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Zi-Chao Liu, Jin-Yang Liang, Xin-Qiang Lan, Tao Li, Jia-Rui Zhang, Fang Zhao, Geng Li, Pei-Yi Chen, Yun Zhang, Wen-Hui Lee, Feng Zhao. Comparative analysis of diverse toxins from a new pharmaceutical centipede, Scolopendra mojiangica. Zoological Research, 2020, 41(2): 138-147. doi: 10.24272/j.issn.2095-8137.2020.019
Citation: Zi-Chao Liu, Jin-Yang Liang, Xin-Qiang Lan, Tao Li, Jia-Rui Zhang, Fang Zhao, Geng Li, Pei-Yi Chen, Yun Zhang, Wen-Hui Lee, Feng Zhao. Comparative analysis of diverse toxins from a new pharmaceutical centipede, Scolopendra mojiangica. Zoological Research, 2020, 41(2): 138-147. doi: 10.24272/j.issn.2095-8137.2020.019

Comparative analysis of diverse toxins from a new pharmaceutical centipede, Scolopendra mojiangica

doi: 10.24272/j.issn.2095-8137.2020.019
Funds:  This work was supported by grants from the Chinese National Natural Science Foundation (81860696, 31560596, 81373945, and 31360516), Yunnan Applied Basic Research Projects (2016FD076), Key Research Program of the Chinese Academy of Sciences (KJZD-EW-L03), "Yunling Scholar" Program, Yunnan Provincial Training Programs of Youth Leader in Academic and Technical Reserve Talent (2019HB058), and Puer University (2017XJKT12 & CXTD011)
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  • As the oldest venomous animals, centipedes use their venom as a weapon to attack prey and for protection. Centipede venom, which contains many bioactive and pharmacologically active compounds, has been used for centuries in Chinese medicine, as shown by ancient records. Based on comparative analysis, we revealed the diversity of and differences in centipede toxin-like molecules between Scolopendra mojiangica, a substitute pharmaceutical material used in China, and S. subspinipes mutilans. More than 6 000 peptides isolated from the venom were identified by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and inferred from the transcriptome. As a result, in the proteome of S. mojiangica, 246 unique proteins were identified: one in five were toxin-like proteins or putative toxins with unknown function, accounting for a lower percentage of total proteins than that in S. mutilans. Transcriptome mining identified approximately 10 times more toxin-like proteins, which can characterize the precursor structures of mature toxin-like peptides. However, the constitution and quantity of the toxin transcripts in these two centipedes were similar. In toxicity assays, the crude venom showed strong insecticidal and hemolytic activity. These findings highlight the extensive diversity of toxin-like proteins in S. mojiangica and provide a new foundation for the medical-pharmaceutical use of centipede toxin-like proteins.
  • #Authors contributed equally to this work
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Comparative analysis of diverse toxins from a new pharmaceutical centipede, Scolopendra mojiangica

doi: 10.24272/j.issn.2095-8137.2020.019
Funds:  This work was supported by grants from the Chinese National Natural Science Foundation (81860696, 31560596, 81373945, and 31360516), Yunnan Applied Basic Research Projects (2016FD076), Key Research Program of the Chinese Academy of Sciences (KJZD-EW-L03), "Yunling Scholar" Program, Yunnan Provincial Training Programs of Youth Leader in Academic and Technical Reserve Talent (2019HB058), and Puer University (2017XJKT12 & CXTD011)

Abstract: As the oldest venomous animals, centipedes use their venom as a weapon to attack prey and for protection. Centipede venom, which contains many bioactive and pharmacologically active compounds, has been used for centuries in Chinese medicine, as shown by ancient records. Based on comparative analysis, we revealed the diversity of and differences in centipede toxin-like molecules between Scolopendra mojiangica, a substitute pharmaceutical material used in China, and S. subspinipes mutilans. More than 6 000 peptides isolated from the venom were identified by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and inferred from the transcriptome. As a result, in the proteome of S. mojiangica, 246 unique proteins were identified: one in five were toxin-like proteins or putative toxins with unknown function, accounting for a lower percentage of total proteins than that in S. mutilans. Transcriptome mining identified approximately 10 times more toxin-like proteins, which can characterize the precursor structures of mature toxin-like peptides. However, the constitution and quantity of the toxin transcripts in these two centipedes were similar. In toxicity assays, the crude venom showed strong insecticidal and hemolytic activity. These findings highlight the extensive diversity of toxin-like proteins in S. mojiangica and provide a new foundation for the medical-pharmaceutical use of centipede toxin-like proteins.

#Authors contributed equally to this work
Zi-Chao Liu, Jin-Yang Liang, Xin-Qiang Lan, Tao Li, Jia-Rui Zhang, Fang Zhao, Geng Li, Pei-Yi Chen, Yun Zhang, Wen-Hui Lee, Feng Zhao. Comparative analysis of diverse toxins from a new pharmaceutical centipede, Scolopendra mojiangica. Zoological Research, 2020, 41(2): 138-147. doi: 10.24272/j.issn.2095-8137.2020.019
Citation: Zi-Chao Liu, Jin-Yang Liang, Xin-Qiang Lan, Tao Li, Jia-Rui Zhang, Fang Zhao, Geng Li, Pei-Yi Chen, Yun Zhang, Wen-Hui Lee, Feng Zhao. Comparative analysis of diverse toxins from a new pharmaceutical centipede, Scolopendra mojiangica. Zoological Research, 2020, 41(2): 138-147. doi: 10.24272/j.issn.2095-8137.2020.019
    • As one of the oldest and most important predatory arthropods, the centipede has a fossil record that extends back 420 million years (Undheim & King, 2011). Approximately 3 300–3 500 centipede species have been found, with distribution worldwide and in most provinces of China (Rong et al., 2015). Centipede venom, which is secreted from venom glands in the first pair of limbs (Edgecombe & Giribet, 2007), is essential for survival, not only for subduing and killing prey but also for defense against predators.

      Animal venom has long been considered a rich source of pharmacological and novel therapeutics (Kalia et al., 2015; Smith et al., 2013; Zhang, 2015). Furthermore, dried centipedes have been used medicinally for centuries, as shown in ancient Chinese medical records. Recently, an increasing number of studies have shown that centipede venom contains various functional components, including a rich reservoir of structural and pharmacological peptides (Hakim et al., 2015; Undheim et al., 2015, 2016). In addition, because of their excellent chemical and pharmacological activities, particularly as neurotoxins and ion channel inhibitors, centipede toxins have received further attention (Liu et al., 2012; Yang et al., 2012, 2013, 2015). Several antimicrobial peptides and specific toxins have also been identified in centipede venom (Chen et al., 2014; Hou et al., 2013; Peng et al., 2010; Yang et al., 2012). Interestingly, centipede toxins are expressed outside the venom gland and are involved in gene recruitment processes (Zhao et al., 2018a). These venom peptides have significant chemical, thermal, and biological stability, which enable researchers to adapt their functions for therapeutic use.

      Therefore, centipede venom research is of great interest for investigating putative toxins. These toxins can act on a range of molecular targets, including voltage-gated sodium (NaV), potassium (KV), and calcium (CaV) channels (Liu et al., 2012; Yang et al., 2012). However, biochemical studies on centipede toxins are not nearly as extensive as studies on other venomous animals, such as snakes, spiders, and scorpions (Undheim et al., 2016), and complete data on centipede venom toxins, peptides, and protein sequences are currently limited to a small number of species (Hakim et al., 2015; Undheim et al., 2016). One potential reason is that most centipede species are considered too small to obtain enough venom for activity testing or high-throughput drug screening. Omics analysis of venom or venom glands is one approach for probing toxin molecular diversity. Specifically, to identify new putative proteins and enable comparison across species, large-scale sequencing of a broad array of centipede venom should be applied to further confirm the complexity of venom (Gonzalez-Morales et al., 2014; Liu et al., 2012; Rong et al., 2015).

      Previous centipede research has mainly focused on Scolopendra mutilans (Zhao et al., 2018a), and occasionally on S. subspinipes subspinipes, S. viridis, and S. dehaani (Liu et al., 2012). To date, however, no comprehensive research has been reported on the new pharmaceutical centipede, S. mojiangica (Wang et al., 1997), which is used as a substitute medicinal material in traditional Chinese medicine. Therefore, a fully integrated approach combining transcriptomics and proteomics is essential for understanding the differences among pharmaceutical centipedes, including venom composition and toxin diversity. Here, in-depth proteotranscriptomic analyses (combined proteomic and transcriptomic analyses) were used to study centipede venom, and the protein/peptide composition of the dissected venom gland from S. mojiangica was described. Complete comparative analyses of the protein compounds and toxin distribution in the venom or venom gland of S. mojiangica and S. mutilans were also presented based on RNA-Seq and MS datasets.

    • Adult S. mojiangica (both sexes) were collected from Mojiang (N23°27', E101°41'), Yunnan Province, China. All centipede (S. mojiangica) studies were reviewed and approved by the Animal Care and Use Committee of Puer University (ACUP. 531068520180126, approved on 17 September 2018).

    • The venom of S. mojiangica was collected as per our previous method. Briefly, a 3 V alternating current (AC) was used to stimulate the venom glands in the first pair of centipede limbs (Liu et al., 2012). The venom samples were stored at −20 °C until use. A 300 mg S. mojiangica venom sample was solubilized in 3 mL of Tris-HCl buffer. The venom solution was then loaded on a Sephacryl S-100HR (HiprepTM26/60, 71-1247-00-EG, GE Healthcare, USA) gel filtration column with a flow rate of 0.5 mL/h. Thirteen peaks (named P1−13) were obtained from this procedure (Supplementary Figure S1).

      The proteins/peptides contained in the venom were pre-denatured with 500 µL of 25 nmol/L NH4HCO3 and separated with a 3 kDa cut-off ultrafiltration tube. The low molecular weight (<3 kDa) proteins/peptides were collected and desalinated before peptidomic analysis. Proteins/peptides with molecular weights greater than 3 kDa were applied to SDS-PAGE gels for separation. One half of each sample was mixed with extraction buffer (0.25% acetic acid and protease inhibitor cocktail) and disrupted with a sonicator (Hielscher Ultrasound Technology, Germany). To further separate these samples, 12% gel with protein ladder (Thermo, ref. 26614, USA) SDS-PAGE was used, followed by staining with GelCode Blue Stain (Thermo ref. 24592, USA) and destaining with Milli-Q water (Millipore, USA). We excised six bands from each lane for in-gel trypsin digestion. Samples were extracted with 100% acetonitrile, desalinated, lyophilized, and stored at −80 °C until further electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analysis.

    • A total of 260 mg of venom gland was preserved in liquid nitrogen after collection from S. mojiangica until use. RNA extraction and cDNA library construction were performed according to our previous work (Zhao et al., 2014a, 2014b). cDNA from the S. mojiangica venom gland was sequenced using the Illumina HiSeq™ 2000 (USA), and the short-read assembly program SOAPdenovo-Trans (v1.03) was run with default parameters to complete de novo transcriptome assembly. Overlaps with certain lengths and connected paired-end reads were combined in the program to form contigs. The sequence clustering software TGICL was used to splice sequences and remove redundant sequences to produce the complete assembly of contigs of each sample (Pertea et al., 2003), and the longest possible non-redundant unigenes were produced. The TGICL parameters were the same as the parameters used in our previous work (Zhao et al., 2014b).

    • After in-gel digestion, candidate fractionation samples were loaded onto an EASY-nLC HPLC system (Thermo Fisher Scientific, USA) equipped with a binary rapid separation nano-flow pump and ternary loading pump. Mobile phase eluent A (0.1% TFA contained in ddH2O) and mobile phase eluent B (ACN/ddH2O/TFA 90/10/0.08% (v/v/v)) were used. Samples were applied to a Thermo Scientific EASY loading column (2 cm×100 µm, 5 µm −C18, USA) by the auto-sampler and analytical column (75 µm×100 mm, 3 µm −C18), respectively, with a flow rate of 250 nL/min. With linear stepwise gradients (0′−5% B, 5′−5% B, 12.5′−20% B, 62.5′−70% B, 63.5′−99% B, 65′−99% B, 66′−5% B and 72′−5% B), we separated the peptides with the column. Starting at 20% eluent B, 1.25 mL/5 min of each fraction was collected and lyophilized.

      We selected the data-dependent mode of the Q Exactive instrument (Thermo Finnigan, USA), which then switched between full scan MS and MS/MS acquisition automatically. Based on the predictive automatic gain control (AGC) of the previous full scan, we accumulated 3×106 target value ions and acquired 70 000 (m/z 200) resolution of full scan MS spectra (m/z 300−1 800) in the Orbitrap. In addition, 15 s was set as the dynamic exclusion value. We isolated and fragmented the 10 most intense multiply charged ions (z≥2) sequentially by higher-energy collisional dissociation (HCD) with a fixed resolution of 17 500 (m/z 200) and an injection time of 60 ms for the MS2 scanning method. The mass spectrometric conditions were as follows: 2 kV spray voltage, no sheath and auxiliary gas flow, 250 °C heated capillary temperature, 27 eV normalized HCD collision energy, and 0.1% underfill ratio. A total of 1×105 counts was set as the ion selection threshold for MS/MS.

    • Using Proteome Discoverer (version 1.4), RAW data files were produced. Mascot v2.2 was used as the search tool to generate peak lists in our transcriptome database. Trypsin was chosen as an enzyme, and two missed cleavages were allowed. The MS/MS search criteria were as follows: MS polypeptide tolerance 2×104 mg/m³ and MS/MS mode 0.1 Da. The aminomethylation of cysteine was statically modified and the oxidation of methionine was dynamically modified. High confidence peptides were used for protein identification, generating a 1% false discovery rate (FDR) threshold. Only unique peptides with high confidence were used for protein identification.

      All unigenes in our centipede database were annotated with BLASTX and searched against known databases, as presented in our previous study (Zhao et al., 2014a, 2014b, 2018a). Unigenes were aligned with high-priority databases and annotated with a given description instead of aligning with a low-priority database. Gene Ontology (GO) annotation was carried out using the Blast2GO (Conesa et al., 2005) software suite v2.5.0. In these searches, the BLASTX cut-off was set to 1e-6. The BLAST tool was used to search the toxin database and annotate the toxin with Tox-Prot in UniProtKB (02 February 2019, 6 822 sequences) and the animal toxin database platform ATDB (He et al., 2008), with the toxins then verified by phylogenetic analyses. The grouped sequences were aligned using MUSCLE v3.8.31 (Edgar, 2010). MrBayes 3.2.7 was used for phylogenetic analyses with maximum likelihood. The values were estimated by ultrafast bootstrap using 10 000 iterations. The resulting trees were analysed with MEGA 7 (Kumar et al., 2016), which was also used to automatically plot expression values and detection in venom.

      Comparative expression analysis was performed as follows: comparison of RNA-Seq data of venom glands of various species was performed using Bowtie v0.12.7 (Langmead et al., 2009) and TopHat v2.0.6 (Trapnell et al., 2009) for mapping. Gene expression values were calculated from the expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) (Trapnell et al., 2010). The FPKM values for genes from every tissue were determined by rSeq (Jiang & Wong, 2009). The graphs and statistical analyses were performed using GraphPad Prism v5.0 (La Jolla, USA) and R v3.3.2. Here, P<0.05 was considered statistically significant.

    • Insect bioassays were performed according to the method in Yang et al. (2012). Freeze-dried crude venom powder was dissolved in insect saline (concentrations in deionized water: 140 mmol/L NaCl, 5 mmol/L KCl, 4 mmol/L NaHCO3, 1 mmol/L MgCl2, 0.75 mmol/L CaCl2, 5 mmol/L HEPES) and injected into grasshoppers (Locusta migratoria manilensis; mass 700–900 mg) and mealworms (Tenebrio molitor larvae; mass 190–210 mg). Ants (Tetramorium spp., adults; mass 35–55 mg) were fed with same venom.

      Using human, mouse, and rabbit red blood cells (RBCs), hemolytic activity was assayed as described previously (Liu et al., 2012; Zhao et al., 2018b). Briefly, serial dilutions of the samples were incubated with washed RBCs (3%) at 37 °C for 30 min and then centrifuged. The resulting supernatant was measured at an absorbance of 540 nm. Maximum hemolysis was determined by adding 1% Triton X-100 to the cell samples.

    • Original Chinese medicinal centipedes include S. mutilans, S. multidens, S. mojiangica, and S. negrocapitis (Wang et al., 1997). Here, we studied the novel substitutional pharmaceutical centipede, S. mojiangica, with comparative analysis of active molecules. Scolopendra mojiangica showed a relatively close relationship to S. negrocapitis, S. mutilans, and S. multidens (Figure 1A), though a smaller body size than S. mutilans, S. dehaani, and S. multidens. Similar to other species, it also uses venom to attack prey and in defense.

      Figure 1.  Proteomic and transcriptomic analyses of new pharmaceutical centipede

      The protocol for isolating venom glands from S. mojiangica was described in our previous study (Liu et al., 2012). Healthy adult centipedes (n=280) without injury were selected, and the venom glands were dissected from their first pair of limbs. After that, 3 V AC was used to stimulate the venom gland and ensure that more toxins were included, so that proteome coverage could be improved. The isolated venom glands were then further processed (Figure 1B). A portion of each sample was used to obtain the proteome by SDS-PAGE analysis. Protein bands from the venom gland were excised for in-gel digestion and subjected to ESI-MS/MS analysis. The remaining portion of each sample was used to extract RNA, followed by RNA-Seq analysis of the transcriptome.

    • A total of 246 proteins were identified in S. mojiangica at 95% coverage by ESI-MS/MS analysis (Supplementary Table S1; Figure 2A). In the proteome, 73.6% of proteins (n=181) were cellular components and 19.1% of proteins (n=47) were unknown functional proteins, which were putative venom toxins. Only 18 proteins were identified as toxin-like proteins, including neurotoxins, K+ channel inhibitors, and blarina toxins (Figure 2B; Table 1). Although we obtained more proteins in S. mojiangica than in S. mutilans and S. viridis with proteomic analysis, the detected toxin-like proteins in S. mojiangica represented a lower percentage of total proteins than those identified in S. mutilans in our previous study (Figure 2C). In the venom proteome, most of the identified proteins showed a molecular weight of less than 50 kDa, similar to the proteome of S. mutilans (Figure 2D). Thus, the centipedes contained notably small functional molecules for potential pharmaceutical use, as expected. Based on peptide detection, 23.2% of proteins consisted of six or more unique peptides (Supplementary Figure S2). In addition, the more enriched the peptides assembled into proteins, the more comprehensive was the proteome obtained.

      Sequence IDGenBank accession No.Sequence descriptionCategoryPeptidesE-ValueMW (kD)Calc. pIFPKM
      ScoMo_singlet48841AT0003236Blarina toxin precursor (EC 3.4.21.-)Blarina toxin91.00E-3721.614.1592.54
      ScoMo_singlet50899AT0003766Mucrofibrase-5 precursor (EC 3.4.21.-)Mucrofibrase-5114.00E-1614.409.933 454.74
      ScoMo_singlet71394AT0002263Pseudechetoxin-like protein precursorPseudechetoxin2769.00E-4228.749.867 195.57
      ScoMo_contig2076gi|429840589K+ channel inhibitorChannel inhibitor6174.00E-16462.769.151.37
      ScoMo_singlet78309AT0000117Latisemin precursorLatisemin4122.00E-2220.897.960.00
      ScoMo_contig4762AT0003236Blarina toxin precursor (EC 3.4.21.-)Blarina toxin1081.00E-4428.586.515 173.32
      ScoMo_singlet45908AT0003741Thrombin-like enzyme contortrixobin (EC 3.4.21.-)Serine proteinase1091.00E-4144.945.081 685.57
      ScoMo_singlet67462AT0000120Pseudecin precursorPseudechetoxin665.00E-3223.718.9114 111.58
      ScoMo_singlet72573AT0000552Hopsarin-D (EC 3.4.21.6)Hopsarin-D931.00E-12185.156.53132.70
      ScoMo_singlet76606AT0000554Trocarin precursor (EC 3.4.21.6)Trocarin383.00E-13884.926.1760.34
      ScoMo_singlet25641AT0000552Hopsarin-D (EC 3.4.21.6)Hopsarin-D465.00E-2027.214.6184.53
      ScoMo_singlet69905AT0000554Trocarin precursor (EC 3.4.21.6)Trocarin144.00E-10740.695.281 245.366
      ScoMo_singlet57737AT0003404Zinc metalloproteinase fibrolase (EC 3.4.24.72)Metalloproteinase204.00E-1635.218.1348.71
      ScoMo_singlet8256AT0000762Alpha-latrocrustotoxinAlpha-latrocrustotoxin10050.486.79136.27
      ScoMo_singlet68890AT0000552Hopsarin-D (EC 3.4.21.6)Hopsarin-D135.00E-7542.037.88161.84
      ScoMo_singlet7846gi|392295725Omega-slptx-ssm2a neurotoxin precursorNeurotoxin118.00E-368.564.9316 647.01
      ScoMo_singlet55496gi|501293796Cathepsin LCathepsin L1801.00E-15537.306.352.83
      ScoMo_singlet39956AT0000554Trocarin precursor (EC 3.4.21.6)Trocarin124E-094.643.795.55
      MW: Molecular Weight; Calc. pI: The calculated isoelectric point (pI); FPKM: Fragments Per Kilobase of exon model per Million mapped fragments.

      Table 1.  Toxin-like proteins/peptides identified from venom proteome of S. mojiangica centipede

      Figure 2.  Profiles of S. mojiangica proteome

    • We acquired 43 381 437 clean reads assembled into 132 597 contigs from the venom gland using the Trinity program. As a result, the transcriptome data consisted of 107 642 putative gene objects (all unigenes) ranging from 101 bp to 9 184 bp, with an average length of 423 bp. The number of unigenes larger than 500 bp was 24 219. The largest unigenes were 9 184 bp in size, and the N50 of the unigenes was 214 bp (Supplementary Figure S3 and Table S2).

      For comparative analysis, the venom gland transcriptome from S. mojiangica showed many transcripts (n=46 571) with high similarity to those of S. mutilans. Notably, however, most transcripts showed low similarity between the two centipede species (Figure 3A). In the transcriptomic expression analysis, the read count of each transcript in S. mojiangica and S. mutilans showed biases for gene expression, with higher expressed transcripts in S. mojiangica (Figure 3B). Functional annotation analyses of these transcripts were combined with Blast searching and phylogenetic analyses to obtain toxin-like unigenes. In total, 410 toxin-like transcripts were identified in the transcriptome of S. mojiangica, more than that identified in S. mutilans (342 transcripts). Furthermore, these transcripts were divided into 34 categories, mainly consisting of alpha-latrocrustotoxin, delta-latroinsectotoxin, ion channel inhibitors, and alpha-latrotoxin (Figure 3C).

      Figure 3.  Identification of toxins from transcriptome of venom gland in centipedes

    • As expected, we identified 34 kinds of toxin-like unigenes (n=342) from the transcriptome of S. mutilans using the same annotation method as that of S. mojiangica (Figure 4A). In total, 11 of these toxin-like unigenes encoded the most transcripts in the two centipedes. With gene expression analyses, most toxin-like unigenes showed no differential expression between S. mojiangica and S. mutilans, except for four toxin-like unigenes (i.e., alpha-latrotoxin, hopsarin-D, metalloproteinase, and trocarin) (Figure 4B).

      Figure 4.  Comparison of toxin-like molecules distributed in centipedes S. mojiangica and S. mutilans

      Finally, we determined the toxicity and performed crude isolation of the centipede venom. The crude centipede venom exhibited strong insecticidal action (Figure 5A), and the crude venom had a similar potency as the venom of S. mutilans. The crude venom and its fractions eluted from the S-100HR column (Supplementary Figure S1; Figure 5B) showed hemolytic activity. The elution of peak 1 (P1) showed high hemolytic activity on human RBCs when 1 mg/mL protein/peptide was incubated for 4 h. In contrast, peaks 3, 5, and 6 (P3, P5, and P6) had lower hemolytic activity than that of P1 and crude venom.

      Figure 5.  Insecticidal activity of crude centipede venom

    • Due to long-term evolutionary fine-tuning, venom toxins exhibit high specificity and potency for molecular targets that are not often found in natural or synthetic small molecules, and thus animal toxins are valuable pharmacological tools (King, 2011, 2013). There are many cases in which venom toxin has been used as a pharmacological molecule, e.g., snake venom, dried toad skin secretions (Chan Su), tarantula venom, and cobra venom used as traditional Ayurvedic, Chinese, Mexican, and Central and South American medicines, respectively (Harvey, 2014; King, 2011). These traditional medicines have been used to treat arthritis, gastrointestinal ailments, asthma, polio, multiple sclerosis, rheumatism, severe pain, and trigeminal neuralgia, or as a diuretic anesthetic and anti-cancer agent. Centipede venom has different biomedical properties and represents a vast reservoir of toxins, similar to venom from other animals. Due to its origins in one of the oldest venomous arthropods, centipede venom displays excellent activities and good prospects for drug development (Undheim et al., 2016; Zhang, 2015). Importantly, the centipede is a traditional Chinese medicine with an application history of more than 2 000 years (Chen & Yu, 1999; Zhao et al., 2018a). In China, pharmaceutically applied centipedes include S. mutilans, S. multidens, S. dehaani, and S. negrocapitis, with S. mojiangica (Wang et al., 1997) very occasionally used as a substitute. Our results showed that the venom toxicity of this centipede is strong in comparison to that of S. mutilans, a commonly used centipede in medicine.

      In our previous study, the centipede showed diverse protein or peptide components, with the most abundant toxins in the venom and torso tissues found to be more highly expressed than other active molecules using our method (Liu et al., 2012; Zhao et al., 2018a). Here, based on proteomic detection, we showed that the toxin-like proteins in S. mojiangica accounted for a lower percentage of total proteins than that in S. mutilans. However, there was a similar constitution and quantity of toxin transcripts in these two centipedes. We used high-throughput ESI-MS/MS and RNA-Seq technology to investigate the diversity of novel venom proteins, especially low-abundance peptides/proteins not detected using conventional methods (Savitski et al., 2005). Most of the detected proteins were identified as potentially active molecules with low molecular weights and unknown functions. In addition, each detected protein contained at least six peptides in the proteome dataset. The proteomic results for S. mojiangica were very similar to the protein detection results for S. mutilans. More than 400 toxin-like proteins/peptides were identified by transcriptome analysis in the centipede, but not detected in the proteome. Thus, most putative toxins in centipede venom may have low levels of expression in S. mojiangica and S. mutilans. In conclusion, centipede venom contains a surprising variety of toxin-like proteins/peptides.

      Regarding toxin distribution, based on transcriptomic analysis, we identified more toxin transcripts in S. mojiangica than in S. mutilans. Most toxins did not show significantly differential expression between S. mojiangica and S. mutilans, including that of ion channel inhibitors and serine proteinases. The centipede S. mojiangica demonstrated higher gene expression of metalloproteinase, trocarin, hopsarin-D, and alpha-latrotoxin compared to S. mutilans. Therefore, S. mojiangica could be substituted for S. mutilans in medical use. These results indicate that S. mojiangica venom could be a rich source of pharmacologically and medically useful compounds.

      Usually, we can obtain approximately 0.2–0.5 mg of crude venom from a single adult S. mutilans centipede over a period of two weeks. However, one adult S. mojiangica yielded less than 0.1 mg of crude venom in the same period. Therefore, it was difficult to study the venom components, including their pharmaceutical activity or medicinal application. In addition to the current annotation methods of centipede toxins, our results revealed that a wide variety of toxin-like active molecules were expressed in the venom gland by combining Blast alignment with the existing toxin databases and phylogenetic reconstruction of toxin relationships. Theoretically, this method may produce false positives, especially for proteins with low abundance and expression when using high-throughput proteomic and transcriptomic analyses with ESI-MS/MS and RNA-Seq technology. However, we used previously established approaches to maximize the search for functional proteins. Our results provide good evidence that the use of this substitute medicinal centipede is an appropriate medical option. Importantly, our data provide important clues to improve the use of the centipede as a traditional Chinese medicine.

    • Here, we used omics techniques to determine the profiles of venom components and toxin-like molecules in a new pharmaceutical centipede, S. mojiangica. We performed in-depth proteomic analysis of venom and deduced full-length protein sequences by combining proteome and transcriptome databases. We obtained more than 400 toxin-like molecules with potent activity. With gene expression and inter-species comparative analysis, we identified a broad and diverse composition of toxin-like molecules, which may play key roles in the functions of centipede venom. Our results indicate that this centipede is valuable for medicinal use and drug development, like other centipede species. Furthermore, our methods could improve the application of the centipede as a traditional Chinese medicine.

    • Supplementary data to this article can be found online.

    • The authors declare that they have no competing interests.

    • F.Z., W.H.L., and Y.Z. conceived the study. F.Z. wrote the paper and performed data and bioinformatics analyses. Z.C.L., J.Y.L., X.Q.L., T.L., J.R.Z., F.Z., G.L., and P.Y.C. performed all experiments. All authors read and approved the final version of the manuscript.

    • We thank the members of our research groups for providing technical assistance and participating in discussions.

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