Toxins

2020

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25 pages, 3265 KiB

Open AccessArticle

Size Matters: An Evaluation of the Molecular Basis of Ontogenetic Modifications in the Composition of Bothrops jararacussu Snake Venom

by Luciana A. Freitas-de-Sousa, Pedro G. Nachtigall, José A. Portes-Junior, Matthew L. Holding, Gunnar S. Nystrom, Schyler A. Ellsworth, Noranathan C. Guimarães, Emilly Tioyama, Flora Ortiz, Bruno R. Silva, Tobias S. Kunz, Inácio L. M. Junqueira-de-Azevedo, Felipe G. Grazziotin, Darin R. Rokyta and Ana M. Moura-da-Silva

Toxins 2020, 12(12), 791; https://doi.org/10.3390/toxins12120791 - 11 Dec 2020

Cited by 24 | Viewed by 4963

Abstract

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom [...] Read more.

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A₂ (PLA₂s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes. Full article

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Figure 1

Figure 1
Distribution of the toxin families in the venom of 19 specimens of Bothrops jararacussu identified from proteome analysis. Relative expression is indicated by the normalized Total Spectrum Count (TSC) in each snake. CTL (C-Type Lectin), PLA2 (Phospholipase A2), SVMP classes PI, PII, and PIII (Snake Venom Metalloproteinase), SVSP (Snake Venom Serine Protease), VEGF (Vascular Endothelial Growth Factor), LAAO (L-amino Acid Oxidase), CRISP (Cysteine-rich secretory protein), HYAL (Hyaluronidase), VNGF (Venom Nerve Growth Factor), SVNUC (Snake Venom Nucleotidase), PDE (Venom Phosphodiesterase), PLB (Phospholipase B) and GLUT (Snake Venom Glutaminyl Cyclase). The higher the snake number, the greater the size of the snake. Full article ">Figure 2
Correlation of the main toxin families of Bothrops jararacussu venom with: (A) the individuals’ snout-vent length (SVL), (B) reproductive status, sex, and place of collection. Relative expression is indicated by the normalized Total Spectrum Count (TSC) in each snake. CTL (C-Type Lectin), PLA2 (Phospholipase A2), SVMP classes PI, PII and PIII (Snake Venom Metalloproteinase), SVSP (Snake Venom Serine Protease), LAAO (L-amino Acid Oxidase), CRISP (Cysteine-rich secretory protein). * p < 0.05. Full article ">Figure 3
Distribution of PLA2 isoforms present in the venoms of 19 individuals from B. jararacussu and functional inferences. Mature deduced protein sequences of PLA2 were aligned together with sequences of PLA2s isolated from other venoms with functions well-established by previous studies using experimental approaches, as follows: a non-toxic phospholipase A2 from the venom of Notechis scutatus scutatus; Bothropstoxin-1 (Q90249.3), Bothropstoxin-2 (P45881) and BthAI (Q8AXY1.1), B. jararacussu venom; acidic PLA2 (Q2HZ28.1), B. erythromelas venom. Notechis PLA2 (P08873.1) sequence was used to root the tree. The maximum likelihood phylogenetic tree was generated using RaxML (v8.2.12; Stamatakis 2014). The heatmap on the right indicates the number of normalized Exclusive Unique Spectrum Counts (EUSC) of MS/MS in each venom sample, which are as indicated at the top of the figure. Bootstrap values are described only when greater than 75. Full article ">Figure 4
Distribution of SVMPIII isoforms present in the venoms of 19 individuals from B. jararacussu and functional inferences. Mature deduced protein sequences of SVMPIII were aligned together with sequences of SVMPs isolated from other venoms with functions well-established by previous studies using experimental approaches, as follows: BjussuMP-1P (Q1PHZ4; from B. jararacussu venom; Batroxrhagin, Bothrops atrox venom (ALB00542.1); hemorrhagic factor 3 (HF3), B. jararaca venom (Q98UF9.3); vascular apoptosis-inducing protein 1 (VAP-1), Crotalus atrox venom (Q9DGB9.1); berythractivase, B. erythromelas venom (Q8UVG0.1); VaF1, Vipera ammodytes ammodytes venom (AJC52543). The disintegrin and metalloproteinase domains of Mus musculus ADAM28 (NP034212.1) was used to root the tree. The maximum likelihood phylogenetic tree was generated using RaxML (v8.2.12; Stamatakis 2014). The heatmap on the right indicates the number of normalized Exclusive Unique Spectrum Counts (EUSC) of MS/MS in each venom sample, which are as indicated at the top of the figure. Bootstrap values are described only when greater than 75. Full article ">Figure 5
Comparisons of gene transcription (TMM normalized) rates in the venom gland and protein abundance (normalized EUSC—Exclusive Unique Spectrum Count) in the venom of individual B. jararacussu snakes. Each spot represents detected transcription and translation levels for a specific toxin family from one specimen. Dotted lines indicate a hypothetical correspondence between transcript and protein abundances. Full article ">Figure 6
Comparison between levels of transcripts (TMM normalized) and protein abundance (normalized EUSC—Exclusive Unique Spectrum Count) of the isoforms PLA002, SVMPIII002, and SVMPIII009 in the venoms of B. jararacussu snakes. Dotted lines indicate a hypothetical correspondence between transcript and protein abundances. Full article ">Figure 7
Functional activities of Bothrops jararacussu venom samples. (A)—Metalloproteinase activity (SVMP) was measured by fluorometric assays using the substrate Abz-AGLA-EDDnp. (B)—Phospholipase A2 (PLA2) activity was tested using the 4-nitro-3- [octanoyloxy] benzoic acid substrate. (C)—Procoagulant activity was evaluated by the amount of venom (µg) capable of inducing the coagulation of human citrated plasma in up to 60 s and is represented by the reciprocal Minimum Coagulant Dose (1/MCD). (D)—For hemorrhagic activity, 10 µg of each pool of venoms were injected in the dorsum of mice, and the hemorrhagic lesions were measured three hours after injection. (E)—The myotoxic activity was assayed using 50 µg of pools of venoms injected intramuscularly into the gastrocnemius muscle in Swiss mice, and after 3 h, the sera were assayed for creatine–kinase activity. (F)—For the evaluation of lethal activity, samples containing 200 μg of each venom in a final volume of 200 μL were injected intraperitoneally into groups of four mice. Phosphate-buffered saline (PBS)—was the control in all in vivo experiments. The results are representative of two independent experiments. * p < 0.05. Full article ">

2018

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2 pages, 216 KiB

Open AccessAddendum

Addendum: Aird, S.D. et al. Coralsnake Venomics: Analyses of Venom Gland Transcriptomes and Proteomes of Six Brazilian Taxa. Toxins 2017, 9(6), 187

by Steven D. Aird, Nelson Jorge Da Silva, Lijun Qiu, Alejandro Villar-Briones, Vera Aparecida Saddi, Mariana Pires de Campos Telles, Miguel L. Grau and Alexander S. Mikheyev

Toxins 2018, 10(5), 172; https://doi.org/10.3390/toxins10050172 - 24 Apr 2018

Viewed by 3744

Abstract

Following publication of this paper, Dr. Daniel Dashevsky discovered to our chagrin, that the transcriptomic datasets uploaded to the DNA Databank of Japan (DDBJ) contained numerous complete 3FTx sequences that were not included in our paper.[...] Full article

2017

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14820 KiB

Open AccessArticle

Enter the Dragon: The Dynamic and Multifunctional Evolution of Anguimorpha Lizard Venoms

by Ivan Koludarov, Timothy NW Jackson, Bianca op den Brouw, James Dobson, Daniel Dashevsky, Kevin Arbuckle, Christofer J. Clemente, Edward J. Stockdale, Chip Cochran, Jordan Debono, Carson Stephens, Nadya Panagides, Bin Li, Mary-Louise Roy Manchadi, Aude Violette, Rudy Fourmy, Iwan Hendrikx, Amanda Nouwens, Judith Clements, Paolo Martelli, Hang Fai Kwok and Bryan G. Fry Show full author list Hide full author list

Toxins 2017, 9(8), 242; https://doi.org/10.3390/toxins9080242 - 6 Aug 2017

Cited by 38 | Viewed by 31895

Abstract

While snake venoms have been the subject of intense study, comparatively little work has been done on lizard venoms. In this study, we have examined the structural and functional diversification of anguimorph lizard venoms and associated toxins, and related these results to dentition [...] Read more.

While snake venoms have been the subject of intense study, comparatively little work has been done on lizard venoms. In this study, we have examined the structural and functional diversification of anguimorph lizard venoms and associated toxins, and related these results to dentition and predatory ecology. Venom composition was shown to be highly variable across the 20 species of Heloderma, Lanthanotus, and Varanus included in our study. While kallikrein enzymes were ubiquitous, they were also a particularly multifunctional toxin type, with differential activities on enzyme substrates and also ability to degrade alpha or beta chains of fibrinogen that reflects structural variability. Examination of other toxin types also revealed similar variability in their presence and activity levels. The high level of venom chemistry variation in varanid lizards compared to that of helodermatid lizards suggests that venom may be subject to different selection pressures in these two families. These results not only contribute to our understanding of venom evolution but also reveal anguimorph lizard venoms to be rich sources of novel bioactive molecules with potential as drug design and development lead compounds. Full article

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19648 KiB

Open AccessArticle

Coralsnake Venomics: Analyses of Venom Gland Transcriptomes and Proteomes of Six Brazilian Taxa

by Steven D. Aird, Nelson Jorge Da Silva, Lijun Qiu, Alejandro Villar-Briones, Vera Aparecida Saddi, Mariana Pires de Campos Telles, Miguel L. Grau and Alexander S. Mikheyev

Toxins 2017, 9(6), 187; https://doi.org/10.3390/toxins9060187 - 8 Jun 2017

Cited by 69 | Viewed by 9880

Abstract

Venom gland transcriptomes and proteomes of six Micrurus taxa (M. corallinus, M. lemniscatus carvalhoi, M. lemniscatus lemniscatus, M. paraensis, M. spixii spixii, and M. surinamensis) were investigated, providing the most comprehensive, quantitative data on Micrurus venom [...] Read more.

Venom gland transcriptomes and proteomes of six Micrurus taxa (M. corallinus, M. lemniscatus carvalhoi, M. lemniscatus lemniscatus, M. paraensis, M. spixii spixii, and M. surinamensis) were investigated, providing the most comprehensive, quantitative data on Micrurus venom composition to date, and more than tripling the number of Micrurus venom protein sequences previously available. The six venomes differ dramatically. All are dominated by 2–6 toxin classes that account for 91–99% of the toxin transcripts. The M. s. spixii venome is compositionally the simplest. In it, three-finger toxins (3FTxs) and phospholipases A₂ (PLA₂s) comprise >99% of the toxin transcripts, which include only four additional toxin families at levels ≥0.1%. Micrurus l. lemniscatus venom is the most complex, with at least 17 toxin families. However, in each venome, multiple structural subclasses of 3FTXs and PLA₂s are present. These almost certainly differ in pharmacology as well. All venoms also contain phospholipase B and vascular endothelial growth factors. Minor components (0.1–2.0%) are found in all venoms except that of M. s. spixii. Other toxin families are present in all six venoms at trace levels (<0.005%). Minor and trace venom components differ in each venom. Numerous novel toxin chemistries include 3FTxs with previously unknown 8- and 10-cysteine arrangements, resulting in new 3D structures and target specificities. 9-cysteine toxins raise the possibility of covalent, homodimeric 3FTxs or heterodimeric toxins with unknown pharmacologies. Probable muscarinic sequences may be reptile-specific homologs that promote hypotension via vascular mAChRs. The first complete sequences are presented for 3FTxs putatively responsible for liberating glutamate from rat brain synaptosomes. Micrurus C-type lectin-like proteins may have 6–9 cysteine residues and may be monomers, or homo- or heterodimers of unknown pharmacology. Novel KSPIs, 3× longer than any seen previously, appear to have arisen in three species by gene duplication and fusion. Four species have transcripts homologous to the nociceptive toxin, (MitTx) α-subunit, but all six species had homologs to the β-subunit. The first non-neurotoxic, non-catalytic elapid phospholipase A₂s are reported. All are probably myonecrotic. Phylogenetic analysis indicates that the six taxa diverged 15–35 million years ago and that they split from their last common ancestor with Old World elapines nearly 55 million years ago. Given their early diversification, many cryptic micrurine taxa are anticipated. Full article

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Graphical abstract

2250 KiB

Open AccessArticle

Venom Profiling of a Population of the Theraphosid Spider Phlogius crassipes Reveals Continuous Ontogenetic Changes from Juveniles through Adulthood

by Renan C. Santana, David Perez, James Dobson, Nadya Panagides, Robert J. Raven, Amanda Nouwens, Alun Jones, Glenn F. King and Bryan G. Fry

Toxins 2017, 9(4), 116; https://doi.org/10.3390/toxins9040116 - 25 Mar 2017

Cited by 20 | Viewed by 8486

Abstract

Theraphosid spiders (tarantulas) are venomous arthropods found in most tropical and subtropical regions of the world. Tarantula venoms are a complex cocktail of toxins with potential use as pharmacological tools, drugs and bioinsecticides. Although numerous toxins have been isolated from tarantula venoms, little [...] Read more.

Theraphosid spiders (tarantulas) are venomous arthropods found in most tropical and subtropical regions of the world. Tarantula venoms are a complex cocktail of toxins with potential use as pharmacological tools, drugs and bioinsecticides. Although numerous toxins have been isolated from tarantula venoms, little research has been carried out on the venom of Australian tarantulas. We therefore investigated the venom profile of the Australian theraphosid spider Phlogius crassipes and examined whether there are ontogenetic changes in venom composition. Spiders were divided into four ontogenic groups according to cephalothorax length, then the venom composition of each group was examined using gel electrophoresis and mass spectrometry. We found that the venom of P. crassipes changes continuously during development and throughout adulthood. Our data highlight the need to investigate the venom of organisms over the course of their lives to uncover and understand the changing functions of venom and the full range of toxins expressed. This in turn should lead to a deeper understanding of the organism’s ecology and enhance the potential for biodiscovery. Full article

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Figure 1

Figure 1
Combined liquid chromatography (LC) chromatograms of specimens from the (a) OXS – Olkola extra small group, (b) OS – Olkola small group, (c) OM – Olkola medium group, and (d) OL – Olkola large group. Each colour represent a different specimen. Vertical axis is Intensity and horizontal axis is Time (minutes). Full article ">Figure 1 Cont.
Combined liquid chromatography (LC) chromatograms of specimens from the (a) OXS – Olkola extra small group, (b) OS – Olkola small group, (c) OM – Olkola medium group, and (d) OL – Olkola large group. Each colour represent a different specimen. Vertical axis is Intensity and horizontal axis is Time (minutes). Full article ">Figure 1 Cont.
Combined liquid chromatography (LC) chromatograms of specimens from the (a) OXS – Olkola extra small group, (b) OS – Olkola small group, (c) OM – Olkola medium group, and (d) OL – Olkola large group. Each colour represent a different specimen. Vertical axis is Intensity and horizontal axis is Time (minutes). Full article ">Figure 2
Principal component analysis and discriminant analysis (PCA-DA) analysis of Phlogius crassipes population from Olkola Aboriginal land. Tarantulas are grouped according to cephalothorax size. Each dot represents a specimen. Full article ">Figure 3
Left. 1D SDS PAGE gel of representatives from a population of Phlogius crassipes. Left lane is molecular marker (protein ladder) followed by OL, OM, OS and OXS specimens, respectively. Right. Clustering analyses with Rho similarity and 10,000 bootstrap values from one-dimensional gel electrophoresis of P. crassipes individuals of four different sizes. Full article ">Figure 4
Simple Correspondence Analysis of representatives of group size from a population of P. crassipes. Each number corresponds to a single venom compound identified by Protein Pilot. Axes correspond to the two dimensions created by the analysis. Full article ">

2016

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1760 KiB

Open AccessArticle

Exon Shuffling and Origin of Scorpion Venom Biodiversity

by Xueli Wang, Bin Gao and Shunyi Zhu

Toxins 2017, 9(1), 10; https://doi.org/10.3390/toxins9010010 - 26 Dec 2016

Cited by 15 | Viewed by 7911

Abstract

Scorpion venom is a complex combinatorial library of peptides and proteins with multiple biological functions. A combination of transcriptomic and proteomic techniques has revealed its enormous molecular diversity, as identified by the presence of a large number of ion channel-targeted neurotoxins with different [...] Read more.

Scorpion venom is a complex combinatorial library of peptides and proteins with multiple biological functions. A combination of transcriptomic and proteomic techniques has revealed its enormous molecular diversity, as identified by the presence of a large number of ion channel-targeted neurotoxins with different folds, membrane-active antimicrobial peptides, proteases, and protease inhibitors. Although the biodiversity of scorpion venom has long been known, how it arises remains unsolved. In this work, we analyzed the exon-intron structures of an array of scorpion venom protein-encoding genes and unexpectedly found that nearly all of these genes possess a phase-1 intron (one intron located between the first and second nucleotides of a codon) near the cleavage site of a signal sequence despite their mature peptides remarkably differ. This observation matches a theory of exon shuffling in the origin of new genes and suggests that recruitment of different folds into scorpion venom might be achieved via shuffling between body protein-coding genes and ancestral venom gland-specific genes that presumably contributed tissue-specific regulatory elements and secretory signal sequences. Full article

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Graphical abstract

Graphical abstract
Full article ">Figure 1
The fold diversity of scorpion venom components. (A) Representative structure of three different types of peptides: MMTX (PDB: 2RTZ) (CSαβ fold), λ-MeuTx-1 (ICK fold) [<a href="#B7-toxins-09-00010" class="html-bibr">7</a>], and the α-helical Meucin-24 (PDB: 2KFE); (B) Representative structures of scorpion venom-derived proteases and protease inhibitors: The chymotrypsin-like protease MmChTP whose structure was modelled on SWISS-MODEL (<a href="http://www.expasy.org" target="_blank">www.expasy.org</a>) using the template of a mannose-binding lectin-associated serine proteinase-3 (PDB: 4KKD); the Kunitz-type protease inhibitor LmKTT-1a (PDB: 2M01). Full article ">Figure 2
Representative gene structures of scorpion venom-derived neurotoxins and antimicrobial peptides. UTR, untranslated region; SP, signal peptide; MP, mature peptide; PP, propeptide. Functional classes: KCT, potassium channel toxin; SCT, sodium channel toxin; CCT, chloride channel toxin; DFN, defensin; RyR, ryanodine receptor; AMP, antimicrobial peptide. MM, Mesobuthus martensii; ME, M. eupeus; CE, Centruroides exilicauda; and OC, Opistophthalmus carinatus. Full article ">Figure 3
Representative gene structures of scorpion venom-derived proteases (MmChTP and CsEChTP) and protease inhibitors (MmPI-1, MmPI-2a, MmPI-2b, and CsEPI-1). MM, M. martensii; CE, C. exilicauda. Full article ">Figure 4
The hypothetical evolutionary model for the origin of scorpion venom biodiversity. Exon shuffling is proposed as a major evolutionary mechanism mediating the origin of venom proteins from ancestral body proteins, in which a venom gland-specific ancestral gene is considered as a donor providing two necessary elements for venom gland-specific expression: a promoter and a secretory signal. Full article ">

3135 KiB

Open AccessArticle

Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones

by Jason Macrander and Marymegan Daly

Toxins 2016, 8(12), 368; https://doi.org/10.3390/toxins8120368 - 8 Dec 2016

Cited by 35 | Viewed by 6775

Abstract

Sea anemones (Cnidaria, Anthozoa, and Actiniaria) use toxic peptides to incapacitate and immobilize prey and to deter potential predators. Their toxin arsenal is complex, targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Among these, actinoporins are one [...] Read more.

Sea anemones (Cnidaria, Anthozoa, and Actiniaria) use toxic peptides to incapacitate and immobilize prey and to deter potential predators. Their toxin arsenal is complex, targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Among these, actinoporins are one of the better characterized toxins; these venom proteins form a pore in cellular membranes containing sphingomyelin. We used a combined bioinformatic and phylogenetic approach to investigate how actinoporins have evolved across three superfamilies of sea anemones (Actinioidea, Metridioidea, and Actinostoloidea). Our analysis identified 90 candidate actinoporins across 20 species. We also found clusters of six actinoporin-like genes in five species of sea anemone (Nematostella vectensis, Stomphia coccinea, Epiactis japonica, Heteractis crispa, and Diadumene leucolena); these actinoporin-like sequences resembled actinoporins but have a higher sequence similarity with toxins from fungi, cone snails, and Hydra. Comparative analysis of the candidate actinoporins highlighted variable and conserved regions within actinoporins that may pertain to functional variation. Although multiple residues are involved in initiating sphingomyelin recognition and membrane binding, there is a high rate of replacement for a specific tryptophan with leucine (W112L) and other hydrophobic residues. Residues thought to be involved with oligomerization were variable, while those forming the phosphocholine (POC) binding site and the N-terminal region involved with cell membrane penetration were highly conserved. Full article

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Figure 1

Figure 1
(A) Maximum Likelihood tree of actinoporins and actinoporin-like proteins produced in FastTree2. Numbers on branches represent bootstrap values of 1000 replicates. Bootstrapping values greater than 50 are shown at the nodes. Branch labels of clustered gene groups represent lineage common names followed by percent identity (identical amino acid residues) within gene cluster and percent identity when compared to actinoporins in bold. Labeled individual branches show GenBank accession followed by species, and protein name (if applicable). Labels denoted with PREDICTED or GENOME indicate that they were derived bioinformatically in GenBank and are not validated proteins. Branch labels with Genbank accession and species for sea anemones are indicated in bold. The colored box indicates which actinoporin sequences were used in subsequent analyses; (B) Phylogenetic tree with branch colors depicting superfamily associations (Blue: Edwardsioidea, Yellow: Actinostoloidea, Red: Actinioidea, Green: Metridioidea) [<a href="#B42-toxins-08-00368" class="html-bibr">42</a>]. Full article ">Figure 2
(A) Functionally important residues identified on EqII [<a href="#B43-toxins-08-00368" class="html-bibr">43</a>]. Functional sites are as follows: (B) site of bend when N-terminus comes into contact with the cell membrane, (POC) residues involved with the POC binding site, (O) residues involved with oligomerization, (S) key sphingomyelin binding site. Colors are used to aid in determining the orientation between the two views shown; (B) Characterization of amino acid variation for the different gene clusters (Clade 1, Clade 2M, Clade 2A) identified in our analysis. SeqLogo graphs for residues that have been identified previously as functionally important above the alignment with the size of each amino acid residue representing the frequency in which these residues occurred in the alignment. Numbers along the bottom correspond to positions of specific amino acid residues in EqII; (C) Maximum Likelihood actinoporin gene tree produced in FastTree2. Colored branches depict superfamily associations (see <a href="#toxins-08-00368-f001" class="html-fig">Figure 1</a>, Yellow: Actinostoloidea, Red: Actinioidea, Green: Metridioidea). Bootstrapping values greater than 50 are shown at the nodes. Branch labels include GenBank ID (when applicable) and the species from which the toxin gene was derived. Bold labels indicate that the mature protein sequence was recovered. Sequences derived from genomic data are indicated with “G” following species in sequence IDs. The superfamily association for Actineria villosa may be incorrect and is noted with an asterisk. Full article ">Figure 3
Edmundson wheel projections were determined in HeliQuest. Associated Sequence IDs are shown below the species name. Full article ">Figure 3 Cont.
Edmundson wheel projections were determined in HeliQuest. Associated Sequence IDs are shown below the species name. Full article ">

1044 KiB

Open AccessArticle

A Tricky Trait: Applying the Fruits of the “Function Debate” in the Philosophy of Biology to the “Venom Debate” in the Science of Toxinology

by Timothy N. W. Jackson and Bryan G. Fry

Toxins 2016, 8(9), 263; https://doi.org/10.3390/toxins8090263 - 7 Sep 2016

Cited by 30 | Viewed by 7811

Abstract

The “function debate” in the philosophy of biology and the “venom debate” in the science of toxinology are conceptually related. Venom systems are complex multifunctional traits that have evolved independently numerous times throughout the animal kingdom. No single concept of function, amongst those [...] Read more.

The “function debate” in the philosophy of biology and the “venom debate” in the science of toxinology are conceptually related. Venom systems are complex multifunctional traits that have evolved independently numerous times throughout the animal kingdom. No single concept of function, amongst those popularly defended, appears adequate to describe these systems in all their evolutionary contexts and extant variations. As such, a pluralistic view of function, previously defended by some philosophers of biology, is most appropriate. Venom systems, like many other functional traits, exist in nature as points on a continuum and the boundaries between “venomous” and “non-venomous” species may not always be clearly defined. This paper includes a brief overview of the concept of function, followed by in-depth discussion of its application to venom systems. A sound understanding of function may aid in moving the venom debate forward. Similarly, consideration of a complex functional trait such as venom may be of interest to philosophers of biology. Full article

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2305 KiB

Open AccessArticle

Canopy Venom: Proteomic Comparison among New World Arboreal Pit-Viper Venoms

by Jordan Debono, Chip Cochran, Sanjaya Kuruppu, Amanda Nouwens, Niwanthi W. Rajapakse, Minami Kawasaki, Kelly Wood, James Dobson, Kate Baumann, Mahdokht Jouiaei, Timothy N. W. Jackson, Ivan Koludarov, Dolyce Low, Syed A. Ali, A. Ian Smith, Andrew Barnes and Bryan G. Fry

Toxins 2016, 8(7), 210; https://doi.org/10.3390/toxins8070210 - 8 Jul 2016

Cited by 7 | Viewed by 7099

Abstract

Central and South American pitvipers, belonging to the genera Bothrops and Bothriechis, have independently evolved arboreal tendencies. Little is known regarding the composition and activity of their venoms. In order to close this knowledge gap, venom proteomics and toxin activity of species [...] Read more.

Central and South American pitvipers, belonging to the genera Bothrops and Bothriechis, have independently evolved arboreal tendencies. Little is known regarding the composition and activity of their venoms. In order to close this knowledge gap, venom proteomics and toxin activity of species of Bothriechis, and Bothrops (including Bothriopsis) were investigated through established analytical methods. A combination of proteomics and bioactivity techniques was used to demonstrate a similar diversification of venom composition between large and small species within Bothriechis and Bothriopsis. Increasing our understanding of the evolution of complex venom cocktails may facilitate future biodiscoveries. Full article

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Figure 1
Taxonomical relationships of American pitvipers, based on Castoe 2006 [<a href="#B65-toxins-08-00210" class="html-bibr">65</a>]. Full article ">Figure 2
1D SDS page. MW = molecular weight marker; aur = Bothriechis aurifer; lat = Bothriechis lateralis; mar = Bothriechis marchi; sch = Bothriechis schlegelli; bil = Bothrops bilineata; tan = Bothrops taeniata; asp = Bothrops asper; neu = Bothrops neuwiedi bolivianus. Annotation indicates the dominant type in a region. However, other toxin types may also be present (see <a href="#app1-toxins-08-00210" class="html-app">Supplementary File 1</a> for full annotation). Full article ">Figure 3
2D SDS page analysis of (A) Bothriechis aurifer; (B) Bothriechis lateralis; (C) Bothriechis marchi; (D) Bothriechis schlegelli; (E) Bothrops bilineata; (F) Bothrops taeniata; (G) Bothrops asper and (H) Bothrops neuwiedi bolivianus. pI range is 3–10 (left to right) and molecular weight markers are as for <a href="#toxins-08-00210-f002" class="html-fig">Figure 2</a>. Full article ">Figure 4
Metalloprotease activity of venom—Metalloprotease activity of venom (10 ng/µL) was measured based on its ability to cleave a fluorogenic peptide substrate (Mca-PLGL-Dpa-AR-NH2, 10 µM final). aur = Bothriechis aurifer; lat = Bothriechis lateralis; mar = Bothriechis marchi; sch = Bothriechis schlegelii; bil = Bothrops bilineata; tan = Bothrops taeniata; asp = Bothrops asper; neu = Bothrops neuwiedi bolivianus. Full article ">Figure 5
Crude venom zymography gel analysis using casein (A) and gelatin (B) as protein substrates. MW = molecular weight marker; aur = Bothriechis aurifer; lat = Bothriechis lateralis; mar = Bothriechis marchi; sch = Bothriechis schlegelii; bil = Bothrops bilineata; tan = Bothrops taeniata; asp = Bothrops asper; neu = Bothrops neuwiedi bolivianus. Full article ">Figure 6
Phospholipase A2 enzymatic activity profiling measured by means of absorbance over time at 1 µg for (A) Bothriechis species; (B) arboreal Bothrops species and (C) terrestrial Bothrops species. Full article ">

223 KiB

Open AccessArticle

Tempo and Mode of the Evolution of Venom and Poison in Tetrapods

by Richard J. Harris and Kevin Arbuckle

Toxins 2016, 8(7), 193; https://doi.org/10.3390/toxins8070193 - 23 Jun 2016

Cited by 24 | Viewed by 7775

Abstract

Toxic weaponry in the form of venom and poison has evolved in most groups of animals, including all four major lineages of tetrapods. Moreover, the evolution of such traits has been linked to several key aspects of the biology of toxic animals including [...] Read more.

Toxic weaponry in the form of venom and poison has evolved in most groups of animals, including all four major lineages of tetrapods. Moreover, the evolution of such traits has been linked to several key aspects of the biology of toxic animals including life-history and diversification. Despite this, attempts to investigate the macroevolutionary patterns underlying such weaponry are lacking. In this study we analyse patterns of venom and poison evolution across reptiles, amphibians, mammals, and birds using a suite of phylogenetic comparative methods. We find that each major lineage has a characteristic pattern of trait evolution, but mammals and reptiles evolve under a surprisingly similar regime, whilst that of amphibians appears to be particularly distinct and highly contrasting compared to other groups. Our results also suggest that the mechanism of toxin acquisition may be an important distinction in such evolutionary patterns; the evolution of biosynthesis is far less dynamic than that of sequestration of toxins from the diet. Finally, contrary to the situation in amphibians, other tetrapod groups show an association between the evolution of toxic weaponry and higher diversification rates. Taken together, our study provides the first broad-scale analysis of macroevolutionary patterns of venom and poison throughout tetrapods. Full article

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2296 KiB

Open AccessFeature PaperArticle

Is Hybridization a Source of Adaptive Venom Variation in Rattlesnakes? A Test, Using a Crotalus scutulatus × viridis Hybrid Zone in Southwestern New Mexico

by Giulia Zancolli, Timothy G. Baker, Axel Barlow, Rebecca K. Bradley, Juan J. Calvete, Kimberley C. Carter, Kaylah De Jager, John Benjamin Owens, Jenny Forrester Price, Libia Sanz, Amy Scholes-Higham, Liam Shier, Liam Wood, Catharine E. Wüster and Wolfgang Wüster

Toxins 2016, 8(6), 188; https://doi.org/10.3390/toxins8060188 - 16 Jun 2016

Cited by 30 | Viewed by 11376

Abstract

Venomous snakes often display extensive variation in venom composition both between and within species. However, the mechanisms underlying the distribution of different toxins and venom types among populations and taxa remain insufficiently known. Rattlesnakes (Crotalus, Sistrurus) display extreme inter- and [...] Read more.

Venomous snakes often display extensive variation in venom composition both between and within species. However, the mechanisms underlying the distribution of different toxins and venom types among populations and taxa remain insufficiently known. Rattlesnakes (Crotalus, Sistrurus) display extreme inter- and intraspecific variation in venom composition, centered particularly on the presence or absence of presynaptically neurotoxic phospholipases A₂ such as Mojave toxin (MTX). Interspecific hybridization has been invoked as a mechanism to explain the distribution of these toxins across rattlesnakes, with the implicit assumption that they are adaptively advantageous. Here, we test the potential of adaptive hybridization as a mechanism for venom evolution by assessing the distribution of genes encoding the acidic and basic subunits of Mojave toxin across a hybrid zone between MTX-positive Crotalus scutulatus and MTX-negative C. viridis in southwestern New Mexico, USA. Analyses of morphology, mitochondrial and single copy-nuclear genes document extensive admixture within a narrow hybrid zone. The genes encoding the two MTX subunits are strictly linked, and found in most hybrids and backcrossed individuals, but not in C. viridis away from the hybrid zone. Presence of the genes is invariably associated with presence of the corresponding toxin in the venom. We conclude that introgression of highly lethal neurotoxins through hybridization is not necessarily favored by natural selection in rattlesnakes, and that even extensive hybridization may not lead to introgression of these genes into another species. Full article

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Graphical abstract

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Full article ">Figure 1
Ordination of specimens of C. scutulatus, C. atrox, C. viridis and putative hybrids along the first two axes of a principal components analysis of nine morphological characters. All specimens from the eastern slope of the Peloncillo Mountains in southwestern New Mexico are labeled as hybrids irrespective of morphological or genetic profile. The first and second principal components represent 41.5% and 23.7% of the total variance, respectively. Full article ">Figure 2
Neighbor-joining tree of individual ND4 sequences. Tip labels with yellow background indicate specimens from the putative hybrid zone in SW New Mexico. Full article ">Figure 3
(A) Bayesian population clustering of individuals of C. atrox, C. scutulatus, C. viridis and putative hybrids based on allele frequencies of four single copy nuclear genes. Above the Structure clustering, rows of boxes indicate mtDNA haplotype affinities (same colors as nuclear structuring), and the presence (black) or absence (grey) of the genes coding for the basic and acidic subunits of Mojave toxin (MTX), and above the confirmed presence or absence of the corresponding proteins in the venom. White spaces indicate absence of data. Black arrows indicate specimens morphologically intermediate between C. scutulatus and C. atrox. (B) Equivalent analysis excluding C. atrox to emphasize hybrid zone between C. scutulatus and C. viridis. Full article ">Figure 4
Sampling localities, hybrid status and MTX status of individuals of C. scutulatus, C. viridis and their hybrids. Diamonds indicate MTX+ve, circles MTX−ve individuals, the degree of shading of the symbols indicates the proportion of the genome attributed to C. scutulatus in the Structure analysis. Full article ">Figure 5
Examples of reverse-phase high performance liquid chromatography (RP-HPLC) chromatograms from different venoms included in the study. A. Crotalus scutulatus type II venom with MTX but lacking snake venom metalloproteinases (SVMPs); sample 4311, nr. Rodeo, Hidalgo Co., NM. B. Crotalus scutulatus × viridis, hybrid containing MTX and SVMPs; sample 4687, nr. Cotton City, Hidalgo Co., NM. C. Crotalus viridis, typical venom lacking MTX but containing SVMPs; sample 4590, nr. Animas, Hidalgo Co., NM. D. Crotalus atrox, typical venom lacking MTX but containing SVMPs; sample 4594, nr. Benson, Cochise Co., AZ. Full article ">

2015

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701 KiB

Open AccessReview

Cabinet of Curiosities: Venom Systems and Their Ecological Function in Mammals, with a Focus on Primates

by Johanna E. Rode-Margono and K. Anne-Isola Nekaris

Toxins 2015, 7(7), 2639-2658; https://doi.org/10.3390/toxins7072639 - 17 Jul 2015

Cited by 32 | Viewed by 16824

Abstract

Venom delivery systems (VDS) are common in the animal kingdom, but rare amongst mammals. New definitions of venom allow us to reconsider its diversity amongst mammals by reviewing the VDS of Chiroptera, Eulipotyphla, Monotremata, and Primates. All orders use modified anterior dentition as [...] Read more.

Venom delivery systems (VDS) are common in the animal kingdom, but rare amongst mammals. New definitions of venom allow us to reconsider its diversity amongst mammals by reviewing the VDS of Chiroptera, Eulipotyphla, Monotremata, and Primates. All orders use modified anterior dentition as the venom delivery apparatus, except Monotremata, which possesses a crural system. The venom gland in most taxa is a modified submaxillary salivary gland. In Primates, the saliva is activated when combined with brachial gland exudate. In Monotremata, the crural spur contains the venom duct. Venom functions include feeding, intraspecific competition, anti-predator defense and parasite defense. Including mammals in discussion of venom evolution could prove vital in our understanding protein functioning in mammals and provide a new avenue for biomedical and therapeutic applications and drug discovery. Full article

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Venom system of vampire bats. Common vampire bat Desmodus rotundus (a); with specialized tongue as indicated by the arrow (b); and teeth (c) Illustrations: Kathleen Reinhardt. Full article ">Figure 2
European water shrew Neomys fodiens (a); with concave incisor surfaces (as indicated by the arrow) that help with flow and injection of venom (b). Illustrations: Kathleen Reinhardt. Full article ">Figure 3
Venom system of solenodons. Hispaniolan solenodon Solenodon paradoxus (a); with deeply grooved lower canines (as indicated by the arrow) that aid in flow and injection of venom (b). Illustrations: Kathleen Reinhardt. Full article ">Figure 4
Venom system of the platypus. Platypus Ornithorhynchus anatinus (a); with crural spur as indicated by the arrow (b). Illustrations: Kathleen Reinhardt. Full article ">Figure 5
Venom system of slow lorises. Javan slow loris Nycticebus javanicus showing warning coloration of face (a); Javan slow loris displaying defense position (b); brachial gland as indicated by the arrow (c); tooth comb as indicated by the arrow (d); Illustrations: Kathleen Reinhardt. Full article ">

634 KiB

Open AccessReview

Facing Hymenoptera Venom Allergy: From Natural to Recombinant Allergens

by Amilcar Perez-Riverol, Débora Lais Justo-Jacomini, Ricardo De Lima Zollner and Márcia Regina Brochetto-Braga

Toxins 2015, 7(7), 2551-2570; https://doi.org/10.3390/toxins7072551 - 9 Jul 2015

Cited by 27 | Viewed by 8031

Abstract

Along with food and drug allergic reactions, a Hymenoptera insect Sting (Apoidea, Vespidae, Formicidae) is one of the most common causes of anaphylaxis worldwide. Diagnoses of Hymenoptera venom allergy (HVA) and specific immunotherapy (SIT) have been based on the use of crude venom [...] Read more.

Along with food and drug allergic reactions, a Hymenoptera insect Sting (Apoidea, Vespidae, Formicidae) is one of the most common causes of anaphylaxis worldwide. Diagnoses of Hymenoptera venom allergy (HVA) and specific immunotherapy (SIT) have been based on the use of crude venom extracts. However, the incidence of cross-reactivity and low levels of sensibility during diagnosis, as well as the occurrence of nonspecific sensitization and undesired side effects during SIT, encourage the search for novel allergenic materials. Recombinant allergens are an interesting approach to improve allergy diagnosis and SIT because they circumvent major problems associated with the use of crude venom. Production of recombinant allergens depends on the profound molecular characterization of the natural counterpart by combining some “omics” approaches with high-throughput screening techniques and the selection of an appropriate system for heterologous expression. To date, several clinically relevant allergens and novel venom toxins have been identified, cloned and characterized, enabling a better understanding of the whole allergenic and envenoming processes. Here, we review recent findings on identification, molecular characterization and recombinant expression of Hymenoptera venom allergens and on the evaluation of these heterologous proteins as valuable tools for tackling remaining pitfalls on HVA diagnosis and immunotherapy. Full article

2014

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2598 KiB

Open AccessReview

Quo Vadis Venomics? A Roadmap to Neglected Venomous Invertebrates

by Bjoern Marcus Von Reumont, Lahcen I. Campbell and Ronald A. Jenner

Toxins 2014, 6(12), 3488-3551; https://doi.org/10.3390/toxins6123488 - 19 Dec 2014

Cited by 86 | Viewed by 22729

Abstract

Venomics research is being revolutionized by the increased use of sensitive -omics techniques to identify venom toxins and their transcripts in both well studied and neglected venomous taxa. The study of neglected venomous taxa is necessary both for understanding the full diversity of [...] Read more.

Venomics research is being revolutionized by the increased use of sensitive -omics techniques to identify venom toxins and their transcripts in both well studied and neglected venomous taxa. The study of neglected venomous taxa is necessary both for understanding the full diversity of venom systems that have evolved in the animal kingdom, and to robustly answer fundamental questions about the biology and evolution of venoms without the distorting effect that can result from the current bias introduced by some heavily studied taxa. In this review we draw the outlines of a roadmap into the diversity of poorly studied and understood venomous and putatively venomous invertebrates, which together represent tens of thousands of unique venoms. The main groups we discuss are crustaceans, flies, centipedes, non-spider and non-scorpion arachnids, annelids, molluscs, platyhelminths, nemerteans, and echinoderms. We review what is known about the morphology of the venom systems in these groups, the composition of their venoms, and the bioactivities of the venoms to provide researchers with an entry into a large and scattered literature. We conclude with a short discussion of some important methodological aspects that have come to light with the recent use of new -omics techniques in the study of venoms. Full article

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697 KiB

Open AccessArticle

The Finding of a Group IIE Phospholipase A₂ Gene in a Specified Segment of Protobothrops flavoviridis Genome and Its Possible Evolutionary Relationship to Group IIA Phospholipase A₂ Genes

by Kazuaki Yamaguchi, Takahito Chijiwa, Naoki Ikeda, Hiroki Shibata, Yasuyuki Fukumaki, Naoko Oda-Ueda, Shosaku Hattori and Motonori Ohno

Toxins 2014, 6(12), 3471-3487; https://doi.org/10.3390/toxins6123471 - 18 Dec 2014

Cited by 6 | Viewed by 7194

Abstract

The genes encoding group IIE phospholipase A₂, abbreviated as IIE PLA₂, and its 5' and 3' flanking regions of Crotalinae snakes such as Protobothrops flavoviridis, P. tokarensis, P. elegans, and Ovophis okinavensis, were found and [...] Read more.

The genes encoding group IIE phospholipase A₂, abbreviated as IIE PLA₂, and its 5' and 3' flanking regions of Crotalinae snakes such as Protobothrops flavoviridis, P. tokarensis, P. elegans, and Ovophis okinavensis, were found and sequenced. The genes consisted of four exons and three introns and coded for 22 or 24 amino acid residues of the signal peptides and 134 amino acid residues of the mature proteins. These IIE PLA₂s show high similarity to those from mammals and Colubridae snakes. The high expression level of IIE PLA₂s in Crotalinae venom glands suggests that they should work as venomous proteins. The blast analysis indicated that the gene encoding OTUD3, which is ovarian tumor domain-containing protein 3, is located in the 3' downstream of IIE PLA₂ gene. Moreover, a group IIA PLA₂ gene was found in the 5' upstream of IIE PLA₂ gene linked to the OTUD3 gene (OTUD3) in the P. flavoviridis genome. It became evident that the specified arrangement of IIA PLA₂ gene, IIE PLA₂ gene, and OTUD3 in this order is common in the genomes of humans to snakes. The present finding that the genes encoding various secretory PLA₂s form a cluster in the genomes of humans to birds is closely related to the previous finding that six venom PLA₂ isozyme genes are densely clustered in the so-called NIS-1 fragment of the P. flavoviridis genome. It is also suggested that venom IIA PLA₂ genes may be evolutionarily derived from the IIE PLA₂ gene. Full article

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1221 KiB

Open AccessReview

Tracing Monotreme Venom Evolution in the Genomics Era

by Camilla M. Whittington and Katherine Belov

Toxins 2014, 6(4), 1260-1273; https://doi.org/10.3390/toxins6041260 - 2 Apr 2014

Cited by 15 | Viewed by 20036

Abstract

The monotremes (platypuses and echidnas) represent one of only four extant venomous mammalian lineages. Until recently, monotreme venom was poorly understood. However, the availability of the platypus genome and increasingly sophisticated genomic tools has allowed us to characterize platypus toxins, and provides a [...] Read more.

The monotremes (platypuses and echidnas) represent one of only four extant venomous mammalian lineages. Until recently, monotreme venom was poorly understood. However, the availability of the platypus genome and increasingly sophisticated genomic tools has allowed us to characterize platypus toxins, and provides a means of reconstructing the evolutionary history of monotreme venom. Here we review the physiology of platypus and echidna crural (venom) systems as well as pharmacological and genomic studies of monotreme toxins. Further, we synthesize current ideas about the evolution of the venom system, which in the platypus is likely to have been retained from a venomous ancestor, whilst being lost in the echidnas. We also outline several research directions and outstanding questions that would be productive to address in future research. An improved characterization of mammalian venoms will not only yield new toxins with potential therapeutic uses, but will also aid in our understanding of the way that this unusual trait evolves. Full article

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1208 KiB

Open AccessArticle

Elapid Snake Venom Analyses Show the Specificity of the Peptide Composition at the Level of Genera Naja and Notechis

by Aisha Munawar, Maria Trusch, Dessislava Georgieva, Diana Hildebrand, Marcel Kwiatkowski, Henning Behnken, Sönke Harder, Raghuvir Arni, Patrick Spencer, Hartmut Schlüter and Christian Betzel

Toxins 2014, 6(3), 850-868; https://doi.org/10.3390/toxins6030850 - 28 Feb 2014

Cited by 19 | Viewed by 12038

Abstract

Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms—that of the African cobra, N. m. mossambica (genus Naja [...] Read more.

Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms—that of the African cobra, N. m. mossambica (genus Naja), and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis). A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F) cytotoxins, bradykinin-potentiating peptides (BPPs) and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI)-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467) from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes. Full article

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(A) Size-exclusion chromatography of N. m. mossambica venom on a Superdex C-75 10/300 column at pH 5.0; (B) size-exclusion chromatography of the N. scutatus from Kangaroo Island venom on a Superdex G-75 16/60 column at pH 5.0. Full article ">Figure 2
SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of the fractions, 1–7, from the size exclusion chromatography of N. m. mossambica venom. Full article ">Figure 3
Further purification by fast protein liquid chromatography (FPLC) of Peak 5 (<a href="#toxins-06-00850-f001" class="html-fig">Figure 1</a>A) with a Resource-S column (1 mL) at pH 5.5. Full article ">Figure 4
(A) Sequence alignment of cytotoxin 1 (P01467), cytotoxin 3 (P01470) and cytotoxin 4 (P01452) isolated from N. m. mossambica venom. The cysteine residues are shaded black, and the disulfide bonds are indicated. The variable amino acid residues are shaded grey; (B) the sequence alignment of the Kunitz inhibitors isolated from the N. scutatus (Kangaroo Island) venom shows a variation in the reactive site residues. The same peptides, named protease inhibitor tigerin 1 (Q61TB3) and tigerin 3 (B5KL32), were found by transcriptome analysis in the Notechis scutatus scutatus venom gland [<a href="#B10-toxins-06-00850" class="html-bibr">10</a>]. Full article ">Figure 5
ESI-TOF-MS spectrum of cytotoxin-1, P01467 (isolated from N. m. mossambica venom) showing multiply charged ions. Full article ">Figure 6
MALDI TOF/TOF MS of the representative peptides isolated from N. m. mossambica. (A) At m/z 1214.6581 (M + H)+; (B) at m/z 1276.6380 (M + H)+. Full article ">Figure 7
SDS-PAGE of fractions 1–6 from the size exclusion chromatographic separation (<a href="#toxins-06-00850-f001" class="html-fig">Figure 1</a>B) of the crude venom of N. scutatus (Kangaroo Island). Full article ">Figure 8
Purification of Peak 4 (<a href="#toxins-06-00850-f001" class="html-fig">Figure 1</a>B) with a SOURCE™ 5RPC column by HPLC. Inset shows MALDI-TOF-MS of the peptide (tigerin-3, B5KL32) eluting at 19.319 min. Full article ">

2013

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9748 KiB

Open AccessArticle

Venom Down Under: Dynamic Evolution of Australian Elapid Snake Toxins

by Timothy N. W. Jackson, Kartik Sunagar, Eivind A. B. Undheim, Ivan Koludarov, Angelo H. C. Chan, Kate Sanders, Syed A. Ali, Iwan Hendrikx, Nathan Dunstan and Bryan G. Fry

Toxins 2013, 5(12), 2621-2655; https://doi.org/10.3390/toxins5122621 - 18 Dec 2013

Cited by 53 | Viewed by 16117

Abstract

Despite the unparalleled diversity of venomous snakes in Australia, research has concentrated on a handful of medically significant species and even of these very few toxins have been fully sequenced. In this study, venom gland transcriptomes were sequenced from eleven species of small [...] Read more.

Despite the unparalleled diversity of venomous snakes in Australia, research has concentrated on a handful of medically significant species and even of these very few toxins have been fully sequenced. In this study, venom gland transcriptomes were sequenced from eleven species of small Australian elapid snakes, from eleven genera, spanning a broad phylogenetic range. The particularly large number of sequences obtained for three-finger toxin (3FTx) peptides allowed for robust reconstructions of their dynamic molecular evolutionary histories. We demonstrated that each species preferentially favoured different types of α-neurotoxic 3FTx, probably as a result of differing feeding ecologies. The three forms of α-neurotoxin [Type I (also known as (aka): short-chain), Type II (aka: long-chain) and Type III] not only adopted differential rates of evolution, but have also conserved a diversity of residues, presumably to potentiate prey-specific toxicity. Despite these differences, the different α-neurotoxin types were shown to accumulate mutations in similar regions of the protein, largely in the loops and structurally unimportant regions, highlighting the significant role of focal mutagenesis. We theorize that this phenomenon not only affects toxin potency or specificity, but also generates necessary variation for preventing/delaying prey animals from acquiring venom-resistance. This study also recovered the first full-length sequences for multimeric phospholipase A₂ (PLA₂) ‘taipoxin/paradoxin’ subunits from non-Oxyuranus species, confirming the early recruitment of this extremely potent neurotoxin complex to the venom arsenal of Australian elapid snakes. We also recovered the first natriuretic peptides from an elapid that lack the derived C-terminal tail and resemble the plesiotypic form (ancestral character state) found in viper venoms. This provides supporting evidence for a single early recruitment of natriuretic peptides into snake venoms. Novel forms of kunitz and waprin peptides were recovered, including dual domain kunitz-kunitz precursors and the first kunitz-waprin hybrid precursors from elapid snakes. The novel sequences recovered in this study reveal that the huge diversity of unstudied venomous Australian snakes are of considerable interest not only for the investigation of venom and whole organism evolution but also represent an untapped bioresource in the search for novel compounds for use in drug design and development. Full article

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1909 KiB

Open AccessArticle

A Proteomics and Transcriptomics Investigation of the Venom from the Barychelid Spider Trittame loki (Brush-Foot Trapdoor)

by Eivind A. B. Undheim, Kartik Sunagar, Volker Herzig, Laurence Kely, Dolyce H. W. Low, Timothy N. W. Jackson, Alun Jones, Nyoman Kurniawan, Glenn F. King, Syed A. Ali, Agostino Antunes, Tim Ruder and Bryan G. Fry

Toxins 2013, 5(12), 2488-2503; https://doi.org/10.3390/toxins5122488 - 13 Dec 2013

Cited by 61 | Viewed by 11191

Abstract

Although known for their potent venom and ability to prey upon both invertebrate and vertebrate species, the Barychelidae spider family has been entirely neglected by toxinologists. In striking contrast, the sister family Theraphosidae (commonly known as tarantulas), which last shared a most recent [...] Read more.

Although known for their potent venom and ability to prey upon both invertebrate and vertebrate species, the Barychelidae spider family has been entirely neglected by toxinologists. In striking contrast, the sister family Theraphosidae (commonly known as tarantulas), which last shared a most recent common ancestor with Barychelidae over 200 million years ago, has received much attention, accounting for 25% of all the described spider toxins while representing only 2% of all spider species. In this study, we evaluated for the first time the venom arsenal of a barychelid spider, Trittame loki, using transcriptomic, proteomic, and bioinformatic methods. The venom was revealed to be dominated by extremely diverse inhibitor cystine knot (ICK)/knottin peptides, accounting for 42 of the 46 full-length toxin precursors recovered in the transcriptomic sequencing. In addition to documenting differential rates of evolution adopted by different ICK/knottin toxin lineages, we discovered homologues with completely novel cysteine skeletal architecture. Moreover, acetylcholinesterase and neprilysin were revealed for the first time as part of the spider-venom arsenal and CAP (CRiSP/Allergen/PR-1) were identified for the first time in mygalomorph spider venoms. These results not only highlight the extent of venom diversification in this neglected ancient spider lineage, but also reinforce the idea that unique venomous lineages are rich pools of novel biomolecules that may have significant applied uses as therapeutics and/or insecticides. Full article

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Figure 1
Magnetic resonance imaging of Trittame loki venom glands. Full article ">Figure 2
Phylogenetic reconstruction of Trittame loki and related inhibitor cystine knot (ICK)/knottin peptide toxins, conserved ancestral cysteines are shown in black, newly evolved cysteines are in red. Sequences obtained in this study are in green. Signal peptides are shown in lowercase. Full article ">Figure 3
Sequence alignment of spider venom colipase venom peptides: (1) Trittame loki COLIPASE-1; (2) D2Y2E5 Haplopelma hainanum; (3) Q5D233 Hadronyche infensa; (4) Q5D231 Hadronyche sp. (strain 20); (5) Q5D232 Hadronyche sp. (strain 20); (6) B1P1J0 Chilobrachys jingzhao; and (7) B1P1J2 Chilobrachys jingzhao. Signal peptides are shown in lowercase. Full article ">Figure 4
Sequence alignment of spider venom CAP (CRiSP/Allergen/PR-1) venom peptides: (1) Trittame loki CAP-1; and (2) A9QQ26 Lycosa singoriensis. Signal peptides are shown in lowercase. Full article ">Figure 5
Sequence alignment of spider venom kunitz venom peptides: (1) Trittame loki KUNITZ-1; and (2) E7D1N7 Latrodectus hesperus. Signal peptides are shown in lowercase. Full article ">Figure 6
Sequence alignment of the Trittame loki venom acetylcholinesterase and the non-venom homologue P56161 Anopheles stephensi. Signal peptides are shown in lowercase. Full article ">Figure 7
Sequence alignment of the Trittame loki venom neprilysin and the snake venom convergent neprilysin homologue T1E4Z0 Crotalus horridus. Signal peptides are shown in lowercase. Full article ">

5767 KiB

Open AccessArticle

Evolution Stings: The Origin and Diversification of Scorpion Toxin Peptide Scaffolds

by Kartik Sunagar, Eivind A. B. Undheim, Angelo H. C. Chan, Ivan Koludarov, Sergio A. Muñoz-Gómez, Agostinho Antunes and Bryan G. Fry

Toxins 2013, 5(12), 2456-2487; https://doi.org/10.3390/toxins5122456 - 13 Dec 2013

Cited by 75 | Viewed by 14315

Abstract

The episodic nature of natural selection and the accumulation of extreme sequence divergence in venom-encoding genes over long periods of evolutionary time can obscure the signature of positive Darwinian selection. Recognition of the true biocomplexity is further hampered by the limited taxon selection, [...] Read more.

The episodic nature of natural selection and the accumulation of extreme sequence divergence in venom-encoding genes over long periods of evolutionary time can obscure the signature of positive Darwinian selection. Recognition of the true biocomplexity is further hampered by the limited taxon selection, with easy to obtain or medically important species typically being the subject of intense venom research, relative to the actual taxonomical diversity in nature. This holds true for scorpions, which are one of the most ancient terrestrial venomous animal lineages. The family Buthidae that includes all the medically significant species has been intensely investigated around the globe, while almost completely ignoring the remaining non-buthid families. Australian scorpion lineages, for instance, have been completely neglected, with only a single scorpion species (Urodacus yaschenkoi) having its venom transcriptome sequenced. Hence, the lack of venom composition and toxin sequence information from an entire continent’s worth of scorpions has impeded our understanding of the molecular evolution of scorpion venom. The molecular origin, phylogenetic relationships and evolutionary histories of most scorpion toxin scaffolds remain enigmatic. In this study, we have sequenced venom gland transcriptomes of a wide taxonomical diversity of scorpions from Australia, including buthid and non-buthid representatives. Using state-of-art molecular evolutionary analyses, we show that a majority of CSα/β toxin scaffolds have experienced episodic influence of positive selection, while most non-CSα/β linear toxins evolve under the extreme influence of negative selection. For the first time, we have unraveled the molecular origin of the major scorpion toxin scaffolds, such as scorpion venom single von Willebrand factor C-domain peptides (SV-SVC), inhibitor cystine knot (ICK), disulphide-directed beta-hairpin (DDH), bradykinin potentiating peptides (BPP), linear non-disulphide bridged peptides and antimicrobial peptides (AMP). We have thus demonstrated that even neglected lineages of scorpions are a rich pool of novel biochemical components, which have evolved over millions of years to target specific ion channels in prey animals, and as a result, possess tremendous implications in therapeutics. Full article

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1117 KiB

Open AccessArticle

Three-Fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of Snake Venom Toxins

by Kartik Sunagar, Timothy N. W. Jackson, Eivind A. B. Undheim, Syed. A. Ali, Agostinho Antunes and Bryan G. Fry

Toxins 2013, 5(11), 2172-2208; https://doi.org/10.3390/toxins5112172 - 18 Nov 2013

Cited by 103 | Viewed by 12163

Abstract

Three-finger toxins (3FTx) represent one of the most abundantly secreted and potently toxic components of colubrid (Colubridae), elapid (Elapidae) and psammophid (Psammophiinae subfamily of the Lamprophidae) snake venom arsenal. Despite their conserved structural similarity, they perform a diversity of biological functions. Although they [...] Read more.

Three-finger toxins (3FTx) represent one of the most abundantly secreted and potently toxic components of colubrid (Colubridae), elapid (Elapidae) and psammophid (Psammophiinae subfamily of the Lamprophidae) snake venom arsenal. Despite their conserved structural similarity, they perform a diversity of biological functions. Although they are theorised to undergo adaptive evolution, the underlying diversification mechanisms remain elusive. Here, we report the molecular evolution of different 3FTx functional forms and show that positively selected point mutations have driven the rapid evolution and diversification of 3FTx. These diversification events not only correlate with the evolution of advanced venom delivery systems (VDS) in Caenophidia, but in particular the explosive diversification of the clade subsequent to the evolution of a high pressure, hollow-fanged VDS in elapids, highlighting the significant role of these toxins in the evolution of advanced snakes. We show that Type I, II and III α-neurotoxins have evolved with extreme rapidity under the influence of positive selection. We also show that novel Oxyuranus/Pseudonaja Type II forms lacking the apotypic loop-2 stabilising cysteine doublet characteristic of Type II forms are not phylogenetically basal in relation to other Type IIs as previously thought, but are the result of secondary loss of these apotypic cysteines on at least three separate occasions. Not all 3FTxs have evolved rapidly: κ-neurotoxins, which form non-covalently associated heterodimers, have experienced a relatively weaker influence of diversifying selection; while cytotoxic 3FTx, with their functional sites, dispersed over 40% of the molecular surface, have been extremely constrained by negative selection. We show that the a previous theory of 3FTx molecular evolution (termed ASSET) is evolutionarily implausible and cannot account for the considerable variation observed in very short segments of 3FTx. Instead, we propose a theory of Rapid Accumulation of Variations in Exposed Residues (RAVER) to illustrate the significance of point mutations, guided by focal mutagenesis and positive selection in the evolution and diversification of 3FTx. Full article

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Figure 1

Figure 1
Bayesian molecular phylogeny of representative three-finger toxins. Uniprot [<a href="#B49-toxins-05-02172" class="html-bibr">49</a>] accession numbers are given for each. Cysteine framework variation is displayed, with ancestral cysteines in black and newly evolved cysteines in red. Full article ">Figure 2
Bayesian molecular phylogeny and structural and functional evolution of +2C/−2C Type II (long-chain) α-neurotoxins. Pseudonaja/Oxyuranus −2C and +2C sequences are coloured purple and green, respectively. Sequences presented (uniprot): (1) R4FIT5 Pseudonaja modesta, (2) R4FIU6 Pseudonaja modesta, (3) A8HDK6 Pseudonaja textilis, (4) R4FK68 Pseudonaja modesta, (5) A8HDK8 Oxyuranus microlepidotus, (6) A7X4Q3 Oxyuranus microlepidotus, (7) A8HDK7 Oxyuranus microlepidotus, (8) A7X4R0 Oxyuranus microlepidotus, (9) A8HDK9 Oxyuranus scutellatus, (10) Q9W7J5 Pseudonaja textilis, (11) R4FIT0 Pseudonaja modesta, (12) R4G7K3 Pseudonaja modesta, (13) R4G321 Pseudonaja modesta, (14) R4G2J4 Pseudonaja modesta, (15) R4G319 Pseudonaja modesta and (16) R4FK75 Pseudonaja modesta. Full article ">Figure 3
Molecular evolution of plesiotypic 3FTxs. Three-dimensional homology models of various three-finger toxins (top: surface-fill; bottom: wireframe), depicting the locations of positively selected sites are presented. Site-model 8 computed omega and the total number of positively selected sites detected by its Bayes Empirical Bayes (BEB) approach (PP ≥ 0.95) are also indicated. Full article ">Figure 4
Molecular evolution of derived 3FTx. Three-dimensional homology models of various three-finger toxins (top: surface-fill; bottom: wireframe), depicting the locations of positively selected sites are presented. Site-model 8 computed omega and the total number of positively selected sites detected by its Bayes Empirical Bayes (BEB) approach (PP ≥ 0.95) are also indicated. Full article ">Figure 5
Surface accessibility of three-finger toxins. A plot of amino acid positions (x-axis) against accessible surface area (ASA) ratio (y-axis) indicating the locations of amino acids (exposed or buried) in the crystal structure of various three-finger toxins is presented. Positively selected residues are presented as large dots, while the remaining sites are presented as small dots in the plot. Residues with an ASA ratio greater than 50% are considered to be exposed (ASA of 40%–50% are likely to have exposed side chains), while those with a ratio lesser than 20% are considered to be buried to the surrounding medium. Three-dimensional structures of various 3FTx types, depicting the locations of positively selected (PS) sites along with model 8 omega values and the number of exposed and buried positively selected sites are also presented (for details see <a href="#toxins-05-02172-t005" class="html-table">Table 5</a>). Full article ">Figure 6
Structural and functional evolution of plesiotypic 3FTxs. Sequence alignment and homology models depicting structurally and functionally important residues and hypermutable sites of various three-finger toxins are shown. Extremely well-conserved residues implicated in structural/functional roles, and hypermutable sites are shaded. Sequences presented (uniprot): A. (1) M9T1L2 Aspidites melanocephalus, (2) M9T271 Aspidites melanocephalus, (3) M9SZR1 Cylindrophis ruffus and (4) M9SZV9 Cylindrophis ruffus; B. (1) A5X2W8 Sistrurus catenatus edwardsii, (2) B4Y146 Sistrurus catenatus edwardsii, (3) A5X2W7 Sistrurus catenatus edwardsii, (4) B4Y143 Sistrurus catenatus edwardsii, (5) B4Y144 Sistrurus catenatus edwardsii and (6) M9T2J4 Azemiops feae; C. (1) Q06ZW0 Boiga dendrophila, (2) A0S864 Boiga irregularis, (3) A0S865 Boiga irregularis, 4) A7X3V0 Telescopus dhara and (5) A7X3S0 Trimorphodon biscutatus; D. (1) Q6IZ95 Bungarus candidus, (2) Q9PW19 Bungarus multicinctus, (3) Q9YGH9 Bungarus multicinctus, (4) Q8AY51 Bungarus candidus, (5) Q2VBN2 Ophiophagus hannah and 6) Q9YGI2 Naja atra. Full article ">Figure 7
Structural and functional evolution of derived α-neurotoxins and κ-neurotoxins. Sequence alignment and homology models depicting structurally and functionally important residues and hypermutable sites of various three-finger toxins are shown. Extremely well-conserved residues implicated in structural/functional roles, and hypermutable sites are shaded. Sites that are shown to be structurally/functionally important but exhibit less than 70% identity are indicated by arrow heads in the alignment. Sequences presented (uniprot): (A). (1) B5G6F6 Oxyuranus microlepidotus, (2) H8PG58 Suta nigriceps, (3) P60770 Naja atra, (4) P10455 Laticauda colubrina, (5) B2BRS2 Austrelaps labialis and (6) F8J2H2 Drysdalia coronoides; (B). (1) F8J2E0 Drysdalia coronoides, (2) Q8UW29 Hydrophis hardwickii, (3) P01384 Notechis scutatus, (4) P82662 Ophiophagus hannah, (5) R4G2L3 Suta fasciata and (6) R4G2E5 Brachyurophis roperi; C. (1) R4G2S0 Cacophis squamulosus, (2) R4G7H6 Furina ornata, (3) R4G332 Pseudonaja modesta, (4) Q9W7K2 Pseudonaja textilis, (5) R4G7F3 Brachyurophis roperi and (6) R4G7M0 Vermicella annulata; D. (1) Q8AY56 Bungarus candidus, (2) Q8AY55 Bungarus candidus, (3) P15817 Bungarus multicinctus, (4) P01398 Bungarus multicinctus, (5) O12962 Bungarus multicinctus and (6) Q9W729 Bungarus multicinctus. Full article ">Figure 8
Structural and functional evolution of cytotoxins. Sequence alignment and homology models depicting A. the locations of putative functional residues, and B. hydrophobic regions of cytotoxins are presented. Extremely well-conserved residues implicated in structural/functional roles, and hypermutable sites are shaded. Sequences presented (uniprot): (1) P60301 Naja atra, (2) P60303 Naja kaouthia, (3) Q9DGH9 Naja kaouthia, (4) P60301 Naja sputatrix, (5) Q02454 Naja sputatrix and (6) O93471 Naja sputatrix. Full article ">

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Open AccessArticle

Atractaspis aterrima Toxins: The First Insight into the Molecular Evolution of Venom in Side-Stabbers

by Yves Terrat, Kartik Sunagar, Bryan G. Fry, Timothy N. W. Jackson, Holger Scheib, Rudy Fourmy, Marion Verdenaud, Guillaume Blanchet, Agostinho Antunes and Frederic Ducancel

Toxins 2013, 5(11), 1948-1964; https://doi.org/10.3390/toxins5111948 - 28 Oct 2013

Cited by 18 | Viewed by 9172

Abstract

Although snake venoms have been the subject of intense research, primarily because of their tremendous potential as a bioresource for design and development of therapeutic compounds, some specific groups of snakes, such as the genus Atractaspis, have been completely neglected. To date [...] Read more.

Although snake venoms have been the subject of intense research, primarily because of their tremendous potential as a bioresource for design and development of therapeutic compounds, some specific groups of snakes, such as the genus Atractaspis, have been completely neglected. To date only limited number of toxins, such as sarafotoxins have been well characterized from this lineage. In order to investigate the molecular diversity of venom from Atractaspis aterrima—the slender burrowing asp, we utilized a high-throughput transcriptomic approach completed with an original bioinformatics analysis pipeline. Surprisingly, we found that Sarafotoxins do not constitute the major ingredient of the transcriptomic cocktail; rather a large number of previously well-characterized snake venom-components were identified. Notably, we recovered a large diversity of three-finger toxins (3FTxs), which were found to have evolved under the significant influence of positive selection. From the normalized and non-normalized transcriptome libraries, we were able to evaluate the relative abundance of the different toxin groups, uncover rare transcripts, and gain new insight into the transcriptomic machinery. In addition to previously characterized toxin families, we were able to detect numerous highly-transcribed compounds that possess all the key features of venom-components and may constitute new classes of toxins. Full article

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Figure 1

Figure 1
Functional annotation of Atractaspis transcriptomes. (a) Differences of subsystem’s annotation of reads between Normalized and Non-Normalized libraries. (b) Gene Ontology classification of reads covering 80% of assembled contigs (Non-Normalized library). Full article ">Figure 2
Network analysis of putative toxins. The network includes 6036 non redundant Toxins or associated venom protein classified by Uniprot (ToxProtDb) and 637 partial & full-length putative toxins from the present study. Minimal e-value for edge connexion is set to 1E−10. Full article ">Figure 3
gene network analysis of snake’s 3FTXs and A. aterrima 3FTXs consensus sequence. Minimal e-value for edge connexion is set to 1E-20. Full article ">Figure 4
Three-dimensional homology model of Atractaspis 3FTx, depicting the locations of positively selected sites (shown in red) detected by site-model 8. The omega value and the number of positively selected sites (Model 8, PP ≥ 0.95, depicting the locations of poach) are also indicated. Full article ">Figure 5
Annotation of the most abundant transcripts. Transcripts are sorted according to their abundance. In red are putative toxins, in grey are protein of unknown function and in black sequences that do not match these two categories. Full article ">Figure 6
EST processing workflow. Full article ">

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