A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Effect of VEGF on neurological impairment and prognosis of acute cerebral infarction patients: A retrospective case-control study.

OBJECTIVE: Due to the complex pathological mechanism of acute cerebral infarction, the role of vascular endothelial growth factor (VEGF) on the disease is not clear. Therefore, a retrospective case-control study was performed to explore the effect of VEGF on neurological impairment and prognosis of acute cerebral infarction patients. METHOD: A total of 100 patients with acute cerebral infarction admitted to our hospital from April 2021 to April 2022 were selected. Blood samples from all patients would be routinely collected to detect the expression of serum VEGF. Pearson chi-square, Spearman correlation and univariate Logistic regression were used to analyze the clinical data to explore the relationship between VEGF expression and basic information, stroke degree, quality of life, and prognosis of patients. To determine whether VEGF can provide relevant basis for the early prevention and prognostic treatment of acute cerebral infarction. And multivariate logistic regression was used to calculate the odds ratio between each variable and VEGF expression. RESULTS: Pearson chi-square test and Spearman correlation coefficient showed that sex, degree of stroke, limb convulsions, loss of consciousness, hemiplegia, aphasia, mental functioning score, overall quality of life score, and short-term prognosis were significantly correlated with VEGF expression in 100 patients. Univariate logistic regression was used to describe the ORs and 95% confidence interval of subjects at the univariate level, and the degree of stroke (OR = 83.333, P < 0.001), tic of limbs (OR = 26.316, P < 0.001), loss of consciousness (OR = 23.256, P < 0.001), hemiplegia (OR = 62.500, P < 0.001), aphasia (OR = 76.923, P < 0.001), mental functioning score (OR = 7.937, P < 0.001), overall quality of life score (OR = 5.464, P < 0.001), short-term prognosis (OR = 37.037, P < 0.001) was significantly correlated with the high expression of VEGF. CONCLUSIONS: The level of serum VEGF was positively correlated with neurological impairment degree and prognosis in patients with acute cerebral infarction, the more severe the degree of stroke and the worse the prognosis.

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.

Population Source of Third-Generation Oriental Armyworm in Jilin, China, Determined by Entomology Radar, Trajectory Analysis, and Mitochondrial COI Sequences.

The armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae), is an important polyphagous pest with a strong migratory ability. Recently, third-generation larvae have become an increasingly serious pest threat in Jilin Province of northeast China. To investigate the population source of this species, scanning entomological radar observations and insect mitochondrial cytochrome oxidase I (COI) genes were used in this study. Five main results were found: (1) The peak period in captured second-generation moths was from mid to late July. The temperature and wind speeds were optimum for the moths to have migrated. Strong southwesterly winds occurred during the peak migration period. (2) Radar observations indicated that most of the moths' migration took place at a height of 600 m, often in a dense layer which formed at heights of 350-800 m. (3) Analyses of adult ovarian development and larval haplotypes showed third-generation larvae were progeny of both locally produced progeny and immigrant moths. (4) Based on our back-tracking and haplotype analyses, immigration led to an outbreak originated in the same source area to the southwest. (5) Emigration of second-generation moths was confirmed by both radar observation and mtDNA analysis. Forward trajectories indicated that the moths were capable of immigrating far from their overwintering range. These results are useful for improving the forecasting systems of this insect pest species.

Myco-Synergism Boosts Herbivory-Induced Maize Defense by Triggering Antioxidants and Phytohormone Signaling.

BACKGROUND: Biocontrol strategies are the best possible and eco-friendly solution to develop resistance against O furnacalis and improve the maize yield. However, the knowledge about underlying molecular mechanisms, metabolic shifts, and hormonal signaling is limited. METHODS: Here, we used an axenic and a consortium of entomopathogenic Beauveria bassiana OFDH1-5 and a pathogen-antagonistic Trichoderma asperellum GDFS1009 in maize and observed that consortium applications resulted in higher chlorophyll contents and antioxidants activities [superoxide dismutase (SOD), peroxidase (POD), proline, protease, and polyphenol oxidase (PPO)] with a decrease in O. furnacalis survival. We performed a comprehensive transcriptome and an untargeted metabolome profiling for the first time at a vegetative stage in fungal inoculated maize leaves at 0-, 12-, 24-, 48-, and 72-h post insect infestation. RESULTS: The consortium of B. bassiana and T. asperellum leads to 80-95% of O. furnacalis mortality. A total of 13,156 differentially expressed genes were used for weighted gene coexpression network analysis. We identified the six significant modules containing thirteen candidate genes [protein kinase (GRMZM2G025459), acyl-CoA dehydrogenase (GRMZM5G864319), thioredoxin gene (GRMZM2G091481), glutathione S-transferase (GRMZM2G116273), patatin-like phospholipase gene (GRMZM2G154523), cytochrome P450 (GRMZM2G139874), protease inhibitor (GRMZM2G004466), (AC233926.1_FG002), chitinase (GRMZM2G453805), defensin (GRMZM2G392863), peroxidase (GRMZM2G144153), GDSL- like lipase (AC212068.4_FG005), and Beta-glucosidase (GRMZM2G031660)], which are not previously reported that are highly correlated with Jasmonic acid - Ethylene (JA-ET) signaling pathway and antioxidants. We detected a total of 130 negative and 491 positive metabolomic features using a ultrahigh-performance liquid chromatography ion trap time-of-flight mass spectrometry (UHPLC-QTOF-MS). Intramodular significance and real time-quantitative polymerase chain reaction (RT-qPCR) expressions showed that these genes are the true candidate genes. Consortium treated maize had higher jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) levels. CONCLUSION: Our results provide insights into the genetics, biochemicals, and metabolic diversity and are useful for future biocontrol strategies against ACB attacks.

Influence of voltine ecotype and geographic distance on genetic and haplotype variation in the Asian corn borer.

Diapause is an adaptive dormancy strategy by which arthropods endure extended periods of adverse climatic conditions. Seasonal variation in larval diapause initiation and duration in Ostrinia furnacalis may influence adult mating generation number (voltinism) across different local environments. The degree to which voltine ecotype, geographic distance, or other ecological factors influence O. furnacalis population genetic structure remains uncertain. Genetic differentiation was estimated between voltine ecotypes collected from 8 locations. Mitochondrial haplotypes were significantly different between historically allopatric univoltine and bivoltine locations, but confounded by a strong correlation with geographic distance. In contrast, single nucleotide polymorphism (SNP) genotypes show low but significant levels of variation and a lack of influence of geographic distance between allopatric voltine locations. Regardless, 11 of 257 SNP loci were predicted to be under selection, suggesting population genetic homogenization except at loci proximal to factors putatively under selection. These findings provide evidence of haplotype divergent voltine ecotypes that may be maintained in allopatric and sympatric areas despite relatively high rates of nuclear gene flow, yet influence of voltinism on maintenance of observed haplotype divergence remains unresolved.

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.

Geographic Distribution of Bacillus thuringiensis Cry1F Toxin Resistance in Western Bean Cutworm (Lepidoptera: Noctuidae) Populations in the United States.

The western bean cutworm (WBC), Striacosta albicosta (Lepidoptera: Noctuidae), can be a severe pest of transgenic corn in the western Plains and Great Lakes regions of North America, including on hybrids expressing the Bacillus thuringiensis (Bt) Cry1F toxin. The level and geographic distribution of Cry1F resistance are not completely known. Neonate S. albicosta from 10 locations between Nebraska and New York state were subjected to dose-response trypsin-activated native Cry1F toxin overlay bioassays. In 2017, the mean estimated lethal concentration causing 50% larval mortality (LC50) ranged from 15.1 to 18.4 µg Cry1F cm-2, and were not significantly different among locations. In 2018, LC50 estimates at Scottsbluff, NE (22.0 µg Cry1F cm-2) and Watertown, NY (21.7 µg Cry1F cm-2) were significantly higher when compared to locations in Michigan (15.8 µg Cry1F cm-2). Significantly lower 14-day larval weight among survivors was correlated with higher Cry1F dose. Results from this study indicate that S. albicosta survivorship on purified Bt Cry1F toxin shows a relatively even distribution across the native and range expansion areas where seasonal field infestations typically occur.

Influence of genetic diversity of seventeen Beauveria bassiana isolates from different hosts on virulence by comparative genomics.

BACKGROUND: Beauveria bassiana (B. bassiana) is a famous entomopathogenic fungus that could parasitize on hundreds of insect species, which are being used as an environmentally friendly mycoinsecticide. Nevertheless, the possible effect of genetic diversity of these B. bassiana isolates from different hosts on virulence has not been explored before. In order to explore that issue, we compared the genome sequences among seventeen B. bassiana isolates from 17 different insects using whole genome re-sequencing, with B. bassiana strain ARSEF 2860 as the reference genome. RESULTS: There were a total of 10,098 missense mutated genes, 720 positively selected genes were identified in 17 strains of B. bassiana. Among these, two genes with high frequency mutations encode the toxin-producing non-ribosomal peptide synthase (NRPS) protein. Seven genes undergoing positive selection were enriched in the two-component signaling pathway that is known to regulate the fungal toxicity. In addition, the domain changes of three positively selected genes are also directly related to the virulence plasticity. Besides, the functional categorization of mutated genes showed that most of them involved in the biological functions of toxic proteins involved in. CONCLUSIONS: Based on our data, our results indicate that several mutated genes and positively selected genes may underpin virulence of B. bassiana towards hosts during infection process, which provide an insight into the potential effects of natural variation on the virulence of B. bassiana, which will be useful in screening out potential virulence factors in B. bassiana.

Influence of host plant, geography and pheromone strain on genomic differentiation in sympatric populations of Ostrinia nubilalis.

Patterns of mating for the European corn borer (Ostrinia nubilalis) moth depend in part on variation in sex-pheromone blend. The ratio of (E)-11- and (Z)-11-tetradecenyl acetate (E11- and Z11-14:OAc) in the pheromone blend that females produce and males respond to differs between strains of O. nubilalis. Populations also vary in female oviposition preference for and larval performance on maize (C4) and nonmaize (C3) host plants. The relative contributions of sexual and ecological trait variation to the genetic structure of O. nubilalis remains unknown. Host-plant use (13 C/14 C ratios) and genetic differentiation were estimated among sympatric E and Z pheromone strain O. nubilalis males collected in sex-pheromone baited traps at 12 locations in Pennsylvania and New York between 2007 and 2010. Among genotypes at 65 single nucleotide polymorphism marker loci, variance at a position in the pheromone gland fatty acyl-reductase (pgfar) gene at the locus responsible for determining female pheromone ratio (Pher) explained 64% of the total genetic differentiation between males attracted to different pheromones (male response, Resp), providing evidence of sexual inter-selection at these unlinked loci. Principal coordinate, Bayesian clustering, and distance-based redundancy analysis (dbRDA) demonstrate that host plant history or geography does not significantly contribute to population variation or differentiation among males. In contrast, these analyses indicate that pheromone response and pgfar-defined strain contribute significantly to population genetic differentiation. This study suggests that behavioural divergence probably plays a larger role in driving genetic variation compared to host plant-defined ecological adaptation.

Introgression between divergent corn borer species in a region of sympatry: Implications on the evolution and adaptation of pest arthropods.

The Asian corn borer, Ostrinia furnacalis, and European corn borer, O. nubilalis (Lepidoptera: Crambidae), cause damage to cultivated maize in spatially distinct geographies and have evolved divergent hydrocarbons as the basis of sexual communication. The Yili area of Xinjiang Uyghur Autonomous Region in China represents the only known region where O. furnacalis has invaded a native O. nubilalis range, and these two corn borer species have made secondary contact. Genetic differentiation was estimated between Ostrinia larvae collected from maize plants at 11 locations in Xinjiang and genotyped using high-throughput SNP and microsatellite markers. Maternal lineages were assessed by direct sequencing of mitochondrial cytochrome c oxidase subunit I and II haplotypes, and a high degree of genotypic diversity was demonstrated between lineages based on SNP genotypes. Furthermore, historical introgression was predicted among SNP genotypes only at sympatric locations in the Yili area, whereas in Xinjiang populations only O. furnacalis haplotypes were detected and no analogous introgressed genotypes were predicted. Our detection of putative hybrids and historical evidence of introgression defines Yili area as a hybrid zone between the species in normal ecological interactions and furthermore, might indicate that adaptive traits could spread even between seemingly divergent species through horizontal transmission. Results of this study indicate there may be a continuum in the degree of reproductive isolation between Ostrinia species and that the elegance of distinct and complete speciation based on modifications to the pheromone communication might need to be reconsidered.

mRNA-sequencing whole transcriptome analysis of a single cell on the SOLiD system.

We have developed a sequencing-based gene expression profiling assay at single-cell resolution by combining a modified single-cell whole transcriptome amplification method with the next generation sequencing technique, the SOLiD system. Using this assay, we have shown that blastomeres in a four-cell stage embryo have similar gene expression, which is compatible with the fact that they have similar developmental potential. We proved that compared with cDNA microarray technique, our single-cell cDNA SOLiD sequencing assay can detect expression of thousands of more genes. Moreover, for the genes detected by microarray and SOLiD sequencing, our assay detected new transcript variants for a large proportion of them, which confirms unambiguously at single-cell resolution that the transcriptome complexity is higher than expected traditionally. Finally, by using our assay to Dicer knockout (KO) and Ago2 KO oocytes, we showed that a significant amount of transposons were up-regulated abnormally in Dicer/Ago2 KO mature oocytes compared with wild-type controls.

mRNA-Seq whole-transcriptome analysis of a single cell.

Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.

Analysis of the 2.0 A crystal structure of the protein-DNA complex of the human PDEF Ets domain bound to the prostate specific antigen regulatory site.

PDEF, a prostate epithelial specific transcription factor, is a member of the Ets family of DNA binding proteins. Here we report a 2.0 A crystal structure of the PDEF Ets domain in complex with a natural, high-affinity DNA binding site in the promoter/enhancer region of the human prostate specific antigen gene. Comparison of the PDEF-DNA complex with other Ets complexes revealed key features that are shared among Ets members, as well as important differences in substrate specification at both the "GGA" core and the flanking regions of the DNA site. The combination of the serine residue at position 308 and the glutamine at position 311 explains the previous observation that the PDEF binds preferentially to a thymine at the +4 position of its binding site. Despite the common essential features that are shared among Ets members, PDEF demonstrates distinct patterns of interactions at different positions of DNA in achieving sequence specific recognition. Collectively, the common and unique interactions with both the DNA bases and the backbone phosphates lead to substrate specificity and individual preference for certain DNA sites.