RESUMEN
Gene duplication followed by nucleotide differentiation is one of the simplest mechanisms to develop new functions for genes. However, the evolutionary processes underlying the divergence of multigene families remain controversial. We used multigene families found within the diversity of toxic proteins in centipede venom to test two hypotheses related to venom evolution: the two-speed mode of venom evolution and the rapid accumulation of variation in exposed residues (RAVER) model. The two-speed mode of venom evolution proposes that different types of selection impact ancient and younger venomous lineages with negative selection being the predominant form in ancient lineages and positive selection being the dominant form in younger lineages. The RAVER hypothesis proposes that, instead of different types of selection acting on different ages of venomous lineages, the different types of selection will selectively contribute to amino acid variation based on whether the residue is exposed to the solvent where it can potentially interact directly with toxin targets. This hypothesis parallels the longstanding understanding of protein evolution that suggests that residues found within the structural or active regions of the protein will be under negative or purifying selection, and residues that do not form part of these areas will be more prone to positive selection. To test these two hypotheses, we compared the venom of 26 centipedes from the order Scolopendromorpha from six currently recognized species from across North America using both transcriptomics and proteomics. We first estimated their phylogenetic relationships and uncovered paraphyly among the genus Scolopendra and evidence for cryptic diversity among currently recognized species. Using our phylogeny, we then characterized the diverse venom components from across the identified clades using a combination of transcriptomics and proteomics. We conducted selection-based analyses in the context of predicted three-dimensional properties of the venom proteins and found support for both hypotheses. Consistent with the two-speed hypothesis, we found a prevalence of negative selection across all proteins. Consistent with the RAVER hypothesis, we found evidence of positive selection on solvent-exposed residues, with structural and less-exposed residues showing stronger signal for negative selection. Through the use of phylogenetics, transcriptomics, proteomics, and selection-based analyses, we were able to describe the evolution of venom from an ancient venomous lineage and support principles of protein evolution that directly relate to multigene family evolution.
Asunto(s)
Artrópodos , Evolución Molecular , Filogenia , Selección Genética , Animales , Artrópodos/genética , Venenos de Artrópodos/genética , Venenos de Artrópodos/química , Familia de Multigenes , Ponzoñas/genética , Ponzoñas/química , América del Norte , Duplicación de Gen , Modelos Moleculares , Conformación ProteicaRESUMEN
Developmental phenotypic changes can evolve under selection imposed by age- and size-related ecological differences. Many of these changes occur through programmed alterations to gene expression patterns, but the molecular mechanisms and gene-regulatory networks underlying these adaptive changes remain poorly understood. Many venomous snakes, including the eastern diamondback rattlesnake (Crotalus adamanteus), undergo correlated changes in diet and venom expression as snakes grow larger with age, providing models for identifying mechanisms of timed expression changes that underlie adaptive life history traits. By combining a highly contiguous, chromosome-level genome assembly with measures of expression, chromatin accessibility, and histone modifications, we identified cis-regulatory elements and trans-regulatory factors controlling venom ontogeny in the venom glands of C. adamanteus. Ontogenetic expression changes were significantly correlated with epigenomic changes within genes, immediately adjacent to genes (e.g., promoters), and more distant from genes (e.g., enhancers). We identified 37 candidate transcription factors (TFs), with the vast majority being up-regulated in adults. The ontogenetic change is largely driven by an increase in the expression of TFs associated with growth signaling, transcriptional activation, and circadian rhythm/biological timing systems in adults with corresponding epigenomic changes near the differentially expressed venom genes. However, both expression activation and repression contributed to the composition of both adult and juvenile venoms, demonstrating the complexity and potential evolvability of gene regulation for this trait. Overall, given that age-based trait variation is common across the tree of life, we provide a framework for understanding gene-regulatory-network-driven life-history evolution more broadly.
Asunto(s)
Venenos de Crotálidos , Serpientes Venenosas , Animales , Venenos de Crotálidos/genética , Venenos de Crotálidos/metabolismo , Epigenómica , Crotalus/genética , Crotalus/metabolismoRESUMEN
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.
Asunto(s)
Escorpiones , Ponzoñas , Animales , Ponzoñas/genética , Escorpiones/genética , Filogenia , Proteómica/métodosRESUMEN
Research on centipede venoms has led to the discovery of a diverse array of novel proteins and peptides, including those with homology to previously discovered toxin families (e.g., phospholipase A2s and pM12a metalloproteases) and novel toxin families not previously detected in venoms (e.g., ß-pore forming toxins and scoloptoxins). Most of this research has focused on centipedes in the order Scolopendromorpha, particularly those in the families Scolopendridae, Cryptopidae, and Scolopocryptopidae. To generate the first high-throughput venom characterization for a centipede in the scolopendromorph family Plutoniumidae, we performed venom-gland transcriptomics and venom proteomics on two Theatops posticus. We identified a total of 64 venom toxins, 60 of which were detected in both the venom-gland transcriptome and venom proteome and four of which were only detected transcriptomically. We detected a single highly abundant arylsulfatase B (ARSB) toxin, the first ARSB toxin identified from centipede venoms. As ARSBs have been detected in other venomous species (e.g., scorpions), ARSBs in T. posticus highlights a new case of convergent evolution across venoms. Theatops posticus venom also contained a much higher abundance and diversity of phospholipase A2 toxins compared to other characterized centipede venoms. Conversely, we detected other common centipedes toxins, such as CAPs and scoloptoxins, at relatively low abundances and diversities. Our observation of a diverse set of toxins from T. posticus venom, including those from novel toxin families, emphasizes the importance of studying unexplored centipede taxonomic groups and the continued potential of centipede venoms for novel toxin discovery and unraveling the molecular mechanisms underlying trait evolution.
Asunto(s)
Venenos de Artrópodos , Artrópodos , Animales , Quilópodos/metabolismo , Artrópodos/química , Arilsulfatasas/metabolismo , Fosfolipasas/metabolismo , Venenos de Artrópodos/química , TranscriptomaRESUMEN
Biological specialization reduces the size of niche space while increasing efficiency in the use of available resources. Specialization often leads to phenotypic changes via natural selection aligning with niche space constraints. Commonly observed changes are in size, shape, behavior, and traits associated with feeding. One often selected trait for dietary specialization is venom, which, in snakes, often shows variation dependent on diet across and within species. The Neotropical Blunt-headed Treesnake (Imantodes cenchoa) is a highly specialized, rear-fanged, arboreal, lizard hunter that displays a long thin body, enlarged eyes, and a large Duvernoy's gland. However, toxin characterization of I. cenchoa has never been completed. Here, we use RNA-seq and mass spectrometry to assemble, annotate, and analyze the venom gland transcriptomes of four I. cenchoa from across their range. We find a lack of significant venom variation at the sequence and expression levels, suggesting venom conservation across the species. We propose this conservation provides evidence of a specialized venom repertoire, adapted to maximize efficiency of capturing and processing lizards. Importantly, this study provides the most complete venom gland transcriptomes of I. cenchoa and evidence of venom specialization in a rear-fanged snake, giving insight into selective pressures of venom across all snake species.
Asunto(s)
Colubridae , Lagartos , Toxinas Biológicas , Animales , Venenos de Serpiente/química , Lagartos/metabolismo , Colubridae/genética , Colubridae/metabolismo , Toxinas Biológicas/metabolismo , FenotipoRESUMEN
Scorpion venoms have long been studied for their peptide discovery potential, with modern high-throughput venom-characterization techniques paving the way for the discovery of thousands of novel putative toxins. Research into these toxins has provided insight into the pathology and treatment of human diseases, even resulting in the development of one compound with Food and Drug Administration (FDA) approval. Although most of this research has focused on the toxins of scorpion species considered medically significant to humans, the venom of harmless scorpion species possess toxins that are homologous to those from medically significant species, indicating that harmless scorpion venoms may also serve as valuable sources of novel peptide variants. Furthermore, as harmless scorpions represent a vast majority of scorpion species diversity, and therefore venom toxin diversity, venoms from these species likely contain entirely new toxin classes. We sequenced the venom-gland transcriptome and venom proteome of two male Big Bend scorpions (Diplocentrus whitei), providing the first high-throughput venom characterization for a member of this genus. We identified a total of 82 toxins in the venom of D. whitei, 25 of which were identified in both the transcriptome and proteome, and 57 of which were only identified in the transcriptome. Furthermore, we identified a unique, enzyme-rich venom dominated by serine proteases and the first arylsulfatase B toxins identified in scorpions.
Asunto(s)
Venenos de Escorpión , Escorpiones , Animales , Humanos , Masculino , Proteoma , Transcriptoma , Péptidos/química , Venenos de Escorpión/toxicidad , Venenos de Escorpión/químicaRESUMEN
As biochemical traits with clear fitness consequences, venoms serve a critical ecological role for the animals that produce them. Understanding how venoms are maintained and regenerated after use will, therefore, provide valuable insight into the ecology of venomous animals. Furthermore, most studies on venomous organisms often require removing animals from the wild and waiting extended periods of time between venom extractions. Uncovering the patterns of venom regeneration across different species will likely lead to the development of more efficient venom extraction protocols, reducing both experimental time and the number of animals required. Using reversed-phase high-performance liquid chromatography, we identified asynchronous regeneration of venom protein component abundances in the centipede Scolopendra viridis, but found no evidence for asynchronous venom regeneration in the scorpion Centruroides hentzi. We also observed high levels of intraspecific venom variation in C. hentzi, emphasizing the importance of testing for intraspecific venom variation in studies evaluating the synchronicity of venom regeneration. Although the regeneration of relative venom protein component abundances is an asynchronous process in S. viridis, we provide evidence that the presence-absence of major venom components is not an asynchronous process and suggest that studies relying on just the presence-absence of individual proteins (e.g. bioprospecting, drug discovery) could use catch-and-release methods of venom extraction to reduce the number of animals removed from the wild.
Asunto(s)
Venenos de Escorpión , Escorpiones , Alérgenos , Animales , Quilópodos , Regeneración , Venenos de Escorpión/química , PonzoñasRESUMEN
The role of natural selection in the evolution of trait complexity can be characterized by testing hypothesized links between complex forms and their functions across species. Predatory venoms are composed of multiple proteins that collectively function to incapacitate prey. Venom complexity fluctuates over evolutionary timescales, with apparent increases and decreases in complexity, and yet the causes of this variation are unclear. We tested alternative hypotheses linking venom complexity and ecological sources of selection from diet in the largest clade of front-fanged venomous snakes in North America: the rattlesnakes, copperheads, cantils, and cottonmouths. We generated independent transcriptomic and proteomic measures of venom complexity and collated several natural history studies to quantify dietary variation. We then constructed genome-scale phylogenies for these snakes for comparative analyses. Strikingly, prey phylogenetic diversity was more strongly correlated to venom complexity than was overall prey species diversity, specifically implicating prey species' divergence, rather than the number of lineages alone, in the evolution of complexity. Prey phylogenetic diversity further predicted transcriptomic complexity of three of the four largest gene families in viper venom, showing that complexity evolution is a concerted response among many independent gene families. We suggest that the phylogenetic diversity of prey measures functionally relevant divergence in the targets of venom, a claim supported by sequence diversity in the coagulation cascade targets of venom. Our results support the general concept that the diversity of species in an ecological community is more important than their overall number in determining evolutionary patterns in predator trait complexity.
Asunto(s)
Crotalinae/genética , Dieta/tendencias , Venenos de Serpiente/genética , Adaptación Biológica/genética , Animales , Crotalinae/metabolismo , Dieta/veterinaria , Expresión Génica/genética , América del Norte , Filogenia , Conducta Predatoria/fisiología , Proteómica/métodos , Selección Genética/genética , Venenos de Serpiente/metabolismo , Diente/metabolismo , Transcriptoma/genéticaRESUMEN
MOTIVATION: Next-generation sequencing has become exceedingly common and has transformed our ability to explore nonmodel systems. In particular, transcriptomics has facilitated the study of venom and evolution of toxins in venomous lineages; however, many challenges remain. Primarily, annotation of toxins in the transcriptome is a laborious and time-consuming task. Current annotation software often fails to predict the correct coding sequence and overestimates the number of toxins present in the transcriptome. Here, we present ToxCodAn, a python script designed to perform precise annotation of snake venom gland transcriptomes. We test ToxCodAn with a set of previously curated transcriptomes and compare the results to other annotators. In addition, we provide a guide for venom gland transcriptomics to facilitate future research and use Bothrops alternatus as a case study for ToxCodAn and our guide. RESULTS: Our analysis reveals that ToxCodAn provides precise annotation of toxins present in the transcriptome of venom glands of snakes. Comparison with other annotators demonstrates that ToxCodAn has better performance with regard to run time ($>20x$ faster), coding sequence prediction ($>3x$ more accurate) and the number of toxins predicted (generating $>4x$ less false positives). In this sense, ToxCodAn is a valuable resource for toxin annotation. The ToxCodAn framework can be expanded in the future to work with other venomous lineages and detect novel toxins.
Asunto(s)
Algoritmos , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Venenos de Serpiente/genética , Serpientes/genética , Toxinas Biológicas/genética , Animales , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Filogenia , Venenos de Serpiente/química , Venenos de Serpiente/metabolismo , Serpientes/clasificación , Serpientes/metabolismo , Especificidad de la Especie , Toxinas Biológicas/química , Toxinas Biológicas/metabolismoRESUMEN
Variation in gene regulation is ubiquitous, yet identifying the mechanisms producing such variation, especially for complex traits, is challenging. Snake venoms provide a model system for studying the phenotypic impacts of regulatory variation in complex traits because of their genetic tractability. Here, we sequence the genome of the Tiger Rattlesnake, which possesses the simplest and most toxic venom of any rattlesnake species, to determine whether the simple venom phenotype is the result of a simple genotype through gene loss or a complex genotype mediated through regulatory mechanisms. We generate the most contiguous snake-genome assembly to date and use this genome to show that gene loss, chromatin accessibility, and methylation levels all contribute to the production of the simplest, most toxic rattlesnake venom. We provide the most complete characterization of the venom gene-regulatory network to date and identify key mechanisms mediating phenotypic variation across a polygenic regulatory network.
Asunto(s)
Venenos de Crotálidos/genética , Crotalus/genética , Genoma/genética , Anotación de Secuencia Molecular , Animales , Regulación de la Expresión Génica/genética , Genotipo , Transcriptoma/genética , Secuenciación Completa del GenomaRESUMEN
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.
Asunto(s)
Tamaño Corporal/genética , Bothrops/genética , Venenos de Crotálidos/genética , Proteómica/métodos , Transcriptoma/genética , Animales , Venenos de Crotálidos/análisis , Venenos de Crotálidos/química , Femenino , Ontología de Genes , Masculino , Análisis de Secuencia de ADN/métodosRESUMEN
Ontogenetic shifts in venom occur in many snakes but establishing their nature as gradual or discrete processes required additional study. We profiled shifts in venom expression from the neonate to adult sizes of two rattlesnake species, the eastern diamondback and the timber rattlesnake. We used serial sampling and venom chromatographic profiling to test if ontogenetic change occurs gradually or discretely. We found evidence for gradual shifts in overall venom composition in six of eight snakes, which sometimes spanned more than two years. Most chromatographic peaks shift gradually, but one quarter shift in a discrete fashion. Analysis of published diet data showed gradual shifts in overall diet composition across the range of body sizes attained by our eight study animals, while the shifts in abundance of different prey classes varied in form from gradual to discrete. Testosterone concentrations were correlated with the change in venom protein composition, but the relationship is not strong enough to suggest causation. Venom research employing simple juvenile versus adult size thresholds may be failing to account for continuous variation in venom composition lifespan. Our results imply that venom shifts represent adaptive matches to dietary shifts and highlight venom for studies of alternative gene regulatory mechanisms.
Asunto(s)
Venenos de Crotálidos/metabolismo , Crotalus/metabolismo , Ecosistema , Proteínas de Reptiles/metabolismo , Testosterona/metabolismo , Factores de Edad , Animales , Tamaño Corporal , Venenos de Crotálidos/genética , Crotalus/genética , Crotalus/crecimiento & desarrollo , Dieta , Regulación del Desarrollo de la Expresión Génica , Proteínas de Reptiles/genéticaRESUMEN
Sexually dimorphic traits are widespread across metazoans and are often the result of sex-specific inheritance or sex-based differences in gene expression. Intersexual differences have even been observed in invertebrate venoms, although the identification of these differences has been limited to the more well-studied groups, such as scorpions and spiders, where sex-based differences in morphology and behavior are apparent. Recent studies on centipede venom have identified evidence of intraspecific variation, but intersexual differences have not been reported. To investigate the potential for sex-based differences in centipede venom composition, we performed reversed-phase high performance liquid chromatography (RP-HPLC) analyses on five male and 15 female eastern bark centipedes (Hemiscolopendra marginata) from the Apalachicola National Forest in northern Florida. After detecting a significant sex-based difference in H. marginata venom composition, we completed a high-throughput venom-gland transcriptomic and venom proteomic analysis of one male and one female to determine the genetic basis for differences in venom composition. We identified 47 proteomically confirmed toxins and 717 nontoxin transcripts in H. marginata venom-glands. Of these proteomically confirmed toxins, the most abundantly expressed in the male venom included ion channel-modulating toxins and toxins so divergent from any characterized homologs that they could not be given a functional classification, whereas the most abundantly expressed in the female venom were γ-glutamyl transferases and CAPs (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins). These differences were then confirmed by performing replicate LC-MS/MS analyses on the venom from an additional three male and three female H. marginata. Our RP-HPLC and high-throughput transcriptomic and proteomic approach resulted in not only an in-depth characterization of H. marginata venom, but represents the first example of sex-based variation in centipede venoms.
Asunto(s)
Venenos de Artrópodos/química , Artrópodos/química , Caracteres Sexuales , Animales , Proteínas de Artrópodos/química , Venenos de Artrópodos/genética , Artrópodos/genética , Cromatografía Líquida de Alta Presión , Femenino , Masculino , Análisis de Componente Principal , Proteómica , TranscriptomaRESUMEN
Many venom proteins have presumably been convergently recruited by taxa from diverse venomous lineages. These toxic proteins have characteristics that allow them to remain stable in solution and have a high propensity for toxic effects on prey and/or potential predators. Despite this well-established convergent toxin recruitment, some toxins seem to be lineage specific. To further investigate the toxic proteins found throughout venomous lineages, venom proteomics and venom-gland transcriptomics were performed on two individual red bark centipedes (Scolopocryptops sexspinosus). Combining the protein phenotype with the transcript genotype resulted in the first in-depth venom characterization of S. sexspinosus, including 72 venom components that were identified in both the transcriptome and proteome and 1468 nontoxin transcripts identified in the transcriptome. Ten different toxin families were represented in the venom and venom gland with the majority of the toxins belonging to metalloproteases, CAPS (cysteine-rich secretory protein, antigen 5, and pathogenesis-related 1 proteins), and ß-pore-forming toxins. Nine of these toxin families shared a similar proteomic structure to venom proteins previously identified from other centipedes. However, the most highly expressed toxin family, the adamalysin-like metalloproteases, has until now only been observed in the venom of snakes. We confirmed adamalysin-like metalloprotease activity by means of in vivo functional assays. The recruitment of an adamalysin-like metalloprotease into centipede venom represents a striking case of convergent evolution.
Asunto(s)
Venenos de Artrópodos/enzimología , Artrópodos/enzimología , Artrópodos/genética , Metaloproteasas/química , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Venenos de Artrópodos/genética , Evolución Molecular , Metaloproteasas/genética , Proteoma , TranscriptomaRESUMEN
Sex-biased genes are expressed at higher levels in one sex and contribute to phenotypic differences between males and females, as well as overall phenotypic variation within and among populations. Venom has evolved primarily for predation and defense, making venom expression a highly variable phenotype as a result of local adaptation. Several scorpion species have shown both intraspecific and intersexual venom variation, and males have been observed using venom in courtship and mating, suggesting the existence of venom-specific, sex-biased genes that may contribute to population divergence. We used reversed-phase high-performance liquid chromatography (RP-HPLC), Agilent protein bioanalyzer chips, nano-liquid chromatography mass spectrometry (nLC/MS/MS), and median lethal dose (LD50) assays in fruit flies (Drosophila melanogaster) and banded crickets (Gryllodes sigillatus) to investigate proteomic and functional venom variation within and among three Florida populations of the Hentz striped scorpion (Centruroides hentzi). We found significant venom variation among populations, with females, not males, being responsible for this divergence. We also found significant variation in venom expression within populations, with males contributing more to within population variation than females. Our results provide evidence that male and female scorpions experience different natural and sexual selective pressures that have led to the expression of sex-biased venom genes and that these genes may be consequential in population divergence.
Asunto(s)
Venenos de Escorpión/química , Venenos de Escorpión/toxicidad , Escorpiones/metabolismo , Animales , Drosophila melanogaster , Femenino , Variación Genética , Gryllidae , Dosificación Letal Mediana , Masculino , Proteoma , Venenos de Escorpión/genética , Escorpiones/genética , Factores SexualesRESUMEN
Scorpions are an ancient and diverse venomous lineage, with over 2200 currently recognized species. Only a small fraction of scorpion species are considered harmful to humans, but the often life-threatening symptoms caused by a single sting are significant enough to recognize scorpionism as a global health problem. The continued discovery and classification of new species has led to a steady increase in the number of both harmful and harmless scorpion species. The purpose of this review is to update the global record of medically significant scorpion species, assigning each to a recognized sting class based on reported symptoms, and provide the major toxin classes identified in their venoms. We also aim to shed light on the harmless species that, although not a threat to human health, should still be considered medically relevant for their potential in therapeutic development. Included in our review is discussion of the many contributing factors that may cause error in epidemiological estimations and in the determination of medically significant scorpion species, and we provide suggestions for future scorpion research that will aid in overcoming these errors.
Asunto(s)
Distribución Animal , Venenos de Escorpión/química , Escorpiones/clasificación , Escorpiones/fisiología , Toxinas Biológicas/química , Animales , BiodiversidadRESUMEN
Of the 14 extant scorpion families, Buthidae has the most thoroughly characterized venoms. Most of this characterization, however, has been limited to species with medically significant stings, including members of the Centruroides genus, which have caused human deaths (e.g., Centruroides sculpturatus). To understand the origin and evolution of highly toxic venoms, we should also characterize the more harmless venoms of close relatives. We used Illumina sequencing to separately characterize the venom-gland transcriptomes of a male and female Hentz striped scorpion (Centruroides hentzi) and performed independent quantitative mass-spectrometry analysis of the venom from each individual, providing the first full venom characterization of a Centruroides species that poses no serious threat to humans. We identified 59 venom proteins that were proteomically confirmed, 63 additional transcripts that were identified on the basis of homology to known toxins, and 355 nontoxins expressed in the venom-glands. The most abundant toxins belonged to the Na+ and K+-channel toxin classes. Antimicrobial peptides and peptidases were also identified, along with a large group of venom proteins that could not be classified based on homology, suggesting C. hentzi is a source of previously untapped toxin diversity.