Transcriptomic response of wolf spider, Pardosa pseudoannulata, to transgenic rice expressing Bacillus thuringiensis Cry1Ab protein
- Juan Wang†1,
- Yuande Peng†2,
- Kaifu Xiao1,
- Baoyang Wei1,
- Jilin Hu1,
- Zhi Wang1Email authorView ORCID ID profile,
- Qisheng Song3 and
- Xuguo Zhou4
© The Author(s). 2017
Received: 6 February 2016
Accepted: 23 December 2016
Published: 18 January 2017
Bacillum thuringiensis (Bt) toxin produced in Cry1-expressing genetically modified rice (Bt rice) is highly effective to control lepidopteran pests, which reduces the needs for synthetic insecticides. Non-target organisms can be exposed to Bt toxins through direct feeding or trophic interactions in the field. The wolf spider Pardosa pseudoannulata, one of the dominant predators in South China, plays a crucial role in the rice agroecosystem. In this study, we investigated transcriptome responses of the 5th instar spiders fed on preys maintained on Bt- and non-Bt rice.
Comparative transcriptome analysis resulted in 136 differentially expressed genes (DEGs) between spiderlings preying upon N. lugens fed on Bt- and non-Bt rice (Bt- and non-Bt spiderlings). Functional analysis indicated a potential impact of Bt toxin on the formation of new cuticles during molting. GO and KEGG enrichment analyses suggested that GO terms associated with chitin or cuticle, including “chitin binding”, “chitin metabolic process”, “chitin synthase activity”, “cuticle chitin biosynthetic process”, “cuticle hydrocarbon biosynthetic process”, and “structural constituent of cuticle”, and an array of amino acid metabolic pathways, including “alanine, asparatate and glutamate metabolism”, “glycine, serine and theronine metabolism”, “cysteine and methionine metabolism”, “tyrosine metabolism”, “phenylalanine metabolism and phenylalanine”, and “tyrosine and tryptophan biosynthesis” were significantly influenced in response to Cry1Ab.
The Cry1Ab may have a negative impact on the formation of new cuticles during molting, which is contributed to the delayed development of spiderlings. To validate these transcriptomic responses, further examination at the translational level will be warranted.
KeywordsPardosa pseudoannulata Cry1Ab Development RNA-Seq Chitin Cuticle
Genetically modified (GM) technology has reshaped the agricultural industry since its insertion in the late1990s . From 1996 to 2012, the global acreage of GM crops has increased dramatically from 1.7 to 160.4 million hectares . The ecological benefits from rapid development and adoption of GM crops include a significant reduction in both insecticide and herbicide usage and greenhouse gas emissions . A meta-analysis in 2014 showed a 37% reduction in synthetic pesticide use, 22% increase in crop yield, and 68% increase in farmer profits .
Besides yield and profit gains and environmental benefits, non-monetary incentives include time savings, ease of use, and more flexibility in planning . With limited arable land in China, GM technology provides a potential solution to improve agricultural productivity and sustainability. Currently, transgenic Bacillum thuringiensis (Bt) cotton, resistant to Lepidoptera pests, is the most successful commercial GM crop in China . In 2012, acreage of Bt cotton has reached 3.59 million hectares, representing 80% of total cotton area in China. Bt cotton increased yield by 10%, reduced insecticide use by 60% and generated additional US $220 profit per hectare on average . Even with the success of Bt cotton, consumers still have doubts about GM crops, partially due the lack of knowledge regarding the ecological risks [5, 8, 9]. Bt rice is facing the same challenges for the public acceptance.
The community structure of a rice field is primarily composed of soil organisms, rice, insect herbivores, predators, and parasitoids. While insect herbivores are exposed to Bt toxins by direct feeding, other community members can access Bt toxins through trophic interactions. Previous risk assessment studies showed no harmful effect of Bt rice on diversity, dominant species and abundance of non-target arthropods among the arthropod community in the field [10, 11]. Laboratory studies, on one hand, did not detect adverse impacts of Bt rice on non-target arthropods. For example, the developmental time, fecundity and survival rate of herbivorous insects Nilaparvata lugens and Sogatella furcitera were unaffected when exposed to Cry1C, Cry2A, and Cry1AC proteins, respectively [12, 13]. No significant effects were found on life history traits for predators as well, including Chrysoperia sinica, Propylea japonica, Cyrtorhinus lividipennis, and Ummeliata insecticeps [14–17]. On the other hand, some reports show non-target organisms may be susceptible to Bt toxins. A significant longer developmental time of Pirata subpiraticus was recorded when it prayed on Bt rice fed Cnaphalocrocis medinalis . Significantly lower catalase activity was found in Fosomia candida fed on Bt rice in comparison to those fed on non-Bt rice . Due to the varing degradation of Bt toxin protein in soils with different physicochemical properties [20, 21], researchers did not find consistent differences in soil microorganism communities between Bt and non Bt rice fields . For parasitoids, effects of Bt rice is also inconsistent, which depends on the host species, target or non-target insects . As a whole, risk assessment of Bt rice has been focusing on the organismal level impacts, suborganismal impacts are largely unknown. The advent of genomics era, however, allows us to evaluate ecological risks of transgenic Bt rice on non-target organism at the transcription and translational level.
The wolf spider Pardosa pseudoannulata is one of the dominant predators in South China, playing a crucial role in maintaining the stability of the rice agroecosystem . In this study, we carried out a comparative transcriptome analysis of the 5th instar spiders fed on N. lugens maintained on Bt- and non-Bt rice, respectively. Developmental time from the 2nd to 8th instars was recorded to reveal the potential impacts of Bt rice on P. pseudoannulata and to correlate the biological impacts with differentially expressed genes.
Plant materials and Nilaparvata lugens preparation
Transgenic Shanyou 63 rice expressing Cry1Ab protein (test group) and its non-transgenic parental wild type Shanyou 63 rice (control group) were obtained from the Life Science College, Hunan Normal University. Both rice varieties were grown under nylon nets (3 × 2 × 1 m3) without insecticide application during the entire experimental period.
Nilaparvata lugens were collected from farmland in the Hunan Academy of Agricultural Science and reared on non-transgenic parental wild type, Shanyou 63, allowing for natural colonization. The newly moulted 2nd instar N. lugens nymphs were then transplanted to transgenic and control rice lines. After 15-day feeding, N. lugens was collected and used as spider diets 
Spider sample collection
Female spiders with egg sacs were collected from the experimental farmland in the Hunan Academy of Agricultural Science. Pardosa pseudoannulata larvae were collected immediately after hatch and placed individually in a glass tube with a moist cotton ball separately (12 × 100 mm). Spiders in the test and control group were fed daily with N. lugens consumed Bt and non-Bt rice, respectively. All tubes were marked and maintained in an artificial climate chamber (30 °C, 70% RH and L:D 10:14 photoperiod). Developmental time of each spiderling at each instar was recorded until sexual maturity was reached. In this analysis, 120 spiders were raised for the developmental time recording (three biological replicates of 20 spiders each for 2 groups). Observation was made twice a day at 9 am and 9 pm, respectively.
Quantification of the Bt toxin, Cry1Ab, in spiderling
An enzyme-linked immunosorbent assay (ELISA) was conducted for Cry1Ab protein detection using a Qualipate kit for Cry1Ab/Cry1Ac (EnviroLogix, US). For each treatment, five 5th instar spiderlings were weighed as a group (test group, 0.0384 g, control group, 0.0316 g), homogenized in 1 ml PBS buffer and centrifuged for 20 min at 2,000 g. The supernatant was used to determine Cry1Ab concentration. Spectrophotometric measurements for three technical replications were obtained using a microplate reader (BioTek, ELX 800) at 450 nm. Purified Cry1Ab toxin (EnviroLogix, US) at concentrations of 0, 2, 4, 8, 16, and 24 ng/L was used to generate a standard curve. Three biological replicates were performed in ELISA assays.
RNA isolation and Illumina sequencing
A total of ten 5th instar spiderlings from the test and control groups were collected, respectively, on the ninth day after moulting and submitted to Oebiotech Enterprise (Shanghai) for RNA extraction and sequencing. Total RNA was extracted from each sample using TRIzol (Invitrogen Corp, USA) according to the manufacturer’s instructions. RNA quality was assessed using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies Inc, Rockland, DE, USA) using a standard of1.8 ≤ OD260/OD280 ≤ 2.1 and was further confirmed by agarose gel electrophoresis.
RNA sequencing libraries were constructed and sequenced on flow cells using an Illumina Hiseq 2000 platform. Clean reads were assembled using the de novo transcriptome assembler Trinity after removing adaptor sequences, low quantity reads (reads with ambiguous bases N), and duplicate sequences . The libraries were established and unigenes of length greater than 200 bp were subjected to subsequent sequence annotation analysis. All raw reads were deposited in the NCBI Sequence Read Archive (Accession number: SRR2024874, SRR2024877).
All unigenes were compared to those available in the NCBI non-redundant protein (Nr) database and Swiss-prot database using Blastx with an E-value cutoff of 10-5. The Blast2GO program and WEGO software were used to obtain GO annotation for all unigenes [26, 27]. KEGG (Kyoto encyclopedia of genes and genomes database) metabolic pathway annotation and COG (clusters of orthologous group) classification of unigenes were determined by Blastx searching against KEGG and COG databases [28, 29]. The best aligning results were used to determine potential function of the unigenes.
Identification of differentially expressed genes (DEGs)
The FPKM (number of reads per kb of exon region per million mapped reads) method was used for quantifying gene expression levels and was able to eliminate the influence of different gene lengths and sequencing levels in the calculation of gene expression . The DEGseq software package (http://www.bioconductor.org/packages/2.6/bioc/html/DEGseq.html) was used to screen differentially expressed genes (DEGs) based on a statistical analysis of negative binomial distribution and to quantify the gene expression levels with baseMean values . A threshold for false discovery rate of <0.01 and absolute value of log2 (fold change) ratio > 2 were used to determine significant differences in gene expression.
Functional annotation of DEGs
All DEGs were searched against five public databases, Swiss prot, Nr, COG, GO, and KEGG. The hypergeometric test was used to find significantly enriched GO terms in DEGs based on GO annotation. The calculated p value then underwent Bonferroni Correction, using corrected p value ≤ 0.001 as a threshold. GO terms fulfilling this condition are defined as significantly enriched GO terms in DEGs. Similarly, pathway enrichment analysis was conducted to identify significantly enriched metabolic pathways or signal transduction pathways in DEGs, using p value ≤ 0.01 as a threshold.
Quantitative real-time PCR analysis
Transcriptome results were verified using quantitative real-time PCR (qPCR). Total RNA was isolated from each sample with TRIzol (Invitrogen, USA) and subjected to DNase I treatment (Promega, USA) according to the manufacturers’ protocols. cDNA was synthesized with a RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas Lithuania) and qPCR was performed using the ABI 7900 HT system (ABI, USA) with a reaction volume of 25 μl containing 1 μl of 1:10 diluted cDNA in ddH2O, 12.5 μl of 2 × SYBR Green Master Mix (ABI, USA) and 100 nM of each of the primers. The qPCR conditions were 94 °C for 3 min, followed by 40 cycles of 94 °C for 30 s for denaturation, 55 °C for 30 s for annealing and 72 °C for 30 s for extension. The experiment was repeated three times, and expression levels of each gene were normalized to 18S ribosomal RNA (18S rRNA, GenBank accession number: X13457, primers: 5’-AGATGCCCTTAGATGTCCGG-3’, 5’-AAGGGCAGGGACGTAATCAA-3’). All primers were designed using the primer 3.0 program (http://bioinfo.ut.ee/primer3-0.4.0/) .
Data on developmental time of spiderlings and qPCR were analysed using a t-test with SPSS 17.0 software. Significant differences at p < 0.05 were designated with *, and data were presented as the mean ± SE.
Bt rice affects the developmental time of P. pseudoannulata
Illumina sequencing and de novo assembly
Sequences of mRNAs pooled from the whole body of spiderlings were analyzed using an Illumina 2000 platform and resulted in 48,243,314 and 42,798,756 raw reads for non-Bt and Bt spiderlings, respectively. Trinity software was used for de novo assembly according to standard parameters. The assembly yielded 217,017 total transcripts (≥200 bp) with an average length of 612 bp, and the unigene dataset included 169,703 sequences with an average length of 537 bp. All unigenes were used for the annotation.
Annotation of all assembled unigenes
A total of 169,703 unigene sequences were subjected to blast searching against five public available databases, including Nr, Swiss-prot, COG, GO and KEGG, with a cutoff E < 10−5. Of these, 39,727 (23.4%) could be matched to Nr, 31,039 (18.3%) to Swiss-prot, 7,111 (4.2%) to KEGG, 28,646 (16.9%) to COG and 33,652 (19.8%) unigenes to GO database.
Identification of DEGs and functional analysis
Enriched pathways of DEGs between Bt- and non-Bt spiderdlings
Fatty acid elongation
Alanine, aspartate and glutamate metabolism
Glycine, serine and threonine metabolism
Cysteine and methionine metabolism
Arginine and proline metabolism
Phenylalanine, tyrosine and tryptophan biosynthesis
Carbon fixation in photosynthetic organisms
Isoquinoline alkaloid biosynthesis
Tropane, piperidine and pyridine alkaloid biosynthesis
Biosynthesis of unsaturated fatty acids
2-Oxocarboxylic acid metabolism
Fatty acid metabolism
Biosynthesis of amino acids
PPAR signaling pathway
Real-time PCR assays
To validate RNA-seq results, five DEGs were randomly selected for qPCR analysis. These genes were homologous to structural constituents of cuticle, chitin binding and chitin metabolic process (Additional file 2: Table S2). The expression profile of all five DEGs was consistent with RNA-seq data (Fig. 1-b).
Bt toxins can be transferred via the food web and accumulate in organisms to different degrees . The level of Bt toxin protein in predators mainly depends on expression patterns of Bt-protein in plants, and the feeding behavior of the herbivore . Our tritrophic bioassay indicated the accumulative Cry1Ab content in 5th instar spdierling was 1.451 ng/g when P. pseudoannulata was preyed on N. lugens maintained on Cry1Ab rice. Although this protein level is slightly lower than those in Ummeliata insecticeps (2.04 ng/g) , it is still informative. Developmental time of Bt spiderlings was significantly prolonged, which is consistent with Pirata subpiraticus . However, spiderlings were able to recover from the effect of Bt rice at a later instar. Similar to other arthropods, P. pseudoannulata must molt periodically to grow. The formation of new cuticle is a vital step during molting of arthropods . We speculated that the delayed development of spiderlings may be due to the disruption of chitin synthesis (formation of the new cuticle) during molting.
Comparative transcriptome analysis identified 136 DEGs between Bt- and non-Bt spiderlings (FDR < 0.001, Log2foldchange > 2). Furthermore, GO annotation and enrichment analysis both suggested potential impacts of Bt rice on the chitin synthesis and cuticle formation (Fig. 3-c, Fig. 6). As with other arthropods, the exoskeleton of spider is made of cuticle, of which one of the primary component is chitin . The molting process in spiders involves activation of hypodermal cells, secretion of exuvial fluid and apolysis, activation of enzymes in the exuvial fluid, and secretion of the new cuticle . Functional analysis of DEGs suggested a disruption of new cuticle formation during molting. In addition, GO and KEGG enrichment analyses indicated that GO terms associated with chitin or cuticle, including “chitin binding”, “chitin metabolic process”, “chitin synthase activity”, “cuticle chitin biosynthetic process”, “cuticle hydrocarbon biosynthetic process”, and “structural constituent of cuticle”, and an array of amino acid metabolic pathways, including “alanine, asparatate and glutamate metabolism”, “glycine, serine and theronine metabolism”, “cysteine and methionine metabolism”, “tyrosine metabolism”, “phenylalanine metabolism and phenylalanine”, and “tyrosine and tryptophan biosynthesis” were significantly affected in response to Cry1Ab.
The advent of Genomic Era offers new transcriptome resources for the study of wolf spiders. Meng et al. sequenced cephalothoraxes of P. pseudoannulata adults and identified genes involved in insecticide metabolism and detoxification, including P450s, GSTs, AChEs, AChRs, GABA receptors, and GluCI . Xiao et al. carried out RNA-Seq analysis in P. pseudoannulata and revealed an array of genes responding to temperature stress . In this study, we focused on the genes corresponding to ingested Bt toxins. As a non-model animal without a reference genome, omics resources, such as transcriptomes, lay the foundation for future functional genomic research.
The Cry1Ab may have a negative impact on the formation of new cuticles during molting, which is contributed to the delayed development of spiderlings. To validate these transcriptomic responses, further examination at the translational level will be warranted.
Cluster of Orthologous Groups of proteins
Differential expression genes
Number of reads per kb of exon region per million mapped reads
Kyoto Encyclopedia of Genes and Genomes Database
- PBS buffer:
Phosphate buffer solution
The authors would like to thank the Oebiotech Enterprise (Shanghai) for their technical assistance.
This work was supported by the National Natural Science Foundation of P. R. China (No. 31071943, 318 31272339), the Scientific Research Key Fund of Hunan Provincial Science and Technology Department (No.319 10A054), and Agricultural Science and Technology Innovation Program of China (No. CAAS-ASTIP-2016-IBFC).
Availability of data and materials
The data set supporting the results of this article are available in the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/) repositories, SRR2024874, and SRR2024877.
ZW designed the whole study. JW, YDP, and KFX performed all the experiments and analyzed data. BYW collected and analyzed the data. JLH contributed to preparation for the experiment. JW was responsible for the paper writing. QSS, YDP, and XGZ revised and enhanced the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The ethical approval was not required. Materials used in this study were unregulated common arthropod spiders, Pardosa pseudoannulata, and insect pests, Nilaparvata lugens.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Bawa AS, Anilakumar KR. Genetically modified foods: safety, risks and public concerns-a review. J Food Sci Technol. 2013;50(6):1035–46.View ArticleGoogle Scholar
- Kamle S, Ali S. Genetically modified crops: Detection strategies and biosafety issues. Gene. 2013;522(2):123–32.View ArticleGoogle Scholar
- Brookes G, Barfoot P. Environmental impacts of genetically modified (GM) crop use 1996-2013: Impacts on pesticide use and carbon emissions. GM Crops Food. 2015;6(2):103–33.View ArticleGoogle Scholar
- Klumper W, Qaim M. A meta-analysis of the impacts of genetically modified crops. PLoS One. 2014;9(11):e111629.View ArticleGoogle Scholar
- Lucht JM. Public acceptance of plant biotechnology and GM crops. Viruses. 2015;7(8):4254–81.View ArticleGoogle Scholar
- Wu K. Suppression of cotton bollworm in multiple crops in China in areas with Bt toxin-containing cotton. Science. 2009;321(5896):1676–8.View ArticleGoogle Scholar
- Li Y, Peng Y, Hallerman EM, Wu K. Biosafety management and commercial use of genetically modified crops in China. Plant Cell Rep. 2014;33(4):565–73.View ArticleGoogle Scholar
- Han F, Zhou D, Liu X, Cheng J, Zhang Q, Shelton AM. Attitudes in China about crops and foods developed by biotechnology. PLoS One. 2015;10(9):e0139114.View ArticleGoogle Scholar
- Qiu J. Controversy of GM crops in China. Natl Sci Rev. 2014;1(3):466–70.View ArticleGoogle Scholar
- Bai YY, Yan RH, Ye GY, Huang F, Wangila DS, Wang JJ, Cheng JA. Field response of aboveground non-target arthropod community to transgenic Bt-Cry1Ab rice plant residues in postharvest seasons. Transgenic Res. 2012;21(5):1023–32.View ArticleGoogle Scholar
- Lu ZB, Tian JC, Han NS, Hu C, Peng YF, Stanley D, Ye GY. No direct effects of two transgenic Bt rice lines, T1C-19 and T2A-1, on the arthropod communities. Environ Entomol. 2014;43(5):1453–63.View ArticleGoogle Scholar
- Lu ZB, Liu YE, Han NS, Tian JC, Peng YF, Hu C, Guo YY, Ye GY. Transgenic cry1C or cry2A rice has no adverse impacts on the life-table parameters and population dynamics of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). Pest Manag Sci. 2015;71(7):937–45.View ArticleGoogle Scholar
- Qiang FU, Wang F, Dong LI, Yao Q, Lai F, Zhang Z. Effects of insect-resistant transgenic rice lines MSA and MSB on non-target pests Nilaparvata lugens and Sogatella fucifera. Acta Entomol Sin. 2003;46(6):697–704.Google Scholar
- Bai YY, Jiang MX, Cheng JA, Wang D. Effects of Cry1Ab Toxin on Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) through its prey, Nilaparvata lugens Stål (Homoptera: Delphacidae), feeding on transgenic Bt rice. Environ Entomol. 2006;35(4):1130–6.View ArticleGoogle Scholar
- Chen M, Liu ZC, Ye GY, Shen ZC, Hu C, Peng YF, Altosaar I, Shelton AM. Impacts of transgenic cry1Ab rice on non-target planthoppers and their main predator Cyrtorhinus lividipennis (Hemiptera: Miridae)-A case study of the compatibility of Bt rice with biological control. Biol Control. 2007;42(2):242–50.View ArticleGoogle Scholar
- Li Y, Chen X, Hu L, Romeis J, Peng Y. Bt rice producing Cry1C protein does not have direct detrimental effects on the green lacewing Chrysoperla sinica (Tjeder). Toxicol Environ Chem. 2014;33(6):1391–7.View ArticleGoogle Scholar
- Li Y, Wang Y, Romeis J, Liu Q, Lin K, Chen X, Peng Y. Bt rice expressing Cry2Aa does not cause direct detrimental effects on larvae of Chrysoperla sinica. Ecotoxicology. 2013;22(9):1413–21.View ArticleGoogle Scholar
- Chen M, Ye GY, Liu ZC, Qi F, Cui H, Peng YF, Shelton AM. Analysis of Cry1Ab toxin bioaccumulation in a food chain of Bt rice, an herbivore and a predator. Ecotoxicology. 2009;18(2):230–8.View ArticleGoogle Scholar
- Yuan Y, Ke X, Chen F, Krogh PH, Ge F. Decrease in catalase activity of Folsomia candida fed a Bt rice diet. Environ Pollut. 2011;159(12):3714–20.View ArticleGoogle Scholar
- Stotzky G. Persistence and biological activity in soil of the insecticidal proteins from Bacillus thuringiensis, especially from transgenic plants. Plant Soil. 2004;266(1-2):77–89.View ArticleGoogle Scholar
- Koskella J, Stotzky G. Microbial utilization of free and clay-bound insecticidal toxins from bacillus thuringiensis and their retention of insecticidal activity after incubation with microbes. Appl Environ Microbiol. 1997;63(9):3561–8.Google Scholar
- Li Y, Hallerman EM, Liu Q, Wu K, Peng Y. The development and status of Bt rice in China. Plant Biotechnol J. 2016;14(3):839–48.View ArticleGoogle Scholar
- Zhang G, Zhang W, Gu D. The structure and dynamics of main arthropod predator community in paddy field. Suppl J Sun Yasten Univer. 1995;2:33–40.
- Tian YX, Zhou Y, Xiao K, Wang Z, Chen JJ, Lu XY, Song QS. Effect of Cry1Ab protein on hemocytes of the wolf spider Pardosa pseudoannulata. Biocontrol Sci Technol. 2013;23(4):423–32.View ArticleGoogle Scholar
- Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29(7):644–52.View ArticleGoogle Scholar
- Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics. 2005;21(18):3674–6.View ArticleGoogle Scholar
- Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, et al. WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 2006;34(Web Server issue):W293–7.View ArticleGoogle Scholar
- Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.View ArticleGoogle Scholar
- Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikolskaya AN, et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics. 2003;4:41.View ArticleGoogle Scholar
- Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):511–5.View ArticleGoogle Scholar
- Wang L, Feng Z, Wang X, Wang X, Zhang X. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics (Oxford, England). 2010;26(1):136–8.View ArticleGoogle Scholar
- Andreas U, Ioana C, Triinu K, Jian Y, Faircloth BC, et al. Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012;40(15):e115.View ArticleGoogle Scholar
- Gene Ontology C. The Gene Ontology project in 2008. Nucleic Acids Res. 2008;36(Database issue):D440–4.Google Scholar
- Rivals I, Personnaz L, Taing L, Potier MC. Enrichment or depletion of a GO category within a class of genes: which test. Bioinformatics (Oxford, England). 2007;23(4):401–7.View ArticleGoogle Scholar
- Wang J, Peng YD, He C, et al. Cry1Ab-expressing rice did not influence expression of fecundity-related genes in the wolf spider Pardosa pseudoannulata. Gene. 2016;592(1):1–7.
- De Schrijver A, Devos Y, De Clercq P, Gathmann A, Romeis J. Quality of laboratory studies assessing effects of Bt-proteins on non-target organisms: minimal criteria for acceptability. Transgenic Res. 2016;25(4):1–17.
- Tian JC, Liu ZC, Chen M, Chen Y, Chen XX, Peng YF, Hu C, Ye GY. Laboratory and field assessments of prey-mediated effects of transgenic Bt rice on Ummeliata insecticeps (Araneida: Linyphiidae). Environ Entomol. 2010;39(4):1369–77.View ArticleGoogle Scholar
- Gnatzy W, Romer F. Cuticle: Formation, moulting and control. In: Bereiter-Hahn J, Matoltsy AG, Richards KS, editors. Biology of Integument. Berlin Heidelberg: Springer-Verlag; 1984. p. 638–84.View ArticleGoogle Scholar
- Al-Sawalmih A, Li C, Siegel S, Fabritius H, Yi S, Raabe D, Fratzl P, Paris O. Microtexture and chitin/calcite orientation relationship in the mineralized exoskeleton of the american lobster. Adv Funct Mater. 2008;18(20):3307–14.View ArticleGoogle Scholar
- Foelix R. Biology of spiders. In. Cambridge, Massachusetts, and London, Engand: Harvard University Press; 1982.Google Scholar
- Meng X, Zhang Y, Bao H, Liu Z. Sequence analysis of insecticide action and detoxification-related genes in theinsect pest natural enemy Pardosa pseudoannulata. PLoS One. 2015;10(4):e0125242.View ArticleGoogle Scholar
- Xiao R, Wang L, Cao Y, Zhang G. Transcriptome response to temperature stress in the wolf spider Pardosa pseudoannulata (Araneae: Lycosidae). Ecol Evol. 2016;6(11):3540–54.View ArticleGoogle Scholar