further development. The effects on immediate survival, maturation success to adulthood, and female fecundity were studied. Eggs, young larvae, late pupae and newly emerged adults had relatively higher immediate survival rates than the other experimental groups. Heat treatment at the egg and larval stages had no impact on development to adulthood and on female fecundity, while it significantly reduced the survival rate of larvae but not of eggs. Brief exposure to high temperature during the early pupal stage and as adults depressed female fecundity whereas exposure during the late pupal stage had no effect.

Nanoparticle-loaded microcapsules providing effective UV protection for Cry1Ac.

AIM: To prepare several novel microcapsules using chitosan (Cs) and Alginate (Alg) as coating materials, and nano-ZnO, nano-SiO2, nano-TiO2 as UV protective agents for improving UV resistance of Cry1Ac. METHODS: Microcapsules were prepared by the layer-by-layer (LbL) self-assembly technique and electrostatic adsorption. The morphologies were observed by scanning electron microscopy (SEM), and the stability under UV radiation was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bioassay. RESULTS: SEM showed that nano-ZnO and nano-TiO2 could be adsorbed on the negatively charged MC with the outermost layer being Alg, while nano-SiO2 could be adsorbed on the positively charged MC with Cs as the outermost layer. SDS-PAGE and bioassay showed that nano-ZnO and nano-SiO2 could provide effective UV protection after 8 h UV irradiation (p > 0.05), and nano-TiO2 could provide effective UV protection after 4 h UV irradiation (p > 0.05). CONCLUSION: The microcapsules loaded with nanoparticles provided excellent UV resistance for Cry1Ac.

Genome-Wide Analysis of WRKY Gene Family and the Dynamic Responses of Key WRKY Genes Involved in Ostrinia furnacalis Attack in Zea mays.

WRKY transcription factors comprise one of the largest gene families and serve as key regulators of plant defenses against herbivore attack. However, studies related to the roles of WRKY genes in response to herbivory are limited in maize. In this study, a total of 128 putative maize WRKY genes (ZmWRKYs) were identified from the new maize genome (v4). These genes were divided into seven subgroups (groups I, IIa-e, and III) based on phylogenomic analysis, with distinct motif compositions in each subgroup. Syntenic analysis revealed that 72 (56.3%) of the genes were derived from either segmental or tandem duplication events (69 and 3, respectively), suggesting a pivotal role of segmental duplication in the expansion of the ZmWRKY family. Importantly, transcriptional regulation prediction showed that six key WRKY genes contribute to four major defense-related pathways: L-phenylalanine biosynthesis II and flavonoid, benzoxazinoid, and jasmonic acid (JA) biosynthesis. These key WRKY genes were strongly induced in commercial maize (Jingke968) infested with the Asian corn borer, Ostrinia furnacalis, for 0, 2, 4, 12 and 24 h in the field, and their expression levels were highly correlated with predicted target genes, suggesting that these genes have important functions in the response to O. furnacalis. Our results provide a comprehensive understanding of the WRKY gene family based on the new assembly of the maize genome and lay the foundation for further studies into functional characteristics of ZmWRKY genes in commercial maize defenses against O. furnacalis in the field.

Sexual-biased gene expression of olfactory-related genes in the antennae of Conogethes pinicolalis (Lepidoptera: Crambidae).

BACKGROUND: Conogethes pinicolalis (Lepidoptera: Crambidae), is similar to Conogethes punctiferalis (yellow peach moth) and its host plant is gymnosperms, especially for masson pine. So far, less literature was reported on this pest. In the present study, we sequenced and characterized the antennal transcriptomes of male and female C. pinicolalis for the first time. RESULTS: Totally, 26 odorant-binding protein (OBP) genes, 19 chemosensory protein (CSP) genes, 55 odorant receptor (OR) genes and 20 ionotropic receptor (IR) genes were identified from the C. pinicolalis antennae transcriptome and amino sequences were annotated against homologs of C. punctiferalis. The neighbor-joining tree indicated that the amino acid sequence of olfactory related genes is highly homologous with C. punctiferalis. Furthermore, the reference genes were selected, and we recommended the phosphate dehydrogenase gene (GAPDH) or ribosomal protein 49 gene (RP49) to verify the target gene expression during larval development stages and RP49 or ribosomal protein L13 gene (RPL13) for adult tissues. CONCLUSIONS: Our study provides a starting point on the molecular level characterization between C. pinicolalis and C. punctiferalis, which might be supportive for pest management studies in future.

Aphid fecundity and defenses in wheat exposed to a combination of heat and drought stress.

Plants are routinely subjected simultaneously to different abiotic and biotic stresses, such as heat, drought, and insect infestation. Plant-insect interactions in such complex stress situations are poorly understood. We evaluated the performance of the grain aphid (Sitobion avenae) in wheat (Triticum aestivum L.) exposed to a combination of heat and drought stresses. We also performed assays of the relative water content, nutritional quality, and responses of phytohormone signaling pathways. Lower relative water content and accumulation of soluble sugars and amino acids were observed in plants exposed to combined heat and drought stress. These conditions increased abscisic acid levels in the absence of aphids, as well as leading to higher levels of jasmonate-dependent transcripts. The grain aphid infestation further increased abscisic acid levels and the abundance of jasmonic acid- and salicylic acid-dependent defenses under the combined stress conditions. Aphids reared on plants grown under drought stress alone showed lower net reproductive rates, intrinsic rates of increase, and finite rates of increase compared with aphids reared on plants in the absence of stress. The heat-treated plants also showed a decreased aphid net reproductive rate. These findings demonstrate that exposure to a combination of stresses enhances plant defense responses against aphids as well as altering nutritional quality.

Identification of Cry1Ah-binding proteins through pull down and gene expression analysis in Cry1Ah-resistant and susceptible strains of Ostrinia furnacalis.

Bacillus thuringiensis produces insecticidal Cry toxins used in the control of multiple insect pests. Evolution of insect resistance to Bt toxins endangers the use of Cry toxins for pest control. Analysis of the Cry1Ah-binding proteins from brush border membrane vesicles (BBMV) of Ostrinia furnacalis, Asian corn borer (ACB) from the Cry1Ah-resistant (ACB-AhR) and susceptible (ACB-BtS) strains was performed by an improved pull down assay that includes coupling Cry1Ah to NHS-activated Sepharose combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our data show that Cry1Ah bound to alkaline phosphatase (ALP), cadherin-like (CAD), actin, aminopeptidase-N (APN), prophenoloxidase (proPO), serine proteinase inhibitor (SPI), immulectin, and V-ATPase and to other proteins that were not previously characterized as Cry-binding proteins in ACB-BtS strain. Analysis of Cry1Ah-pulled down proteins of the BBMV from ACB-AhR revealed that Cry1Ah toxin did not bind to ALP in ACB-AhR strain, suggesting that this protein may correlate with the resistant phenotype of this strain. Additionally, we analyzed the expression of representative genes coding for Cry1Ah-binding proteins such as ALP, APN, CAD, proPO, SPI, and immulectin by qRT-PCR. ACB-AhR showed increased expression levels of proPO (7.5 fold), ALP (6.2 fold) and APN (1.4 fold) in comparison to ACB-BtS strain. In contrast, the cad gene showed slight decreased expression in ACB-AhR strain (0.7 fold) compared with ACB-BtS strain. Our data suggest that differences in the susceptibility to Cry1Ah toxin in the ACB-AhR strain may be associated with reduced ALP binding sites and with an increased immune response. This study also brings evidence of a possible binding interaction of Cry1Ah toxin to immune related proteins like proPO.

Synergistic Effect of Beauveria bassiana and Trichoderma asperellum to Induce Maize (Zea mays L.) Defense against the Asian Corn Borer, Ostrinia furnacalis (Lepidoptera, Crambidae) and Larval Immune Response.

Ostrinia furnacalis, is the major pest of maize causing significant yield losses. So far, many approaches have been used to increase the virulence of entomopathogenic fungal isolates. The current study is an attempt to estimate synergistic effect of Beauveria bassiana and Trichoderma asperellum in order to explore larval immune response through RNA sequencing and differentially expression analysis. In vivo synergism was examined in seven proportions (B. bassiana: T. asperellum = 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, 2:1) and in the in vitro case, two inoculation methods were applied: seed coating and soil drenching. Results revealed significant decrease in plant damage and high larval mortality in fungal treatments. Fungal isolates mediated the plant defense by increasing proline, superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and protease activities. Seed coating method was proved to be the most effective in case of maize endophytic colonization. In total, 59 immune-related differentially expressed genes DEGs were identified including, cytochrome P450, heat shock protein, ABC transporter, cadherin, peptidoglycan recognition protein (PGRP), cuticlular protein, etc. Further, transcriptomic response was confirmed by qRT-PCR. Our results concluded that, coculture of B. bassiana and T. asperellum has the synergistic potential to suppress the immune response of O. furnacalis and can be used as sustainable approach to induce plant resistance through activation of defense-related enzymes.

The Asian corn borer Ostrinia furnacalis feeding increases the direct and indirect defence of mid-whorl stage commercial maize in the field.

The Asian corn borer (Ostrinia furnacalis Guenée) is a destructive pest of maize (Zea mays L.). Despite large-scale commercial maize production, little is known about the defensive responses of field-grown commercial maize to O. furnacalis herbivory, and how these responses result in direct and indirect defence against this pest. To elucidate the maize transcriptome response to O. furnacalis feeding, leaves of maize hybrid Jingke968 were infested with O. furnacalis for 0, 2, 4, 12 and 24 h. Ostrinia furnacalis feeding elicited stronger and more rapid changes in the defence-related gene expression (i.e. after 2 h), and more differentially expressed genes (DEGs) were up-regulated than down-regulated at all times post-induction (i.e. 2, 4, 12 and 24 h) in the O. furnacalis pre-infested maize plants. KEGG pathway analysis indicated that the DEGs in the O. furnacalis pre-infested maize are involved in benzoxazinoids, phytohormones, volatiles, and other metabolic pathways related to maize resistance to herbivores. In addition, the maize leaves previously infested by O. furnacalis for 24 h showed an obvious inhibition of the subsequent O. furnacalis performance, and maize volatiles induced by O. furnacalis feeding for 24 and 48 h attracted the parasitic wasp, Macrocentrus cingulum Brischke. The increased direct and indirect defences induced by O. furnacalis feeding were correlated with O. furnacalis-induced phytohormones, benzoxazinoids, and volatiles. Together, our findings provide new insights into how commercial maize orchestrates its transcriptome and metabolome to directly and indirectly defend against O. furnacalis at the mid-whorl stage in the field.

Analysis of cross-resistance to Vip3 proteins in eight insect colonies, from four insect species, selected for resistance to Bacillus thuringiensis insecticidal proteins.

Bacillus thuringiensis Vip3 proteins are synthesized and secreted during the vegetative growth phase. They are activated by gut proteases, recognize and bind to midgut receptors, form pores and lyse cells. We tested the susceptibility to Vip3Aa and Vip3Ca of Cry1A-, Cry2A-, Dipel- and Vip3-resistant insect colonies from different species to determine whether resistance to other insecticidal proteins confers cross-resistance to Vip3 proteins. As expected, the colonies resistant to Cry1A proteins, Dipel (Helicoverpa armigera, Trichoplusia ni, Ostrinia furnacalis and Plodia interpunctella) or Cry2Ab (H. armigera and T. ni) were not cross-resistant to Vip3 proteins. In contrast, H. armigera colonies resistant to Vip3Aa or Vip3Aa/Cry2Ab showed cross-resistance to the Vip3Ca protein. Moreover, the Vip3Ca protein was highly toxic to O. furnacalis (LC50 not significantly different from that of Cry1Ab), whereas the Vip3Aa protein only showed moderate growth inhibition at the highest concentration tested (100 µg/g of diet). These results extend the cross-resistance studies between Vip3 and Cry proteins, show for the first time cross-resistance between proteins within the Vip3 subfamily, and points to O. furnacalis as a target for the Vip3Ca protein.

Binding affinity of five PBPs to Ostrinia sex pheromones.

BACKGROUND: Pheromone binding proteins (PBPs) of male Lepidoptera function in chemical communication, mate attraction and recognition. Directional selection was previously predicted between PBP3 orthologs of Ostrinia furnacalis and Ostrinia nubilalis were interpreted as being involved in sexual isolation. RESULTS: In vitro assays show that recombinant male OfurPBP3 bound O. furnacalis sex pheromones, Z-12-tetradecenyl acetate (Z12-14:OAc) and E-12-tetradecenyl acetate (E12-14:OAc), as well as to ECB pheromones Z11- and E11-14:OAc. Recombinant OfurPBP4 and OfurPBP5 bound E11- and Z11-14:OAc with greater affinity compared to Z12- and E12-14:OAc, and OfurPBP4 incapable of binding with E12-14:OAc. In silico molecular docking predicted OfurPBP3 residues Phe12, Ile52, Leu94, Ile113 within a hydrophobic ligand-binding pocket and may participate in E12- and Z12-14:OAc binding. Independent site-directed mutagenesis experiments demonstrated that Ser12, Asn52, Arg94, and Asn113 residues variants caused an approximately 1.7- to 4.6-fold reduction in OfurPBP3 affinity for Z12- and E12-14:OAc, and a 2.7- to 8.4-fold decrease in affinity towards E11- and Z11-14:OAc. CONCLUSIONS: Five PBPs of O. furnacalis play important functions in Ostrinia pheromones binding. These four amino acids may play a role in binding of sex pheromone, but this study does not address questions regarding specific response between males of O. furnacalis and O. nubilalis. Additional studies are required determine the role, if any, PBPs play in the evolution of sex pheromone communication.

Molecular cloning, expression profile, odorant affinity, and stability of two odorant-binding proteins in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae).

The polyembryonic endoparasitoid wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is deployed successfully as a biocontrol agent for corn pest insects from the Lepidopteran genus Ostrinia in Europe and throughout Asia, including Japan, Korea, and China. The odorants are recognized, bound, and solubilized by odorant-binding protein (OBP) in the initial biochemical recognition steps in olfaction that transport them across the sensillum lymph to initiate behavioral response. In the present study, we examine the odorant-binding effects on thermal stability of McinOBP2, McinOBP3, and their mutant form that lacks the third disulfide bonds. Real-time PCR experiments indicate that these two are expressed mainly in adult antennae, with expression levels differing by sex. Odorant-binding affinities of aldehydes, terpenoids, and aliphatic alcohols were measured with circular dichroism spectroscopy based on changes in the thermal stability of the proteins upon their affinities to odorants. The obtained results reveal higher affinity of trans-caryophelle, farnesene, and cis-3-Hexen-1-ol exhibits to both wild and mutant McinOBP2 and McinOBP3. Although conformational flexibility of the mutants and shape of binding cavity make differences in odorant affinity between the wild-type and mutant, it suggested that lacking the third disulfide bond in mutant proteins may have chance to incorrect folded structures that reduced the affinity to these odorants. In addition, CD spectra clearly indicate proteins enriched with α-helical content.

Biopolymer microencapsulations of Bacillus thuringiensis crystal preparations for increased stability and resistance to environmental stress.

Parasporal crystals synthesized by Bacillus thuringiensis (Bt) have been widely used as microbial pesticides because of their toxicity to the larval stages of specific insects. However, parasporal crystals can be damaged by environmental stresses, such as high temperature, ultraviolet radiation, and desiccation. To reduce environmental susceptibility of parasporal crystals and extend the duration of their activity, we developed a new type of protection by making microcapsules of crystals (MCs). The microcapsules were self-assembled by alternate deposition (layer by layer) of low-cost chitosan and sodium alginate (or sodium carboxymethyl cellulose) on the crystal surface. Crystal toxins (Cry1Ac) were released from microcapsules at pH values above 9.0. Bioassay results demonstrated that microencapsulated preparations had larvicidal toxicity equivalent to the non-encapsulated form. Microencapsuled crystals were protected from environmental stresses such as high temperature and desiccation. The results indicate that microcapsule protection can enhance the efficacy of Bt in pest control, especially to Lepidoptera larvae that have a alkaline midgut.

Effects of transgenic cry1Ie maize on non-lepidopteran pest abundance, diversity and community composition.

Non-lepidopteran pests are exposed to, and may be influenced by, Bt toxins when feeding on Bt maize that express insecticidal Cry proteins derived from Bacillus thuringiensis (Bt). In order to assess the potential effects of transgenic cry1Ie maize on non-lepidopteran pest species and ecological communities, a 2-year field study was conducted to compare the non-lepidopteran pest abundance, diversity and community composition between transgenic cry1Ie maize (Event IE09S034, Bt maize) and its near isoline (Zong 31, non-Bt maize) by whole plant inspections. Results showed that Bt maize had no effects on non-lepidopteran pest abundance and diversity (Shannon-Wiener diversity index, Simpson's diversity index, species richness, and Pielou's index). There was a significant effect of year and sampling time on those indices analyzed. Redundancy analysis indicated maize type, sampling time and year totally explained 20.43 % of the variance in the non-lepidopteran pest community composition, but no association was presented between maize type (Bt maize and non-Bt maize) and the variance. Nonmetric multidimensional scaling analysis showed that sampling time and year, rather than maize type had close relationship with the non-lepidopteran pest community composition. These results corroborated the hypothesis that, at least in the short-term, the transgenic cry1Ie maize had negligible effects on the non-lepidopteran pest abundance, diversity and community composition.