Identification and Phylogenetic Analysis of Tityus pachyurus and Tityus obscurus Novel Putative Na+-Channel Scorpion Toxins

Background: Colombia and Brazil are affected by severe cases of scorpionism. In Colombia the most dangerous accidents are caused by Tityus pachyurus that is widely distributed around this country. In the Brazilian Amazonian region scorpion stings are a common event caused by Tityus obscurus. The main objective of this work was to perform the molecular cloning of the putative Na+-channel scorpion toxins (NaScTxs) from T. pachyurus and T. obscurus venom glands and to analyze their phylogenetic relationship with other known NaScTxs from Tityus species. Methodology/Principal Findings: cDNA libraries from venom glands of these two species were constructed and five nucleotide sequences from T. pachyurus were identified as putative modulators of Na+-channels, and were named Tpa4, Tpa5, Tpa6, Tpa7 and Tpa8; the latter being the first anti-insect excitatory b-class NaScTx in Tityus scorpion venom to be described. Fifteen sequences from T. obscurus were identified as putative NaScTxs, among which three had been previously described, and the others were named To4 to To15. The peptides Tpa4, Tpa5, Tpa6, To6, To7, To9, To10 and To14 are closely related to the a-class NaScTxs, whereas Tpa7, Tpa8, To4, To8, To12 and To15 sequences are more related to the b-class NaScTxs. To5 is possibly an arthropod specific toxin. To11 and To13 share sequence similarities with both a and b NaScTxs. By means of phylogenetic analysis using the Maximum Parsimony method and the known NaScTxs from Tityus species, these toxins were clustered into 14 distinct groups. Conclusions/Significance: This communication describes new putative NaScTxs from T. pachyurus and T. obscurus and their phylogenetic analysis. The results indicate clear geographic separation between scorpions of Tityus genus inhabiting the Amazonian and Mountain Andes regions and those distributed over the Southern of the Amazonian rainforest. Based on the consensus sequences for the different clusters, a new nomenclature for the NaScTxs is proposed

Purification and molecular mass determination of Tf2.

<p>(A) Chromatography by RP-HPLC of 1 mg of <i>T</i>. <i>fasciolatus</i> crude venom. Fractionation was performed on a C18 analytical column, using a linear gradient from 0% solvent A (0.12% TFA in water) to 60% solvent B (0.10% TFA in acetonitrile) over 60 min at a flow rate of 1 mL/min, with detection at a wavelength of 216 and 280nm. The component eluted at 38.5% of acetonitrile corresponds to Tf2. (B-D) Three additional chromatographic protocols performed to obtain pure Tf2. (B) Linear gradient of B solvent, from 25 to 45% B in 40 minutes, at room temperature (22°C). (C) Linear gradient of B solvent, from 25 to 45% of B in 40 minutes, at 45°C. (D) Linear gradient of B solvent, from 30 to 40% of B in 40 minutes, at 45°C. Inset on (A) shows mass spectrometry analysis of Tf2 by micrOTOF-Q II, presenting the monoisotopic distribution of the +7 charged ion ([M+7H]⁷⁺ = 993.7050), which is equivalent to [M+H]⁺ 6949.9350 Da.</p

Structural comparison between Tf2 and other Na_v channel toxins.

<p>(A) Structural alignment between Tf2 (in blue) and five other Na_v channel scorpion toxins—Ts1, Ts2, CssII, CssIV, and AaHII (in gray). (B) Comparison of electrostatic potentials between the toxins Tf2, Ts1, Ts2, CssII, CssIV, and AaHII. The figure shows charge distribution along the toxin surface, divided into faces A and B. Shown in red are acidic residues whereas blue represents basic residues; in white, neutral regions are shown.</p

Sequence alignment of the domain II paddle motif in 8 mammalian Na_v channel isoforms.

<p>Figure shows a sequence alignment of the domain II paddle motif as found in 8 mammalian Na_v channel isoforms. As a reference, the number in italic indicates the coordinates of the first Gly residue in hNa_v1.1. Although the Ile in hNa_v1.3 (position 830 according to hNa_v1.3 coordinates, 840 according to hNa_v1.1 coordinates) differs from the Phe found in other neuronal isoforms, this residue is not present within the paddle motif and may not be accessible to Tf2. The Ser at position 842 (hNa_v1.3 coordinates—indicated in red) is unique among hNa_v1.1–1.3.</p

Tf2 sequence and alignment with scorpion Na_v channel toxins.

<p>(A) The nucleotide sequence of Tf2 was obtained by HiSeq (Illumina, USA). Signal peptide is underlined, mature peptide is highlighted in gray, and the amidation set point is marked in italic. (B) Multiple sequence alignment of Tf2 with other Na_v channel toxins. Toxins are presented by their short names and UniProt KB codes. Capital letters denote amino acids. Lower-case letters denote: h, hydrophobic; s, small; b, big; p, polar; t, tiny; a, aromatic; l, aliphatic. Positive (+) and negative (-) amino acid residues that are part of the consensus sequence are also colored. Cys residues are shaded in black. aa means amino acid residues, and %Id is the percentage of sequence identity with Tf2.</p

Toxins from <i>T. pachyurus</i> and <i>T. obscurus</i> and their average molecular masses and retention times (RT).

<p>Swiss-Prot accession numbers are present when available.</p

Consensus sequences for the 14 proposed subfamilies of NaTxs from scorpions of the genus <i>Tityus</i>.

<p>Amino acid sequences were obtained by classical proteomic approach and/or predicted by cDNA library constructions from scorpion venom glands. Acidic and basic residues are shown in red and blue, respectively. Cys residues are shown in bold.</p

High performance liquid chromatography separation of 1.0 mg soluble venom from <i>T. obscurus</i>.

<p>This was performed in an analytical C18 reversed phase column equilibrated with solution A (water in 0.1% TFA), using a gradient from 0 to 60% solution B (acetonitrile in 0.12% TFA) over 60 min, with a flow rate of 1 mL/min and absorbance at 216 nm. Fractions labeled in the chromatogram (Tc49b to To7) were detected at both protein and transcript levels.</p

Phylogenetic analysis of NaScTxs from <i>T. pachyurus</i>, <i>T. obscurus</i> and other scorpions from <i>Tityus</i> genus.

<p>Filled symbols indicate toxins from scorpions inhabiting the Northern region of the Amazon Basin: square for <i>T. pachyurus</i>, circle for <i>T. obscurus</i>, triangle for <i>T. discrepans</i>, and inverted triangle for <i>T. zulianus</i> toxins. Open symbols indicate toxins from scorpions inhabiting the Southern part of the Amazon Basin: square for <i>T. bahiensis</i>, triangle for <i>T. stigmurus</i>, inverted triangle for <i>T.costatus</i>, circle for <i>T. serrulatus</i>, and rhombus for <i>T. fasciolatus</i> toxins.</p

Oxidative and biochemical responses in Brycon amazonicus anesthetized and sedated with Myrcia sylvatica (G. Mey.) DC. and Curcuma longa L. essential oils

Objective: To investigate the effects of rapid anesthesia and long-term sedation with the essential oils (EOs) of Myrcia sylvatica (EOMS) and Curcuma longa (EOCL) on biochemical and oxidative parameters in matrinxã. Study design: Prospective, randomized, laboratory experiment. Animals: A total of 72 matrinxã (Brycon amazonicus) adults weighing 404.8 ± 27.9 g were divided into eight groups of nine fish. Methods: Biochemical and oxidative effects were investigated in plasma and tissues of matrinxã subjected to rapid anesthesia (5 minutes) or long-term sedation (360 minutes, simulating the practice of transport) with EOMS (200 μL L−1 and 10 μL L−1, respectively) and EOCL (500 μL L−1 and 40 μL L−1, respectively). Results: Transport simulation without sedation or anesthesia increased lipid peroxidation levels in the gills and kidney of fish in the control group. Anesthesia and sedation with EOs decreased cortisol concentrations and increased lactate concentrations compared with controls. Lipid peroxidation was lower in the brain, gills, liver and kidney of sedated and anesthetized fish, than in the control group. Anesthesia with EOs increased the activity of superoxide dismutase and glutathione-S-transferase in the brain, and catalase in the liver and gills, compared with controls. Long-term sedation with EOs increased superoxide dismutase, glutathione peroxidase and glutathione reductase activities in the brain, catalase in the liver, glutathione peroxidase and glutathione reductase in the gills and superoxide dismutase in the kidney. In general, nonprotein thiols content and total reactive antioxidant potential of tissues were higher after anesthesia and sedation with EOs compared with the control group. Conclusions and clinical relevance: The concentrations of EOMS and EOCL used were effective at preventing a stress response and excess of reactive oxygen species formation. For these reasons, these substances may be recommended for use in the transportation of fish to improve survival and animal welfare.Fil: Saccol, Etiane M. H.. Universidad Federal de Santa Maria; BrasilFil: Londero, Érika P.. Universidad Federal de Santa Maria; BrasilFil: Bressan, Caroline A.. Universidad Federal de Santa Maria; BrasilFil: Salbego, Joseânia. Universidad Federal de Santa Maria; BrasilFil: Gressler, Luciane T.. Universidad Federal de Santa Maria; BrasilFil: Silva, Lenise V. F.. Universidade Federal do Pará; BrasilFil: Mourão, Rosa H. V.. Universidade Federal do Pará; BrasilFil: Oliveira, Ricardo B.. Universidad Federal de Santa Maria; BrasilFil: Llesuy, Susana Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Baldisserotto, Bernardo. Universidad Federal de Santa Maria; BrasilFil: Pavanato, Maria A.. Universidad Federal de Santa Maria; Brasi