Venom gland transcriptomic and proteomic analyses have improved our knowledge on the diversity of the heterogeneous components present in scorpion venoms. However, most of these studies have focused on species from the family Buthidae. To gain insights into the molecular diversity of the venom components of scorpions belonging to the family Superstitioniidae, one of the neglected scorpion families, we performed a transcriptomic and proteomic analyses for the species Superstitionia donensis. The total mRNA extracted from the venom glands of two specimens was subjected to massive sequencing by the Illumina protocol, and a total of 219,073 transcripts were generated. We annotated 135 transcripts putatively coding for peptides with identity to known venom components available from different protein databases. Fresh venom collected by electrostimulation was analyzed by LC-MS/MS allowing the identification of 26 distinct components with sequences matching counterparts from the transcriptomic analysis. In addition, the phylogenetic affinities of the found putative calcins, scorpines, La1-like peptides and potassium channel κ toxins were analyzed. The first three components are often reported as ubiquitous in the venom of different families of scorpions. Our results suggest that, at least calcins and scorpines, could be used as molecular markers in phylogenetic studies of scorpion venoms.

Venom glands and soluble venom from the Mexican scorpion Centruroides limpidus (Karsch, 1879) were used for transcriptomic and proteomic analyses, respectively. An RNA-seq was performed by high-throughput sequencing with the Illumina platform. Approximately 80 million reads were obtained and assembled into 198,662 putative transcripts, of which 11,058 were annotated by similarity to sequences from available databases. A total of 192 venom-related sequences were identified, including Na⁺ and K⁺ channel-acting toxins, enzymes, host defense peptides, and other venom components. The most diverse transcripts were those potentially coding for ion channel-acting toxins, mainly those active on Na⁺ channels (NaScTx). Sequences corresponding to β- scorpion toxins active of K⁺ channels (KScTx) and λ-KScTx are here reported for the first time for a scorpion of the genus Centruroides. Mass fingerprint corroborated that NaScTx are the most abundant components in this venom. Liquid chromatography coupled to mass spectometry (LC-MS/MS) allowed the identification of 46 peptides matching sequences encoded in the transcriptome, confirming their expression in the venom. This study corroborates that, in the venom of toxic buthid scorpions, the more abundant and diverse components are ion channel-acting toxins, mainly NaScTx, while they lack the HDP diversity previously demonstrated for the non-buthid scorpions. The highly abundant and diverse antareases explain the pancreatitis observed after envenomation by this species.

Centruroides baergi is a scorpion species distributed in the biogeographical province of the Balsas Basin in Mexico. Health officials have reported acute envenomation in human populations living on the western side of this scorpion's range, but none in the eastern region. This disparity in toxicity suggested that there may be two distinct species. We used two different approaches, including venom analysis and morphometric specimens from both regions, to test our hypothesis. We performed chromatographic, electrophoretic, and mass spectrometry analysis to identify the known β-toxins involved in the intoxication. The most remarkable finding was the absence of Cb3 β-toxin in the eastern population. Consequently, the LD₅₀ of the eastern population was lower than that of the western population. We analyzed linear and ratio body measurements with parametric and nonparametric statistics to test species limits. These analyses indicated that all putative populations of C. baergi are significantly similar, suggesting that they may represent a single species. Unexpectedly, the population of scorpions in the center of the study area, Suchixtlahuaca, previously identified as C. baergi, showed significant morphological and venom composition differences. We provided empirical evidence of an abrupt change of highly toxic peptides around the 98th meridian that limits populations of Centruroides baergi to the east of Mexico.

From the monotypic family Superstitioniidae, the scorpion Superstitionia donensis presents a high diversity of transcripts encoding non-disulfide-bridged peptides (NDBPs). In this study, five peptide sequences inferred from a previous venom gland transcriptomic analysis were selected based on sequence similarity and physicochemical properties associated with previously antimicrobial peptides. An additional peptide was designed to evaluate the effect of changes on selected physicochemical properties on its hemolytic and antimicrobial activity. The selected peptides were synthesized using Merrifield Solid-Phase peptide synthesis and evaluated form antimicrobial activity against Gram-negative and Gram-positive bacteria, belonging to the ESKAPE group and yeasts of the Candida and Cryptococcus genera, as well as for hemolytic and cytotoxic activity in mammalian cells. Some peptides showed antimicrobial activity against selected microbial strains, including Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Candida krusei, and Candida tropicalis, while limited or no activity was observed against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida glabrata. Notably, treatment with some selected peptides improved the survival of Galleria mellonella larvae infected with S. aureus, supporting their functionality in vivo. Peptide toxicity was also evaluated in human erythrocytes and two human cell lines, showing that some peptides with antimicrobial activity also exhibited hemolytic effects; however, they showed low cytotoxicity to cell lines at antimicrobial concentrations. Despite observed hemolytic activity, the relatively low cytotoxicity against human cell lines suggests potential for improvement through structural refinement. These results demonstrate that S. donensis NDBPs exhibit antimicrobial activity and hemolytic effect, and their sequences may serve as templates for further modification. Furthermore, the findings reveal that antimicrobial potency and cytotoxicity are influenced by sequence similarity, net charge, and hydrophobicity. Nevertheless, the physicochemical property values required to identify an optimal antimicrobial peptide remain poorly defined, as several parameters exhibit overlap between high- and low-performing candidates, which highlights a key challenge for their therapeutic development.