A Pungent and Painful Toxin.

In this issue of Cell, King et al. (2019) have discovered a cell penetrating peptide isolated from the venom of the Australian Black Rock scorpion that activates the TRPA1 receptor in a unique way to induce pain. Their findings offer new insights into how animals evolved venoms to target specific ion channel functions.

A Cell-Penetrating Scorpion Toxin Enables Mode-Specific Modulation of TRPA1 and Pain.

TRPA1 is a chemosensory ion channel that functions as a sentinel for structurally diverse electrophilic irritants. Channel activation occurs through an unusual mechanism involving covalent modification of cysteine residues clustered within an amino-terminal cytoplasmic domain. Here, we describe a peptidergic scorpion toxin (WaTx) that activates TRPA1 by penetrating the plasma membrane to access the same intracellular site modified by reactive electrophiles. WaTx stabilizes TRPA1 in a biophysically distinct active state characterized by prolonged channel openings and low Ca2+ permeability. Consequently, WaTx elicits acute pain and pain hypersensitivity but fails to trigger efferent release of neuropeptides and neurogenic inflammation typically produced by noxious electrophiles. These findings provide a striking example of convergent evolution whereby chemically disparate animal- and plant-derived irritants target the same key allosteric regulatory site to differentially modulate channel activity. WaTx is a unique pharmacological probe for dissecting TRPA1 function and its contribution to acute and persistent pain.

Whole transcriptome sequencing reveals the activity of the PLA2 family members in Androctonus crassicauda (Scorpionida: Buthidae) venom gland.

Phospholipase A2 is the most abundant venom gland enzyme, whose activity leads to the activation of the inflammatory response by accumulating lipid mediators. This study aimed to identify, classify, and investigate the properties of venom PLA2 isoforms. Then, the present findings were confirmed by chemically measuring the activity of PLA2. The sequences representing PLA2 annotation were extracted from the Androctonus crassicauda transcriptome dataset using BLAS searches against the local PLA2 database. We found several cDNA sequences of PLA2 classified and named by conducting multiple searches as platelet-activating factor acetylhydrolases, calcium-dependent PLA2s, calcium-independent PLA2s, and secreted PLA2s. The largest and smallest isoforms of these proteins range between approximately 70.34 kDa (iPLA2) and 17.75 kDa (cPLA2). Among sPLA2 isoforms, sPLA2GXIIA and sPLA2G3 with ORF encoding 169 and 299 amino acids are the smallest and largest secreted PLA2, respectively. These results collectively suggested that A. crassicauda venom has PLA2 activity, and the members of this protein family may have important biological roles in lipid metabolism. This study also revealed the interaction between members of PLA2s in the PPI network. The results of this study would greatly help with the classification, evolutionary relationships, and interactions between PLA2 family proteins in the gene network.

Identification of an arthropod molecular target for plant-derived natural repellents.

Arthropods maintain ecosystem balance while also contributing to the spread of disease. Plant-derived natural repellents represent an ecological method of pest control, but their direct molecular targets in arthropods remain to be further elucidated. Occupying a critical phylogenetic niche in arthropod evolution, scorpions retain an ancestral genetic profile. Here, using a behavior-guided screening of the Mesobuthus martensii genome, we identified a scorpion transient receptor potential (sTRP1) channel that senses Cymbopogon-derived natural repellents, while remaining insensitive to the synthetic chemical pesticide DEET. Scrutinizing orthologs of sTRP1 in Drosophila melanogaster, we further demonstrated dTRPγ ion channel as a chemosensory receptor of natural repellents to mediate avoidance behavior. This study sheds light on arthropod molecular targets of natural repellents, exemplifying the arthropod­plant adaptation. It should also help the rational design of insect control strategy and in conserving biodiversity.

Dual Functions of labial Resolve the Hox Logic of Chelicerate Head Segments.

Despite an abundance of gene expression surveys, comparatively little is known about Hox gene function in Chelicerata. Previous investigations of paralogs of labial (lab) and Deformed (Dfd) in a spider have shown that these play a role in tissue maintenance of the pedipalp segment (lab-1) and in patterning the first walking leg identity (Dfd-1), respectively. However, extrapolations of these data across chelicerates are hindered by the existence of duplicated Hox genes in arachnopulmonates (e.g., spiders and scorpions), which have resulted from an ancient whole genome duplication (WGD) event. Here, we investigated the function of the single-copy ortholog of lab in the harvestman Phalangium opilio, an exemplar of a lineage that was not subject to this WGD. Embryonic RNA interference against lab resulted in two classes of phenotypes: homeotic transformations of pedipalps to chelicerae, as well as reduction and fusion of the pedipalp and leg 1 segments. To test for combinatorial function, we performed a double knockdown of lab and Dfd, which resulted in a homeotic transformation of both pedipalps and the first walking legs into cheliceral identity, whereas the second walking leg is transformed into a pedipalpal identity. Taken together, these results elucidate a model for the Hox logic of head segments in Chelicerata. To substantiate the validity of this model, we performed expression surveys for lab and Dfd paralogs in scorpions and horseshoe crabs. We show that repetition of morphologically similar appendages is correlated with uniform expression levels of the Hox genes lab and Dfd, irrespective of the number of gene copies.

Convergent evolution of giant size in eurypterids.

Eurypterids-Palaeozoic marine and freshwater arthropods commonly known as sea scorpions-repeatedly evolved to remarkable sizes (over 0.5 m in length) and colonized continental aquatic habitats multiple times. We compiled data on the majority of eurypterid species and explored several previously proposed explanations for the evolution of giant size in the group, including the potential role of habitat, sea surface temperature and dissolved sea surface oxygen levels, using a phylogenetic comparative approach with a new tip-dated tree. There is no compelling evidence that the evolution of giant size was driven by temperature or oxygen levels, nor that it was coupled with the invasion of continental aquatic environments, latitude or local faunal diversity. Eurypterid body size evolution is best characterized by rapid bursts of change that occurred independently of habitat or environmental conditions. Intrinsic factors played a major role in determining the convergent origin of gigantism in eurypterids.

Late Ordovician eurypterid preserves oldest euchelicerate musculature in pyrite.

Pyritization of soft tissues of invertebrates is rare in the fossil record. In New York State, it occurs in black shales of the Lorraine Group (Late Ordovician), the best-known example of which is Beecher's Trilobite Bed. Exceptional preservation at the quarry where this bed is exposed allowed detailed examination of trilobite and ostracod soft-tissue anatomy. Here, we present the first example of a eurypterid (sea scorpion) currently ascribed to Carcinosomatidae from this deposit that also preserves the first evidence for mesosomal musculature in eurypterids. This specimen demonstrates that eurypterid musculature can be preserved in pyrite and evidences the oldest example of euchelicerate muscles within the fossil record. Sulfur isotope data illustrate that pyrite rapidly replicated muscle tissue in the early burial environment, prior to the pyritization of biomineralized exoskeleton and cuticular trilobite limbs. This discovery therefore expands the limited fossil record of euchelicerate musculature, while extending the taphonomic scope for preservation of detailed internal structures, more broadly, within arthropods.

Profiling the Linear Peptides of Venom from the Brazilian Scorpion Tityus serrulatus: Structural and Functional Characterization.

Scorpion venoms are a rich source of bioactive peptides, most of which are neurotoxic, with 30 to 70 amino acid residues in their sequences. There are a scarcity of reports in the literature concerning the short linear peptides found in scorpion venoms. This type of peptide toxin may be selectively extracted from the venom using 50% (v/v) acetonitrile. The use of LC-MS and MS/MS enabled the detection of 12 bioactive short linear peptides, of which six were identified as cryptides. These peptides were shown to be multifunctional, causing hemolysis, mast cell degranulation and lysis, edema, pain, and anxiety, increasing the complexity of the envenomation mechanism. Apparently, the natural functions of these peptide toxins are to induce inflammation and discomfort in the victims of scorpion stings.

First biochemical and behavioural analysis of the response of the scorpion Urophonius brachycentrus (Thorell: 1876) upon exposure to an organophosphate.

Scorpionism is an increasing public health problem in the world. Although no specific methodology or product is currently available for the control of those arachnids, the use of insecticides could be an effective tool. Chlorpyrifos is one of the insecticides used, but to date, whether scorpions recognise surfaces with that insecticide and how it affects their physiology and/or biochemistry is unknown. In the present study, we observed that scorpions recognise surfaces with 0.51 and 8.59 µg/cm2 of chlorpyrifos and avoid those areas. The 0.51 µg/cm2 concentration produced a decrease in acetylcholinesterase and an increase in catalase, superoxide dismutase and glutathione S-transferase, whereas the 8.59 µg/cm2 concentration evoked a decrease in acetylcholinesterase and an increase in catalase and glutathione S-transferase. Using the comet assay, we observed that the insecticide at 0.17, 0.51 and 8.59 µg/cm2 caused DNA damage. Finally, we found that the insecticide does not generate significant variations in glutathione peroxidase, glutathione reductase, the amount of protein or lipid peroxidation. The present results offer a comprehensive understanding of how scorpions respond, both at the biochemical and behavioural levels, when exposed to insecticides.

Study of the Acute Toxicity of Scorpion Leiurus macroctenus Venom in Rats.

Background: The expansion of the territory of human habitation leads to inevitable interference in the natural range of distribution of one or another species of animals, some of which may be dangerous for human life. Scorpions-the Arachnida class and order Scorpiones-can be considered as such typical representatives. Scorpions of the Buthidae family pose a particular danger to humans. However, LD50 has not yet been defined for many species of this family, in particular, new representatives of the genus Leiurus. Leiurus macroctenus is a newly described species of scorpion distributed in Oman, and the toxicity of its venom is still unknown. Estimating the LD50 of the venom is the first and most important step in creating the antivenom and understanding the medical significance of the researched animal species. The purpose of this study was to determine the lethal dose (LD100), the maximum tolerated dose (LD0), and the average lethal dose (LD50) in rats when using Leiurus macroctenus scorpion venom. Methods and Results: 15 sexually mature scorpions were used in the study, which were kept in the same conditions and milked by a common method (electric milking). For the study, 60 male rats were used, which were injected intramuscularly with 0.5 ml of venom solution with a gradual increase in the dose (5 groups, 10 rats in each), and 10 rats were injected intramuscularly with physiological solution as control group. LD calculations were done using probit analysis method in the modification of the method by V.B. Prozorovsky. The LD0 of Leiurus macroctenus scorpion venom under the conditions of intramuscular injection was 0.02 mg/kg, LD100 was 0.13 mg/kg, and LD50 was 0.08 ± 0.01 mg/kg. Conclusions: The analysis of scientific publications and other sources of information gives reason to believe that Leiurus macroctenus has one of the highest values of LD50 not only among scorpions but also among all arthropods in the world. All these point to the significant clinical importance of this species of scorpion and require further research that will concern the toxic effect of its venom on various organ systems. Determining the LD50 of the venom for new scorpion species is crucial for creating effective antivenoms and understanding the medical implications of envenomation by this species.

Features of immune reactivity of the spleen and mechanisms of organ damage under the influence of animal venom toxins including scorpions (review).

OBJECTIVE: Aim: To establish features of immune reactivity of the spleen and mechanisms of organ damage under the influence of animal venom toxins including scorpions. PATIENTS AND METHODS: Materials and Methods: A thorough literature analysis was conducted on the basis of PubMed, Google Scholar, Web of Science, and Scopus databases. When processing the search results, we chose the newest publications up to 5 years old or the most thorough publications that vividly described the essence of our topic. CONCLUSION: Conclusions: Spleen plays a leading role in the implementation of the body's defense processes, the elimination of structural elements affected by toxins, and the restoration of immune homeostasis. Its participation in the formation of the immune response can be accompanied by qualitative and quantitative changes in histological organization. Morpho-functional changes in the spleen under the action of animal venom toxins currently require careful study, because from the information available in the literature today, it is not possible to clearly construct a complete picture of lesions of certain components of the organ at the microscopic or submicroscopic levels. Therefore, this direction of research in the medical field is currently relevant, taking into account the existence of a large number of poisonous animals, including scorpions.

[Site-specific PCR identification of animal species and formula particles of Scorpio].

Scorpio, a commonly used animal medicine in China, is derived from Buthus martensii as recorded in the Chinese Pharmacopoeia. China harbors rich species of Scorpionida and adulterants exist in the raw medicinal material and deep-processed products of Scorpio. The microscopic characteristics of the deep-processed products may be incomplete or lost during processing, which makes the identification difficult. In this study, the maximum likelihood(ML) tree was constructed based on the morphology and cytochrome C oxidase subunit I(COⅠ) to identify the species of Scorpio products. The results showed that the main adulterant of Scorpio was Lychas mucronatus. According to the specific SNP sites in the COⅠ sequence of B. martensii, the stable primers were designed for the identification of the medicinal material and formula granules of Scorpio. The polymerase chain reaction(PCR) at the annealing temperature of 61 ℃ and 30 cycles produced bright specific bands at about 150 bp for both B. martensii and its formula particles and no band for adulterants. The adaptability of the method was investigated, which showed that the bands at about 150 bp were produced for Scorpio medicinal material, lyophilized powder, and formula granules, and commercially available formula granules. The results showed that the established method could be used to identify the adulterants of Scorpio and its formula granules, which could help to improve the quality control system and ensure the safe clinical application of Scorpio formula granules.

[Research progress on chemical constituents and pharmacological activities of small molecule compounds in scorpions].

Scorpions, a group of oldest animals with wide distribution in the world, have a long history of medicinal use. Scorpio, the dried body of Buthus martensii, is a rare animal medicine mainly used for the treatment of liver diseases, spasm, and convulsions in children in China. The venom has been considered as the active substance of scorpions. However, little is known about the small molecules in the venom of scorpions. According to the articles published in recent years, scorpions contain amino acids, fatty acids, steroids, and alkaloids, which endow scorpions with antimicrobial, anticoagulant, metabolism-regulating, and antitumor activities. This paper summarizes the small molecule chemical components and pharmacological activities of scorpions, with a view to providing valuable information for the discovery of new active molecules and the clinical use of scorpions.

Genome-wide identification, structural homology analysis, and evolutionary diversification of the phospholipase D gene family in the venom gland of three scorpion species.

BACKGROUND: Venom phospholipase D (PLDs), dermonecrotic toxins like, are the major molecules in the crude venom of scorpions, which are mainly responsible for lethality and dermonecrotic lesions during scorpion envenoming. The purpose of this study was fivefold: First, to identify transcripts coding for venom PLDs by transcriptomic analysis of the venom glands from Androctonus crassicauda, Hottentotta saulcyi, and Hemiscorpius lepturus; second, to classify them by sequence similarity to known PLDs and motif extraction method; third, to characterize scorpion PLDs; fourth to structural homology analysis with known dermonecrotic toxins; and fifth to investigate phylogenetic relationships of the PLD proteins. RESULTS: We found that the venom gland of scorpions encodes two PLD isoforms: PLD1 ScoTox-beta and PLD2 ScoTox-alpha I. Two highly conserved regions shared by all PLD1s beta are GAN and HPCDC (HX2PCDC), and the most important conserved regions shared by all PLD2s alpha are two copies of the HKDG (HxKx4Dx6G) motif. We found that PLD1 beta is a 31-43 kDa acidic protein containing signal sequences, and PLD2 alpha is a 128 kDa basic protein without known signal sequences. The gene structures of PLD1 beta and PLD2 alpha contain 6 and 21 exons, respectively. Significant structural homology and similarities were found between the modeled PLD1 ScoTox-beta and the crystal structure of dermonecrotic toxins from Loxosceles intermedia. CONCLUSIONS: This is the first report on identifying PLDs from A. crassicauda and H. saulcyi venom glands. Our work provides valuable insights into the diversity of scorpion PLD genes and could be helpful in future studies on recombinant antivenoms production.

Varying Modes of Selection Among Toxin Families in the Venoms of the Giant Desert Hairy Scorpions (Hadrurus).

Venoms are primarily believed to evolve under strong diversifying selection resulting from persistent coevolution between predator and prey. Recent research has challenged this hypothesis, proposing that venoms from younger venomous lineages (e.g., snakes and cone snails) are governed predominantly by diversifying selection, while venoms from older venomous lineages (e.g., centipedes, scorpions, and spiders) are under stronger purifying selection. However, most research in older lineages has tested selection at more diverse phylogenetic scales. Although these tests are important for evaluating broad macroevolutionary trends underlying venom evolution, they are less equipped to detect species-level evolutionary trends, which likely have large impacts on venom variation seen at more diverse phylogenetic scales. To test for selection among closely related species from an older venomous lineage, we generated high-throughput venom-gland transcriptomes and venom proteomes for four populations of Giant Desert Hairy Scorpions (Hadrurus), including three Hadrurus arizonensis populations and one Hadrurus spadix population. We detected significant episodic and pervasive diversifying selection across a highly abundant toxin family that likely has a major role in venom function ([Formula: see text]KTxs), providing a contrast to the stronger purifying selection identified from other studies on scorpion venoms. Conversely, we detected weak episodic diversifying and/or stronger purifying selection in four toxin families (non-disulfide bridged peptides, phospholipase A2s, scorpine-like antimicrobial peptides, and serine proteases), most of which were less abundant and likely have ancillary functional roles. Finally, although we detected several major toxin families at disproportionate transcriptomic and/or proteomic abundances, we did not identify significant sex-based variation in Hadrurus venoms.

Old and cold: Diverse phylogenomic datasets support an ancient transantarctic dispersive route on the scorpion family Bothriuridae in temperate Gondwana.

In this contribution we try to unveil the diversification process of Bothriuridae in temperate Gondwana through dated phylogenomic analyses using UCE and transcriptomics, and including in the analyses species of genera Urophonius and Cercophonius, the most closely related genera of Bothriuridae from South America and Australia respectively. Additionally we explored the hypothesis that the winter activity period of some species of Urophonius, as well as the cold environmental preferences of this genus, could be related to the climatic conditions of the time frame and area in which it evolved. Genus Urophonius was recovered as sister group to Cercophonius using amino acids and UCE. The time frame obtained for the split between South American and Australian bothriurids is 94 Ma., which suggests a dispersal event through temperate Gondwana, before the final breakup of the land bridge of South America-Antarctica-Australia ca. 35 Ma. The split between summer and winter species of Urophonius, taking place at 64 Ma, is considered representative to the turnover time from the summer activity period to the winter activity period in some species of the genus. This time frame is compatible with a period of global warming of the late Cretaceous greenhouse episode that could have triggered this change.

Phylogenomics of Scorpions Reveal Contemporaneous Diversification of Scorpion Mammalian Predators and Mammal-Active Sodium Channel Toxins.

Scorpions constitute a charismatic lineage of arthropods and comprise more than 2500 described species. Found throughout various tropical and temperate habitats, these predatory arachnids have a long evolutionary history, with a fossil record that began in the Silurian. While all scorpions are venomous, the asymmetrically diverse family Buthidae harbors nearly half the diversity of extant scorpions, and all but one of the 58 species that are medically significant to humans. However, the lack of a densely sampled scorpion phylogeny has hindered broader inferences of the diversification dynamics of scorpion toxins. To redress this gap, we assembled a phylogenomic data set of 100 scorpion venom gland transcriptomes and genomes, emphasizing the sampling of highly toxic buthid genera. To infer divergence times of venom gene families, we applied a phylogenomic node dating approach for the species tree in tandem with phylostratigraphic bracketing to estimate the minimum ages of mammal-specific toxins. Our analyses establish a robustly supported phylogeny of scorpions, particularly with regard to relationships between medically significant taxa. Analysis of venom gene families shows that mammal-active sodium channel toxins (NaTx) have independently evolved in five lineages within Buthidae. Temporal windows of mammal-targeting toxin origins are correlated with the basal diversification of major scorpion mammal predators such as shrews, bats, and rodents. These results suggest an evolutionary model of relatively recent diversification of buthid NaTx homologs in response to the diversification of scorpion predators. [Adaptation; arachnids; phylogenomic dating; phylostratigraphy; venom.].

Comparative biology of spatial navigation in three arachnid orders (Amblypygi, Araneae, and Scorpiones).

From both comparative biology and translational research perspectives, there is escalating interest in understanding how animals navigate their environments. Considerable work is being directed towards understanding the sensory transduction and neural processing of environmental stimuli that guide animals to, for example, food and shelter. While much has been learned about the spatial orientation behavior, sensory cues, and neurophysiology of champion navigators such as bees and ants, many other, often overlooked animal species possess extraordinary sensory and spatial capabilities that can broaden our understanding of the behavioral and neural mechanisms of animal navigation. For example, arachnids are predators that often return to retreats after hunting excursions. Many of these arachnid central-place foragers are large and highly conducive to scientific investigation. In this review we highlight research on three orders within the Class Arachnida: Amblypygi (whip spiders), Araneae (spiders), and Scorpiones (scorpions). For each, we describe (I) their natural history and spatial navigation, (II) how they sense the world, (III) what information they use to navigate, and (IV) how they process information for navigation. We discuss similarities and differences among the groups and highlight potential avenues for future research.

Congruence between ultraconserved element-based matrices and phylotranscriptomic datasets in the scorpion Tree of Life.

Scorpions are ancient and historically renowned for their potent venom. Traditionally, the systematics of this group of arthropods was supported by morphological characters, until recent phylogenomic analyses (using RNAseq data) revealed most of the higher-level taxa to be non-monophyletic. While these phylogenomic hypotheses are stable for almost all lineages, some nodes have been hard to resolve due to minimal taxonomic sampling (e.g. family Chactidae). In the same line, it has been shown that some nodes in the Arachnid Tree of Life show disagreement between hypotheses generated using transcritptomes and other genomic sources such as the ultraconserved elements (UCEs). Here, we compared the phylogenetic signal of transcriptomes vs. UCEs by retrieving UCEs from new and previously published scorpion transcriptomes and genomes, and reconstructed phylogenies using both datasets independently. We reexamined the monophyly and phylogenetic placement of Chactidae, sampling an additional chactid species using both datasets. Our results showed that both sets of genome-scale datasets recovered highly similar topologies, with Chactidae rendered paraphyletic owing to the placement of Nullibrotheas allenii. As a first step toward redressing the systematics of Chactidae, we establish the family Anuroctonidae (new family) to accommodate the genus Anuroctonus.

Animals evoking fear in the Cradle of Humankind: snakes, scorpions, and large carnivores.

Theories explain the presence of fears and specific phobias elicited by animals in contemporary WEIRD (Western, educated, industrialized, rich, and democratic) populations by their evolutionary past in Africa. Nevertheless, empirical data about fears of animals in the Cradle of Humankind are still fragmentary. To fill this gap, we examined which local animals are perceived as the most frightening by Somali people, who inhabit a markedly similar environment and the region where humans have evolved. We asked 236 raters to rank 42 stimuli according to their elicited fear. The stimuli were standardized pictures of species representing the local fauna. The results showed that the most frightening animals were snakes, scorpions, the centipede, and large carnivores (cheetahs and hyenas). These were followed up by lizards and spiders. Unlike in Europe, spiders represent less salient stimuli than scorpions for Somali respondents in this study. This conforms to the hypothesis suggesting that fear of spiders was extended or redirected from other chelicerates.