The first report on transcriptome analysis of the venom gland of Iranian scorpion, Hemiscorpius lepturus
Graphical abstract
Introduction
Scorpions belong to the phylum arthropoda and the class arachnida that are distributed around the world. At present, there are 1500 species of scorpion and only 12% of them are venomous and cause major health problems (Chippaux and Goyffon, 2008). Scorpion venom consists of mixture of toxins that specifically acts on ion channel and receptor function (de la Vega et al., 2010). Moreover, venom of scorpion contains enzymes (phospholipases, serine proteases, metalloproteases and hyalorunidases), proteins and peptides (antimicrobial peptides and toxic peptides acting on ion channels) (Abdel-Rahman et al., 2016). In general, scorpion toxins are categorized into two classes; peptides with cystein amino acid residue and disulfide bridges (disulfide-bridged peptides; DBPs) and peptides without cystein and disulfide bridges (non-disulfide-bridged peptides; NDBPs) (Almaaytah et al., 2012). According to literature, diversity of non-disulfide-bridged peptides are more than disulfide-bridged peptides (Zeng et al., 2004). Disulfide-bridged peptides mainly act on ion channels and fall into four groups depending on the type of ion channel (sodium, potassium, calcium and chloride) that they affect (Chen et al., 2003, Chen et al., 2005, Possani et al., 1999). In Iran, there are six dangerous scorpions which are categorized into two families of Hemiscorpiidae and Buthidae (Yardehnavi et al., 2014). Hemiscorpius lepturus belongs to the Hemiscorpiidae family and is distributed in southern regions of Iran (Yardehnavi et al., 2014). Ten to fifteen percent (10–15%) of scorpionism in warm seasons and almost all cases in winter are related to H. lepturus [10]. H. lepturus, the most dangerous scorpion of Iran, is responsible for 67% of mortality and morbidity of scorpionism [10]. Therefore, venom of H. lepturus is very potent and possesses hemotoxin and cytotoxins. For instance, a potassium channel inhibitor called Hemitoxin (HTX), isolated from H. lepturus venom. Hemitoxin comprises 8 cystein residue, categorized in disulfide-bounded peptides and represents approximately 0.1% of crude venom (Srairi-Abid et al., 2008). Hemicalcin is another peptide isolated from H. lepturus venom. Hemicalcin is a calcium channel blocker and represents 0.6% of H. lepturus crude venom (Shahbazzadeh et al., 2007). Heminecrolysin is a 32 kDa protein with dermonecrotic activity, isolated from H. lepturus venom that functions through lysophosholipase D activity (Borchani et al., 2011, Borchani et al., 2013). Recently, a protein called Hemilipin (Phospholipase A2), isolated from H. lepturus venom, inhibited angiogenesis both in vitro and in vivo (Jridi et al., 2015). It has been demonstrated that in venom of non-Buthidae family, there are potassium channel toxins, calcium channel toxins and antimicrobial peptides (AMPs). Antimicrobial peptides are potent component of scorpion venom with activity on Gram-negative and Gram-positive bacteria, Fungi as well as viruses (Almaaytah et al., 2012). Two antimicrobial peptides called Smp24 and Smp43, identified in venom of Scorpio mauruspalmatus, demonstrated antibacterial activity via disruption of cell membrane (Harrison et al., 2016). A peptide named TistH, identified and characterized from the venom of Tityus stigmurus, exhibited antimicrobial and anti-fungal activity (Machado et al., 2016). In spite of defensive function of scorpion venom against hunters, some components of scorpion venom could be used as potential molecules for drug development (Ortiz et al., 2015). Nevertheless, a little percent of scorpion venom components have been identified in literature (Kastin, 2013). Most researches have been focused on the identification of scorpion toxins that results in animal envenomation. Therefore, most of venom components are neglected and remained unknown. Progress in proteomic analysis has contributed to rapid and high throughput identification of venom component (de la Vega et al., 2010). Although, with such analysis, some venom components with low concentration in total venom still remain unknown (Luna-Ramírez et al., 2013). Transcriptome analysis of venom gland is a novel approach for the identification of whole venom components such as proteins, toxic peptides as well as enzymes. Until now, transcriptome and proteome analysis of various scorpion venom (including Buthidae and non-Buthidae family) have been reported in many studies and their results showed toxic component of the venom (Almeida et al., 2012, Alvarenga et al., 2012, Batista et al., 2004, Batista et al., 2006, D'Suze et al., 2009, He et al., 2013, Luna-Ramírez et al., 2015, Ma et al., 2012, Ma et al., 2009, Ma et al., 2010, Morgenstern et al., 2011, Quintero-Hernández et al., 2015, Rendón-Anaya et al., 2012, Ruiming et al., 2010, Schwartz et al., 2007). Advent of Next Generation Sequencing (NGS) technology provided high throughput and cost-effective sequencing for various goals such as genome sequencing, expression as well as transcriptome studies (Angeloni et al., 2011).
In this study, Illumina Next Generation Sequencing was carried out for transcriptome analysis of venom gland of Iranian dangerous Scorpion; Hemiscorpius lepturus. As earlier mentioned, only a few components of H. lepturus venom have been identified and most of them are unknown. Therefore, identifying the whole venom component of H. lepturus was the main aim of the current study. For that aim, cDNA library of the venom gland of H. lepturus was constructed and sequenced by Source BioScience (England, Nottingham). The volume of transcriptome data was about 13.5 GB, which indicates the existence of high numbers of genes in transcriptome of venom gland of H. lepturus. Thereafter, venom genes involved in biological process, molecular function and cellular component were investigated. Indeed, 101 transcripts of venom coding 69 full-unique sequences of venom proteins, peptides and enzymes were found. To the best of our knowledge, this is the first report of transcriptome analysis of venom gland of H. lepturus. Results released here, opens new platform for researches involve in venomics studies and drug development from natural sources.
Section snippets
Sample preparation and RNA isolation
The H. lepturus scorpion was collected from Khuzestan, south-west of Iran in winter 2013. Each species of scorpions are maintained in individual polyethylene cages. All challenges were according to ethical guidelines of world health organization (WHO) method (World Health Organization, 2010). Captured scorpions were maintained in serpentarium of Pasteur Institute of Iran, Karaj, Iran. Two days after venom milking, total RNA was isolated from fresh tissue of scorpion glands (100 samples)
Illumina sequencing and annotations
Total RNA extracted from the venom gland of H. lepturus mRNA was purified, enriched and fragmented. Complementary DNA was synthesized and sequenced by Illumina HiSeq 2000 with 100bp read length. Bioinformatics analysis was performed on assembled sequences and RNA-seq data was submitted to SRA of NCBI with the following accession number SRP070907. Summary of statistics after sequencing are presented in Table 1. 171, 976 raw transcripts containing 179, 399 entries due to multiple hits of some
Conclusion
In this study, for the first time, transcriptome of venom gland of Iranian scorpion, H. lepturus (belonging to the Hemiscorpiidae family) was analyzed and whole components (new toxins and peptides) of its venom were identified. However, some venom component of H. lepturus like; Hemicalcin, Hemilipin and Hemitoxin were previously identified. The results of this study could be a reference for venomics studies of other scorpions of Hemiscorpiidae family. It is expected that identification of new
Conflict of interest
The authors have declared that no competing interests exist.
Ethical statement
The H. lepturus scorpion was collected from Khuzestan, south-west of Iran in winter 2013. Each species of scorpions are maintained in individual polyethylene cages. All challenges were according to ethical guidelines of world health organization (WHO) method (World Health Organization, 2010). Captured scorpions were maintained in serpentarium of Pasteur Institute of Iran, Karaj, Iran.
Acknowledgment
This study financially supported by Pasteur Institute of Iran, Tehran, Iran. The authors thereby acknowledge with deep respect the Pasteur Institute of Iran, Tehran, Iran for funding this study.
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