Venom gland transcriptomic and venom proteomic

Toxicon 133 (2017) 95e109

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Venom gland transcriptomic and venom proteomic analyses of the scorpion Megacormus gertschi Díaz-Najera, 1966 (Scorpiones: Euscorpiidae: Megacorminae) n ~ ez-Lo pez a, 2, 1, Jimena I. Cid-Uribe a, 2, Fernando Z. Zamudio a, Carlos E. Santiba b Cesar V.F. Batista , Ernesto Ortiz a, **, Lourival D. Possani a, * noma de M Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Auto exico, Avenida Universidad 2001, Apartado Postal 510-3, Cuernavaca, Morelos, 62210, Mexico mica, Instituto de Biotecnología, Universidad Nacional Auto noma de M Laboratorio Universitario de Proteo exico, Avenida Universidad 2001, Apartado Postal 510-3, Cuernavaca, Morelos, 62210, Mexico a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 March 2017 Received in revised form 20 April 2017 Accepted 1 May 2017 Available online 3 May 2017

The soluble venom from the Mexican scorpion Megacormus gertschi of the family Euscorpiidae was obtained and its biological effects were tested in several animal models. This venom is not toxic to mice at doses of 100 mg per 20 g of mouse weight, while being lethal to arthropods (insects and crustaceans), at doses of 20 mg (for crickets) and 100 mg (for shrimps) per animal. Samples of the venom were separated by high performance liquid chromatography and circa 80 distinct chromatographic fractions were obtained from which 67 components have had their molecular weights determined by mass spectrometry analysis. The N-terminal amino acid sequence of seven protein/peptides were obtained by Edman degradation and are reported. Among the high molecular weight components there are enzymes with experimentally-confirmed phospholipase activity. A pair of telsons from this scorpion species was dissected, from which total RNA was extracted and used for cDNA library construction. Massive sequencing by the Illumina protocol, followed by de novo assembly, resulted in a total of 110,528 transcripts. From those, we were able to annotate 182, which putatively code for peptides/proteins with sequence similarity to previously-reported venom components available from different protein databases. Transcripts seemingly coding for enzymes showed the richest diversity, with 52 sequences putatively coding for proteases, 20 for phospholipases, 8 for lipases and 5 for hyaluronidases. The number of different transcripts potentially coding for peptides with sequence similarity to those that affect ion channels was 19, for putative antimicrobial peptides 19, and for protease inhibitor-like peptides, 18. Transcripts seemingly coding for other venom components were identified and described. The LC/MS analysis of a trypsin-digested venom aliquot resulted in 23 matches with the translated transcriptome database, which validates the transcriptome. The proteomic and transcriptomic analyses reported here constitute the first approach to study the venom components from a scorpion species belonging to the family Euscorpiidae. The data certainly show that this venom is different from all the ones described thus far in the literature. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Euscorpiidae Megacormus gertschi Proteome Scorpion Transcriptome

1. Introduction * Corresponding author. ** Corresponding author. n ~ ez-Lo pez), jcidu@ibt. E-mail addresses: [email protected] (C.E. Santiba unam.mx (J.I. Cid-Uribe), [email protected] (F.Z. Zamudio), [email protected] (C.V.F. Batista), [email protected] (E. Ortiz), [email protected] (L.D. Possani). 1 Present address: Department of Zoology, University of Wisconsin e Madison, 430 Lincoln Drive, Madison, WI 53706, USA. 2 Contributed equally to this work. http://dx.doi.org/10.1016/j.toxicon.2017.05.002 0041-0101/© 2017 Elsevier Ltd. All rights reserved.

Scorpions of the genus Megacormus Karsch 1881, a genus member of the family Euscorpiidae represented by four species, are ~ ez-Lo pez et al., 2016a). These species endemic to Mexico (Santib an inhabit pine-oak forests and tropical rainforests in the Sierra Madre taro, Oriental (in the Mexican states of Tamaulipas, Hidalgo, Quere Puebla, Oaxaca and Veracruz) at altitudes ranging from 300 to n ~ ez-Lo pez et al., 2016a; 2300 m above mean sea level (Santiba

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n ~ ez-Lo pez et al. / Toxicon 133 (2017) 95e109 C.E. Santiba

n and Alvarez-Padilla, Gonz alez-Santilla 2014). They are considered corticolous and lapidicolous species (Prendini, 2001) because they live under bark, logs or stones in the ground (Supplementary Fig. 1 shows a photo of this scorpion). Not a single report has yet been filed on human intoxication from these species, suggesting that either they represent no danger to people, or haven't been correctly identified in envenomation cases. Likewise, given their restricted habitat preferences, accidents ending with stung humans are rare, most of them due to encounters while manipulating firewood in small town localities within their distribution area [CESL, personal observation]. However, other members of the family Euscorpiidae (such as some species of Euscorpius in Italy) cause more frequent accidents with humans although not considered as medical emergencies (Dutto et al., 2010). There is no report on the composition of the venom from species belonging to the family Euscorpiidae of Mexico. Ma et al. (2009, 2012) analyzed the venom gland transcriptome of Scorpiops jendeki and Scorpiops margerisonae, species supposedly belonging to this family. However, recent phylogenomic analysis showed that scorpion species of genus Scorpiops belong to the family Scorpiopidae (Sharma et al., 2015). Thus, it seems that there are some discrepancies on the taxonomic points of view regarding scorpions of these two families of scorpions. However, to the best of our knowledge this is the first report on venom components of any scorpion of the family Euscorpiidae found in Mexico. The Next Generation Sequencing (NGS) allows obtaining the whole venom gland transcriptomes at low cost, providing information on several transcripts coding for toxins and other venom n-Anaya et al., 2015). peptides (Luna-Ramírez et al., 2015; Rendo Venom gland transcriptome analyses have become more common n ~ ez-Lo pez and Possani, nowadays (summarized recently in Santiba 2015) with nine of the twenty extant scorpion families already studied (including some species from families Buthidae, Bothriuridae, Caraboctonidae, Hemiscorpiidae, Hormuridae, Scorpionidae, Scorpiopidae, Urodacidae, Vaejovidae and Superstitioniidae). These studies have shown the presence of transcripts seemingly coding for an extensive diversity of peptides with multiple potential functions: ion channel modifiers, factors activating lipolysis, phospholipases A2, serine-proteases, metalloproteinases, peptides with antimicrobial, antiviral and anti-parasitic activities, among others (Ma et al., 2009, 2012; Rodríguez de la Vega et al., 2010; He et al., 2013; Ortiz et al., 2014; Luna-Ramírez et al., 2015; Mille et al., ndez et al., 2015; 2015; Oliveira et al., 2015; Quintero-Herna n ~ ez-Lo pez and Possani, 2015; Santiba n ~ ez-Lo pez et al., Santiba 2016a,b; Zhong et al., 2017 Kazemi-Lomdeasht et al., 2017). While these studies have deduced peptide sequences potentially expressed in the venom of those species, only in a few cases proteomic analyses and mass spectrometric analyses have validated the actual presence of the peptides in the venoms (Smith and Alewood, 2015). According to Smith and Alewood (2015) the only transcriptome analyses validated at the proteome level are those of the following species: Heterometrus petersii (Ma et al., 2010), Pandinoides cavimanus (previously known as Pandinus cavimanus) (Diego-García et al., 2012), Urodacus yaschenkoi (Luna-Ramírez et al., 2013), Scorpio maurus palmatus (Abdel-Rahman et al., 2013) [which was recently elevated to the species' level under the name Scorpio palmatus (Talal et al., 2015)], Centruroides tecomanus (Valdez-Velazquez et al., 2013), and Superstitionia donensis n ~ ez-Lo pez et al., 2016b). Nevertheless, as pointed out by (Santiba Luna-Ramírez et al. (2015), the correlation between the deduced peptide sequences and their predicted molecular weights versus those obtained by proteomic analysis may be limited because of several reasons, including: variation among different specimens of the same species, the impact of the protocols for venom extraction and characterization on the stability/integrity of the venom

components, or the post-translational modification of the expressed proteins. In this contribution, we report the first proteomic characterization of the venom from the Mexican scorpion M. gertschi collected in the State of Hidalgo. The effects of the venom on various animal models were assayed (mammals, insects and crustaceans). The venom was separated by HPLC and the main fractions were collected. To most of them (67) a molecular mass was assigned by mass spectrometry. The N-terminal sequence of a few peptides (seven) was determined by Edman degradation. A transcriptomic analysis of the venom gland was conducted by RNA-Seq. We report 182 annotated venom gland transcripts, among which we found sequences putatively coding for toxins, peptides of nontoxin nature, enzymes, and other peptides/proteins with an unknown biological function. This database certainly increases our knowledge on venom peptides from poorly studied non-buthid families of scorpions. A summary of this work was presented as a poster, during the American Section Congress of IST held in Miami on 2016. 2. Material and methods 2.1. Biological material Scorpion specimens were collected in El Salto Jacala, Hidalgo, Mexico on October 2013 and May 2016. The permits for collection were issued by SEMARNAT (SGPA/DGVS/02483 of March 18, 2005, and Scientific Permit FAUT-0175 granted to Oscar Francke, see acknowledgments). The scorpions were maintained in plastic boxes with a permanent water supply and were routinely fed with crickets. The specimens were classified based on the available literature (Sissom, 1994). Species names follow current classifican ~ ez-Lo pez et al., 2016a). tion (Sharma et al. 2015; Santiba 2.2. RNA extraction, RNA-seq and venom gland transcriptome assembly The telsons from two specimens were dissected under RNAsefree conditions and pooled into a single tube. RNA was isolated using the SV Total RNA Isolation System Kit (Promega) following the protocol provided by the manufacturer. Briefly, the dissected telsons were manually macerated to homogeneity with a Kontes microtube pellet pestle rod (Daigger) in a 1.5 mL microtube with the provided RNA Lysis Buffer. After dilution with the RNA Dilution Buffer the sample was heated at 70 C for 3 min, and then centrifuged to discard all cellular debris. The cleared lysate was mixed with 95% ethanol and transferred to one of the spin baskets supplied by the kit. After washing with the RNA Wash Solution, the sample was treated with the provided DNAse for 15 min and then washed twice with the RNA Wash Solution. After centrifugation, total RNA was recovered in Nuclease-Free Water. The RNA was quantified with a Nanodrop 1000 (Thermo Scientific) and its integrity was confirmed using a 2100 Bioanalyzer (Agilent Technologies). A complementary DNA (cDNA) library was constructed from the obtained total RNA, using the Illumina TruSeq Stranded mRNA Sample Preparation Kit, following the protocol provided by the supplier. Automated DNA sequencing was performed at the Massive DNA Sequencing Facility in the Institute of Biotechnology (Cuernavaca, Mexico) with a Genome Analyzer IIx (Illumina), using a 72 bp paired-end sequencing scheme over cDNA fragments of 200e400 bp. Each library consisted of two fastq files (forward and reverse reads), from which the adaptors were clipped-off. The quality of cleaned raw reads was assessed by means of the FastQC program (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/).


The short reads were assembled into contigs in a de novo fashion with the Trinity software (v. 2.0.3), using the standard protocol (Grabherr et al., 2011), executing the strand-specific parameter and normalizing reads. To assess the quality of the assembly, basic statistics for the number of gene models and isoforms as well as the contiguity were obtained by running the TrinityStats.pl script. The assembled contigs were used as queries to search the UniProt database (http://www.uniprot.org) with the blastx algorithm of the BLAST program. Then, they were analyzed with Trinotate (https://trinotate.github.io/, Grabherr et al., 2011). Signal peptides were predicted using the Signal P 4.0 server (http://www.cdbs.dtu. dk/services/SignalP/) and propeptides were determined with the ProP 1.0 (http://www.cbs.dtu.dk/services/ProP/) and spiderP (www.arachnoserver.org/spiderP.html) servers. The theoretical molecular weights of the putative mature peptides were obtained using the ProtParam server (http://web.expasy.org/protparam). Subsequent UnitProt and GenBank searches using the blastx algorithm were conducted with those selected transcripts that showed sequence similarity to venom components. From these new searches, matching sequences with lower Expect (E) values, higher query cover values and higher percentages of identity were chosen as the main input sequences for the multiple alignments reported here. They were enriched with seemingly lower-quality matching sequences (according to the above parameters), but which have been deduced/isolated from scorpions, arachnids or arthropods (in that order or priority), to give them a counterweight based on their phylogenetic relationship. Multiple sequence alignments of the relevant M. gertschi transcript-derived sequences with the corresponding input sets were obtained using the online server of MAFFT ver. 7.0 (Katoh and Standley, 2013) at http://mafft. cbrc.jp/alignment/server/. Alignments were edited with Jalview (Waterhouse et al., 2009) and Adobe Illustrator CS6. 2.3. GenBank accession numbers This Sequence Read Archive (SRA) project has been deposited at DBJ/EMBL/GenBank with accession number for the master record SUB1490154. The version described in this paper corresponds to the first assembly of the first version as a Transcriptome or Gene expression TSA, i.e., SUB1615117. 2.4. Venom extraction, fractionation and molecular mass determination Venom from M. gertschi was obtained in the laboratory by electrical stimulation (15e25 V for 3 s) applied at the articulation of the telson of the specimens. The venom was collected in Eppendorf Lo-bind™ tubes and dissolved in double-distilled water. Then it was centrifuged at 14,000 g for 15 min at 4 C. The soluble supernatant was either lyophilized or stored at 20 C, and later separated by high-performance liquid chromatography, essentially as described earlier [e.g. (Valdez-Velazquez et al., 2013)]. Briefly, whole soluble venom was injected into a C4 reverse-phase semipreparative column (Vydac, Hisperia, CA, USA) and separated by using a linear gradient from solvent A (0.12% trifluoroacetic acid, TFA, in water) to 60% solvent B (0.10% TFA in acetonitrile) for 90 min. The fractions obtained by HPLC were dried on a Savant SpeedVac (Thermo Scientific, San Jose, CA) and reconstituted in 60% acetonitrile with 1% acetic acid and directly applied (5 mL) into a Thermo Scientific LCQ Fleet™ ion trap mass spectrometer (San Jose, CA). The spray voltage was set to 1.6 kV and the capillary temperature was set to 180 C. All spectra were obtained in the positiveion mode. In addition, 20 mg of total venom were reduced with 25 mM dithiothreitol (DTT) at 65 C for 25 min and then alkylated with

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25 mM iodoacetamide at room temperature, in the dark, for 20 min. Following this, the sample was digested with trypsin, desalted by ZipTip-C18 and concentrated in the SpeedVac. The complex mixture of tryptic peptides was applied into an LC/MS system composed of a nano-flow HPLC pump and an Orbitrap Velos mass spectrometer (Thermo Scientific) with a nano-electrospray ion source. For online peptide fractionation, an in-house made RP C18 micro-column was first washed with buffer A (0.1% formic acid) and then ran with a 5% to 60% gradient of buffer B (acetonitrile and 0.1% formic acid) for 120 min. The flow was maintained at 300 nL/min along the LC/MS analysis. The eluting peptides were detected in full scan mode and all m/z values were selected for fragmentation by CID and HCD using the 10 most intense signals of the spectra. The raw file generated was analyzed with the Protein Discoverer 1.4 program using the Sequest HT as the search engine and the in silico translated M. gertschi RNA-Seq database as the reference. The main search parameters were: cysteines alkylated with iodoacetamide as static modification and oxidation of methionines as dynamic modification (two missed cleavages were accepted). Accuracies of 10 ppm for fragment ions and 0.4 Da for precursor ions were preestablished. 2.5. Venom bioassays and enzymatic activity Since this is the first report in the literature where the venom of scorpions of the family Euscorpiidae is studied, it was important to have a general idea towards which kind of animals has the venom evolved to be toxic, or to cause harm. It is known that scorpions have evolved their venoms for capturing prey or defending themn ~ ez-Lo pez et al., 2016a; but see also selves from predators (Santiba Zhang et al., 2015a). The soluble venom of this scorpion was tested in three animal models: mouse (Mus musculus, strain CD1, 25 to 30 days old, and 20 grams each) was chosen for mammals, cricket (Acheta domestica) for insects and freshwater shrimp (Cambarellus montezumae) for crustaceans. The rationale for choosing these species was based on previously reported data in the literature from toxicity assays conducted with scorpion venoms, which have demonstrated the existence of different types of peptides that work as species-specific toxins (Possani et al., 1999). Lethality tests were conducted with protocols that followed the approved guidelines of the Animal Welfare Committee of our Institute. The protein content of the venom was estimated based on the absorbance at l ¼ 280 nm. It was assumed that one unit of absorbance at this wavelength corresponds to a protein concentration of 1 mg/mL. The phospholipase activity was assayed by the egg yolk technique of Habermann and Hardt (Habermann and Hardt, 1972). 3. Results and discussion 3.1. Biological effects of the venom Assays with the soluble venom on three animal models showed dissimilar results. Three mice of the strain CD1 injected intraperitoneally with 100 mg of whole soluble venom per 20 g of body weight showed no symptoms of intoxication, suggesting that this venom is not toxic for mammals. Usually, soluble venom from scorpions of the family Buthidae has a median lethal dose (LD50) for mice in the order of 3e5 mg per 20 g of body weight (Zamudio et al., 1992; Licea et al., 1996). On the contrary, injection of crickets with 20 mg of venom per animal was lethal (experiment performed in triplicate). The animals showed progressive paralysis and died within 2 h after injection. Crickets injected with the same volume of just water remained unaltered, showing the same normal behavior as the non-injected control animals. The three venom-injected freshwater shrimps (30, 50 and 100 mg per animal) also showed

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symptoms of intoxication, but the amount of venom needed to achieve lethality was higher than for crickets. The shrimp injected with 100 mg of venom (same amount used for mice) died within the first 5 h after injection. The intoxicated animals showed similar envenomation symptoms as the crickets, starting with progressive paralysis. Thus, the venom from M. gertschi is not toxic to mammals at doses that might be lethal to crickets and shrimps. This correlates with the reported lack of envenomation symptoms in stung humans, as mentioned in the Introduction. In conclusion, at the tested doses, the venom from M. gertschi resulted toxic to insects and crustaceans while not to mammals. These scorpions feed on other arthropods, reason why natural selection has contributed to the development of specific hunting tools (venom with arthropodspecific toxins). 3.2. Total RNA isolation The extraction of RNA from the telsons of the M. gerstchi scorpions yielded 18.3 mg of total RNA. In the electropherogram obtained with the Bioanalyzer the peak corresponding to the 28S ribosomal RNA was absent, making impossible to obtain the RNA Integrity Number (RIN). However, since a concentrated sharp peak was detected for the 18S ribosomal RNA, and no signs of degradation were observed, the integrity of the RNA sample was considered adequate for downstream applications. The lack of the 28S ribosomal RNA has been reported before in RNA extractions from insects, a phenomenon known as “the hidden break” (Winnebeck et al., 2010) and we have previously observed the same particularity with total RNAs purified from scorpions (unpublished results). Similar findings were seeing in triatomids of the family Reduviidae (Winnebeck et al., 2010; Majerowicz et al., 2011) and gasteropds of the genus Conus (Figueroa-Montiel et al., 2016). 3.3. M. gertschi venom gland global transcriptome analysis After sequencing, assembly and cleaning, 39,638,145 reads were obtained corresponding to 110,528 transcripts, with an N50 of 1367 bp. From these transcripts, a total of 95,422 where identified matching sequences listed in databases. A subgroup of 22,419 was annotated, of which 3565 matched known arthropod sequences. Remarkably, only 210 were identified as being from arachnids and 68 specifically from scorpions. These two low numbers could just reflect the fact that the number of annotated sequences in the databases for these taxonomical Class and Order is still scarce. Fig. 1 shows the most abundant GO-term categories found in the transcriptome analysis of the venom gland of M. gertschi. 3.4. The repertoire of venom gland transcripts in M. gertschi Following the scheme presented in other venom gland transcriptome analyses (e.g. Luna-Ramírez et al., 2015; Quinterondez et al., 2015; Santiba n ~ ez-Lo pez et al., 2016b), here we Herna report 182 sequences putatively coding for the following peptides and proteins recognized as venom components by sequence similarity from databases, PFAM, or the available literature. (Supplementary Table S1 and Fig. 2): 3.4.1. Toxins The ion channel-acting toxins are the best-studied scorpion venom components, not only for historical reasons related to their clinical relevance, but also because they have shown themselves as valuable tools for studying the physiology and functioning mechanism of their targeted channels. Toxins that affect sodium, potassium, calcium and chloride channels have been found in the scorpion venoms thus far. Considering that toxins belonging to the

distinct families share high sequence similarity, their dissimilar affinity and specificity for different channel subtypes and their contrasting toxicity for diverse groups of animals (e.g. mammals and arthropods) is striking (Laraba-Djebari et al., 2015). We found that transcripts potentially coding for ion channel-acting toxins are poorly represented in the M. gertschi venom gland transcriptome in terms of sequence diversity, comprising only 10% of the 182 transcripts mentioned above (Fig. 2). This did not come as a surprise, since other transcriptomic analyses from non-Buthidae species have reached similar results [e.g. (Luna-Ramírez et al., 2015; ndez et al., 2015)]. Quintero-Herna 3.4.1.1. Sodium channel toxins. Toxins affecting Naþ channels are responsible for the neurotoxic symptoms during envenomation n ~ ez-Lo pez and (Rodríguez de la Vega and Possani, 2005; Santiba Possani, 2015). The clear majority of peptides that affect the normal physiological function of Naþ channels have been discovered in the venom of species belonging to the family Buthidae, versus only a few peptides found in the venom of non-buthid scorpions [e.g. in Anuroctonus phaiodactylus (Valdez-Cruz et al., 2004), S. palmatus (Abdel-Rahman et al., 2013), U. yaschenkoi (Luna-Ramírez et al., 2013) and some vaejovid species (Quinterondez et al., 2015)]. Therefore, our results coincide with the Herna low representativeness previously reported for Naþ toxin-coding transcripts in non-buthid venom glands. We found four transcripts with a conserved PFAM domain related to sodium toxins: a) component labeled mgc80741_g1_i1 with 41% identity with the mature chain of toxin Acra II-2, deduced from a cDNA cloned from Androctonus crassicauda (Caliskan et al., 2006, Fig. 3A); b) component mgc28936_g1_i1 with similarity to Toxin CngtIV, deduced from the cDNA cloned from Centruroides noxius (Becerril et al., 1993, Fig. 3B); c) component mgc28714_g2_i4 with similarity to the precursor of Defensin-1 deduced from the venom transcriptome analysis of Androctonus bicolor (Zhang et al., 2015b); and d) component mgc16052_g1_i1 with similarity to Lipolysis-activating venom peptide (LVP) 1-alpha chain deduced from the cDNA cloned from Lychas mucronatus (Zhao et al., 2010). The LVPs are known to share sequence similarity to sodium channel-specific toxins, but the lack of a cysteine in their sequence results in a reduced number of intra-chain disulfide linkages and a distinct inter-chain linkage (Soudani et al., 2005). 3.4.1.2. Potassium channel toxins. As suggested by earlier transcriptome analyses, potassium channel toxins are abundant in scorpion venoms. These peptides vary in length (from 23 to more than 64 amino acids) and are classified based on their primary amino acid sequences and the cysteine pairing. Initially, three types of Kþ-channel blocking peptides were described (Tytgat et al., 1999): a) the a-KTx, that usually contain 30-40 amino acid residues; b) the longer b-KTx, containing 60e64 residues; and c) the gKTx, which are blockers of ERG-Kþ-channels. Currently there are 174 sequences from different peptides subdivided into at least 30 subfamilies, reported to be specific for Kþ-channels (http:// kaliumdb.org/) and (Luna-Ramírez et al., 2015; Kuzmenkov et al., 2016). Unlike the transcriptomic analysis of U. yaschenkoi where 17 different potassium channel toxins were reported (Luna-Ramírez et al., 2015); only eight sequences in the present analysis had hits with potassium channel toxins. Four sequences had domain PFAM (protein families database) for short potassium channel toxin: with putative potassium channel blocking activity. a) mgc25969_g1_i1 with similarity to potassium channel toxin precursor deduced from the transcriptome analysis of venom gland of U. yaschenkoi (LunaRamírez et al., 2015); b) mgc21622_g1_i1 with similarity to potassium channel toxin deduced from the transcriptome analysis of


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Fig. 1. The GO distribution of the annotated sequences. The sequences from the venom gland transcriptome of M. gertschi were annotated according to Gene Ontology (GO) terms. The category designated by GO as “Biological process” was the most diverse.

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Fig. 2. Distribution of the annotated transcripts according to protein families and subfamilies.

Fig. 3. Sodium channel-specific toxins. Sequence alignment of translated precursors found in the transcriptome analysis of the venom gland of M. gertschi with similarity to previously reported sequences corresponding to sodium channel toxins. The predicted signal peptides and mature sequences are delimited with the upper horizontal bars. UnitProt entry numbers are given in brackets. A) Precursor mgc80741_g1_i1; Toxin Acra1 from Androctonus crassicauda (P0C292); Toxin Acra II-2 from A. crassicauda (P0C296); Dortoxin from Parabuthus transvaalicus (P0C1B7); and Bestoxin from P. transvaalicus (P0C1B6). B) Precursor mgc28936_g1_i1, CSab Cer 1 from Cercophonis squama (T1DMR2); CSab Cer 2 from Cercophonis squama (T1DEJ4); Toxin CngtIV from Centruroides noxius (P45665); Toxin Cn11 from Centruroides noxius (P58296); and CsE1 from Centruroides sculpturatus (P01491).


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Fig. 4. Potassium channel-specific toxins. Sequence alignment of the proposed mature peptide from transcript mgc21622_g1_i1 (a possible Kþ channel toxin) with similar sequences from other scorpions. (Uniprot entry numbers are given in brackets). Proposed mature peptide mgc21622_g1_i1; KTx 6.6 from Opistophthalmus carinatus (Q6XLL9); KTx 6.14 from Hoffmannihadrurus gertschi (P84864); KTx 6.15 from Hemiscorpius lepturus (P85528); KTx 6.2 from Scorpio palmatus (P80719); KTx 6.3 from Heterometrus spinifer (P59867); KTx 6.11 from Opisthacanthus madagascarensis (P0C194); KTx 6.21 from Urodacus yaschenkoi (P0DL37); KTx 6.12 from Anuroctonus phaiodactylus (P0C166); KTx 6.1 from Pandinus imperator (Q10726); and KTx 6.17 from Opisthacanthus cayaporum (P86116). Four sequences had the PFAM domain for long-chain potassium channel toxins and scorpinelike peptides. Component mgc25471_g1_i1 had similarity to the precursor of the potassium channel toxin deduced from the transcriptome analysis of the venom gland of H. lepturus; and component mgc25298_g1_i1 had similarity to another potassium channel toxin deduced from the transcriptome analysis of the venom gland of H. lepturus. The other two sequences had similarity to scorpine-like peptides and will be described in the next section.

venom gland of Hemiscorpius lepturus (Kazemi-Lomdeasht et al., 2017); c) mgc27885_g1_i1 showing similarity to the venom peptide HtKTx3 deduced from the transcriptome analysis of venom gland of Hadogenes troglodytes (Zhong et al., 2017); and d) mgc29973_g1_i2 with similarity to the potassium channel toxin alpha-KTx 23.1 peptide obtained from the venom of Vaejovis smithi (previously known as Vaejovis mexicanus smithi; Gurrola et al., 2012; Varga et al., 2012). Fig. 4 shows the amino acid sequence of the mature peptide of the last clone (mgc21622_g1_i1) together with the other amino acid sequences of the known peptides from sub-family a-KTx 6 of non-buthid scorpions.

3.4.1.3. Scorpine-like peptides. The scorpine-like peptides have two structural and functional domains: one with amino acid sequence similarity to antimicrobial peptides, whereas the other is similar to potassium channel toxins (Wu et al., 2007; Luna-Ramírez et al., n ~ ez-Lo pez et al., 2015; Quintero-Hern andez et al., 2015; Santiba 2016b; Luna-Ramírez et al., 2016). Here two sequences encoding putative scorpine-like peptides are listed: a) component mgc22639_g1_i1 (see Fig. 5A) had similarity to the precursor of the scorpine-like peptide Ev37, deduced from the cDNA cloned from Euscorpiops validus (Feng et al., 2013); and b) component mgc19218_g1_i1 (Fig. 5B) which codes for a type 2 scorpine with

Fig. 5. Scorpine-like peptides. Sequence alignment of translated M. gertschi precursors with similarity to scorpine-like peptides and their matching sequences from databases. The predicted signal peptides and mature sequences are delimited with the upper horizontal bars. UnitProt entry numbers are given in brackets. A) Component mgc22639_g1_i1; Ev37 from Euscorpiops validus (P0DL47); Hg scorpine from Hoffmanihadrurus gertschi (Q0GY41); Scorpine from S. palmatus as reported in (Abdel-Rahman et al., 2013); and Ctrycontig44 from Chaerilus tryznai as reported in (He et al., 2013). B) Component mgc19218_g1_i1; Hg scorpine-like 2 from H. gertschi (P0C8W5); CSab-Uro-3 from Urodacus manicatus (T1DEJ8); antimicrobial peptide scorpine-like 2 from Urodacus yaschenkoi (L0GCW2).

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similarity to the precursor of the Hg Scorpine-like 2 deduced from the transcriptome analysis of the venom gland of Hoffmannihadrurus gertschi (then referred as Hadrurus gertschi) (Schwartz et al., 2007). Two additional sequences are included in this Fig. 5B: CSabUro-3 from Urodacus manicatus (Sunagar et al., 2013); and the antimicrobial peptide Scorpine-like-2 from U. yaschenkoi (LunaRamírez et al., 2015). It is worth mentioning that only the nonbuthid scorpine-like peptides were included in Fig. 5, for comparison purposes. Our results were consistent with the number of scorpine-like peptides found in transcriptomic analyses published before (Luna-Ramírez et al., 2015; Quintero-Hern andez et al., 2015). 3.4.1.4. Calcins. These peptides are structurally different from those affecting other ion channels (i.e. sodium, potassium and chloride) by the presence of a three-dimensional structure known as the Inhibitor Cystine Knot (ICK) motif, similar to the peptides found in snail or spider venoms affecting calcium channels ~ ez(Narasimhan et al., 1994; Luna-Ramírez et al., 2015; Santib an pez et al., 2016b), instead of a cysteine-stabilized a/b (CS-ab) Lo ~ ez-Lo pez and Possani, 2015). Calcins recognize motif (Santib an ryanodine-sensitive calcium channels (RyRs) of the endoplasmic and sarcoplasmic reticula of skeletal and cardiac muscle (Valdivia et al., 1992; Xiao et al., 2016). These peptides have been isolated from the venom of buthids and non-buthids; however, they are more abundant in non-buthid scorpions. As described in detail in Section 3.5, the presence of a calcin-like peptide was identified both by Edman degradation and transcriptomic analysis. Identical Nterminal amino acid sequences and molecular masses were identified. In the transcriptomic analysis of the venom of M. gertschi we found six sequences corresponding to putative calcins: a) mgc24976_g1_i1 similar to Opicalcin-2 deduced from the cDNA cloned from Opistophthalmus carinatus (Zhu et al., 2003); b) mgc24976_g1 _i2 with similarity to venom peptide HtRyRTx1 obtained from the transcriptome analysis of the venom gland of H. troglodytes (Zhong et al., 2017); c) mgc26477_g1_i1 had similarity to venom peptide HtSTx deduced of transcriptome analysis of the venom gland of H. troglodytes (Zhong et al., 2017); d) mgc27145_g4_i1 with similarity to venom peptide HtRyRTx1 obtained of transcriptome analysis of the venom gland of H. troglodytes (Zhong et al., 2017); e) mgc28763_g1_i3 showing similarity to Opicalcin-1 deduced from the cDNA cloned from O. carinatus (Zhu et al., 2003) and f) mgc29132_g1_i3 which showed similarity to toxin protein deduced from the transcriptome analysis of venom gland of H. lepturus (Kazemi-Lomdeasht et al.,

2017) as shown in Fig. 6. The transcripts contain a signal peptide (underlined sequences) a propeptide (in italics) and the mature peptide (in boldface) that starts with the amino acid asparagine at position 44. The mature peptide has only 32 amino acid residues. These sequences were also similar to the precursor of a calcin deduced from the transcriptome analysis of the venom gland of S. jendeki (Ma et al., 2009). Seven other sequences were included in Fig. 6 for comparative purposes, identified in non-buthid scorpions. It is worth mentioning that another similar sequence was identified (mgc15702_g1) with similarity to the precursor of the U8 agatoxin Ao1a deduced from the genomic analysis of the horseshoe crab Limulus polyphemus, which is a toxin affecting Ca2þ channels, but is not a calcin. 3.4.1.5. Defensins. Arthropod defensins are widely distributed and abundant 3e4 kDa peptides stabilized by three disulfide bridges, with antimicrobial activity (Cociancich et al., 1993; Ganz and Lehrer, 1994; White et al., 1995; Froy and Gurevitz, 2004). Defensins share a common ancestor with scorpion toxins (Froy and Gurevitz, 2004), which has been proved experimentally (i.e. Zhu et al., 2014). We found four components with sequence similarity to Defensin 1, deduced from the transcriptome analysis of the venom gland of A. bicolor (Zhang et al., 2015b; see Table S1). 3.4.2. Non-disulfide-bridged peptides (NDBPs) While disulfide linkages stabilize most of the toxins and venom peptides, there is another large group of peptides in the scorpion venom called the Non-Disulfide-Bridged Peptides (NDBPs). As the name suggests, these peptides lack cysteines. For the NDBPs multiple biological activities have been demonstrated, such as antimicrobial, cytolytic, anti-inflammatory, among others (Almaaytah and Albalas, 2014). Due to these activities, with potential clinical and therapeutic applicability, the NDBPs have recently attracted major attention from researchers (Ortiz et al., 2015). These peptides are apparently more abundant in the venoms of the non-buthid scorndez et al., 2015), with some studies even pions (Quintero-Herna suggesting that they could represent a third of all the peptides in the venom (Valdez-Velazquez et al., 2013). Our analysis revealed the presence of fourteen sequences with identities to those of the NDBPs peptides, from which nine sequences shows identities to short antimicrobial peptides; one sequence is similar to mediumlength antimicrobial peptide and three sequences shows similarity to long chain multifunctional peptides. The short antimicrobial peptide are: a) mgc23353_g1_i1 with similarity to the precursor of

Fig. 6. Peptides similar to calcins. Sequence alignment of precursor from transcript mgc29132_g1_i3 (a possible calcin) and similar sequences. UniProt or Genbank entry numbers are given in brackets: Calcin from Scorpiops margerisonae (FD664670); Hadrucalcin from Hoffmannihadrurus gertschi (B8QG00); CaTx 20 from Urodacus yaschenkoi (L0GBR1); Opicalcin 2 from Opistophthalmus carinatus (P60253); Calcin from Heterometrus petersii (FD664155); toxin protein from Hemiscorpius lepturus (API81327). The predicted signal peptide is underlined; the mature peptide is in boldface and the propeptide is in italics.


103

Fig. 7. Peptides similar to vejovine (NDBP peptides). Sequence alignment of component mgc16627_g2_i1 (NDBP) and a few known examples from the literature (UniProt entry numbers in brackets): Vejovine from Vaejovis mexicanus (F1AWB0); Peptide Con13 from Opisthacanthus cayaporum (C7C1L2); Peptide Con22 from Urodacus yaschenkoi (L0GBQ6); Heterin1 from Heterometrus spinifer (A0A0C4G489). The predicted signal peptide is underlined; the mature peptide is in boldface and the propeptide is in italics. For Heterin1, the SpiderP algorithm could not predict a propeptide region, therefore, the complete sequence beyond the signal peptide is indicated in boldface.

Amp1 deduced from a cDNA clone of the venom gland of Mesomexovis punctatus (then referred as Vaejovis punctatus); b) mgc26729_g1_i1 with similarity to the precursor of Amphiphatic peptide CT2, deduced from cDNA cloned from the venom gland of V. smithi (Ramírez-Carreto et al., 2012); c) mgc28123_g1_i1 with similarity to the precursor of Hp1036 deduced from the transcriptome analysis of the venom gland of H. petersii (Hong et al., 2014) which has antibacterial and antiviral properties (Ma et al., 2010; Hong et al., 2014)]; d) mgc29210_g5_i1 had similarity to Amphipathic peptide CT2 deduced from cDNA of venom gland of Mesomexovis subcristatus (then referred as Vaejovis subcristatus; Ramírez-Carreto et al., 2012). The transcripts: mgc30131_g5_i1, mgc30131_g5_i2, mgc30131_g5_i4 and mgc30131_g5_i5 all show identities with the precursor of the amphipathic peptide OcyC2 (Silva et al., 2009), deduced de cDNA cloned of venom gland from Opisthacanthus cayaporum. Finally, mgc30131_g5_i3 had similarity to venom toxin obtained from transcriptome analysis of venom gland of H. lepturus (Kazemi-Lomdeasht et al., 2017). The unique peptide with similarity to a medium-length antimicrobial peptide was mgc17460_g1_i1 which is similar to Amp2 precursor deduced from a cDNA clone from the venom gland of M. punctatus (RamírezCarreto et al., 2015). The sequences related to long chain multifunctional peptide were: a) transcript mgc16627_g2_i1, which has hits with the precursor of venom peptide HtAPx of transcriptome analysis of the venom gland of H. troglodytes (Zhong et al., 2017) and b) components mgc22255_g1_i1 and mgc20285_g1_i1, which show similarity to the precursor of Vejovine, an antibacterial peptide (with activity against Gram negatives), deduced from the cDNA ndez-Aponte et al., of venom gland of Vaejovis mexicanus (Herna 2011). Fig. 7 shows the amino acid sequence deduced from one of the best characterized NDBPs. In the same figure, three additional sequences are shown with more than 50% identity to vejovine. 3.4.3. Protease inhibitors 3.4.3.1. Ascaris-type peptides. These peptides have been found in different organisms. They are serine protease inhibitors, and have been proposed to modulate protease activity in the scorpion venoms, thus protecting toxins and other components from unwanted degradation (Chen et al., 2013). They were first isolated from the parasite Ascaris suum (Babin et al., 1984) and have a common structure with short b strands in two b sheets, stabilized by five disulfide bridges (Gronenborn et al., 1990; Chen et al., 2013). Three sequences were obtained from the transcriptome analysis of the venom gland of M. gertschi (shown in Supplementary Table S1): a) two components mgc20934_g1_i1 and mgc21207_g1_i1 with similarity to Papilin, deduced from the genomic analysis from

Stegodyphus mimosarum and b) component mgc29407_g1_i1, showing similarity to the precursor of venom peptide HtPi2 deduced from the transcriptome analysis from scorpion H. troglodytes (Zhong et al., 2017). 3.4.3.2. Kunitz-type peptides. These peptides have a dual function as trypsin inhibitors and as blockers of the Kv1.3 potassium channels, although very weak (Chen et al., 2012). Our results showed the presence of three sequences (shown in Supplementary Table S1): a) mgc27030_g2_i1, which had similarity to fused toxin protein-like isoform X3-3 deduced from genome analysis from Alligator sinensis; b) mgc21255_g1_i1, which had hits with the precursor of Kunitz-type serine protease inhibitor Hg1 deduced from the transcriptome of H. gertschi (Schwartz et al., 2007; Chen et al., 2012); c) mgc27030_g1_i3 which had hits with the precursor of Kunitz-type carboxypeptidase inhibitor Kci-1deduced from the transcriptome of A. bicolor (Zhang et al., 2015b). 3.4.3.3. Serpins. Serine protease inhibitors (Serpins) haven been isolated from the saliva of ticks, and play an important role in inflammation and blood coagulation (Rubin, 1996). We found six transcripts with similarity to putative venom serpin-like proteinase inhibitor deduced of transcriptome analysis from the venom gland of Tityus obscurus; three sequences, which had similarity to leukocyte elastase inhibitor-like deduced from genome analysis of spider Parasteatoda tepidariorum; and one sequence with similarity to serpin peptidase inhibitor-like protein, a partial sequence deduced from cDNA cloned from the venom gland of O. cayaporum (Silva et al., 2009). 3.4.4. Enzymes Of the proteins found in this scorpion venom, enzymes are among the most abundant (Laraba-Djebari et al., 2015). Of the 182 annotated sequences in the M. gertschi transcriptome, 46% represent transcripts coding for enzymes (Fig. 2), including lipases, phospholipases, hyaluronidases, metallopeptidases, and serine proteinases (Supplementary Table S1). Interestingly, not in line with the findings in other venom gland transcriptomes of non-buthid scorpion species, for M. gertschi, metalloproteases were the most represented class of enzymes for which putative transcripts were found (19.8%; Fig. 2). Within these transcripts we report (but also see Supplementary Table S1) two components (mgc30053_g1_i10 and mgc30053_g1_i15) with a respective similarity to the precursor of a putative metalloproteinase, deduced from the transcriptome analysis of venom gland from T. obscurus and one component (mgc30053_g1_i12) with similarity to another putative metalloproteinase also deduced

104


Fig. 8. La1-like peptides. Sequence alignment of precursors from transcripts mgc22696_g1_i1, mgc24114_g1_i1, mgc25682_g1_i1, mgc26985_g1_i1 and mgc28255_g2_i1, (putative La1-like components) and similar sequences (UniProt or GenBank entry numbers are given in brackets): La1-like protein 13 from Urodacus yaschenkoi (L0GCJ1); La1-like protein 15 from U. yaschenkoi (L0GB04); La1 from Liocheles australasiae (P0C5F3); La1-like from Scorpiops jendeki (GH548227); PcavCulster15 from Pandinoides cavimanus (H2CYP1); Vx1 protein from Heterometrus spinifer (K7WMX6). The predicted signal peptide is underlined and the mature peptide is in boldface. For mgc26985_g1_i1 only, a small propeptide is predicted, as indicated in italics.

from transcriptome analysis of venom gland from T. obscurus. In addition, 12 transcripts with different percentages of similarity to six different precursors of angiostensin-converting enzymes were found. These putative enzymes were deduced from the genome analysis of L. polyphemus and of the transcriptome analysis of scorpion T. obscurus. The second most abundant enzyme-coding transcripts were those of phospholipases (29%; Fig. 2). Actually, the venom phospholipase activity was confirmed experimentally using the egg yolk technique mentioned under Material and Methods (Habermann and Hardt, 1972). As little as 2 mg of soluble venom gave positive results within the first minute of application, confirming its strong capacity to hydrolyze phospholipids. Two components (see Supplementary Table S1) were found with identities with the precursor of the phospholipase A2 deduced from the transcriptome analysis of the venom gland of H. gertschi (Schwartz et al., 2007). Two transcripts had matching hits with putative phospholipase A2 deduced from the transcriptome analysis of the venom gland from T. obscurus, or transcripts that translate to proteins with similarity to the precursor of phospholipase D3 deduced from the genome analysis of L. polyphemus, and to the precursor of patatinlike phospholipase from the genome analysis of L. polyphemus; (see Supplementary Table 1). Finally, the fourth most abundant group of enzymes (9%; Fig. 2) were those putatively coded by five transcripts. These enzymes present a conserved hyaluronidase domain and share sequence similarity to the precursor of a venom toxin (API81375) deduced from the transcriptome analysis of H. lepturus (Kazemi-Lomdeasht et al., 2017). We found eight transcripts with a domain related to lipases, five of those transcripts showed similarity to a putative lipase deduced from the transcriptome analysis of the venom gland of T. obscurus (JAT91088). We found two transcripts showing similarities to pancreatic lipase-related protein 2-like, deduced from the genome analysis of L. polyphemus (XP_013779480), and finally, one transcript similar to pancreatic lipase-related protein 2-like, a partial

sequence deduced from genome P. tepidariorum (XP_015926134).

analysis

of

the

spider

3.4.5. La1-like peptides La1 was isolated from the venom of Liocheles australasiae where it is the most abundant component (Miyashita et al., 2007). This peptide (Uniprot accession P0C5F3) consists of a long chain of 73 amino acids stabilized by four disulfide bridges, and defines a family of scorpion venom peptides (Zeng et al., 2013). In the transcriptome analysis of the venom gland of M. gertschi, five transcripts were found which could code for components with similarity to this type of peptides: a) mgc26985_g1_i1 had similarity to the precursor of venom peptide HtLa3 deduced from the transcriptomic analysis of the venom gland of H. troglodytes (Zhong et al., 2017); b) mgc28255_g2_i1 had hits with similarity to the precursor of the venom peptide HtLa15 deduced from the transcriptome analysis of the venom gland of the same species; and c) mgc24114_g1_i1 had hits with similarity to the precursor of toxin OcyC11, deduced from the cDNA cloned (though the peptide itself was later isolated) from the venom gland of O. cayaporum (Silva et al., 2009; Schwartz et al., 2008); d) mgc22696_g1_i1 had similarity to the precursor of toxin protein deduced from the transcriptomic analysis of the venom gland of H. lepturus (KazemiLomdeasht et al., 2017); e) mgc25682_g1_i1 had similarity to the precursor of toxin protein deduced from the cDNA cloned of the venom gland of Hottentotta judaicus. Fig. 8 shows the amino acid sequence of the La1-like peptides found in M. gertschi, together with six additional sequences found in non-buthid scorpions. The results from the transcriptome analyses of two species of Scorpiops (Ma et al., 2009, 2012) and of U. yaschenkoi (Luna-Ramírez et al., 2015) revealed a high representation of transcripts similar to La1. In this communication, it was found that transcripts similar to La1 are poorly represented in the M. gertschi venom gland. As a final commentary, it is important to note that the biological function of the La1-like peptides is not yet well established (Miyashita et al., 2007).


105

Fig. 9. HPLC separation of the soluble venom.

3.4.6. Other venom components 3.4.6.1. Insulin-like growth factor binding protein (IGFBP) family. The IGFBP family comprises a group of structurally and evolutionarily related vertebrate secreted proteins which bind and modulate the biological activity of IGFs. Proteins of this type are putatively encoded by 9.3% of the transcripts identified in the transcriptome analysis (see Supplementary Table S1). Within the peptide sequences deduced by in silico translation we found 15 with sequence similarity with the precursor of a venom toxin deduced from the transcriptome analysis of the venom gland of H. lepturus (KazemiLomdeasht et al., 2017). We also found one sequence with similarity to the precursor of venom peptide Htglin beta deduced from the transcriptome analysis of H. troglodytes (Zhong et al., 2017). One sequence with similarity to the precursor of single insulin-like growth factor-binding domain protein-2, deduced from the genome analysis from P. tepidariorum. 3.4.6.2. CAP superfamily. The cysteine-rich secretory proteins, including the pathogenesis-related 1 proteins and the antigen 5, are found in a wide range of organisms While some of these peptides have extracellular endocrine or paracrine functions, others potentially act as proteases or protease inhibitors (Gibbs et al., 2008). Allergens are also included in this superfamily and have been found in the venom of several arthropods (insects, arachnids and myriapods). In our analysis, we found six sequences potentially coding for components related to this superfamily. Component mgc23073_g1_i1 shared similarity with the partial sequence of the precursor of a putative cysteine-rich protein, deduced from the transcriptome analysis of the venom gland of T. obscurus. Components mgc16519_g1_i1mgc29225_g1 and mgc29609_g1_i1 _i1 had hits with two venom toxins deduced from the transcriptome analysis of the venom gland of H. lepturus (Kazemi-Lomdeasht et al., 2017); component mgc28593_g1_i1 had similarity to a conserved hypothetical protein deduced from the genome analysis of the tick Ixodes scapularis.

3.4.6.3. Other proteins. Thirteen transcripts (7.1% of the total 182 annotated sequences in the M. gertschi transcriptome) code for proteins with different functions of those above-described (Fig. 2). Among them, we found three transcripts with similarity to the precursor of a hypothetical protein deduced from the transcriptome analysis of the venom gland of Pandinoides cavimanus (previously known as Pandinus cavimanus) (Diego-García et al., 2012); two transcripts with similarity to the partial precursor of a putative secreted protein, deduced from the transcriptome analysis of the venom gland of O. cayaporum (Silva et al., 2009); one transcript with similarity to the precursor of venom peptide HtfTx2 deduced from the transcriptome analysis of the venom gland of H. troglodytes (Zhong et al., 2017); and one transcript with similarity to the precursor of venom peptide HtTxB deduced from the transcriptome analysis of the venom gland of H. troglodytes (Zhong et al., 2017). 3.5. Venom fractionation, MS fingerprint and direct Edman sequencing The soluble venom was separated by high performance liquid chromatography (HPLC). Over 80 distinct fractions were recovered, as shown in Fig. 9. Whole soluble venom (2 mg) was injected into a C4 reversephase semi-preparative column and separated by using a linear gradient from solvent A (0.12% trifluoroacetic acid, TFA, in water) to 60% solvent B (0.10% TFA in acetonitrile) for 90 min. Mass spectrometry analysis of these fractions allowed to assign a molecular mass to 67 distinct components, as can be seen in Table 1. Components with more than 2 Da molecular weight (MW) difference were considered chemically distinct. A number of components assumed to have single identities due to their molecular masses eluted in overlapping, adjacent HPLC fractions. A few fractions eluting earlier from the column (from 3.28 to


106

5.66 min) comprise about 8% of the total venom. In these fractions, seven different components with molecular masses between 342 and 950 Da were found (Table 1). None of these fractions, when submitted to automatic Edman sequencing, gave any clear amino acid pattern, meaning that they are either not peptides or that their N-terminal amino acids are blocked. Two fractions eluting at 20.21 and 25.01 min from the HPLC column accounted for about 50% of the total material absorbing at l ¼ 230 nm. This is one of the most important differences of the Euscorpiidae scorpion venom, here described, as compared to venoms from scorpions belonging to other families. The use of absorbance at this wavelength for quantitation is probably not the best method to compare relative concentrations of venom components in this species. Further research is needed to determine the chemical nature of these abundant components, including the determination of their molar absorption coefficients, in order to have a more precise estimation of their relative concentrations in the venom. The structure and function of these components are not known, but we are presently conducting mass spectrometry and nuclear magnetic resonance studies to solve their chemical structure. Apart from the low molecular weight components mentioned above, a few other individual components were subjected to direct Edman degradation in order to determine their N-terminal sequences. However, due to the relative large amounts required for this procedure, most of the peptide sequences generated in this work were obtained by tandem mass spectrometry (MS/MS), a more sensitive technique. A component with an MW of 3815 Da was detected at two closely-related retention times: 24.88 min and 26.58 min, coeluting with other, unrelated molecular species (Table 1). This component, when submitted to Edman sequencing gave the amino acid sequence: NXIAHLQRXRKNN…, where X corresponds to possible cysteine residues. An identical amino acid sequence was found after translation of one of the transcripts identified in the transcriptomic analysis (see Section 3.4.1.4 above): NCIAHLQRCRKNNDCCSKNCKRRGTQPEQRCR, with a predicted MW for the folded molecule, with all the disulfide bonds formed, of 3814.4 Da. Thus, this component is confirmed by both proteomic and transcriptomic analysis. A BLAST search with this amino acid sequence shows that it corresponds to a calcin. Calcins are peptides that bind to ryanodine-sensitive calcium channels (Xiao et al.,

2016). Six other venom components were directly sequenced by automatic Edman degradation (See Supplementary Table S2). A BLAST analysis of the found N-terminal sequences suggests that all but one of these components, share similarity to known scorpion venom components (see last column on Supplementary Table S2). 3.6. LC-MS/MS analysis of the venom An aliquot of the soluble venom was reduced, alkylated and digested with trypsin. After treatment, the sample was analyzed by LC/MS for sequence determination, as described in Material and Methods. The MS/MS-determined sequences were collated with the in silico-translated M. gertschi RNA-Seq database. Table 2 shows the 23 proteins found by LC-MS/MS analysis using the transcriptome database for the identification of peptides. The identifiers for the transcripts, a description of the deduced peptides, their theoretical MW, and the parameters leading to their identification are shown. The identified matching peptides are reported in detail in Supplementary Table S3. The sequence coverage goes from 4 to 50% for the different proteins. Most of the sequences obtained by mass spectrometry show similarities to known venom components (see Description in Table 2). It is remarkable that, notwithstanding the very limited number of venom peptides subjected to direct Edman sequencing, one of them, a putative scorpine-like peptide, was detected by both sequencing approaches (peptide with RT ¼ 58.87 min in Table S2 and translated transcript mgc19218_g1_i1 in Table 2 and Table S1). Thus, if we take into consideration the calcin-like peptide described in Section 3.4.1.4, the 23 peptides with partial sequences determined by spectrometry (Supplementary Table S3), and the six additional sequences listed in Table S2, all together, the proteomic analysis permitted the identification of 30 peptides/proteins from the M. gertschi venom. 4. Conclusion The venom from M. gertschi is not toxic to mammals (mice) but is lethal to arthropods (crickets and shrimps) at the doses assayed. HPLC processing of the whole soluble venom permitted the separation and identification of at least 67 components, of which proteomic analysis by direct Edman degradation and mass

Table 1 Mass fingerprint from the HPLC-separated fractions of the soluble venom (RT ¼ HPLC retention time as can be seen in Fig. 9). RT

Experimental Molecular Weight

RT

Experimental Molecular Weight

3.28 3.90 4.90 5.66 10.79 13.44 14.01 16.07 17.12 19.10 20.21 20.40 21.40 23.16 23.70 24.47 24.88 25.01 26.58 26.93 27.47 28.10

342, 494, 646, 798, 950 231 268 269 266 391 251, 500 281 318 555 288 295, 709 318 3815 3815, 2863 307 323, 388, 3815 337, 4188 337, 4188, 3815 3398 3484 3484

29.24 30.39 30.94 34.70 35.61 36.68 40.54 42.26 44.86 45.58 46.50 47.43 48.29 52.24 54.55 58.87 61.93 63.71 66.92 71.34 77.19 82.20

817 639 625 946 461, 706, 764, 918, 1050, 1073, 1094 957 1244 1230, 1050 4238, 7918, 4021 1333, 4238, 3110 1361, 1217 1347 1437 8375, 2976 8374, 6369 9255 5061, 4626 13128 5375, 11370 31862, 31741, 31615 1915 12374


107

Table 2 Proteins found by LC-MS/MS analysis using the transcriptome database for the identification of peptides. Transcript Code

Description (Similar sequence by Blast; Protein type, see Table S3; Peptide size)

Score

Coverage (%)

Peptides Identified

Theoretical MW [kDa]

mgc30677_g2_i2

Angiotensin-converting enzyme-like (ID: XP_013773749); Metalloprotease; 595 aa Angiotensin-converting enzyme-like (ID: XP_013773749); Metalloprotease; 636 aa Angiotensin-converting enzyme-like (ID: XP_013773749); Metalloprotease; 603 aa Venom toxin (ID: API81339); Phospholipase A2; 221 aa Venom toxin (ID: API81358); CAP superfamily; 416 aa 50 -nucleotidase-like (ID: XP_013774694); Other protein; 549 aa Putative angiotensin-converting enzyme (ID: JAT91086); Metalloprotease; 575 aa No identify; Other protein; 101 aa Hg scorpine like 2(ID: P0C8W5); Potassium channel toxin; 85 aa Angiotensin-converting enzyme-like (ID: XP_013773749); Metalloprotease; 583 aa Angiotensin-converting enzyme-like (ID: XP_013773749); Metalloprotease; 601 aa Hypothetical protein (ID: AEX09195); Other protein; 104 aa Hypothetical protein (ID: AEX09195); Other protein; 139 aa Angiotensin-converting enzyme-like (ID: XP_013773749); Metalloprotease; 567 aa Angiotensin-converting enzyme-like (ID: XP_013773749); Metalloprotease; 562 aa Venom toxin (ID: API81352); CAP superfamily; 384 aa Leucine-rich repeat-containing protein 15, partial (ID: KFM61094); Other protein; 392 aa Venom toxin (ID: API81375.1); Hyaluronidase; 382 aa Venom peptide httxb (ID: AOF40192); Other protein; 100 aa Angiotensin-converting enzyme-like (ID: XP_013773749); Metalloprotease; 577 aa Prothoracicostatic peptide (ID: XP_013771913); Other protein; 142 aa Putative vesicle coat complex copii subunit sfb3 (ID: JAU02117); Other protein; 60 aa Endophilin-B1 (ID: KFM77626); Other protein; 291 aa

628.90

45.1

20

68.8

528.93

46.23

16

72.0

469.32

41.18

18

69.8

393.07 363.47 227.77

24.58 33.26 31.33

4 11 10

24.8 47.2 61.5

173.82

21.61

8

67.9

135.72 135.57

40.34 31.73

3 3

11.3 9.3

131.56

18.9

6

68.0

120.35

13.87

5

69.6

90.32 81.41 65.06

50.39 19.02 13.78

4 2 6

11.8 16.4 66.5

49.46

10.3

4

65.9

47.43 39.72

13.4 11.98

3 3

43.3 44.3

35.06 24.86 24.35

7.29 21.26 4.14

2 1 2

44.3 11.2 67.4

22.10

9.09

2

16.6

19.81

8.59

1

5.7

16.51

15.81

2

33.5

mgc30260_g1_i1 mgc30677_g2_i3 mgc30555_g1_i1 mgc29609_g1_i1 mgc30535_g1_i1 mgc30662_g1_i1 mgc26577_g1_i1 mgc19218_g1_i1 mgc28910_g1_i1 mgc29379_g1_i1 mgc20919_g1_i1 mgc25939_g2_i1 mgc30452_g1_i1 mgc30452_g3_i1 mgc16519_g1_i1 mgc29104_g1_i1 mgc30556_g1_i1 mgc19440_g1_i1 mgc30677_g3_i1 mgc23003_g1_i2 mgc24364_g1_i1 mgc30840_g2_i1

spectrometry identified 30 distinct protein/peptide components. Over 50% of the whole soluble venom is made of low molecular weight non-peptide/protein components, of unknown function. The RNAseq results obtained in this study allowed the identification of 182 annotated sequences potentially coding for venom peptides/proteins. These components comprehend different types of proteins/peptides, such as: enzymes, toxins, non-disulfide bridged peptides, scorpines, La1-like peptides and other venom components. We must note that a large number of transcripts could not be associated to known sequences or activities, and thus remain unannotated. This is a limitation of the transcriptome approach that remarks the need for the further functional characterization of the venom components from this and other scorpion species. Based on the venom profile, and the transcriptome and proteomic analyses of M. gerschi, we can conclude that the venom composition in this species is quite different from those reported for other non-buthids studied to date. The database that we generated will certainly contribute to the knowledge of the evolution of the venom arsenal in scorpions, and might indicate the presence of potential candidates for biotechnological applications. Funding This work was partially supported by grant 237864 from Fondo n para la Educacio n SEP-CONACyT, and grant Sectorial de Investigacio n General de Asuntos del Personal Academico IN203416 from Direccio of the National Autonomous University of Mexico, awarded to LDP.

CESL and JICU are recipients of postdoctoral and PhD program scholarships from CONACyT (No. 237864 and No. 404460, respectively). Conflict of interest The authors declare no conflict of interest. Author contributions Conceived and designed the experiments: CESL, EO, LDP. Performed the experiments: JICU, FZZ, EO, CVFB. Analyzed the data: CESL, JICU, FZZ, CVFB, EO, LDP. Contributed reagents/materials/ analysis tools: CESL, LDP, CVFB. Wrote the paper: CESL, JICU, FZZ, CVFB, EO, LDP. Data submission: CESL, JICU. Ethical statement The protocols which involved the use of animals were approved by the Institution Committee for the care and use of laboratory animals of the Institute of Biotechnology of the National Autonomous University of Mexico. Acknowledgements The authors are grateful to Dr. Oscar Francke from the Institute of Biology, UNAM for support during collection of the scorpions

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used in this work (Scientific Permit FAUT-0175, from SEMARNAT). The technical support of Erika Patricia Meneses Romero is zquez Castro, greatly acknowledged. We are grateful to Gloria T. Va nica Jime nez Jacinto at the DNA Ricardo A. Grande Cano and Vero Massive Sequencing and the Bioinformatics Units from the Instituto de Biotecnología, UNAM for their technical support. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.toxicon.2017.05.002. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2017.05.002. References ndez, V., Possani, L.D., 2013. Venom proteomic Abdel-Rahman, M.A., Quintero-Herna and venomous glands transcriptomic analysis of the Egyptian scorpion Scorpio maurus palmatus (Arachnida: Scorpionidae). Toxicon 74, 193e207. http:// dx.doi.org/10.1016/j.toxicon.2013.08.064. Almaaytah, Ammar, Albalas, Qosay, 2014. Scorpion venom peptides with no disulfide bridges: a review. Peptides 51, 35e45. http://dx.doi.org/10.1016/ j.peptides.2013.10.021. Babin, D.R., Peanasky, R.J., Goos, S.M., 1984. The isoinhibitors of chymotrypsin/ elastase from Ascaris lumbricoides: the primary structure. Arch. Biochem. Biophys. 232, 143e161. http://dx.doi.org/10.1016/0003-9861(84)90530-7. Becerril, B., Vazquez, A., Garcia, C., Corona, M., Bolivar, F., Possani, L.D., 1993. Cloning and characterization of cDNAs that code for Na(þ)-channel-blocking toxins of the scorpion Centruroides noxius Hoffmann. Gene 128 (2), 165e171. Caliskan, F., García, B.I., Coronas, F.I., Batista, C.V., Zamudio, F.Z., Possani, L.D., 2006. Characterization of venom components from the scorpion Androctonus crassicauda of Turkey: peptides and genes. Toxicon 48 (1), 12e22. http://dx.doi.org/ 10.1016/j.toxicon.2006.04.003. Chen, Z., Wang, B., Hu, J., Yang, W., Cao, Z., Zhuo, R., Li, W., Wu, Y., 2013. SjAPI, the first functionally characterized Ascaris-type protease inhibitor from animal venoms. PloS One 8 (3), e57529. http://dx.doi.org/10.1371/ journal.pone.0057529. Chen, Z.Y., Hu, Y.T., Yang, W.S., He, Y.W., Feng, J., Wang, B., Zhao, R.M., Ding, J.P., Li, W.X., Wu, Y.L., 2012. Hg1, novel peptide inhibitor specific for Kv1.3 channels from first scorpion kunitz.type potassium channel toxin family. J. Biol. Chem. 287, 13813e13821. http://dx.doi.org/10.1074/jbc.M112.343996. Cociancich, S., Goyffon, M., Bontems, F., Bulet, P., Bouet, F., Menez, A., Hoffmann, J., 1993. Purification and characterization of a scorpion defensin, a 4kDa antibacterial peptide presenting structural similarities with insect defensins and scorpion toxins. Biochem. Biophys. Res. Commun. 194 (1), 17e22. Diego-García, E., Peigneur, S., Clynen, E., Marien, T., Czech, L., Schoofs, L., 2012. Molecular diversity of the telson and venom components from Pandinus cavimanus (Scorpionidae Latreille 1802): transcriptome, venomics and function. Proteomics 12 (2), 313e328. http://dx.doi.org/10.1002/pmic.201100409. Dutto, M., Dutto, L., Scaglione, N., Bertero, M., 2010. Euscorpius (Scorpiones, Euscorpiidae): three cases of stings in northwestern Italy. J. Venom. Anim. Toxins Incl. Trop. Dis. 16 (4), 659e663. http://dx.doi.org/10.1590/S167891992010000400018. Feng, J., Yu, C., Wang, M., Li, Z., Wu, Y., Cao, Z., Li, W., He, X., Han, S., 2013. Expression and characterization of a novel scorpine-like peptide Ev37, from the scorpion Euscorpiops validus. Protein Expr. Purif. 88, 127e133. http://dx.doi.org/10.1016/ j.pp.2012.12.004. ~ as, S., Pimienta, G., Ortiz, E., Figueroa-Montiel, A., Ramos, M.A., Mares, R.E., Duen Possani, L.D., Licea-Navarro, A., 2016. In Silico identification of protein disulfide isomerase gene families in the De Novo assembled transcriptomes of four different species of the genus Conus. PLoS ONE 11 (2), e0148390. http:// dx.doi.org/10.1371/journal.pone.0148390. Froy, O., Gurevitz, M., 2004. Arthropod defensins illuminate the divergence of scorpion neurotoxins. J. Pept. Sci. Off. Publ. Eur. Pept. Soc. 10 (12), 714e718. Ganz, T., Lehrer, R.I., 1994. Defensins. Curr. Opin. Immunol. 6 (4), 584e589. Gibbs, Gerard M., Roelants, Kim, O'Bryan, Moira K., 2008. The CAP superfamily: cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteinsdroles in reproduction, cancer, and immune defense. Endocr. Rev. 29 (7), 865e897. http://dx.doi.org/10.1210/er.2008-0032. lez-Santilla n, E., Alvarez-Padilla, Gonza F., 2014. The male of Megacormus granosus (Gervais, 1844) with comments on its hemispermatophore (Scorpiones, Euscorpiidae). Zookeys 504, 75e91. http://dx.doi.org/10.3897/ zookyes.504.9027. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B.W., Nusbaum, C.,

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