Genetic Diversity within Scorpio Genus (Scorpion: Scorpionidae) from Iran: Preliminary Evidence based on 16srRNA Sequence.

The yellow digger scorpion, Scorpio maurus, is a medically important scorpion for which little is known about its genetic diversity. Polymerase chain reaction products of 16srRNA gene fragments were generated from scorpion specimens named SmKh1 and SmKh2. These sequences showed high similarity with the only partial sequence of S. maurus isolate SCA1 large subunit ribosomal RNA gene available in the Genbank database. The drawing of the phylogeny tree showed two clusters, A and B. The two specimens (SmKh1 and SmKh2), which are placed in sub-cluster A2, were provided from Behbahan, Iran, and they have the closest relationship with the only sequence of S. maurus (MW281771), which is also collected from Behbahan. It is noteworthy that the two sequences obtained from S. maurus scorpions recorded from Miandoab (MK170444) and Mahabad (KU705354), which are in sub-cluster A1, are more similar to the scorpions isolated from the Mediterranean basin than those collected from Behbahan. This issue is probably due to the fact that patterns of genetic diversity are a reflection of variation in gene flow, which is also influenced by factors such as territorial barriers and geographical distances. We conclude that the scorpions of this study accompanied by similar scorpions in the Mediterranean basin, belong to the same species despite the insignificant differences.

Transcriptome Sequencing and Comparison of Venom Glands Revealed Intraspecific Differentiation and Expression Characteristics of Toxin and Defensin Genes in Mesobuthus martensii Populations.

Mesobuthus martensii, a famous and important Traditional Chinese Medicine has a long medical history and unique functions. It is the first scorpion species whose whole genome was sequenced worldwide. In addition, it is the most widespread and infamous poisonous animal in northern China with complex habitats. It possesses several kinds of toxins that can regulate different ion channels and serve as crucial natural drug resources. Extensive and in-depth studies have been performed on the structures and functions of toxins of M. martensii. In this research, we compared the morphology of M. martensii populations from different localities and calculated the COI genetic distance to determine intraspecific variations. Transcriptome sequencing by RNA-sequencing of the venom glands of M. martensii from ten localities and M. eupeus from one locality was analyzed. The results revealed intraspecific variation in the expression of sodium channel toxin genes, potassium channel toxin genes, calcium channel toxin genes, chloride channel toxin genes, and defensin genes that could be related to the habitats in which these populations are distributed, except the genetic relationships. However, it is not the same in different toxin families. M. martensii and M. eupeus exhibit sexual dimorphism under the expression of toxin genes, which also vary in different toxin families. The following order was recorded in the difference of expression of sodium channel toxin genes: interspecific difference; differences among different populations of the same species; differences between sexes in the same population, whereas the order in the difference of expression of potassium channel toxin genes was interspecific difference; differences between both sexes of same populations; differences among the same sex in different populations of the same species. In addition, there existed fewer expressed genes of calcium channel toxins, chloride channel toxins, and defensins (no more than four members in each family), and their expression differences were not distinct. Interestingly, the expression of two calcium channel toxin genes showed a preference for males and certain populations. We found a difference in the expression of sodium channel toxin genes, potassium channel toxin genes, and chloride channel toxin genes between M. martensii and M. eupeus. In most cases, the expression of one member of the toxin gene clusters distributed in series on the genome were close in different populations and genders, and the members of most clusters expressed in same population and gender tended to be the different. Twenty-one toxin genes were found with the MS/MS identification evidence of M. martensii venom. Since scorpions were not subjected to electrical stimulation or other special treatments before conducting the transcriptome extraction experiment, the results suggested the presence of intraspecific variation and sexual dimorphism of toxin components which revealed the expression characteristics of toxin and defensin genes in M. martensii. We believe this study will promote further in-depth research and use of scorpions and their toxin resources, which in turn will be helpful in standardizing the identification and medical applications of Quanxie in traditional Chinese medicine.

Novel Mutant Phospholipase D from Hemiscorpius lepturus Acts as A Highly Immunogen in BALB/c Mice Against the Lethality of Scorpion Venom.

Hemiscorpius lepturus (H. lepturus) which belongs to the Scorpionidae family, is the deadliest scorpion in Iran. It causes pathological manifestations like dermonecrosis, hemolysis, renal failure, necrotic ulcers, and in some cases, even death. The venom of this scorpion is well-known for its cytotoxic effects in comparison with the other venomous scorpions which show significant neurotoxic effects. Due to the painless nature of the sting of this scorpion, the clinical symptoms occur in victims 24 to 72 h post-sting. In our previous studies during the last decade, we demonstrated that the medical complications are attributable to the presence of phospholipase D (PLD) as a major toxin in the venom. With the purpose of designing and constructing a vaccine against H. lepturus for humans, animal model experiments were performed. To achieve this goal, non-toxic PLD was developed by mutation of two critical catalytic residues-His12 and His48-into alanines and the product was then denominated mut-rPLD1. The in-vivo tests showed that the mice immunized with interval doses of 10 µg of mut-rPLD1, were completely protected against 10× the LD100 of the venom. In conclusion, this mutant may be an effective vaccine candidate against scorpion envenomation by H. lepturus in future clinical studies.

De novo transcriptomic analysis of the venomous glands from the scorpion Heterometrus spinifer revealed unique and extremely high diversity of the venom peptides.

Scorpion, as an ancient species, has been widely used on dozens of human diseases in traditional Chinese Medicine. Although the scorpion venom from the Buthidae family with the potent toxicity attracts more interests, toxins from the non-Buthidae family draw great attention as well because of its abundance and complexity even without harm to mammals. Moreover, several toxic components of scorpion venom have been identified as valuable scaffolds for the drug design and development. Using the Next Generation Sequencing (NGS) technique, here we reported the transcriptome of the venomous glands of Heterometrus spinifer, a non-Buthidae scorpion that only a few toxic and complete components have been identified known-to-date. The total mRNA extracted from the venomous glands of H. spinifer was subjected to illumina sequencing with a strategy of de novo assembly, and a total of 54 189 transcripts were unigenes from a total of 88 311 600 determined reads. We annotated 18 567 (34.26%) unigenes from NR database, 12 258 (22.62%) from SWISSPROT database, 11 161 (20.60%) from GO database, 10 159 (18.75%) from COG database and 5059 (9.34%) from KEGG database, respectively. 2843 unigenes were further selected against the toxin-related sub-database of SWISSPROT. After removing the redundancy, 13 common toxin-related subfamilies with 62 unigenes were manually confirmed, including 8 K-toxins, 1 calcin, 3 Imperatoxin I-like, 2 La1-like, 1 scorpin-like, 3 antimicrobial peptides, two types of protease inhibitors such as 8 Kunitz-type protease inhibitors and 3 Ascaris-type protease inhibitors, and 33 proteases including 16 serine proteinases, 7 phospholipases, 5 metalloproteases, 3 hyaluronidases and 2 phosphatases. Our report is the first transcriptomic analyses of venomous glands from the scorpion H. spinifer, serving as a public information platform for the development of novel bio-therapeutics.

Protease inhibitor in scorpion (Mesobuthus eupeus) venom prolongs the biological activities of the crude venom.

It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival.

The transcriptome recipe for the venom cocktail of Tityus bahiensis scorpion.

Scorpion venom is a mixture of peptides, including antimicrobial, bradykinin-potentiating and anionic peptides and small to medium proteins, such as ion channel toxins, metalloproteinases and phospholipases that together cause severe clinical manifestation. Tityus bahiensis is the second most medically important scorpion species in Brazil and it is widely distributed in the country with the exception of the North Region. Here we sequenced and analyzed the transcripts from the venom glands of T. bahiensis, aiming at identifying and annotating venom gland expressed genes. A total of 116,027 long reads were generated by pyrosequencing and assembled in 2891 isotigs. An annotation process identified transcripts by similarity to known toxins, revealing that putative venom components represent 7.4% of gene expression. The major toxins identified are potassium and sodium channel toxins, whereas metalloproteinases showed an unexpected high abundance. Phylogenetic analysis of deduced metalloproteinases from T. bahiensis and other scorpions revealed a pattern of ancient and intraspecific gene expansions. Other venom molecules identified include antimicrobial, anionic and bradykinin-potentiating peptides, besides several putative new venom components. This report provides the first attempt to massively identify the venom components of this species and constitutes one of the few transcriptomic efforts on the genus Tityus.

[Identification of Scolopendra subspinipes mutilans and its adulterants using DNA barcode].

In this study, the COI barcode was used to identify the Scolopendra medicinal materials and its adulterants in order to provide a new method for the identification of Scolopendra. Genomic DNA was extracted from the experimental samples. The COI sequences were amplified and sequenced bi-directionally. Sequence alignment and NJ tree construction was carried out by MEGA6.0 software. The results showed that the COI sequences can be obtained from all experimental samples. The average inter-specific K2P distance of Scolopendra was 0.222 and the minimum inter-specific distance was 0.190. All the Scolopendra subspinipes mutilans medicinal samples clustered into a clade in the NJ tree and can be distinguished from its adulterants. In a conclusion, COI can be used to correctly identify Scolopendra medicinal materials, and it will be a potential DNA barcode for identifying other animal medicinal materials.

Heteromtoxin (HmTx), a novel heterodimeric phospholipase A(2) from Heterometrus laoticus scorpion venom.

Heteromtoxin (HmTx) is a group III phospholipase A(2) produced in Heterometrus laoticus, in Thailand. In this study, HmTx was purified from venom by separation chromatography, and the PLA(2) activity of the fractions was determined by lecithin agar assay. The enzyme is an acidic protein with a pI of 5.6 and an apparent molecular weight of 14018.4 Da. The nucleotide sequence of HmTx contains 649 bp, and the mature protein is predicted to have 131 amino acid residues-104 of which make up the large subunit, and 27 of which make up the small subunit. The subunit structure of HmTx is highly similar to that of the other toxin, Pandinus imperator imperatoxin I (IpTx(i)) and to Mesobuthus tamulus phospholipase A(2) (MtPLA(2)). The 3D-structure of HmTx consists of three conserved alpha-helices: h1 (Lys24-His34), h2 (Cys59-Asp71), and h3 (Ala80-Phe89). The beta-sheet consisted of a single stranded anti-parallel beta-sheet (b1.1 at Glu43-Lys45 and b1.2 at Lys48-Asn50) that was highly similar to the conserved sequences (-CGXG-, -CCXXHDXC- and CXCEXXXXXC-) of Apis mellifera (bee) phospholipases.

Molecular and bioinformatical characterization of a novel superfamily of cysteine-rich peptides from arthropods.

The full-length cDNA sequences of two novel cysteine-rich peptides (referred to as HsVx1 and MmKTx1) were obtained from scorpions. The two peptides represent a novel class of cysteine-rich peptides with a unique cysteine pattern. The genomic sequence of HsVx1 is composed of three exons interrupted by two introns that are localized in the mature peptide encoding region and inserted in phase 1 and phase 2, respectively. Such a genomic organization markedly differs from those of other peptides from scorpions described previously. Genome-wide search for the orthologs of HsVx1 identified 59 novel cysteine-rich peptides from arthropods. These peptides share a consistent cysteine pattern with HsVx1. Genomic comparison revealed extensive intron length differences and intronic number and position polymorphisms among the genes of these peptides. Further analysis identified 30 cases of intron sliding, 1 case of intron gain and 22 cases of intron loss occurred with the genes of the HsVx1 and HsVx1-like peptides. It is interesting to see that three HsVx1-like peptides XP_001658928, XP_001658929 and XP_001658930 were derived from a single gene (XP gene): the former two were generated from alternative splicing; the third one was encoded by a DNA region in the reverse complementary strand of the third intron of the XP gene. These findings strongly suggest that the genes of these cysteine-rich peptides were evolved by intron sliding, intron gain/loss, gene recombination and alternative splicing events in response to selective forces without changing their cysteine pattern. The evolution of these genes is dominated by intron sliding and intron loss.

Molecular approaches for structural characterization of a new potassium channel blocker from Tityus stigmurus venom: cDNA cloning, homology modeling, dynamic simulations and docking.

Potassium channels are involved in the maintenance of resting membrane potential, control of cardiac and neuronal excitability, neurotransmitters release, muscle contractility and hormone secretion. The Tityus stigmurus scorpion is widely distributed in Northeastern Brazil and known to cause severe human envenomations, inducing pain, hypoesthesia, edema, erythema, paresthesia, headaches and vomiting. Most potassium channel blocking peptides that have been purified from scorpion venoms contain 30-40 amino acids with three or four disulfide bridges. These peptides belong to α-KTx subfamily. On the other hand, the ß-KTx subfamily is poorly characterized, though it is very representative in some scorpion venoms. A transcriptomic approach of T.stigmurus scorpions developed by our group revealed the repertoire of possible molecules present in the venom, including many toxins of the ß-KTx subfamily. One of the ESTs found, named TSTI0003C has a cDNA sequence of 538 bp codifying a mature protein with 47 amino acid residues, corresponding to 5299 Da. This ß-KTx peptide is a new member of the BmTXKß-related toxins, and was here named TstKMK. The three-dimensional structure of this potassium channel toxin of the T. stigmurus scorpion was obtained by computational modeling and refined by molecular dynamic simulations. Furthermore, we have made docking simulations using a Shaker kV-1.2 potassium channel from rats as receptor model and proposed which amino acid residues and interactions could be involved in its blockade.

Evolution and radiation in the scorpion Buthus elmoutaouakili Lourenco and Qi 2006 (Scorpiones: Buthidae) at the foothills of the Atlas Mountains (North Africa).

When low dispersal ability of an organism meets geographical barriers, the evolution of inter- and intraspecific differentiation is often facilitated. In the Atlas massif of North Africa, the genus Buthus splits into several species and diverges into numerous genetic lineages, often following the orographic structures of mountain systems. Such high mountain ranges often act as barriers for species with reduced mobility even on small spatial scales. To study the effect of orographic structures on organisms with low dispersal ability, we collected 61 individuals of the scorpion species Buthus elmoutaouakili at 18 locations around the southwestern foothills of the High Atlas and Antiatlas and in the Sousse valley (western Morocco). We analyzed intraspecific differentiation patterns within this geographically restricted area of about 100 × 50 km using 452 bp of the cytochrome oxidase I mitochondrial gene. We detected 5 distinct genetic lineages. In a second analysis, we added 61 previously published sequences from Buthus species from Europe and North Africa. Using a molecular clock approach, we detected old splits (4-5 Ma) separating the samples from 1) the western High Atlas and north of these mountains, 2) the Sousse valley and adjoining mountain areas, and 3) the southwestern Antiatlas. Further differentiation happened in the first 2 geographical groups about 3 Ma. Thus, the divergence time estimates based on a Bayesian approach support the onset of differentiation into these main clades along the Pliocene (5-2.3 Ma) when climatic oscillations started and a constant global cooling preceded the glacial-interglacial cycles of the Pleistocene. Further genetic splits into parapatric groups are detectable for the Sousse valley main group in the early Pleistocene. The climatic oscillations of the Pliocene and early Pleistocene might have caused repeated range shifts, expansions, and retractions leading to repeated vicariance, hereby producing the hierarchical structure of genetic differentiation in B. elmoutaouakili. A taxonomic revision, including morphological and molecular data, is needed to assess the status of each of these Buthus scorpion lineages.

Molecular diversity of the telson and venom components from Pandinus cavimanus (Scorpionidae Latreille 1802): transcriptome, venomics and function.

Venom from the scorpion Pandinus cavimanus was obtained by electrical stimulation of the telson (stinger). Total venom was toxic to crickets at 7-30 µg and a paralysis or lethal effect was observed at 30 µg of venom (death at 1.5 µg/mg of cricket). Electrophysiological analyses showed cytolytic activity of total venom on oocytes at 7 µg. HPLC allowed separation of the venom components. A total of 38 fractions from total venom were tested on voltage-gated Na(+) and K(+) channels. Some fractions block K(+) currents in different degrees. By using MS analysis, we obtained more than 700 different molecular masses from telson and venom fractions (by LC-MS/MS and MALDI-TOF MS analyses). The number of disulfide bridges of the telson components was determined. A cDNA library from P. cavimanus scorpion was constructed and a random sequencing screening of transcripts was conducted. Different clones were obtained and were analyzed by bioinformatics tools. Our results reveal information about new genes related to some cellular processes and genes involved in venom gland functions (toxins, phospholipases and antimicrobial peptides). Expressed sequence tags from venom glands provide complementary information to MS and reveal undescribed components related to the biological activity of the venom.

Molecular cloning and electrophysiological studies on the first K(+) channel toxin (LmKTx8) derived from scorpion Lychas mucronatus.

LmKTx8, the first toxic gene isolated from the venom of scorpion Lychas mucronatus by constructing cDNA library method, was expressed and characterized physiologically. The mature peptide has 40 residues including six conserved cysteines, and is classified as one of alpha-KTx11 subfamily. Using patch-clamp recording, the recombinant LmKTx8 (rLmKTx8) was used to test the effect on voltage-gated K(+) channels (Kv1.3) stably expressed in COS7 cells and large conductance-Ca(2+)-activated K(+) (BK) channels expressed in HEK293. The results of electrophysiological experiments showed that the rLmKTx8 was a potent inhibitor of Kv1.3 channels with an IC(50)=26.40+/-1.62nM, but 100nM rLmKTx8 did not block the BK currents. LmKTx8 or its analogs might serve as a potential candidate for the development of new drugs for autoimmune diseases.

Wide phylogenetic distribution of Scorpine and long-chain beta-KTx-like peptides in scorpion venoms: identification of "orphan" components.

Scorpine and toxins specific for potassium channels of the family beta (beta-Ktx) are two types of structurally related scorpion venom components, characterized by an unusually long extended N-terminal segment, followed by a Cys-rich domain with some resemblance to other scorpion toxins. In this communication, we report evidence supporting the ubiquitous presence of Scorpine and beta-KTx-like polypeptides and their precursors in scorpions of the genus Tityus of the family Buthidae, but also included is the first example of such peptides in scorpions from the family Iuridae. Seven new beta-KTxs or Scorpine-like peptides and precursors are reported: five from the genus Tityus (T. costatus, T. discrepans and T. trivittatus) and two from Hadrurus gertschi. The cDNA precursors for all of these peptides were obtained by molecular cloning and their presence in the venoms were confirmed for various peptides. Analysis of the sequences revealed the existence of at least three distinct groups: (1) beta-KTx-like peptides from buthids; (2) Scorpine-like peptides from scorpionid and iurid scorpions; (3) heterogeneous peptides similar to BmTXKbeta of buthids and iurids. The biological function for most of these peptides is not well known; that is why they are here considered "orphan" peptides.

Molecular characterization of a new scorpion venom lipolysis activating peptide: Evidence for disulfide bridge-mediated functional switch of peptides.

Venoms from scorpions contain extremely rich bioactive peptides that often carry diverse functions and are presumably needed to achieve synergistic effects for rapidly immobilizing prey and defending themselves. BotLVP1 is a unique heterodimer protein recently found in the scorpion Buthus occitanus tunetanus venom that is structurally related to scorpion toxins affecting sodium channels (NaScTxs) but exhibits adipocyte lipolysis activity. We have isolated and identified two cDNA clones encoding subunits alpha and beta of a BotLVP1-like peptide (named BmLVP1) from the Chinese scorpion Buthus martensii venom gland and determined the first complete gene structure of this subfamily. These results highlight a genetic link between these lipolysis activating peptides and NaScTxs. Comparison of cDNA and genomic sequences combined with protein structural and functional analysis provides evidence supporting the existence of RNA editing mechanism in scorpion venom glands, which could mediate functional switch of BmLVP1 gene, from adipocyte lipolysis to neurotoxicity, by altering the wrapper disulfide bridge (WDB) pattern of the peptides.

Evolutionary trace analysis of scorpion toxins specific for K-channels.

Scorpion alpha-K(+) channel toxins are a large family of polypeptides with a similar structure but diverse pharmacological activities. Despite many structural and functional data available at present, little progress has been made in understanding the toxin's molecular basis responsible for the functional diversification. In this paper, we report the first complete cDNA sequences of toxins belonging to subfamily 6 and identify five new members, called alpha-KTx 6.6-6.10. By analyzing the rates of mutations that occurred in the corresponding cDNAs, we suggest that accelerated evolution in toxin-coding regions may be associated with the functional diversification of this subfamily. To pinpoint sites probably involved in the functional diversity of alpha-KTx family, we analyzed this family of sequences using the evolutionary trace method. This analysis highlighted one channel-binding surface common for all the members. This surface is composed of one conserved lysine residue at position 29 assisted by other residues at positions 10, 26, 27, 32, 34, and 36. Of them, the positions 29, 32, and 34 have been reported to be the most major determinants of channel specificity. Interestingly, another contrary surface was also observed at a higher evolutionary time cut-off value, which may be involved in the binding of ERG (ether-a-go-go-related gene) channel-specific toxins. The good match between the trace residues and the functional epitopes of the toxins suggested that the evolutionary trace results reported here can be applied to predict channel-binding sites of the toxins. Because, the side-chain variation in the trace positions is strongly linked with the functional alteration and channel-binding surface transfer of alpha-KTx family, we conclude that our findings should also be important for the rational design of new toxins targeting a given potassium channel with high selectivity.

Genomic organization of three neurotoxins active on small conductance Ca2+-activated potassium channels from the scorpion Buthus martensi Karsch.

According to the known primary sequences of three neurotoxins active on small conductance Ca2+-activated potassium channels from the scorpion Buthus martensi Karsch, their corresponding cDNAs were cloned and sequenced using 3'- and 5'-RACE. All of them encoded a signal peptide composed of 28 residues and a mature toxin of 29, 28 and 33 residues, respectively. Their cDNA deduced sequences were totally consistent with those determined, and the C-terminal amidation of one neurotoxin was confirmed. The genomic DNAs of these three toxins were also amplified by PCR, cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptide at the same -10 position upstream from the mature toxin, consisting of 94, 78 and 87 bp, respectively.

Genomic organization of the KTX2 gene, encoding a 'short' scorpion toxin active on K+ channels.

A single intron of 87 bp, close to the region encoding the C-terminal part of the signal peptide, was found in the gene of the 'short' scorpion toxin kaliotoxin 2 of Androctonus australis acting on various types of K+ channels. Its A+T content was particularly high (up to 86%). By walking and ligation-mediated PCR, the promoter sequences of the kaliotoxin 2 gene of Androctonus australis were studied. The transcription unit of the gene is 390 bp long. Consensus sequences were identified. The genes of 'short' scorpion toxins active on K+ channels are organized similarly to those of the 'long' scorpion toxins active on Na+ channels and not like those of structurally related insect defensins, which are intronless.