Chromosome-level genome assembly of the bethylid ectoparasitoid wasp Sclerodermus sp. 'alternatusi'.

The Bethylidae are the most diverse of Hymenoptera chrysidoid families. As external parasitoids, the bethylids have been widely adopted as biocontrol agents to control insect pests worldwide. Thus far, the genomic information of the family Bethylidae has not been reported yet. In this study, we crystallized into a high-quality chromosome-level genome of ant-like bethylid wasps Sclerodermus sp. 'alternatusi' (Hymenoptera: Bethylidae) using PacBio sequencing as well as Hi-C technology. The assembled S. alternatusi genome was 162.30 Mb in size with a contig N50 size of 3.83 Mb and scaffold N50 size of 11.10 Mb. Totally, 92.85% assembled sequences anchored to 15 pseudo-chromosomes. A total of 10,204 protein-coding genes were annotated, and 23.01 Mb repetitive sequences occupying 14.17% of genome were pinpointed. The BUSCO results showed that 97.9% of the complete core Insecta genes were identified in the genome, while 97.1% in the gene sets. The high-quality genome of S. alternatusi will not only provide valuable genomic information, but also show insights into parasitoid wasp evolution and bio-control application in future studies.

Chromosome-level Genome Assembly and Sex-specific Differential Transcriptome of the White-backed Planthopper, Sogatella furcifera.

Background: The white-backed planthopper (WBPH), Sogatella furcifera, causes great damage to many crops (mainly rice) by direct feeding or transmitting plant viruses. The previous genome assembly was generated by second-generation sequencing technologies, with a contig N50 of only 51.5 kb, and contained a lot of heterozygous sequences. Methods: We utilized third-generation sequencing technologies and Hi-C data to generate a high-quality chromosome-level assembly. We also provide a large amount of transcriptome data for full-length transcriptome analysis and gender differential expression analysis. Results: The final assembly comprised 56.38 Mb, with a contig N50 of 2.20 Mb and a scaffold N50 of 45.25 Mb. Fourteen autosomes and one X chromosome were identified. More than 99.5% of the assembled bases located on the 15 chromosomes. 95.9% of the complete BUSCO Hemiptera genes were detected in the final assembly and 16,880 genes were annotated. 722 genes were relatively highly expressed in males, while 60 in the females. Conclusion: The integrated genome, definite sex chromosomes, comprehensive transcriptome profiles, high efficiency of RNA interference and short life cycle substantially made WBPH an efficient research object for functional genomics.

The Genetic Network of Forkhead Gene Family in Development of Brown Planthoppers.

We identified 18 distinct Fox genes in the genome of the brown planthopper, Nilaparvata lugens, and further found a novel insect-specific subfamily that we temporarily named FoxT. A total of 16 genes were highly expressed in the eggs, while NlFoxL2 and NlFoxT are female- and male-specific genes, respectively. Large scale RNAi and RNA-seq analyses were used to reveal the functions and potential targets of NlFoxs. In the eggs, NlFoxA, NlFoxN1 and NlFoxN2 are indispensable to early embryogenesis by regulating different target genes; NlFoxG and NlFoxQ co-regulate NlSix3 for brain development; and NlFoxC, NlFoxJ1 and NlFoxP have complementary effects on late embryogenesis. Moreover, NlFoxA, NlFoxNl and NlFoxQ have pleiotropism. NlFoxA and NlFoxQ regulate the expression of NlCHS1 and cuticular proteins, respectively, thereby participating in the formation of cuticles. NlFoxN1, which regulates the expression of NlKrt9 is involved in the formation of intermediate filament frameworks. Our previous studies have revealed that NlFoxL2 and NlFoxO play important roles in chorion formation and wing polyphenism. Altogether, N. lugens Fox genes exhibit functional diversity in embryonic development and organogenesis. This comprehensive study combines genomics, transcriptomics and phenomics, thereby constructing a complex genetic network that spans the entire life cycle of the brown planthopper.

Molecular characterization and efficacy evaluation of a transgenic corn event for insect resistance and glyphosate tolerance.

A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis (Bt) fusion protein Cry1Ab/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein Cry1Ab/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer (Ostrinia furnacalis), cotton boll worm (Helicoverpa armigera), and armyworm (Mythimna separata). This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm.

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for pest resistance management in China.

Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests.

Although farmers in China have grown transgenic Bt-Cry1Ac cotton to resist the major pest Helicoverpa armigera since 1997 with great success, many secondary lepidopteran pests that are tolerant to Cry1Ac are now reported to cause considerable economic damage. Vip3AcAa, a chimeric protein with the N-terminal part of Vip3Ac and the C-terminal part of Vip3Aa, has a broad insecticidal spectrum against lepidopteran pests and has no cross resistance to Cry1Ac. In the present study, we tested insecticidal activities of Vip3AcAa against Spodoptera litura, Spodoptera exigua, and Agrotis ipsilon, which are relatively tolerant to Cry1Ac proteins. The bioassay results showed that insecticidal activities of Vip3AcAa against these three pests are superior to Cry1Ac, and after an activation pretreatment, Vip3AcAa retained insecticidal activity against S. litura, S. exigua and A. ipsilon that was similar to the unprocessed protein. The putative receptor for this chimeric protein in the brush border membrane vesicle (BBMV) in the three pests was also identified using biotinylated Vip3AcAa toxin. To broaden Bt cotton activity against a wider spectrum of pests, we introduced the vip3AcAa and cry1Ac genes into cotton. Larval mortality rates for S. litura, A. ipsilon and S. exigua that had fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and Bt-Cry1Ac cotton in a laboratory experiment. These results suggested that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for integrated pest management in China.

Effects of Transgenic Bt Rice on Nontarget Rhopalosiphum maidis (Homoptera: Aphididae).

The effects of three transgenic Bacillus thuringiensis (Bt) rice lines, KMD1, KMD2, and G8-7, on biological parameters and population dynamics of nontarget insect, Rhopalosiphum maidis (Fitch) (Homoptera: Aphididae), were investigated in the laboratory and field. No significant differences were found between Bt and non-Bt rice lines for aphid survival. The developmental time of R. maidis that fed on KMD1 and KMD2 did not differ significantly from those of the individuals feeding on the parental variety Xiushui11, but significantly prolonged developmental time was observed on G8-7 as compared with its parental variety Xiushui110. Aphid fecundity was significantly higher on Bt than on parental rice. A 2-yr field survey indicated that Bt rice did not significantly affect the population dynamics of R. maidis in comparison with non-Bt rice. Additionally, guttation droplets of Bt rice and aphids feeding on Bt rice were analyzed for presence of Cry1Ab using ELISA. No Cry1Ab protein was found in aphid adults feeding on Bt rice lines both in the laboratory and field. By using the guttation droplets from the top of rice seedlings, we designed a novel method to collect phloem sap, and found that relatively low concentrations were detected in the guttation droplets from Bt rice lines. In conclusion, although the Bt rice lines tested in this study stimulate the fecundity of R. maidis, the aphid population density did not increase in Bt rice fields.

Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene.

Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, Cry1Ac and Cry1Ig, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (G10) were combined into a single transferred DNA (T-DNA) fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice.

Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation.

BACKGROUND: The brown planthopper, Nilaparvata lugens, the most destructive pest of rice, is a typical monophagous herbivore that feeds exclusively on rice sap, which migrates over long distances. Outbreaks of it have re-occurred approximately every three years in Asia. It has also been used as a model system for ecological studies and for developing effective pest management. To better understand how a monophagous sap-sucking arthropod herbivore has adapted to its exclusive host selection and to provide insights to improve pest control, we analyzed the genomes of the brown planthopper and its two endosymbionts. RESULTS: We describe the 1.14 gigabase planthopper draft genome and the genomes of two microbial endosymbionts that permit the planthopper to forage exclusively on rice fields. Only 40.8% of the 27,571 identified Nilaparvata protein coding genes have detectable shared homology with the proteomes of the other 14 arthropods included in this study, reflecting large-scale gene losses including in evolutionarily conserved gene families and biochemical pathways. These unique genomic features are functionally associated with the animal's exclusive plant host selection. Genes missing from the insect in conserved biochemical pathways that are essential for its survival on the nutritionally imbalanced sap diet are present in the genomes of its microbial endosymbionts, which have evolved to complement the mutualistic nutritional needs of the host. CONCLUSIONS: Our study reveals a series of complex adaptations of the brown planthopper involving a variety of biological processes, that result in its highly destructive impact on the exclusive host rice. All these findings highlight potential directions for effective pest control of the planthopper.

Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice.

Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.

De novo intestine-specific transcriptome of the brown planthopper Nilaparvata lugens revealed potential functions in digestion, detoxification and immune response.

The brown planthopper (Nilaparvata lugens, BPH) is the most serious rice plant pests in Asia. In this study, we performed transcriptome-wide analysis on BPH intestine. We obtained more than 26 million sequencing reads that were then assembled into 53,553 unigenes with a mean size of 388 bp. Based on similarity search with the nucleotide sequences available at NCBI, BPH intestine-specific transcriptome analysis identified 21,405 sequences. Assembled sequences were annotated with gene description, gene ontology and clusters of orthologous group terms. The digestion-, defense- and xenobiotic metabolism-related genes were abundantly detected in the transcripts from BPH intestine. Many novel genes including 33 digestion-related genes, 25 immune responsive genes and 27 detoxification-related genes are first reported here. We investigated the gene expression patterns at the transcript levels in different tissues by quantitative real-time PCR analysis, which revealed that some genes had intestine-specific expression, implicating their potential significance for BPH management.

Integration of insecticidal protein Vip3Aa1 into Beauveria bassiana enhances fungal virulence to Spodoptera litura larvae by cuticle and per Os infection.

The entomopathogenic fungus Beauveria bassiana acts slowly on insect pests through cuticle infection. Vegetative insecticidal proteins (Vip3A) of Bacillus thuringiensis kill lepidopteran pests rapidly, via per os infection, but their use for pest control is restricted to integration into transgenic plants. A transgenic B. bassiana strain (BbV28) expressing Vip3Aa1 (a Vip3A toxin) was thus created to infect the larvae of the oriental leafworm moth Spodoptera litura through conidial ingestion and cuticle adhesion. Vip3Aa1 ( approximately 88 kDa) was highly expressed in the conidial cytoplasm of BbV28 and was detected as a digested form ( approximately 62 kDa) in the larval midgut 18 and 36 h after conidial ingestion. The median lethal concentration (LC(50)) of BbV28 against the second-instar larvae feeding on cabbage leaves sprayed with conidial suspensions was 26.2-fold lower than that of the wild-type strain on day 3 and 1.1-fold lower on day 7. The same sprays applied to both larvae and leaves for their feeding reduced the LC(50) of the transformant 17.2- and 1.3-fold on days 3 and 7, respectively. Median lethal times (LT(50)s) of BbV28 were shortened by 23 to 35%, declining with conidial concentrations. The larvae infected by ingestion of BbV28 conidia showed typical symptoms of Vip3A action, i.e., shrinkage and palsy. However, neither LC(50) nor LT(50) trends differed between BbV28 and its parental strain if the infection occurred through the cuticle only. Our findings indicate that fungal conidia can be used as vectors for spreading the highly insecticidal Vip3A protein for control of foliage feeders such as S. litura.