Expression and functional analysis of cytochrome P450 genes in the wolf spider Pardosa pseudoannulata under cadmium stress

Ecotoxicology and Environmental Safety 172 (2019) 19–25

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Expression and functional analysis of cytochrome P450 genes in the wolf spider Pardosa pseudoannulata under cadmium stress

T

⁎

Juan Wanga,b, Zhiyue Lvc, Ziyan Leib, Zhaoyang Chenb, Bo Lvb, Huilin Yangb, Zhi Wanga, , Qisheng Songd a

College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China College of Bioscience and Biotechnology, Hunan Agriculture University, Changsha, Hunan 410128, China c Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 China d Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Cd transporters Cytochrome P450 enzymes Molting Ecdysteroids Cuticle

Cytochrome P450 enzymes (CYPs), encoded by Halloween genes, mediate the biosynthesis of molting hormone, ecdysteroids, in arthropods. In this report, the effect of heavy metal cadmium (Cd) stress on the expression of cytochrome P450 genes in the wolf spider Pardosa pseudoannulata was analyzed. The results showed the expression levels of genes encoding for Cd transporters including ABC transporters, zinc transporters, calcium channel proteins and calcium binding proteins were inhibited or induced by Cd stress. In addition, the increase in metallothionein (MT) content and glutathione peroxidase (GPX) activity and decrease in total acetylcholine esterase (AChE) activity were also detected. Apparently, these detoxification methods did not completely protect the spider from the cytotoxicity of Cd stress. Increased mortality of P. pseudoannulata was observed when they were under Cd tress. In total 569 CYP genes belonging to 62 CYP subfamilies were obtained from P. pseudoannulata RNA-seq databases. BlaxtX analysis showed that 150, 161, 11, and 40 CYP genes were similar to the genes dib, phm, sad and shd, respectively, which are thought to catalyze the biosynthesis of ecdysteroids. Gene expression analysis suggested that 25 dib encoding genes, 27 phm encoding genes, 2 sad encoding genes, and 6 shd encoding genes were differentially expressed in TS2 vs. S2 comparison (Cd-treated 2nd instar spider vs. 2nd instar spider), respectively. There were 70 dib, 70 phm and 19 shd encoding genes either upregulated or downregulated, while 3 sad encoding genes were upregulated in TS5 vs. S5 (Cd-treated 5nd instar spider vs. 5nd instar spider). Genes related to heme binding and essential for activating the CYPs were also differentially expressed. Expression levels of cuticle related genes were significant differentially expressed, implying the changes in activities of chitin synthases and chitinase. Therefore we assume that unsuccessful molting process may occur on P. pseudoannulata due to influenced ecdysteroids levels, thus increasing mortality of spider.

1. Introduction Heavy metal pollution in agricultural soils is widespread in China due to the excessive use of pesticides and fungicides and industrial effluents and wastewater irrigation (Zhang and Chen, 2017; Yan et al., 2015). Levels of metals, such as copper (Cu), zinc (Zn), and cadmium (Cd), in paddy soils often exceed the national standard (Zhuang et al., 2009). The safety of farmland ecosystems is threatened by increasing pollution from heavy metals (Rizwan et al., 2016; Zhuang et al., 2009).

Heavy metals can be translocated to and accumulate in plants. They can accumulate in herbivores and predators exposed to polluted plants, causing physiological damage (Gall et al., 2015; Vinit-Dunand et al., 2002). Cadmium toxicity can affect seed germination, growth, mineral nutrients, photosynthesis, and grain yield of rice (Adhikari et al., 2006; Ahsan et al., 2007; He, 2010; Aziz et al., 2015; Wang et al., 2011). Food crops, such as wheat, rice, maize, sunflower, and cucumber, can suffer from growth inhibition, oxidative damage, and antioxidant stress in response to Cu exposure (Alhakimi and Hamada, 2011; Azooz et al.,

Abbreviations: CYPs, Cytochrome P450 enzymes; Cd, cadmium; S2, normal 2nd instar spiders; S5, normal 5th instar spiders; TS2, Cd-treated 2nd instar spiders; TS5, Cd-treated 5th instar spiders; RIN, RNA Integrity Number; Nr, NCBI non-redundant protein; Nt, non-redundant nucleotide sequences; KEGG, Kyoto Encyclopedia of Genes and Genomes pathway; Pfam, Protein family; GO, Gene Ontology; KOG, euKaryotic Orthologous Groups; DEGs, differentially expressed genes; phm, phantom; dib, disembodied; sad, shadow; shd, shade; FDR, A threshold for false discovery rate ⁎ Corresponding author. E-mail address: [email protected] (Z. Wang). https://doi.org/10.1016/j.ecoenv.2019.01.034 Received 29 September 2018; Received in revised form 13 December 2018; Accepted 9 January 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

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libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. These libraries were then sequenced on the Illumina sequencing platform (HiSeqTM 2500) and 150 bp/125 bp paired-end reads were generated. Clean reads were assembled to transcript sequences by Trinity (Grabherr et al., 2011). The longest sequences were selected as unigenes based on the similarity and length of sequences.

2012; Barbosa et al., 2013; Kumar et al., 2008; Lin et al., 2013; Thounaojam et al., 2012; Vinit-Dunand et al., 2002; Zheng et al., 2005). Changes of farmland arthropod fitness parameters in response to heavy metal pollutions have been documented. For example, the reproduction of Armadillidium granulatum Brandt was negatively affected by heavy metal exposure (Mazzei et al., 2013). Development time of Lymantria dispar L. larvae fed with an artificial diet containing Cd was significantly prolonged (Mirčić et al., 2013); The growth rates and adult sizes of lycosid spiders were reduced when they were exposed to heavy metals (Eraly et al., 2011). Various hormones, including prothoracicotropic hormone, juvenile hormone, and molting hormone play crucial roles in the growth and development of arthropods (Jindra et al., 2013; Vafopoulou et al., 2012; Zhuo et al., 2014). Heavy metals have been reported as endocrine disruptors that affect the secretion of hormones in crustaceans (Luo et al., 2015; Rodríguez et al., 2007). For example, Cd inhibits ecdysone secretion in the crustacean Gammarus fossarum and the crab Carcinus maenas (Abidi et al., 2016; Bondgaard and Bjerregaard, 2005). Hence, hormone levels can be altered by heavy metal stress and this influences growth and development. Cytochrome P450 (CYPs) in arthropods are involved in the synthesis of steroid hormones, such as ecdysone (Domanitskaya et al., 2014; Kong et al., 2015). The wolf spider Pardosa pseudoannulata is a common species in the paddy fields of south China (Preap et al., 2001). The expression of CYP genes in P. pseudoannulata spiderlings was characterized by means of RNA-Seq analysis, to study the effect of Cd on molting hormone production in this species.

2.4. Gene functional annotation and classification The assembled genes were used in BLAST and searched against public databases including NCBI non-redundant protein (Nr) and nonredundant nucleotide sequences (Nt), Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), Swiss-Prot, Protein family (Pfam), Gene Ontology (GO), and euKaryotic Orthologous Groups (KOG) with an Evalue cutoff of 10−5. Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was implemented by the GOseq R packages based on Wallenius non-central hyper-geometric distribution (Young et al., 2010), which can adjust for gene length bias in DEGs. In addition, the BlastX analysis was conducted to identify genes similar to CYP genes involved in ecdysteroid synthesis in Drosophila melanogaster. The reference proteins used for BlastX were phantom (phm), disembodied (dib), shadow (sad), and shade (shd).

2. Methods 2.5. Gene expression analysis and RT-qPCR analysis

2.1. Insect and spider preparation

All of the clean reads were mapped back onto the assembled transcriptome reference sequences by RSEM (Li and Dewey, 2011). Then, the read count of each gene was obtained and normalized to FPKM (Trapnell et al., 2010). The software DESeq was used to screen differentially expressed genes based on the negative binomial distribution. A threshold for false discovery rate (FDR) of < 0.0001 and a Log2 fold change value > 2 was used to determine significant differences in gene expression. The excess samples after RNA-Seq were used for RT-qPCR analysis. A total of 5 genes were selected for validation and the primer sequences were designed using primer 6.0 (Table S1). The FastQuant RT Kit (with gDNase) (TINGEN, China) was used for synthesis of cDNA. Real-time PCR was conducted using the FastFire qPCR PreMix (SYBR Green, TINGEN, China) by the ABI Step One Plus Real-time PCR System (Thermo Fisher Scientific, USA). The expression levels of mRNAs were normalized to 18S ribosomal RNA and quantified using the 2-△△Ct method (Livak and Schmittgen, 2001).

Fruit flies (Drosophila melanogaster) were provided by Hunan Normal University and reared on a corn meal medium. To obtain Cdcontaining fruit flies, we added a 1 mg/L CdCl2 solution to the corn meal medium to achieve a final CdCl2 concentration of 1 mg/L. The fruit flies were fed with the Cd-corn meal diet for 7 d and these flies were used as the spider diet (13.40 ± 0.399 mg/kg). Female P. pseudoannulata, with egg sacs, were collected from farmland without heavy metal pollution in the Hunan Academy of Agricultural Science (Wang et al., 2018a, 2018b). Spiderlings were collected immediately after hatch and placed individually into glass tubes (12 mm × 100 mm) with a moist cotton to maintain high humidity. Spiders were fed with either normal (control group) or Cdcontaining fruit flies daily, and mortality was recorded for each instars. Among these, the normal 2nd and 5th instars (S2 and S5) and Cd-fruit fly fed instars (TS2 and TS5) were collected for RNA-Seq analysis. 2.2. Determination of Cd content and enzyme activities The Cd content in the fruit fly and spider were analyzed with the Plasma Atomic Emission Spectrometer (ICPE-9000, SHIMADZU CORPORATIOM, JAPAN) as described by Wang et al. (2018a, 2018b). Enzyme-linked immunoabsorbent assay (ELISA) kits including the Insect GPX Elisa kit (Jianglaibio, China), Insect TChE Elisa kit (Jianglaibio, China) and Insect MT Elisa kit (Jianglaibio, China) were applied for determining activities of total cholinesterase (TChE) and glutathion peroxidase (GPX), and content of metallothionein, respectively.

3. Results 3.1. Effect of Cd on detoxifying enzyme activities and mortality ICP analysis showed that the Cd content in 5th instar spiders was 0.26 ± 0.0521 mg/kg. The Cd had a negative influence on mortality of the wolf spider P. pseudoannulata during the period of postembryonic development. There was no difference in mortality between Cd treated and control spiders when they were at 3th, and 4th instars. However, the mortality of 5th, 6th, 7th and 8th instars spiders in the treated group were significant higher than controls (p < 0.05, Fig. 1). Results from the ELISA detection showed that activity of GPX (controls: 12.48 ± 0.197, Cd-treated: 16.91 ± 0.620) and content of MT (controls: 3.92 ± 0.27 ng/g, Cd-treated: 4.79 ± 0.14 ng/g) were significantly increased in 5th instar spiderling under Cd stress, while activity of TChE was decreased (controls: 477.7 ± 13.115, Cd-treated: 382.37 ± 16.58, p < 0.05, Table 1).

2.3. RNA isolation and sequencing Total RNA was extracted using the mirRNA Isolation Kit (Ambion) following the manufacturer's protocol. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The samples with a RNA Integrity Number (RIN) ≥ 7 were subjected to subsequent analysis. Sequencing 20

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Table 2 Expression profiles of CYP genes in P. pseudoannulata.

Fig. 1. Effect of Cd stress on Mortality of P. pseudoannulata spiderling. Table 1 Oxidative stress of P.pseudoannulata under Cd stress.

Total cholinesterase (U) Glutathion peroxidase (U) Metallothionein (ng/g)

Control group

Cd-treated group

477.70 ± 13.115 12.48 ± 0.197 3.92 ± 0.27

382.37 ± 16.58* 16.91 ± 0.620* 4.79 ± 0.14*

* The asterisk indicates a significant difference between Cd-treated spiders and controls (p < 0.05).

3.2. Illumina sequencing and transcriptome assembly A total of 0.90 × 108, 1.03 × 108, 0.96 × 108, and 1.04 × 108 raw reads were generated from two replicate libraries for S2, S5, TS2, and TS5 respectively. After filtering out the reads with low quality and adapters, the percentages of clean reads in all 8 transcriptomes were > 95% for each library (Table S2). Using the Trinity program, a total of 323,548 genes were obtained with an average length of 918 bp. The N50 values of unigenes were 1309 bp (Fig. S1-A). The unigenes with a length of 200–500 bp sequences represented the highest proportion, followed by 500–1000 bp sequences (Fig. S1-B). All clean reads were submitted into NCBI SRA database (Accession number: PRJNA482767) 3.3. Functional annotation of all genes To study the functionality of the obtained genes, all of the genes were annotated in the 7 public databases. In the NR, Nt, KEGG, SwissProt, Pfam, GO, and KOG databases, 99,059, 27,911, 44,460, 70,788, 82,533, 83,011, and 40,580 genes were aligned, respectively. Among them, 119,208 genes were annotated in at least one database, while 10,776 (2.49%) genes were annotated in all of the databases (Table S3). A total of 40,580 gene sequences with significant homology were assigned to 26 KOG categories. The largest three groups included “General function prediction only (6764),” “Signal transduction mechanisms (5873),” and “Posttranslational modification, protein turnover, chaperones (4113)” (Fig. S2-A). For GO annotation, 83,011 genes were categorized into 56 functional groups consisting of 25 biological processes, 20 cellular components, and 11 molecular function subcategories (level 2, Fig. S2-B). In all, 44,460 genes were mapped against 32 KEGG pathways, of which the top three were “Signal transduction (5718),” “Endocrine system (3273),” and “Transport and catabolism (2908)” (level 2, Fig. S2-C).

Subfamilies

Total number of annotation genes

Differentially expressed in TS2 vs S2

Differentially expressed in TS5 vs S5

CYP49a1 CYP2A9 CYP12a4 CYP4c3 CYP18a1 CYP4V2 CYP3A13 CYP3A11 CYP3A24 CYP2J5 CYP4C1 CYP2J2 CYP4CU1 CYP2B11 CYP CYP2J6 CYP2C20 CYP2J1 CYP3A8 CYP3A1 CYP315a1 CYP4V2 CYP3A4 CYP3A30 CYP302a1 Cyp6u1 CYP6a2 Cyp4p2 CYP4p1 CYP4e2 CYP3A21 Cyp309a1 CYP2E1 Cyp28a5 CYP12A5 Cyp9f2 CYP9c1 Cyp6v CYP6t3 Cyp6d4 Cyp6a23 Cyp6a22 Cyp6a19 Cyp6a13 CYP516B1 CYP4V3 CYP4g15 Cyp4ac1 CYP3A29 CYP319A1 Cyp317a1 Cyp312a1 Cyp311a1 CYP306a1 CYP305a1 CYP2D10 CYP2C29 Cyp28d1 CYP1A2 CYP12D1 Cyp12c1 Cyp12b2

61 55 52 46 44 42 37 24 20 19 18 14 13 13 13 10 9 8 4 4 4 3 3 3 3 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

10 7 3 7 12 8 8 3 4 5 4 4 2

23 28 13 13 19 15 13 10 11 15 6 5 3 8 5 7 5 2 3 1

2 1 2 2 1

1

3 1

1

1

1

1

pseudoannulata RNA-seq databases through Nr annotation. A total of 62 CYP enzymes were encoded by these CYP genes, of which the CYP49a1 (61 genes) was the most annotated subfamily, followed by CYP2A9 (55 genes), and CYP12a4 (52 genes, Table 2). The distribution of CYP genes was revealed by a Venn diagram, which showed that 168, 182, 224, and 279 CYP genes were expressed in S2, S5, TS2, and TS5, respectively, while 11 genes were common among all four of the samples (FPKM

3.4. Identification and expression of CYP genes In all, 569 sequences of CYP genes were obtained from P. 21

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> 0.3, Fig. S3A). A total of 87 CYP genes, with 64 upregulated and 23 downregulated, were differentially expressed in TS2 vs. S2, and 211 CYP genes were differentially expressed in TS5 vs. S5, with 140 upregulated and 71 downregulated (Table 2). The distribution of log2 Fold Change values were revealed by a column diagram, which indicated that the fold changes of DEGs in TS5 vs. S5 were larger than those in TS2 vs. S2 (Fig. S3B). There were 21 CYP enzymes encoded by 87 differentially expressed CYP genes (TS2 vs. S2), while 24 CYP enzymes encoded by 211 CYP genes (TS5 vs. S5). Most of these CYP subcategories were shared in the two comparisons, including CYP49a1, CYP2A9, CYP12a4, CYP4c3, and CYP18a1 (Table 2). The RT-qPCR analysis was further conducted to validate the accuracy and reliability of sequencing data. Results indicated that the 3 selected genes encoding CYP 4c3, CYP 18a1, and CYP 12a4 were upregulated in TS2 vs S2. This is consistent with transcriptome analysis. And 2 genes encoding CYP 3A13 and cuticle protein respectively displayed the same expression tendency as the DEGs in the S5 vs. S5 comparison (Table S4). 3.5. CYP genes involved in biosynthesis of ecdysteroids in spiders The BlastX analysis indicated that 362 CYP genes shared the high similarity to four protein encoding genes of D. melanogaster, with an Evalue cutoff of 10−10. Proteins, including dib, phm, sad, and shd, were encoded by 150, 161, 11, and 40 genes, respectively. Gene expression analysis suggested that 25 dib-encoding genes, 27 phm-encoding genes, 2 sad-encoding genes, and 6 shd-encoding genes were differentially expressed in TS2 vs. S2, respectively. There were 70 dib, 70 phm, and 19 shd encoding genes that were up- or down-regulated, while 3 sad-encoding genes were upregulated in TS5 vs. S5 (Fig. 2). 3.6. Analysis of Cd-related genes There were 36,698 (15,460 upregulated and 21,238 downregulated) and 41,989 (24,710 upregulated and 17,279 downregulated) DEGs from two comparisons (TS2 vs S2 and TS5 vs S5), respectively (FDR < 0.0001, Log2 fold-change value > 2). In the two comparisons, four groups of Cd-related DEGs were identified as having the high similarity with ABC transporters, zinc transporters, calcium channel proteins, and calcium binding proteins. A total of 23 ABC transporter encoding genes with 14 downregulated and 9 upregulated were differentially expressed in TS2 vs. S2, while 84 genes were differentially expressed in TS5 vs. S5, with 29 downregulated and 55 upregulated. There were 20 DEGs belonging to zinc transporters that were differentially expressed in TS2 vs. S2, and 41 zinc transporters genes were differentially expressed in TS5 vs. S5. Based on pairwise comparisons, 4 and 14 calcium binding protein encoded genes were differentially expressed in TS2 vs. S2, and TS5 vs. S5, respectively. A total of 7 and 13 calcium channel protein genes were downregulated in TS2 vs. S2, and TS5 vs. S5, while 11 and 17 genes were upregulated in two comparisons, respectively (Table 3).

Fig. 2. The putative biosynthetic pathway of ecdysteroids. Enzymes are referred to by their names in D. melanogaster: Phm, phantom; Dib, disembodied; Sad, shadow; Shd, shade. The biosynthetic pathways of ecdysteroids are depicted with their identified corresponding enzymes and the number of genes that putatively code for four enzymes in the presented transcriptomic analysis. The number of differentially expressed ecdysteroids related to CYP genes in two comparisons are also presented. The upward arrow indicates gene upregulation while the downward arrow indicates gene downregulation.

Table 3 Effects of Cd stress on spider Cd transporter related genes.

3.7. GO enrichment analysis of DEGs GO enrichment analysis was conducted for all of the DEGs. The top 30 enriched GO terms are listed in Fig. 3, and the significantly enriched terms are followed by an asterisk. The “structural constituent of cuticle,” “DNA integration,” and “cytosolic ribosome” were the most significant enriched terms in TS2 vs S2 (Fig. 3A), while “structural constituent of cuticle,” “chitin binding,” and “chitin metabolic process” were the most significant enriched terms in TS5 vs S5 (Fig. 3B). In addition, the term “heme binding” was also significantly enriched in TS5 vs. S5.

ABC transporters Zinc transporters Calcium channel proteins Calcium binding proteins

22

Comparison TS 2 vs. S2

Comparison TS 5 vs. S5

Upregulated

Downregulated

Upregulated

Downregulated

9

14

55

29

11

9

23

18

11

7

17

13

2

2

9

5

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Fig. 3. GO enrichment analysis of differentially expressed genes. A. GO terms enriched in comparison TS2 vs. S2. B. GO terms enriched in comparison TS5 vs. S5. The numbers in the x-axis show the GO categories as listed on the right. Columns marked by asterisk are significantly enriched terms. Numerical values on each column are corrected q values of each enriched term.

23

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4. Discussion

into which the microfibers, made of the polysaccharide chitin, are incorporated (Foelix, 2011). Spider molting involves dissolving the old cuticle and formation of a new cuticle (Foelix, 2011). New chitin synthesis is catalyzed by chitin synthases, while old cuticle is hydrolyzed by the chitinase. Chitinase activity in ecdysozoa is regulated by hormones, such as ecdysteroids (Zhang and Zheng, 2016; Yang et al., 2013). In the present study, expression levels of cuticle related genes were significant differentially expressed, implying the changes in activities of chitin synthases and chitinase. GO terms "structure constituent of cuticle" was the most significant term enriched in TS2 vs S2, while "structure constituent of cuticle," "chitin binding", and "chitin metabolic process" were the top three enriched terms in TS5 vs S5. Therefore, we assume that the unsuccessful molting process may occur on P. pseudoannulata due to altered ecdysteroid levels influenced by Cd stress, thus increasing the mortality of spider.

Heavy metal pollution and the associated risks posed by Cd is a serious problem in China (Zhang et al., 2015). Spiders are predators in paddy fields and play an important role in pest control (Liu et al., 1999). Previous studies suggested that the heavy metals could be translocated and accumulated in spiders via food chain (Gall et al., 2015). Hence the present study was designed to assess the effect of Cd stress on the expression of cytochrome P450 genes in P. pseudoannulata following oxidative stress, through imitating the transfer and bioaccumulation of the heavy metal Cd in food chains, from corn meal medium to D. melanogaster, and to predator P. pseudoannulata. The Cd level of corn meal medium was 1 mg/L, which was equivalent to the middle level of soil environmental pollution in China. ICP analysis showed that the Cd content in the 5th instar P. pseudoannulata was 0.26 ± 0.0521 mg/kg, after they fed with Cd-containing fruit flies. Multiple antioxidant strategies were reported on this spider. In spiders, the metals taken with food are usually neutralized in the form of mineral concretion in midgut cells or bind to metallothioneins (Pal and Rai, 2010). The raise in MT content was detected in the 5th instar P. pseudoannulata, indicating a defensive role of the proteins. Significantly higher activities of GPX was recorded in P. pseudoannulata, implying a key role in scavenging excess oxygen free radical induced by Cd stress. A relationship between metal transporters and Cd exposure has been observed in several species including Drosophila, nematodes, and spiders (Yepiskoposyan et al., 2006; Cui et al., 2007; Li et al., 2016). ABC transporters and zinc transporters were reported to play key roles in the acquisition, distribution, and homeostasis of Cd in organisms (Zhang et al., 2018; Kazemi-Dinan et al., 2014). Calcium channel proteins and calcium binding proteins were also involved in Cd transport (Hinkle et al., 1987). Expression levels of these Cd transporter genes were inhibited or induced by Cd in P. pseudoannulata. However, these detoxification methods do not completely protect against the cytotoxicity of Cd stress. Indirect effects of Cd stress on fitness traits of spiders following oxidative stress were still observed. Growth rates and adult sizes of lycosid spider Pardosa saltans could be reduced, when exposed to heavy metal stress (Eraly et al., 2011). Exposure of P. pseudoannulata to Cd stress resulted in reduced body mass, delayed development, fewer eggs and increased mortality (Li et al., 2016; Wang et al., 2018a, 2018b). Here, we also recorded a significant increase in mortality of P. pseudoannulata under Cd stress. Molting is an essential process for spider growth, which is triggered and controlled by ecdysteroids (Riddiford and Edelman, 2003). The biosynthesis of ecdysteroids is usual catalyzed by several CYP enzymes (Tamar et al., 2012). Genes phm, dib, sad, and shd are Halloween genes identified in D. melanogaster coding for cytochrome P450 enzymes in the ecdysteroidogenic pathway. These enzymes catalyze four sequential hydroxylation reactions of the biosynthetic pathway of ecdysteroids from ketodiol to 20-hydroxyecdysone (Gilbert and Warren, 2005). And, study indicated that Halloween genes involved in 20E biosynthesis were conserved in arthropod (Ventura et al., 2017). Hence, the blastX was applied to obtain CYP450 orthologs from P. pseudoannulata RNAseq databases. We obtained 569 CYP genes that belong to 62 CYP subfamilies from P. pseudoannulata RNA-seq databases through Nr annotation, suggesting a wide variety of CYP enzymes in this species. BlaxtX analysis showed that 150, 161, 11, and 40 genes of P. pseudoannulata were similar to the genes dib, phm, sad, and shd, respectively. Meanwhile, the expression levels of these genes were upregulated or downregulated both in the 2nd and 5th instar spiders in response to Cd exposure. Heme is usually contained in the active site of CYPs and acts as a cofactor (Liu et al., 2013). GO enrichment analysis demonstrated that genes involved in heme binding were enriched in TS5 vs S5, implying the potential influence of Cd on catalytic function of CYPs. Spiders are arthropods that possess an exoskeleton comprised of a many-layered cuticle. The basic component of the cuticle is a protein

5. Conclusions In the presented study, we studied the effect of Cd stress on cytochrome P450 genes in the P. pseudoannulata following oxidative stress. Increased mortality of P. pseudoannulata was observed when they were under Cd tress. Expression levels of Cd transporters including ABC transporters, zinc transporters, calcium channel proteins and calcium binding proteins were altered by Cd. And the increase in MT content and GPX activity and decrease in TChE activity were also detected. The150, 161, 11, and 40 CYP genes were similar to the genes dib, phm, sad, and shd, respectively, which are thought to catalyze the biosynthesis of ecdysteroids. Gene expression analysis suggested that these CYP genes involved in ecdysone biosynthesis were upregulated or downregulated under Cd stress. Genes related to heme binding and essential for activating the CYPs and genes acting in new cuticle formation were both differentially expressed, suggesting that increased mortality of spider may be caused by unsuccessful molting, due to changes in expression levels of CYPs. Acknowledgments We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. Ethics approval and consent to participate Ethical approval was not required. Materials used in this study were an unregulated common spider, P. pseudoannulata, and an insect, D. melanogaster. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Natural Science Foundation of China (No. 31472017, 31272339); The key projects of Hunan Provincial Science and Technology Department (No. 2014FJ2003); The Planned Science and Technology Project of Hunan Province, China (No. 2015RS4036); The research project of Education Department of Hunan Province (No. 15C0666); The Postgraduate Research Projects of Hunan Province, China (CX2017B361); The Open Research Fund of Key Laboratory of Tropical Disease Control and Research, Ministry of Education in China (2018 kfkt03). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2019.01.034. 24

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