RESUMO
Molecular genetic data have recently been incorporated in attempts to reconstruct the ecology of the ancestral snake, though this has been limited by a paucity of data for one of the two main extant snake taxa, the highly fossorial Scolecophidia. Here we present and analyze vision genes from the first eye-transcriptomic and genome-wide data for Scolecophidia, for Anilios bicolor, and A. bituberculatus, respectively. We also present immunohistochemistry data for retinal anatomy and visual opsin-gene expression in Anilios. Analyzed in the context of 19 lepidosaurian genomes and 12 eye transcriptomes, the new genome-wide and transcriptomic data provide evidence for a much more reduced visual system in Anilios than in non-scolecophidian (=alethinophidian) snakes and in lizards. In Anilios, there is no evidence of the presence of 7 of the 12 genes associated with alethinophidian photopic (cone) phototransduction. This indicates extensive gene loss and many of these candidate gene losses occur also in highly fossorial mammals with reduced vision. Although recent phylogenetic studies have found evidence for scolecophidian paraphyly, the loss in Anilios of visual genes that are present in alethinophidians implies that the ancestral snake had a better-developed visual system than is known for any extant scolecophidian.
Assuntos
Lagartos , Transcriptoma , Animais , Evolução Molecular , Lagartos/genética , Mamíferos/genética , Opsinas/genética , Filogenia , Serpentes/genéticaRESUMO
Scorpion venoms are mixtures of proteins, peptides and small molecular compounds with high specificity for ion channels and are therefore considered to be promising candidates in the venoms-to-drugs pipeline. Transcriptomes are important tools for studying the composition and expression of scorpion venom. Unfortunately, studying the venom gland transcriptome traditionally requires sacrificing the animal and therefore is always a single snapshot in time. This paper describes a new way of generating a scorpion venom gland transcriptome without sacrificing the animal, thereby allowing the study of the transcriptome at various time points within a single individual. By comparing these venom-derived transcriptomes to the traditional whole-telson transcriptomes we show that the relative expression levels of the major toxin classes are similar. We further performed a multi-day extraction using our proposed method to show the possibility of doing a multiple time point transcriptome analysis. This allows for the study of patterns of toxin gene activation over time a single individual, and allows assessment of the effects of diet, season and other factors that are known or likely to influence intraindividual venom composition. We discuss the gland characteristics that may allow this method to be successful in scorpions and provide a review of other venomous taxa to which this method may potentially be successfully applied.
Assuntos
Peptídeos/genética , Venenos de Escorpião/genética , Escorpiões/genética , Transcriptoma/genética , Sequência de Aminoácidos/genética , Animais , Perfilação da Expressão Gênica , Peptídeos/classificação , Glândulas Salivares/metabolismoRESUMO
More than 400,000 people each year suffer adverse effects following bites from venomous snakes. However, snake venom is also a rich source of bioactive molecules with known or potential therapeutic applications. Manually 'milking' snakes is the most common method to obtain venom. Safer alternative methods to produce venom would facilitate the production of both antivenom and novel therapeutics. This protocol describes the generation, maintenance and selected applications of snake venom gland organoids. Snake venom gland organoids are 3D culture models that can be derived within days from embryonic or adult venom gland tissues from several snake species and can be maintained long-term (we have cultured some organoids for more than 2 years). We have successfully used the protocol with glands from late-stage embryos and recently deceased adult snakes. The cellular heterogeneity of the venom gland is maintained in the organoids, and cell type composition can be controlled through changes in media composition. We describe in detail how to derive and grow the organoids, how to dissociate them into single cells, and how to cryopreserve and differentiate them into toxin-producing organoids. We also provide guidance on useful downstream assays, specifically quantitative real-time PCR, bulk and single-cell RNA sequencing, immunofluorescence, immunohistochemistry, fluorescence in situ hybridization, scanning and transmission electron microscopy and genetic engineering. This stepwise protocol can be performed in any laboratory with tissue culture equipment and enables studies of venom production, differentiation and cellular heterogeneity.
Assuntos
Técnicas de Cultura de Células/métodos , Organoides/crescimento & desenvolvimento , Venenos de Serpentes/biossíntese , Animais , Antivenenos/genética , Hibridização in Situ Fluorescente/métodos , Venenos de Serpentes/química , Venenos de Serpentes/genética , Serpentes/genéticaRESUMO
Venomous snakes are important subjects of study in evolution, ecology, and biomedicine. Many venomous snakes have alpha-neurotoxins (α-neurotoxins) in their venom. These toxins bind the alpha-1 nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction, causing paralysis and asphyxia. Several venomous snakes and their predators have evolved resistance to α-neurotoxins. The resistance is conferred by steric hindrance from N-glycosylated asparagines at amino acids 187 or 189, by an arginine at position 187 that has been hypothesized to either electrostatically repulse positively charged neurotoxins or sterically interfere with α-neurotoxin binding, or proline replacements at positions 194 or 197 of the nAChR ligand-binding domain to inhibit α-neurotoxin binding through structural changes in the receptor. Here, we analyzed this domain in 148 vertebrate species, and assessed its amino acid sequences for resistance-associated mutations. Of these sequences, 89 were sequenced de novo. We find widespread convergent evolution of the N-glycosylation form of resistance in several taxa including venomous snakes and their lizard prey, but not in the snake-eating birds studied. We also document new lineages with the arginine form of inhibition. Using an in vivo assay in four species, we provide further evidence that N-glycosylation mutations reduce the toxicity of cobra venom. The nAChR is of crucial importance for normal neuromuscular function and is highly conserved throughout the vertebrates as a result. Our research shows that the evolution of α-neurotoxins in snakes may well have prompted arms races and mutations to this ancient receptor across a wide range of sympatric vertebrates. These findings underscore the inter-connectedness of the biosphere and the ripple effects that one adaption can have across global ecosystems.
Assuntos
Resistência a Medicamentos , Evolução Molecular , Junção Neuromuscular/efeitos dos fármacos , Neurotoxinas/toxicidade , Antagonistas Nicotínicos/toxicidade , Receptores Nicotínicos/efeitos dos fármacos , Mordeduras de Serpentes/metabolismo , Venenos de Serpentes/toxicidade , Serpentes/metabolismo , Animais , Sítios de Ligação , Resistência a Medicamentos/genética , Glicosilação , Mutação , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiopatologia , Neurotoxinas/metabolismo , Antagonistas Nicotínicos/metabolismo , Filogenia , Ligação Proteica , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Mordeduras de Serpentes/fisiopatologia , Venenos de Serpentes/metabolismo , Especificidade da EspécieRESUMO
Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.
Assuntos
Técnicas de Cultura de Células/métodos , Organoides/crescimento & desenvolvimento , Venenos de Serpentes/metabolismo , Células-Tronco Adultas/metabolismo , Animais , Cobras Corais/metabolismo , Perfilação da Expressão Gênica/métodos , Organoides/metabolismo , Glândulas Salivares/metabolismo , Venenos de Serpentes/genética , Serpentes/genética , Serpentes/crescimento & desenvolvimento , Células-Tronco/metabolismo , Toxinas Biológicas/genética , Transcriptoma/genéticaRESUMO
BACKGROUND: Venom has evolved in parallel in multiple animals for the purpose of self-defense, prey capture or both. These venoms typically consist of highly complex mixtures of toxins: diverse bioactive peptides and/or proteins each with a specific pharmacological activity. Because of their specificity, they can be used as experimental tools to study cell mechanisms and develop novel medicines and drugs. It is therefore potentially valuable to explore the venoms of various animals to characterize their toxins and identify novel toxin-families. This study focuses on the annotation and exploration of the transcriptomes of six scorpion species from three different families. The transcriptomes were annotated with a custom-built automated pipeline, primarily consisting of Basic Local Alignment Search Tool searches against UniProt databases and filter steps based on transcript coverage. RESULTS: We annotated the transcriptomes of four scorpions from the family Buthidae, one from Iuridae and one from Diplocentridae using our annotation pipeline. We found that the four buthid scorpions primarily produce disulfide-bridged ion-channel targeting toxins, while the non-buthid scorpions have a higher abundance of non-disulfide-bridged toxins. Furthermore, analysis of the "unidentified" transcripts resulted in the discovery of six novel putative toxin families containing a total of 37 novel putative toxins. Additionally, 33 novel toxins in existing toxin-families were found. Lastly, 19 novel putative secreted proteins without toxin-like disulfide bonds were found. CONCLUSIONS: We were able to assign most transcripts to a toxin family and classify the venom composition for all six scorpions. In addition to advancing our fundamental knowledge of scorpion venomics, this study may serve as a starting point for future research by facilitating the identification of the venom composition of scorpions and identifying novel putative toxin families.
Assuntos
Perfilação da Expressão Gênica , Anotação de Sequência Molecular , Escorpiões/genética , Toxinas Biológicas/genética , AnimaisRESUMO
Scorpionfishes (Scorpaenidae) are a relatively common cause of human envenomation. They often enter coastal waters and their stings can be quite hazardous, provoking extreme pain and causing the victims to take days to recover. There are few genomic resources available for the scorpionfishes. In this study, we elucidated the transcriptomic profile of the venom glands from three different scorpionfish species, namely Scorpaenopsis cirrosa, S. neglecta and S. possi. This is the first report of scorpionfish transcriptomes. After functional and pathway annotation, we employed toxin annotation to identify many species-specific (18, 13 and 19 respectively) and overlapping putative toxins among the three species. Our study represents a significant improvement in the genetic information about the venoms from these three species. Moreover, this work also provides an archive for future studies on evolution of fish toxins and can be used for comparative studies of other fishes.
Assuntos
Venenos de Peixe/genética , Peixes/genética , Animais , Glândulas Exócrinas/metabolismo , Venenos de Peixe/química , Peixes/classificação , Filogenia , Alinhamento de Sequência , TranscriptomaRESUMO
While it has been known for a while that some snake species are extremely sensitive to acetaminophen, the underlying mechanism for this toxicity has not been reported. To investigate if essential detoxification enzymes are missing in snake species that are responsible for biotransformation of acetaminophen in other vertebrate species, livers were collected from a variety of snake species, together with samples from alligator, snapping turtle, cat, rat, and cattle. Subcellular fractions were analyzed for enzymatic activities of phenol-type sulfotransferase and UDPglucuronosyltransferase, total glutathione Stransferase, and Nacetyltransferase. The results showed that none of the snake species, together with the cat samples, had any phenol-type glucuronidation activity, and that this activity was much lower in alligator and turtle samples than in the mammalian species. Combined with the lack of Nacetyltransferase activity in snakes and cats, this would explain the accumulation of the aminophenol metabolite, which induces methemoglobinemia and subsequent suffocation of snakes and cats after acetaminophen exposure. While previous investigations have concluded that in cats the gene for the phenol-type glucuronosyltransferase isoform has turned into a pseudogene because of several point mutations, evaluation of genomic information for snake species revealed that they have only 2 genes that may code for glucuronosyltransferase isoforms. Similarity of these genes with mammalian genes is <50%, and suggests that the expressed enzymes may act on other types of substrates than aromatic amines. This indicates that the extreme sensitivity for acetaminophen in snakes is based on a different phylogenetic origin than the sensitivity observed in cats.
Assuntos
Acetaminofen/metabolismo , Poluentes Ambientais/metabolismo , Fígado/enzimologia , Filogenia , Proteínas de Répteis/metabolismo , Serpentes/fisiologia , Acetaminofen/efeitos adversos , Acetaminofen/toxicidade , Acetiltransferases/genética , Acetiltransferases/metabolismo , Agkistrodon/genética , Agkistrodon/fisiologia , Analgésicos não Narcóticos/efeitos adversos , Analgésicos não Narcóticos/metabolismo , Animais , Biotransformação , Boidae/genética , Boidae/fisiologia , Colubridae/genética , Colubridae/fisiologia , Crotalus/genética , Crotalus/fisiologia , Bases de Dados Genéticas , Resistência a Medicamentos , Poluentes Ambientais/toxicidade , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Répteis/genética , Serpentes/genética , Especificidade da Espécie , Sulfotransferases/genética , Sulfotransferases/metabolismo , ToxicocinéticaRESUMO
Currently, biological and organic substances are screened in order to find a new generation of therapeutics active against cancer. Previous research has identified promising candidate peptides in snake venom. In this study, venoms from different snake species (Naja annulifera, Naja kaouthia, Ophiophagus hannah and Echis carinatus) were screened for potential anti-cancer properties using pancreatic tumour cells as the assay system. The cells were incubated with venom and then subjected to the following analyses: (i) in vitro cell death (ii) in vitro migration (iii) in vivo dissemination and (iv) in vivo angiogenesis. For the in vivo assays, the cells, after incubation and labelling, were transplanted into the yolk sac of zebrafish embryos for motility and angiogenesis. The results showed strong effects in cells treated with venoms from Ophiophagus hannah and Echis carinatus in the in vitro assays. In the in vivo assays, venom derived from Ophiophagus hannah had the most potent effects with respect to angiogenesis. These venoms might therefore be considered as candidates for further studies.
Assuntos
Antineoplásicos/farmacologia , Venenos Elapídicos/farmacologia , Neovascularização Patológica/tratamento farmacológico , Venenos de Víboras/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular , Venenos Elapídicos/química , Elapidae , Embrião não Mamífero/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase , Venenos de Serpentes , Venenos de Víboras/química , Viperidae , Peixe-ZebraRESUMO
The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more cytotoxic than those of closely related non-spitting species. While this correlation between spitting and non-spitting was found among African cobras, it was not present among Asian cobras. On the other hand, a consistent positive correlation was observed between cytotoxicity and utilisation of the defensive hooding display that cobras are famous for. Hooding and spitting are widely regarded as defensive adaptations, but it has hitherto been uncertain whether cytotoxicity serves a defensive purpose or is somehow useful in prey subjugation. The results of this study suggest that cytotoxicity evolved primarily as a defensive innovation and that it has co-evolved twice alongside hooding behavior: once in the Hemachatus + Naja and again independently in the king cobras (Ophiophagus). There was a significant increase of cytotoxicity in the Asian Naja linked to the evolution of bold aposematic hood markings, reinforcing the link between hooding and the evolution of defensive cytotoxic venoms. In parallel, lineages with increased cytotoxicity but lacking bold hood patterns evolved aposematic markers in the form of high contrast body banding. The results also indicate that, secondary to the evolution of venom rich in cytotoxins, spitting has evolved three times independently: once within the African Naja, once within the Asian Naja, and once in the Hemachatus genus. The evolution of cytotoxic venom thus appears to facilitate the evolution of defensive spitting behaviour. In contrast, a secondary loss of cytotoxicity and reduction of the hood occurred in the water cobra Naja annulata, which possesses streamlined neurotoxic venom similar to that of other aquatic elapid snakes (e.g., hydrophiine sea snakes). The results of this study make an important contribution to our growing understanding of the selection pressures shaping the evolution of snake venom and its constituent toxins. The data also aid in elucidating the relationship between these selection pressures and the medical impact of human snakebite in the developing world, as cytotoxic cobras cause considerable morbidity including loss-of-function injuries that result in economic and social burdens in the tropics of Asia and sub-Saharan Africa.
Assuntos
Venenos Elapídicos , Neurotoxinas , Animais , Comportamento Animal , Evolução Biológica , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Venenos Elapídicos/toxicidade , Elapidae/fisiologia , Humanos , Músculo Esquelético/inervação , Junção Neuromuscular/efeitos dos fármacos , Neurotoxinas/toxicidade , PigmentaçãoRESUMO
Snake genome sequencing is in its infancy-very much behind the progress made in sequencing the genomes of humans, model organisms and pathogens relevant to biomedical research, and agricultural species. We provide here an overview of some of the snake genome projects in progress, and discuss the biological findings, with special emphasis on toxinology, from the small number of draft snake genomes already published. We discuss the future of snake genomics, pointing out that new sequencing technologies will help overcome the problem of repetitive sequences in assembling snake genomes. Genome sequences are also likely to be valuable in examining the clustering of toxin genes on the chromosomes, in designing recombinant antivenoms and in studying the epigenetic regulation of toxin gene expression.
Assuntos
Genoma , Serpentes/genética , Animais , Sequências Repetitivas de Ácido Nucleico , Toxinas Biológicas/genéticaRESUMO
Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.