No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Studies on ribonucleic acid synthesis in the venom glands of Vipera palaestinae (Ophidia, Reptilia)
Abstract
RNA metabolism in the venom glands of Vipera palaestinae was studied at different stages after manual extraction of the venom (milking). The rate of (32)P incorporation into gland RNA was found to be maximal at 1-4 days after milking in correlation with the height of the secretory epithelium. Venom production attained a maximum only after 8-16 days, in parallel with the accumulation of stable species of cellular RNA.
... Venom was collected, vacuum dried, and stored at −80 • C for future use. Four days after venom was collected and transcription was maximized [50], we sacrificed the animal using an intracoelomic injection of sodium pentobarbitol (100 mg/kg). We then removed the venom glands and stored them separately in either RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at 4 • C overnight or in liquid nitrogen before moving to −80 • C for long-term storage. ...
... The version described in this paper is the first version, GGIP01000000. LD 50 Lethal dose 50 ...
... The version described in this paper is the first version, GGIP01000000. LD 50 Lethal dose 50 ...
Phenotypic diversity generated through altered gene expression is a primary mechanism facilitating evolutionary response in natural systems. By linking the phenotype to genotype through transcriptomics, it is possible to determine what changes are occurring at the molecular level. High phenotypic diversity has been documented in rattlesnake venom, which is under strong selection due to its role in prey acquisition and defense. Rattlesnake venom can be characterized by the presence (Type A) or absence (Type B) of a type of neurotoxic phospholipase A 2 (PLA 2 ), such as Mojave toxin, that increases venom toxicity. Mojave rattlesnakes (Crotalus scutulatus), represent this diversity as both venom types are found within this species and within a single panmictic population in the Sonoran Desert. We used comparative venom gland transcriptomics of nine specimens ofC. scutulatusfrom this region to test whether expression differences explain diversity within and between venom types. Type A individuals expressed significantly fewer toxins than Type B individuals owing to the diversity of C-type lectins (CTLs) and snake venom metalloproteinases (SVMPs) found in Type B animals. As expected, both subunits of Mojave toxin were exclusively found in Type A individuals but we found high diversity in four additional PLA 2 s that was not associated with a venom type. Myotoxinaexpression and toxin number variation was not associated with venom type, and myotoxinahad the highest range of expression of any toxin class. Our study represents the most comprehensive transcriptomic profile of the venom type dichotomy in rattlesnakes andC. scutulatus. Even intra-specifically, Mojave rattlesnakes showcase the diversity of snake venoms and illustrate that variation within venom types blurs the distinction of the venom dichotomy.
... Near the center of the accessory gland, the primary duct becomes extremely narrow and often displays intricate curvatures before entering the secondary duct [3,4]. Despite the numerous studies concerning the morphology and physiology of secretory cells for venom production and secretion by the main venom gland [5][6][7][8][9][10][11][12][13][14][15] and by the accessory gland [2,4,9,[16][17][18][19][20][21], the few studies on the primary duct focused on its morphology [2][3][4]9]. ...
... Toxins 2018, 10, x FOR PEER REVIEW 2 of 13 gland, the primary duct becomes extremely narrow and often displays intricate curvatures before entering the secondary duct [3,4]. Despite the numerous studies concerning the morphology and physiology of secretory cells for venom production and secretion by the main venom gland [5][6][7][8][9][10][11][12][13][14][15] and by the accessory gland [2,4,9,[16][17][18][19][20][21], the few studies on the primary duct focused on its morphology [2][3][4]9]. ...
... In the 15-day group, the number of secretory vesicles and the secretion inside the lumen varied but the morphology was not the same as in the 0-day group (Figure 4, panels E and F), suggesting that the cycle is longer than 15 days. Since the number of secretory vesicles was variable, it seems that during the synthesis and secretion cycle, exocytosis occurs and the secretion is stored in the lumen, as well as in the main venom gland [5][6][7][8][9][10]. After the lumen is completely full, the new synthetized secretory proteins are stored in the secretory vesicles in the cytoplasm, as well as in the accessory gland [20]. ...
Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct’s epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins—mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct’s toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.
... Table 3). The variation in venom expression can possibly be attributed to differences in toxin expression over time [52]. For example, toxin expression is higher after feeding events to replenish the used venom proteins [52]. ...
... The variation in venom expression can possibly be attributed to differences in toxin expression over time [52]. For example, toxin expression is higher after feeding events to replenish the used venom proteins [52]. ...
Many venomous animals express toxins that show extraordinary levels of variation both within and among species. In snakes, most studies of venom variation focus on front-fanged species in the families Viperidae and Elapidae, even though rear-fanged snakes in other families vary along the same ecological axes important to venom evolution. Here we characterized venom gland transcriptomes from 19 snakes across two dipsadine rear-fanged genera (Leptodeira and Helicops, Colubridae) and two front-fanged genera (Bothrops, Viperidae; Micrurus, Elapidae). We compared patterns of composition, variation, and diversity in venom transcripts within and among all four genera. Venom gland transcriptomes of rear-fanged Helicops and Leptodeira and front-fanged Micrurus are each dominated by expression of single toxin families (C-type lectins, snake venom metalloproteinase, and phospholipase A2, respectively), unlike highly diverse front-fanged Bothrops venoms. In addition, expression patterns of congeners are much more similar to each other than they are to species from other genera. These results illustrate the repeatability of simple venom profiles in rear-fanged snakes and the potential for relatively constrained venom composition within genera.
... Structural elements have been revealed by light, confocal and electron microscopy, and neural regulation of secretion via sympathetic autonomic innervation was demonstrated by Kerchove et al. (2004Kerchove et al. ( , 2008. Secretory cells in vipers respond to venom use or manual extraction by rapidly increasing in height and changing from a cuboidal to columnar morphology, with a concomitant enlargement of the rough endoplasmic reticulum, a large increase in gene expression and mRNA levels and extensive protein synthesis (Rotenberg et al., 1971;Mackessy, 1991;Kerchove et al., 2004;. Recently, this highly dynamic system was shown to involve the differential regulation of thousands of genes, including a multiplicity of regulatory responses that coalesce to secrete venom into the ductules and lumen of the venom gland (Perry et al., 2020). ...
... Transcriptomic protocols have assumed that secretory cycles of rear-fanged snake venom glands mirror those of front-fanged snakes, with mRNA levels reaching a maximum at 3-4 days post-extraction (but with no supportive evidence), but even among front-fanged snakes, only a handful of studies have been conducted, most of these prior to the advent of -omic technologies (i.e. Rotenberg et al., 1971;Kochva et al., 1982). Thus, tremendous opportunity lies ahead for the investigation of most rear-fanged snake species, at every technical level currently possible. ...
The venom glands of reptiles, particularly those of front-fanged advanced snakes, must satisfy conflicting biological demands: rapid synthesis of potentially labile and highly toxic proteins, storage in the gland lumen for long periods, stabilization of the stored secretions, immediate activation of toxins upon deployment and protection of the animal from the toxic effects of its own venom. This dynamic system could serve as a model for the study of a variety of different phenomena involving exocrine gland activation, protein synthesis, stabilization of protein products and secretory mechanisms. However, these studies have been hampered by a lack of a long-term model that can be propagated in the lab (as opposed to whole-animal studies). Numerous attempts have been made to extend the lifetime of venom gland secretory cells, but only recently has an organoid model been shown to have the requisite qualities of recapitulation of the native system, self-propagation and long-term viability (>1 year). A tractable model is now available for myriad cell- and molecular-level studies of venom glands, protein synthesis and secretion. However, venom glands of reptiles are not identical, and many differ very extensively in overall architecture, microanatomy and protein products produced. This Review summarizes the similarities among and differences between venom glands of helodermatid lizards and of rear-fanged and front-fanged snakes, highlighting those areas that are well understood and identifying areas where future studies can fill in significant gaps in knowledge of these ancient, yet fascinating systems.
... Although exhaustion of venom supply probably never occurs under natural conditions 5 , it is likely that venom supply decreases upon envenomation of multiple prey items or an intense struggle with a predator. Much research has focused on the diversity of snake venom with respect to regeneration time [6][7][8][9]10 , implying the possibility of variations in whole-venom composition, depending on the stage of replenishment. Thus, understanding the timing of replenishment is a significant step in learning how venom supply might influence venom composition and activity. ...
... Venom was extracted from B. multicinctus at four time points (days 3, 6, 9, and 12) and three temperatures (15,28, and 32 °C) using three biological replicates (B15-3-1 to B32-12-3; Table S1). Time point selection was based on the previous study of replenishment time in elapid and viperid [6][7][8][9] , and temperature depended on the optimum breeding temp (15-30 °C) 21 . Two extreme temperature and a intermediate temperature was selected to investigate dynamic change of venom gland. ...
Venom gland is a highly efficient venom production system to maintain their predatory arsenal. Venom toxins mRNA has been shown to increase abruptly in snake after venom expenditure, while the dynamics of venom accumulation during synthesis are poorly understood. Here, PacBio long-read sequencing, Illumina RNA sequencing (RNA-seq), and label-free proteome quantification were used to investigate the composition landscape and time- and temperature-dependent dynamics changes of the Bungarus multicinctus venom gland system. Transcriptome data (19.5223 Gb) from six adult B. multicinctus tissues were sequenced using PacBio RS II to generate a reference assembly, and average 7.28 Gb of clean RNA-seq data was obtained from venom glands by Illumina sequencing. Differentially expressed genes (DEGs) mainly were protein processing rather than venom toxins. Post-translational modifications provided the evidence of the significantly different proportions of toxins in the venom proteome with the changing of replenishment time and temperature, but constant of venom toxins mRNA in the venom gland transcriptome. Dynamic of toxins and genes involved in venom gland contraction suggesting the formation of the mature venom gland system would take at least 9 days. In addition, 59 toxin processing genes were identified, peptidylprolyl isomerase B of which underwent positive selection in Toxicofera. These results provide a reference for determining the extraction time of venom, production of polyclonal and monoclonal antibody for precise treatment plans of venomous snakebites, and construction of an in vitro synthesis system for snake venom protein.
... This demonstrates the limitation of only using BLASTx to identify venom protein transcripts, and it is most likely the reason that previously examined venom gland transcriptomes report a significant percentage (~40%) of unidentified transcripts [36,58,59]. A high abundance of venom protein transcript expression is commonly observed for snake venom glands [35,57,58], especially at four days following venom extraction when there are high levels of venom protein transcript expression [60]. ...
... With the exception of one amino acid within the signal peptide, 3FTx_02 was found to be identical to database three-finger toxin 6 (KU666933), and a three amino acid difference in the signal peptide between 3FTx_40 and threefinger toxin 4 (KU666931) was also observed. It is likely that these few amino acid differences within the signal peptides are due to the use of a degenerate PCR primer within this region to obtain the sequences of extracellular mRNA transcripts [60]. Therefore, transcripts obtained from the next-generation sequencing (NGS) venom gland tissue transcriptome were found to be identical to those observed within the venom of B. irregularis, with one of these transcripts even corresponding to the mature protein sequence of Irditoxin B, highlighting the application of using venom extracellular mRNA to obtain venom protein transcripts of both abundant and novel toxins [62]. ...
... After activation, the secretory epithelium undergoes morphological and biochemical changes. The secretory cells change their shape from cuboid to columnar, the cisternae of the rough endoplasmic reticulum expands, and the Golgi apparatus becomes well developed (Ben-Shaul et al., 1971;Rotenberg et al., 1971;Oron and Bdolah, 1973). When manual venom extraction is performed, the maximal synthetic activity of the secretory cells and the highest mRNA concentration occur between 4 and 8 days after venom extraction (Kochva, 1987;De Lucca et al., 1974;Carneiro et al., 1991). ...
... During this period, the exocytosis of new toxins synthetized by secretory cells is continuous. Venom production cycle is long, lasting around 30e50 days (Ben-Shaul et al., 1971;De Lucca et al., 1974;Kochva, 1960Kochva, , 1987Rotenberg et al., 1971;Bdolah, 1973, Carneiro et al., 1991;Mackessy, 1991;Yamanouye et al., 1997). Therefore, the secretory cells have two different phases: one is the quiescent stage (when the lumen is full of venom), while the other is the activated stage, when the venom toxins are produced (Kochva, 1960(Kochva, , 1987Mackessy, 1991). ...
... The adult N. kaouthia specimen from Malaysia was captured from the northern region of Peninsular Malaya and the specimen from Thailand was captured from the southern region near Bangkok, Thailand. Snake venom milking was carried out prior to tissue harvesting to stimulate the transcription process, and the snake was allowed to rest for four days to maximise the transcription (Rotenberg, Bamberger & Kochva, 1971). Following euthanasia, the venom glands were swiftly removed and sectioned into dimensions of < 5Â5 mm before preserving them in a RNAlater Ò (Ambion, Texas, USA) solution at a 1:10 volume ratio. ...
... Admittedly, the issue of correlation between transcriptome and proteome is a complex one (Li, Bickel & Biggin, 2014), as the analysis of the individual gene expression could be further affected by practical factors, e.g. the time span between venom collection and gland tissue harvesting. In this study, the venom glands were sampled four days after venom milking as a way to maximise the yield of total mRNA (Rotenberg, Bamberger & Kochva, 1971), although the different genes might be expressed at different rates. ...
Background
The monocled cobra (Naja kaouthia) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes of N. kaouthia from Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology.
Methods
The transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T.
Results and discussion
The toxin transcripts showed high redundancy (41–82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin’s fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes from N. kaouthia which have not been reported hitherto; these include N. kaouthia-specific l-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins.
... The Tropidolaemus wagleri specimen was an adult female snake collected from Penang Island in the northwestern part of Peninsular Malaya. To promote transcription activity of the venom genes, venom milking was performed four days before the venom gland tissue was collected post-euthanasia [63]. The venom gland was sectioned into small pieces measuring 5 × 5 mm, and left overnight in RNAlater ® solution (Ambion, TX, USA) at 4 • C before transferring to −80 • C for storage. ...
The venom proteome of Temple Pit Viper (Tropidolaemus wagleri) is unique among pit vipers, characterized by a high abundance of a neurotoxic peptide, waglerin. To further explore the genetic diversity of its toxins, the present study de novo assembled the venom gland transcriptome of T. wagleri from west Malaysia. Among the 15 toxin gene families discovered, gene annotation and expression analysis reveal the dominating trend of bradykinin-potentiating peptide/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (BPP/ACEI-CNP, 76.19% of all-toxin transcription) in the transcriptome, followed by P-III snake venom metalloproteases (13.91%) and other toxins. The transcript TwBNP01 of BPP/ACEI-CNP represents a large precursor gene (209 amino acid residues) containing the coding region for waglerin (24 residues). TwBNP01 shows substantial sequence variations from the corresponding genes of its sister species, Tropidolaemus subannulatus of northern Philippines, and other viperid species which diversely code for proline-rich small peptides such as bradykinin-potentiating peptides (BPPs). The waglerin/waglerin-like peptides, BPPs and azemiopsin are proline-rich, evolving de novo from multiple highly diverged propeptide regions within the orthologous BPP/ACEI-CNP genes. Neofunctionalization of the peptides results in phylogenetic constraints consistent with a phenotypic dichotomy, where Tropidolaemus spp. and Azemiops feae convergently evolve a neurotoxic trait while vasoactive BPPs evolve only in other species.
... The T. wagleri specimen was an adult snake collected from Penang Island in the north-western part of Peninsular Malaya. To promote transcription activity of the venom genes, venom milking was performed four days before the venom gland tissue was collected post-euthanasia [63]. The venom gland was sectioned into small pieces measuring 5 x 5 mm, and left overnight in RNAlater® solution (Ambion, TX, USA) at 4 °C before transferring to −80 °C for storage. ...
The venom proteome of Temple Pit Viper (Tropidolaemus wagleri) is unique among pit vipers, characterized by a high abundance of a neurotoxic peptide, waglerin. To further explore the genetic diversity of its toxins, the present study de novo assembled the venom gland transcriptome of T. wagleri from west Malaysia. Among the 15 toxin gene families discovered, gene annotation and expression analysis reveal the dominating trend of bradykinin-potentiating peptide/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (BPP/ACEI-CNP, 76.19% of all-toxin transcription) in the transcriptome, followed by P-III snake venom metalloproteases (13.91%) and other toxins. The transcript TwBNP01 of BPP/ACEI-CNP represents a large precursor gene (209 amino acid residues) containing the coding region for waglerin (24 residues). TwBNP01 shows substantial sequence variations from the corresponding genes of its sister species, T. subannulatus of Northern Philippines, and other viperid species which diversely code for proline-rich small peptides such as bradykinin-potentiating peptides (BPPs). The waglerin/waglerin-like peptides, BPPs and azemiopsin are proline-rich, evolving de novo from multiple hyper-mutatable propeptide regions within the orthologous BPP/ACEI-CNP genes. Neofunctionalization of the peptides results in phylogenetic constraints consistent with a phenotypic dichotomy, where Tropidolaemus spp. and Azemiops feae convergently evolve a neurotoxic trait while vasoactive BPPs evolve only in other species.
... Beyond insects, literature suggests that the production and maintenance of chemical defence is metabolically costly. For example, viper snakes needed more than 28 days to fully regenerate their venom (Oron and Bdolah, 1973;Rotenberg et al., 1971), whereas tarantula spiders needed up to 85 days (Perret, 1977). During the first 72 h of venom regeneration, the metabolic rate increased by 11% in the pit viper snakes (McCue, 2006) and by 40% in the scorpion Parabuthus transvaalicus (Nisani et al., 2007). ...
Plants produce chemicals (or plant specialised/secondary metabolites, PSM) to protect themselves against various biological antagonists. Herbivorous insects use plants in two ways: as a food source and as a defence source. Insects can detoxify and sequester PSMs in their bodies as a defence mechanism against predators and pathogens. Here, I review the literature on the cost of PSM detoxification and sequestration in insects. I argue that no-cost meals might not exist for insects feeding on toxic plants and suggest that potential costs could be detected in an ecophysiological framework.
... We extracted venom from the individuals by allowing the snakes to bite a sterile cup covered with parafilm. Four days after the venom extraction, we euthanized the snakes with an injection of sodium pentobarbital (100 mg kg ) and excised the venom glands in order to maximize mRNA transcription (Rotenberg et al. 1971). We preserved the glands in RNAlater at 4 • C, then moved the glands to permanent storage at −80 • C. All protocols involving live snakes followed ASIH guidelines and were approved by the Clemson University Animal Care and Use Committee (Animal Use Protocol 2017-067). ...
Snake venom can vary both among and within species. While some groups of New World pitvipers-such as rattlesnakes-have been well studied, very little is known about the venom of montane pitvipers (Cerrophidion) found across the Mesoamerican highlands. Compared to most well-studied rattlesnakes, which are widely distributed, the isolated montane populations of Cerrophidion may facilitate unique evolutionary trajectories and venom differentiation. Here, we describe the venom gland transcriptomes for populations of C. petlalcalensis, C. tzotzilorum, and C. godmani from Mexico, and a single individual of C. sasai from Costa Rica. We explore gene expression variation in Cerrophidion and sequence evolution of toxins within C. godmani specifically. Cerrophidion venom gland transcriptomes are composed primarily of snake venom metalloproteinases, phospholipase A[Formula: see text]s (PLA[Formula: see text]s), and snake venom serine proteases. Cerrophidion petlalcalensis shows little intraspecific variation; however, C. godmani and C. tzotzilorum differ significantly between geographically isolated populations. Interestingly, intraspecific variation was mostly attributed to expression variation as we did not detect signals of selection within C. godmani toxins. Additionally, we found PLA[Formula: see text]-like myotoxins in all species except C. petlalcalensis, and crotoxin-like PLA[Formula: see text]s in the southern population of C. godmani. Our results demonstrate significant intraspecific venom variation within C. godmani and C. tzotzilorum. The toxins of C. godmani show little evidence of directional selection where variation in toxin sequence is consistent with evolution under a model of mutation-drift equilibrium. Cerrophidion godmani individuals from the southern population may exhibit neurotoxic venom activity given the presence of crotoxin-like PLA[Formula: see text]s; however, further research is required to confirm this hypothesis.
... We were unable to get venom from IBSP89901. We sacrificed each snake four days after venom stimulation (Rotenberg et al., 1971), removed the venom glands, placed each gland in RNAlater (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and preserved the specimen in 10% buffered formalin for five days and then transferred to 70% ethanol for deposition. The specimen in Mexico was collected under a scientific collecting permit to M.B. (SEMARNAT: SGPA/DGVS/01090/17) and handled with approval from Clemson University's IACUC (2017-067). ...
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.
... The C. rhodostoma specimen was an adult snake collected from Kedah, a northern state in Peninsular Malaya. The venom was milked four days prior to venom gland tissue collection to promote transcription [123]. The venom glands were collected following euthanasia and sectioned into dimensions of 5 × 5 mm. ...
In Southeast Asia, the Malayan Pit Viper (Calloselasma rhodostoma) is a venomous snake species of medical importance and bioprospecting potential. To unveil the diversity of its toxin genes, this study de novo assembled and analyzed the venom gland transcriptome of C. rhodostoma from Malaysia. The expression of toxin genes dominates the gland transcriptome by 53.78% of total transcript abundance (based on overall FPKM, Fragments Per Kilobase Million), in which 92 non-redundant transcripts belonging to 16 toxin families were identified. Snake venom metalloproteinase (SVMP, PI > PII > PIII) is the most dominant family (37.84% of all toxin FPKM), followed by phospholipase A2 (29.02%), bradykinin/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (16.30%), C-type lectin (CTL, 10.01%), snake venom serine protease (SVSP, 2.81%), L-amino acid oxidase (2.25%), and others (1.78%). The expressions of SVMP, CTL, and SVSP correlate with hemorrhagic, anti-platelet, and coagulopathic effects in envenoming. The SVMP metalloproteinase domains encode hemorrhagins (kistomin and rhodostoxin), while disintegrin (rhodostomin from P-II) acts by inhibiting platelet aggregation. CTL gene homologues uncovered include rhodocytin (platelet aggregators) and rhodocetin (platelet inhibitors), which contribute to thrombocytopenia and platelet dysfunction. The major SVSP is a thrombin-like enzyme (an ancrod homolog) responsible for defibrination in consumptive coagulopathy. The findings provide insight into the venom complexity of C. rhodostoma and the pathophysiology of envenoming.
... The adult C. rhodostoma specimen was collected from Kedah, a northern state in Peninsular Malaya. The venom was milked four days prior to venom gland tissue collection to promote transcription [118]. The venom glands were collected following euthanasia and sectioned into dimensions of 5 x 5 m. ...
In Southeast Asia, the Malayan Pit Viper (Calloselasma rhodostoma) is a venomous snake species of medical importance and bioprospecting potential. To unveil the diversity of its toxin genes, this study assembled and analyzed the de novo venom gland transcriptome of C. rhodostoma from Malaysia. The expression of toxin genes dominates the gland transcriptome by 53.78% of total transcript abundance (based on overall FPKM), in which 92 non-redundant transcripts belonging to 16 toxin families were identified. Snake venom metalloproteinase (SVMP, PI>PII>PIII) is the most dominant family (37.84% of all toxin FPKM), followed by phospholipase A2 (29.02%), bradykinin/angiotensinogen-converting enzyme inhibitor-C-type natriuretic peptide (16.30%), C-type lectin (CTL, 10.01%), snake venom serine protease (SVSP, 2.81%), L-amino acid oxidase (2.25%) and others (1.78%). The expressions of SVMP, CTL and SVSP correlate with hemorrhagic, anti-platelet and coagulopathic effects in envenoming. The SVMP metalloproteinase domains encode hemorrhagins (kistomin and rhodostoxin), while disintegrin (rhodostomin from P-II) is platelet-inhibitory. CTL gene homologues uncovered include rhodocytin (platelet aggregators) and rhodocetin (platelet inhibitor), which contribute to thrombocytopenia and platelet dysfunction. The major SVSP is a thrombin-like enzyme (ancrod homolog) responsible for defibrination in consumptive coagulopathy. The findings provide insight into the venom complexity of C. rhodostoma and the pathophysiology of envenoming.
... Once the venom was obtained, it was vacuum dried and stored at −80°C for future use. Four days after venom was collected and transcription was maximized (Rotenberg et al., 1971), we euthanized the animal using an intracoelomic injection of 1% tricaine mesylate (MS-222) followed by another 50% injection after anesthesia (Conroy et al., 2009). We subsequently removed the DVGs and stored them separately in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C overnight before moving to −80°C for long-term storage. ...
This thesis can be downloaded from: https://tigerprints.clemson.edu/all_theses/3913/
--------------------------------- The material of this thesis has been published in the following article: Schramer, T. D., Rautsaw, R. M., Bayona Serrano, J. D., Nystrom, G. S., West, T. R., Ortiz-Medina, J. A., Sabido-Alpuche, B., Meneses-Millán, M., Borja, M., Junqueira de Azevedo, I. L. M., Rokyta, D. R., & Parkinson, C. L. (2022). An integrative view of the toxic potential of Conophis lineatus (Dipsadidae: Xenodontinae), a medically relevant rear-fanged snake. Toxicon, 205(2022), 38–52. https://doi.org/10.1016/j.toxicon.2021.11.009
... Morphological changes in the secretory epithelium of venom gland after the expulsion of venom was noticed by Velikii in Vipera ammodytes [17] which was later confirmed by further studies [18,19]. ...
Venomous snakes belonging to the family Viperidae, Elapidae, Colubridae and Hydrophidae, produces snake venom in order to facilitate immobilization and digestion of prey, act as defense mechanism against threats. Venom contains zootoxins which is a highly modified saliva that is either injected via fangs during a bite or spitted. The modified parotid gland, encapsulated in a muscular sheath, present on each side of the head, below and behind the eye, have large alveoli which temporarily stores the secreted venom and later conveyed by a duct to tubular fangs through which venom is injected. Venoms are complex mixtures of more than 20 different compounds, mostly proteins and polypeptides, including proteins, enzymes and substances with lethal toxicity which are either neurotoxic or haemotoxic in action and exert effects on nervous/muscular impulses and blood components. Lots of research are directed to use venoms as important pharmacological molecules for treating various diseases like Alzheimer’s disease, Parkinson’s disease etc.
... Two adult specimens of B. fonsecai were collected in January 2018 in São Paulo state, Brazil, with one individual sampled near Campos do Jordão city and the other sampled near São Bento do Sapucaí city. For all snakes, venom was collected by allowing the snake to bite a sterile cup, and venom glands were excised for transcriptomics after four days when transcription is maximized [42]. The specimens were euthanized with single-step sodium pentobarbital (100 mg/kg) injection following standard approved AVMA guidelines. ...
Interspecific differences in snake venom compositions can result from distinct regulatory mechanisms acting in each species. However, comparative analyses focusing on identifying regulatory elements and patterns that led to distinct venom composition are still scarce. Among venomous snakes, Bothrops cotiara and Bothrops fonsecai represent ideal models to complement our understanding of the regulatory mechanisms of venom production. These recently diverged species share a similar specialized diet, habitat, and natural history, but each presents a distinct venom phenotype. Here, we integrated data from the venom gland transcriptome and miRNome and the venom proteome of B. fonsecai and B. cotiara to better understand the regulatory mechanisms that may be acting to produce differing venom compositions. We detected not only the presence of similar toxin isoforms in both species but also distinct expression profiles of phospholipases A2 (PLA2) and some snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) isoforms. We found evidence of modular expression regulation of several toxin isoforms implicated in venom divergence and observed correlated expression of several transcription factors. We did not find strong evidence for miRNAs shaping interspecific divergence of the venom phenotypes, but we identified a subset of toxin isoforms whose final expression may be fine-tuned by specific miRNAs. Sequence analysis on orthologous toxins showed a high rate of substitutions between PLA2s, which indicates that these toxins may be under strong positive selection or represent paralogous toxins in these species. Our results support other recent studies in suggesting that gene regulation is a principal mode of venom evolution across recent timescales, especially among species with conserved ecotypes.
... They were then kept in isolated containers for 48 h to allow them to recover their toxins and express genes involved in that recovery. We based this estimate on the known timeframe for gene expression in snake venoms (96 h; Rotenberg et al., 1971), since it is not known in bufonids. We conservatively estimated that bufonids would take half of this time to regulate gene expression of their toxins since they primarily synthesize small molecules/metabolites rather than large peptides/proteins like snake venoms. ...
Synthesized chemical defenses have broadly evolved across countless taxa and are important in shaping evolutionary and ecological interactions within ecosystems. However, the underlying genomic mechanisms by which these organisms synthesize and utilize their toxins are relatively unknown. Herein, we use comparative transcriptomics to uncover potential toxin synthesizing genes and pathways, as well as interspecific patterns of toxin synthesizing genes across ten species of North American true toads (Bufonidae). Upon assembly and annotation of the ten transcriptomes, we explored patterns of relative gene expression and possible protein-protein interactions across the species to determine what genes and/or pathways may be responsible for toxin synthesis. We also tested our transcriptome dataset for signatures of positive selection to reveal how selection may be acting upon potential toxin producing genes. We assembled high quality transcriptomes of the bufonid parotoid gland, a tissue not often investigated in other bufonid related RNAseq studies. We found several genes involved in metabolic and biosynthetic pathways (e.g. steroid biosynthesis, terpenoid backbone biosynthesis, isoquinoline biosynthesis, glucosinolate biosynthesis) that were functionally enriched and/or relatively expressed across the ten focal species that may be involved in the synthesis of alkaloid and steroid toxins, as well as other small metabolic compounds that cause distastefulness in bufonids. We hope that our study lays a foundation for future studies to explore the genomic underpinnings and specific pathways of toxin synthesis in toads, as well as at the macroevolutionary scale across numerous taxa that produce their own defensive toxins.
... Once the venom was obtained, it was vacuum dried and stored at − 80 • C for future use. Four days after venom was collected and transcription was maximized (Rotenberg et al., 1971), we euthanized the animal using an intracoelomic injection of 1% tricaine mesylate (MS-222) followed by another 50% injection after anesthesia (Conroy et al., 2009). We subsequently removed the DVGs and stored them separately in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at 4 • C overnight before moving to − 80 • C for long-term storage. ...
[https://doi.org/10.1016/j.toxicon.2021.11.009] Most traditional research on snake venoms has focused on front-fanged snake families (Viperidae, Elapidae, and Atractaspididae). However, venom is now generally accepted as being a much more broadly possessed trait within snakes, including species traditionally considered harmless. Unfortunately, due to historical inertia and methodological challenges, the toxin repertoires of non-front-fanged snake families (e.g., Colubridae, Dipsadidae, and Natricidae) have been heavily neglected despite the knowledge of numerous species capable of inflicting medically relevant envenomations. Integrating proteomic data for validation, we perform a de novo assembly and analysis of the Duvernoy's venom gland transcriptome of the Central American Road Guarder (Dipsadidae: Xenodontinae: Conophis lineatus), a species known for its potent bite. We identified 28 putative toxin transcripts from 13 toxin families in the Duvernoy's venom gland transcriptome, comprising 63.7% of total transcriptome expression. In addition to ubiquitous snake toxin families, we proteomically confirmed several atypical venom components. The most highly expressed toxins (55.6% of total toxin expression) were recently described snake venom matrix metalloproteases (svMMPs), with 48.0% of svMMP expression contributable to a novel svMMP isoform. We investigate the evolution of the new svMMP isoform in the context of rear-fanged snakes using phylogenetics. Finally, we examine the morphology of the venom apparatus using μCT and explore how the venom relates to autecology and the highly hemorrhagic effects seen in human envenomations. Importantly, we provide the most complete venom characterization of this medically relevant snake species to date, producing insights into the effects and evolution of its venom, and point to future research directions to better understand the venoms of ‘harmless’ non-front-fanged snakes.
... The second specimen (SB0022CVR) was collected in September 2016 in Rio Grande do Sul state, Brazil. For both snakes, venom was collected by allowing the snake to bite a sterile cup and venom glands were excised for transcriptomics after 4 days when transcription is maximized [50]. The specimens were euthanized with a single-step sodium pentobarbital (100 mg/kg) injection following standard approved American Veterinary Medical Association (AVMA) guidelines. ...
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.
... The sea snake, H. curtus was an adult specimen from the northern waters of Penang Island west of Peninsular Malaysia. The venom was milked four days prior to venom gland tissue collection to promote transcription [51]. The venom glands were collected following euthanasia and sectioned into dimensions of 5 × 5 mm. ...
Envenomation resulted from sea snake bite is a highly lethal health hazard in Southeast Asia. Although commonly caused by sea snakes of Hydrophiinae, each species is evolutionarily distinct and thus, unveiling the toxin gene diversity within individual species is important. Applying next-generation sequencing, this study investigated the venom-gland transcriptome of Hydrophis curtus (spine-bellied sea snake) from Penang, West Malaysia. The transcriptome was de novo assembled, followed by gene annotation and sequence analyses. Transcripts with toxin annotation were only 96 in number but highly expressed, constituting 48.18% of total FPKM in the overall transcriptome. Of the 21 toxin families, three-finger toxins (3FTX) were the most abundantly expressed and functionally diverse, followed by phospholipases A2. Lh_FTX001 (short neurotoxin) and Lh_FTX013 (long neurotoxin) were the most dominant 3FTXs expressed, consistent with the pathophysiology of envenomation. Lh_FTX001 and Lh_FTX013 were variable in amino acid compositions and predicted epitopes, while Lh_FTX001 showed high sequence similarity with the short neurotoxin from Hydrophis schistosus, supporting cross-neutralization effect of Sea Snake Antivenom. Other toxins of low gene expression, for example, snake venom metalloproteinases and L-amino acid oxidases not commonly studied in sea snake venom were also identified, enriching the knowledgebase of sea snake toxins for future study.
... Collected venoms were lyophilized and stored at -20 C for later use. Each animal was sacrificed four days later when transcription of venom proteins is at its maximum [52], via injection of sodium pentobarbitol (100mg/kg). Venom Dashed red lines denote 95 percentiles based on distribution of nontoxins. ...
Background:
Modularity is the tendency for systems to organize into semi-independent units and can be a key to the evolution and diversification of complex biological systems. Snake venoms are highly variable modular systems that exhibit extreme diversification even across very short time scales. One well-studied venom phenotype dichotomy is a trade-off between neurotoxicity versus hemotoxicity that occurs through the high expression of a heterodimeric neurotoxic phospholipase A2 (PLA2) or snake venom metalloproteinases (SVMPs). We tested whether the variation in these venom phenotypes could occur via variation in regulatory sub-modules through comparative venom gland transcriptomics of representative Black-Speckled Palm-Pitvipers (Bothriechis nigroviridis) and Talamancan Palm-Pitvipers (B. nubestris).
Results:
We assembled 1517 coding sequences, including 43 toxins for B. nigroviridis and 1787 coding sequences including 42 toxins for B. nubestris. The venom gland transcriptomes were extremely divergent between these two species with one B. nigroviridis exhibiting a primarily neurotoxic pattern of expression, both B. nubestris expressing primarily hemorrhagic toxins, and a second B. nigroviridis exhibiting a mixed expression phenotype. Weighted gene coexpression analyses identified six submodules of transcript expression variation, one of which was highly associated with SVMPs and a second which contained both subunits of the neurotoxic PLA2 complex. The sub-module association of these toxins suggest common regulatory pathways underlie the variation in their expression and is consistent with known patterns of inheritance of similar haplotypes in other species. We also find evidence that module associated toxin families show fewer gene duplications and transcript losses between species, but module association did not appear to affect sequence diversification.
Conclusion:
Sub-modular regulation of expression likely contributes to the diversification of venom phenotypes within and among species and underscores the role of modularity in facilitating rapid evolution of complex traits.
... Preparing cDNA libraries for NGS also requires isolating total RNA from venom gland tissue, usually at 4 days following venom extraction, when venom protein transcript expression is the highest [46]; extraneous muscle, blood, and/or connective tissues should be trimmed away from gland tissues before proceeding. Of particular importance is making certain that the tissue processed is of venom gland origin, given the sensitivity of NGS and the presence of venom protein homologs within other tissues [47][48][49]. ...
Studying animal toxin evolution requires sequences of these proteins and peptides, and transcript sequences allow for the construction of cladograms and evaluation of selection pressures from nonsynonymous and synonymous nucleotide mutation ratios. In addition, these translated sequences can be useful as custom databases for peptide identifications within venoms and for better proteomic quantification. Obtaining these transcripts is achieved by sequencing cDNA originating from venom gland tissue or venom. This chapter provides the methodology for (1) targeted sequencing of transcripts from a single venom protein family (RNA isolation and 3′RACE [rapid amplification of cDNA ends]), (2) generation of a venom gland transcriptome with next-generation sequencing (NGS) technology (de novo transcriptome assembly, toxin transcript identification, quantification, and positive selection analysis), and (3) combined high-throughput proteomics to identify secreted venom components. Transcriptomics has become fundamental for studying toxin evolution, but it creates many challenges for scientists who are unfamiliar with working with RNA, managing large NGS datasets and executing the required programs, particularly considering that there is an overabundance of available software in this field and not all perform optimally for venom gland transcriptome assembly. This chapter provides one pipeline for the integration of both low- and high-throughput transcriptomics with proteomics to characterize venoms.
... The adult Malaysian N. sumatrana (NS-M) snake was captured in the south-west region of Peninsular Malaysia. The snake was milked for venom and allowed to rest for four days to maximize transcription [64]. The venom glands were promptly removed after euthanasia and sectioned into dimensions of <5 × 5mm. ...
The equatorial spitting cobra, Naja sumatrana, is a distinct species of medically important venomous snakes, listed as WHO Category 1 in Southeast Asia. The diversity of its venom genes has not been comprehensively examined, although a few toxin sequences annotated to Naja sputatrix were reported previously through cloning studies. To investigate this species venom genes’ diversity, de novo venom-gland transcriptomics of N. sumatrana from West Malaysia was conducted using next-generation sequencing technology. Genes encoding toxins represented only 60 of the 55,396 transcripts, but were highly expressed, contributing to 79.22% of total gene expression (by total FPKM) in the venom-glands. The toxin transcripts belong to 21 families, and 29 transcripts were further identified as full-length. Three-finger toxins (3FTx) composed of long, short, and non-conventional groups, constituted the majority of toxin transcripts (91.11% of total toxin FPKM), followed by phospholipase A2 (PLA2, 7.42%)—which are putatively pro-inflammatory and cytotoxic. The remaining transcripts in the 19 families were expressed at extremely low levels. Presumably, these toxins were associated with ancillary functions. Our findings unveil the diverse toxin genes unique to N. sumatrana, and provide insights into the pathophysiology of N. sumatrana envenoming.
... Snakes with SVL less than 34 cm (males) or 38 cm (females) were consider juveniles 39,40 . We collected venom from the snakes, then four days later collected venom glands from each snake to maximize transcription of venom genes 41 . Venom was collected by either allowing the snakes to bite onto a parafilm-sealed sterile collection cup or by electrostimulation 42 , then dehydrated and stored at −80 °C until analyzed. ...
Changes in gene expression can rapidly influence adaptive traits in the early stages of lineage diversification. Venom is an adaptive trait comprised of numerous toxins used for prey capture and defense. Snake venoms can vary widely between conspecific populations, but the influence of lineage diversification on such compositional differences are unknown. To explore venom differentiation in the early stages of lineage diversification, we used RNA-seq and mass spectrometry to characterize Sidewinder Rattlesnake (Crotalus cerastes) venom. We generated the first venom-gland transcriptomes and complementary venom proteomes for eight individuals collected across the United States and tested for expression differences across life history traits and between subspecific, mitochondrial, and phylotranscriptomic hypotheses. Sidewinder venom was comprised primarily of hemorrhagic toxins, with few cases of differential expression attributable to life history or lineage hypotheses. However, phylotranscriptomic lineage comparisons more than doubled instances of significant expression differences compared to all other factors. Nevertheless, only 6.4% of toxins were differentially expressed overall, suggesting that shallow divergence has not led to major changes in Sidewinder venom composition. Our results demonstrate the need for consensus venom-gland transcriptomes based on multiple individuals and highlight the potential for discrepancies in differential expression between different phylogenetic hypotheses.
... All venoms were centrifuged at 9000 x g for 5 min, frozen at −80°C, lyophilized, and stored at −20°C until use. Four days post-extraction, when mRNA levels are highest [41], rear-fanged snakes were heavily anesthetized with isoflurane and euthanized via skull-cervical severing, and venom gland tissue was then collected. Tissue from each of the venom glands (right and left glands) from A. prasina was placed directly into TRIzol reagent for immediate RNA isolation. ...
High-throughput technologies were used to identify venom gland toxin expression and to characterize the venom proteomes of two rear-fanged snakes, Ahaetulla prasina (Asian Green Vine Snake) and Borikenophis portoricensis (Puerto Rican Racer). Sixty-nine complete toxin-coding transcripts from 12 venom protein superfamilies (A. prasina) and 50 complete coding transcripts from 11 venom protein superfamilies (B. portoricensis) were identified in the venom glands. However, only 18% (A. prasina) and 32% (B. portoricensis) of the translated protein isoforms were detected in the proteome of these venoms. Both venom gland transcriptomes and venom proteomes were dominated by P-III metalloproteinases. Three-finger toxins, cysteine-rich secretory proteins, and C-type lectins were present in moderate amounts, but other protein superfamilies showed very low abundances. Venoms contained metalloproteinase activity comparable to viperid snake venom levels, but other common venom enzymes were absent or present at negligible levels. Western blot analysis showed metalloproteinase and cysteine-rich secretory protein epitopes shared with the highly venomous Boomslang (Dispholidus typus). The abundance of metalloproteinases emphasizes the important trophic role of these toxins. Comprehensive, transcriptome-informed definition of proteomes and functional characterization of venom proteins in rear-fanged snake families help to elucidate toxin evolution and provide models for protein structure-function analyses.
... Prior to gland removal, snakes were extracted of venom. Four days later, when mRNA levels are presumed maximal (Rotenberg et al., 1971), two snakes were anesthetized with isofluorane and then sacrificed by decapitation. ...
... Each individual was anesthetized with a propofol injection (10 mg/kg), and venom expulsion was initiated by means of electrostimulation. After allowing four days for transcription to reach maximum levels (Rotenberg,Bamberger & Kochva, 1971Margres et al., 2015a) to have different adult venom compositions. Abbreviations: A, adult; J, juvenile. ...
The same selective forces that give rise to rapid inter-and intraspecific divergence in snake venoms can also favor differences in venoms across life-history stages. Ontogenetic changes in venom composition are well known and widespread in snakes but have not been investigated to the level of unambiguously identifying the specific loci involved. The eastern diamondback rattlesnake was previously shown to undergo an ontogenetic shift in venom composition at sexual maturity, and this shift accounted for more venom variation than geography. To characterize the genetics underlying the ontogenetic venom compositional change in C. adamanteus, we sequenced adult/juvenile pairs of venom-gland transcriptomes from five populations previously shown to have different adult venom compositions. We identified a total of 59 putative toxin transcripts for C. adamanteus, and 12 of these were involved in the ontogenetic change. Three toxins were downregulated, and nine were upregulated in adults relative to juveniles. Adults and juveniles expressed similar total levels of snake-venom metalloproteinases but differed substantially in their featured paralogs, and adults expressed higher levels of Bradykinin-potentiating and C-type natriuretic peptides, nerve growth factor, and specific paralogs of phospholipases A 2 and snake venom serine proteinases. Juvenile venom was more toxic to mice, indicating that the expression differences resulted in a phenotypically, and therefore potentially ecologically, significant difference in venom function. We also showed that adult and juvenile venom-gland transcriptomes for a species with known ontogenetic venom variation were equally effective at individually providing a full characterization of the venom genes of a species but that any particular individual was likely to lack several toxins in their transcriptome. A full characterization of a species' venom-gene complement therefore requires sequencing more than one individual, although the ages of the individuals are unimportant.
... Another issue to be considered is that venom production has a substantial metabolic cost [17] and a long regeneration cycle [9,18], which indicates that the venom composition is optimized in order to maximize efficacy while metabolic expenditure is minimized [19,20]. Thus, the synthesis and secretion of this variety of protein classes presenting merely ancillary and unessential functions seem contradictory considering that the maintenance of the venom repertoire is considerably metabolic expensive. ...
Snake venoms present a great diversity of pharmacologically active compounds that may be applied as research and biotechnological tools, as well as in drug development and diagnostic tests for certain diseases. The most abundant toxins have been extensively studied in the last decades and some of them have already been used for different purposes. Nevertheless, most of the minor snake venom protein classes remain poorly explored, even presenting potential application in diverse areas. The main difficulty in studying these proteins lies on the impossibility of obtaining sufficient amounts of them for a comprehensive investigation. The advent of more sensitive techniques in the last few years allowed the discovery of new venom components and the in-depth study of some already known minor proteins. This review summarizes information regarding some structural and functional aspects of low abundant snake venom proteins classes, such as growth factors, hyaluronidases, cysteine-rich secretory proteins, nucleases and nucleotidases, cobra venom factors, vespryns, protease inhibitors, antimicrobial peptides, among others. Some potential applications of these molecules are discussed herein in order to encourage researchers to explore the full venom repertoire and to discover new molecules or applications for the already known venom components.
... Two individuals of P. chamissonis were returned to their collecting sites, while the third one was sacrificed three days after extract the venom, as venom glands reach their maximal production of mRNA and protein synthesis between two to four days after the venom extraction (Casewell et al., 2009;Paine et al., 1992;Rotenberg et al., 1971). The sex of the individual, a male, was determined during the dissection. ...
Philodryas chamissonis is a rear-fanged snake endemic to Chile. Its bite produces mild to moderate
symptoms with proteolytic and anti-coagulant effects. Presently, the composition of the venom, as well as, the biochemical and structural characteristics of its toxins, remains unknown. In this study, we cloned and reported the first full-length sequences of five toxin-encoding genes from the venom gland of this species: Type III snake venom metalloprotease (SVMP), snake venom serine protease (SVSP), Cysteinerich secretory protein (CRISP), a and b subunits of C-type lectin-like protein (CLP) and C-type natriuretic peptide (NP). These genes are highly expressed in the venom gland and their sequences exhibited a putative signal peptide, suggesting that these are components of the venom. These putative toxins had different evolutionary relationships with those reported for some front-fanged snakes, being SVMP, SVSP and CRISP of P. chamissonis closely related to the toxins present in Elapidae species, while NP was more related to those of Viperidae species. In addition, analyses suggest that the a and b subunits of CLP of P. chamissonis might have a a-subunit scaffold in common with Viperidae species, whose highly variable C-terminal region might have allowed the diversification in a and b subunits. Our results provide the first molecular description of the toxins possibly implicated in the envenomation of prey and humans by the bite of P. chamissonis
... The venom was lyophilized and stored at -20°C until used. Venom milking was carried out to stimulate the venom gland transcription, while the snake was allowed to rest for four days for the transcription process to be maximized [81]. Following euthanasia, the venom glands were swiftly removed and sectioned into dimensions of < 5x5 mm before preserving them in RNAlater solution at a 1:10 volume ratio. ...
Background:
The king cobra (Ophiophagus hannah) is widely distributed throughout many parts of Asia. This study aims to investigate the complexity of Malaysian Ophiophagus hannah (MOh) venom for a better understanding of king cobra venom variation and its envenoming pathophysiology. The venom gland transcriptome was investigated using the Illumina HiSeq™ platform, while the venom proteome was profiled by 1D-SDS-PAGE-nano-ESI-LCMS/MS.
Results:
Transcriptomic results reveal high redundancy of toxin transcripts (3357.36 FPKM/transcript) despite small cluster numbers, implying gene duplication and diversification within restricted protein families. Among the 23 toxin families identified, three-finger toxins (3FTxs) and snake-venom metalloproteases (SVMPs) have the most diverse isoforms. These 2 toxin families are also the most abundantly transcribed, followed in descending order by phospholipases A2 (PLA2s), cysteine-rich secretory proteins (CRISPs), Kunitz-type inhibitors (KUNs), and L-amino acid oxidases (LAAOs). Seventeen toxin families exhibited low mRNA expression, including hyaluronidase, DPP-IV and 5'-nucleotidase that were not previously reported in the venom-gland transcriptome of a Balinese O. hannah. On the other hand, the MOh proteome includes 3FTxs, the most abundantly expressed proteins in the venom (43 % toxin sbundance). Within this toxin family, there are 6 long-chain, 5 short-chain and 2 non-conventional 3FTx. Neurotoxins comprise the major 3FTxs in the MOh venom, consistent with rapid neuromuscular paralysis reported in systemic envenoming. The presence of toxic enzymes such as LAAOs, SVMPs and PLA2 would explain tissue inflammation and necrotising destruction in local envenoming. Dissimilarities in the subtypes and sequences between the neurotoxins of MOh and Naja kaouthia (monocled cobra) are in agreement with the poor cross-neutralization activity of N. kaouthia antivenom used against MOh venom. Besides, the presence of cobra venom factor, nerve growth factors, phosphodiesterase, 5'-nucleotidase, and DPP-IV in the venom proteome suggests its probable hypotensive action in subduing prey.
Conclusion:
This study reports the diversity and abundance of toxins in the venom of the Malaysian king cobra (MOh). The results correlate with the pathophysiological actions of MOh venom, and dispute the use of Naja cobra antivenoms to treat MOh envenomation. The findings also provide a deeper insight into venom variations due to geography, which is crucial for the development of a useful pan-regional antivenom.
... Snakes were anesthetized with a propofol injection (10 mg/kg) and exposure to isoflurane gas, and venom expulsion was initiated by means of electrostimulation. After allowing four days for transcription to be maximized (Rotenberg et al., 1971), animals were euthanized by injection of sodium pentobarbitol (100 mg/kg), and their venom glands were removed and transferred into RNAlater. The above techniques were approved by the Florida State University Institutional Animal Care and Use Committee (IACUC) under protocol #0924. ...
The genetics underlying adaptive trait evolution describes the intersection between the probability that particular types of mutation are beneficial and the rates they arise. Snake venoms can vary in a directly meaningful manner through coding mutations and regulatory mutations. The amounts of different components determine venom efficacy, but point mutations in coding sequences can also change efficacy and function. The Timber Rattlesnake (Crotalus horridus) has populations that have evolved neurotoxic venom from the typical hemorrhagic rattlesnake venom present throughout most of its range. We identified only a handful of nonsynonymous differences in just five loci between animals with each venom type, and these differences affected lower-abundance toxins. Expression of at least 18 loci encoding hemorrhagic toxins was severely reduced in the production of neurotoxic venom. The entire phospholipase A2 toxin family was completely replaced in the neurotoxic venom, possibly through intergeneric hybridization. Venom paedomorphosis could, at best, explain only some of the loss of expression of hemorrhagic toxins. The number of potential mechanisms for altering venom composition and the patterns observed for C. horridus suggest that rapid venom evolution should occur primarily through changes in venom composition, rather than point mutations affecting coding sequences.
Copyright © 2015. Published by Elsevier Ltd.
... Snake venom glands (VG) are very specialized tissues that possess a high capacity of protein synthesis, storage and secretion. The proportion of secretor granules in viperid venom gland cells has been shown to account for only 4% of the cell volume and mRNA and protein synthesis are rapidly initiated, suggesting that secretion constitutes a fast process (Rotenberg et al. 1971;de Lucca and Imaizumi 1972;Oron and Bdolah 1973). However, the cellular mechanisms regulating protein production/secretion in the VG are essentially unknown. ...
Attempts to reconstruct the evolutionary history of snake toxins in the context of their co-option to the venom gland rarely account for non-venom snake genes that are paralogous to toxins, and which therefore represent important connectors to ancestral genes. In order to re-evaluate this process we conducted a comparative transcriptomic survey on body tissues from a venomous snake. A non-redundant set of 33,000 unigenes (assembled transcripts of reference genes) was independently assembled from six organs of the medically-important viperid snake Bothrops jararaca, providing a reference list of 82 full-length toxins from the venom gland and specific products from other tissues, such as pancreatic digestive enzymes. Unigenes were then screened for non-toxin transcripts paralogous to toxins revealing (i) low level co-expression of ~20% of toxin genes (e.g., BPP, CTL, SVMP, NGF) in body tissues, (ii) the identity of the closest paralogs to toxin genes in eight classes of toxins, (iii) the location and level of paralog expression, indicating that, in general, co-expression occurs in a higher number of tissues and at lower levels than observed for toxin genes and (iv) strong evidence of a toxin gene reverting back to selective expression in a body tissue. In addition, our differential gene expression analyses identify specific cellular processes that make the venom gland a highly specialized secretory tissue. Our results demonstrate that the evolution and production of venom in snakes is a complex process that can only be understood in the context of comparative data from other snake tissues, including the identification of genes paralogous to venom toxins.
© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
... Four days post-extraction, when mRNA levels were presumed maximized [80], both snakes were sacrificed by means of overdosing with isoflurane followed by decapitation. Both glands, which reside immediately below the lateral skin surfaces behind the eyes, were rapidly dissected from the snake, placed on clean Parafilm, and non-gland tissues (fat, connective tissue, muscle) were removed. ...
Largely because of their direct, negative impacts on human health, the venoms of front-fanged snakesof the families Viperidae and Elapidae have been extensively characterized proteomically, transcriptomically,and pharmacologically. However, relatively little is known about the molecular complexityand evolution of the venoms of rear-fanged colubrid snakes, which are, with a few notable exceptions,regarded as harmless to humans. Many of these snakes have venoms with major effects on their preferredprey, and their venoms are probably as critical to their survival as those of front-fanged elapidsand viperids.
We sequenced the venom-gland transcriptomes from a specimen of Hypsiglena (Desert Night Snake;family Colubridae, subfamily Dipsadinae) and of Boiga irregularis (Brown Treesnake; family Colubridae,subfamily Colubrinae) and verified the transcriptomic results proteomically by means ofhigh-definition mass spectrometry. We identified nearly 3,000 nontoxin genes for each species. ForB. irregularis, we found 108 putative toxin transcripts in 46 clusters with <1% nucleotide divergence,and for Hypsiglena we identified 79 toxin sequences that were grouped into 33 clusters. Comparisonsof the venoms revealed divergent venom types, with Hypsiglena possessing a viper-like venom dominatedby metalloproteinases, and B. irregularis having a more elapid-like venom, consisting primarilyof three-finger toxins.
Despite the difficulty of procuring venom from rear-fanged species, we were able to complete all analysesfrom a single specimen of each species without pooling venom samples or glands, demonstratingthe power of high-definition transcriptomic and proteomic approaches. We found a high level of divergencein the venom types of two colubrids. These two venoms reflected the hemorrhagic/neurotoxicvenom dichotomy that broadly characterizes the difference in venom strategies between elapids andviperids.
... annulifera, venom regeneration (both wet mass and protein mass) was linear during the first 20 days, and then approached a plateau after 30 days (Kochva et al., 1982). In Daboia palaestinae, rapid regeneration was followed by a plateau after about 16 days (Rotenberg et al., 1971). ...
... Isolation of cDNA hyaluronidase from Bothrops pauloensis venom gland A venom gland from a B. pauloensis adult snake was dissected three days after venom extraction, when transcription is most stimulated [16]. A cDNA library from Bothrops pauloensis venom gland, formerly referred to as Bothropoides pauloensis, was previously constructed by Rodrigues et al. [17]. ...
Background
Hyaluronate is one of the major components of extracellular matrix from vertebrates whose breakdown is catalyzed by the enzyme hyaluronidase. These enzymes are widely described in snake venoms, in which they facilitate the spreading of the main toxins in the victim’s body during the envenoming. Snake venoms also present some variants (hyaluronidases-like substances) that are probably originated by alternative splicing, even though their relevance in envenomation is still under investigation. Hyaluronidases-like proteins have not yet been purified from any snake venom, but the cDNA that encodes these toxins was already identified in snake venom glands by transcriptomic analysis. Herein, we report the cloning and in silico analysis of the first hyaluronidase-like proteins from a Brazilian snake venom.
Methods
The cDNA sequence of hyaluronidase was cloned from the transcriptome of Bothrops pauloensis venom glands. This sequence was submitted to multiple alignment with other related sequences by ClustalW. A phylogenetic analysis was performed using MEGA 4 software by the neighbor joining (NJ) method.
Results
The cDNA from Bothrops pauloensis venom gland that corresponds to hyaluronidase comprises 1175 bp and codifies a protein containing 194 amino acid residues. The sequence, denominated BpHyase, was identified as hyaluronidase-like since it shows high sequence identities (above 83%) with other described snake venom hyaluronidase-like sequences. Hyaluronidases-like proteins are thought to be products of alternative splicing implicated in deletions of central amino acids, including the catalytic residues. Structure-based sequence alignment of BpHyase to human hyaluronidase hHyal-1 demonstrates a loss of some key secondary structures. The phylogenetic analysis indicates an independent evolution of BpHyal when compared to other hyaluronidases. However, these toxins might share a common ancestor, thus suggesting a broad hyaluronidase-like distribution among venomous snakes.
Conclusions
This work is the first report of a cDNA sequence of hyaluronidase from Brazilian snake venoms. Moreover, the in silico analysis of its deduced amino acid sequence opens new perspectives about the biological function of hyaluronidases-like proteins and may direct further studies comprising their isolation and/or recombinant production, as well as their structural and functional characterization.
Background
Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris).
Results
Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis.
Conclusions
Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.
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., β-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.
Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulation in elapids is largely unexplored. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans- acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). Although there is conservation in high-level mechanistic pathways regulating venom production, there are histone methylation, transcription factor, and microRNA regulatory differences between these two snake families. Histone methyltransferases (KMT2A, KMT2C and KMT2D) and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target different toxin transcripts. Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin evolution between these two snake families, demonstrating multiple toxin genes and regulatory mechanisms converged to underpin a highly venomous phenotype.
Background:
The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored.
Methods:
To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts.
Results:
A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids.
Conclusion:
The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.
Significance
A central question in biology is whether trait differences are the result of variation in gene number, sequence, or regulation. Snake venoms are an excellent system for addressing this question because of their genetic tractability, contributions to fitness, and high evolutionary rates. We sequenced and assembled the genome of the Tiger Rattlesnake to determine whether the simplest rattlesnake venom was the product of a simple or complex genotype. The number of venom genes greatly exceeded the number of venom proteins producing the simple phenotype, indicating regulatory mechanisms were responsible for the production of the simplest, but most toxic, rattlesnake venom. We suggest that the retention of genomic complexity may be the result of shared regulatory elements among gene-family members.
Despite the extensive body of research on snake venom, many facets of snake venom systems, such as the physiology and regulation of the venom gland itself, remain virtually unstudied. Here, we use time series gene expression analyses of the rattlesnake venom gland in comparison with several non-venom tissues to characterize physiological and cellular processes associated with venom production and to highlight key distinctions of venom gland cellular and physiological function. We find consistent evidence for activation of stress response pathways in the venom gland, suggesting that mitigation of cellular stress is a crucial component of venom production. Additionally, we demonstrate evidence for an unappreciated degree of cellular and secretory activity in the steady state venom gland relative to other secretory tissues and identify vacuolar ATPases as the likely mechanisms driving acidification of the venom gland lumen during venom production and storage.
In nature, toxins have evolved as weapons to capture and subdue the prey or to counter predators or competitors. When they are inadvertently injected into humans, they cause symptoms ranging from mild discomfort to debilitation and death. Toxinology is the science of studying venoms and toxins that are produced by a wide variety of organisms. In the past, the structure, function and mechanisms of most abundant and/or most toxic components were characterized to understand and to develop strategies to neutralize their toxicity. With recent technical advances, we are able to evaluate determine the toxin profiles using transcriptomes of venom glands and proteomes of tiny amounts of venom. Enormous amounts of data from these studies have opened tremendous opportunities in many directions of basic and applied research. With lower costs for profiling venoms will further fuel the expansion of toxin database, which in turn will provide greater exciting and bright opportunities in toxin research.
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.
In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A2, cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and l-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A2 and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage.
Significance
In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.
There are over 3,000 species of snakes known to man. These limbless predators have been divided into two groups, the basal snakes (Henophidia) and the advanced snakes (Caenophidia). Venom evolved prior to the advanced snake radiation and, consequently, many use venom to subdue their prey. To do so, venom is injected via the use of a venom delivery system. The venom delivery system includes a postorbital venom gland on each side of the upper jaw that is associated with specialized venom-conducting fangs or teeth. Both the venom gland and fangs are considered to have originated from a common ancestor and are thought to be developmentally linked to one another. Even though the venom gland has a common ancestral origin, it can exhibit considerable morphological variation among the main snake families. Similarly, the fangs can occupy various positions on the upper jaw but are always found on the maxilla. Caenophidians are often referred to by the position of their fangs as either rear- or front-fanged snakes. The vast majority of snakes that are medically important to humans are front-fanged, and this character has evolved independently on at least three occasions. In addition, some front-fanged snakes have evolved a secondary gland associated with the venom system, known as the accessory gland. The venom glands, accessory glands, and fangs of different caenophidian snake families exhibit substantial morphological differences reflecting their evolutionary history. However, further studies are required to fully elucidate the ecological significance of differences in fang position, the function of the accessory gland, and the driving forces underpinning the convergent evolution observed in the snake venom delivery system.
There are over 3,000 species of snakes known to man. These limbless predators have been divided into two groups, the basal snakes (Henophidia) and the advanced snakes (Caenophidia). Venom evolved prior to the advanced snake radiation and, consequently, many use venom to subdue their prey. To do so, venom is injected via the use of a venom delivery system. The venom delivery system includes a postorbital venom gland on each side of the upper jaw that is associated with specialized venom-conducting fangs or teeth. Both the venom gland and fangs are considered to have originated from a common ancestor and are thought to be developmentally linked to one another. Even though the venom gland has a common ancestral origin, it can exhibit considerable morphological variation among the main snake families. Similarly, the fangs can occupy various positions on the upper jaw but are always found on the maxilla. Caenophidians are often referred to by the position of their fangs as either rear- or front-fanged snakes. The vast majority of snakes that are medically important to humans are front-fanged, and this character has evolved independently on at least three occasions. In addition, some front-fanged snakes have evolved a secondary gland associated with the venom system, known as the accessory gland. The venom glands, accessory glands, and fangs of different caenophidian snake families exhibit substantial morphological differences reflecting their evolutionary history. However, further studies are required to fully elucidate the ecological significance of differences in fang position, the function of the accessory gland, and the driving forces underpinning the convergent evolution observed in the snake venom delivery system.
The origin of snake venom has been variously ascribed to different body organs. The idea that the venom virulence depends on the snake’s anger led to a famous controversy in the late 17 th century. Redi, an Italien biologist, ascribed the dangerous nature of the venom to the yellow liquid issuing from the fangs, while the French chemist, Charas, maintained that the virulence lies in the anger of the snake. Needless to say, Redi’s ideas eventually prevailed; they were supported by his own experiments and later by the studies of Fontana (1787), who first described the histology of a viper’s venom gland (for references, see Klauber, 1956; Minton and Minton, 1969).
Synchronization of the secretory cycle in vivo was obtained by injecting isoprenaline as an inducer of secretion. A quantitative correlation between enzyme release, its subsequent reaccumulation, and the sequence of ultrastructural changes was found. At the ultrastructural level secretion was paralleled by depletion of zymogen granules through fusion of the granule membrane with the lumen membrane and discharge of the content. Each zymogen granule membrane, once connected with the lumen, acted as a lumen membrane. Fusion was thus sequential and resulted in a dramatic enlargement of the lumen space. During the entire process the passage between the lumen and the intercellular space remained blocked by the tight junctions, as shown by their impenetrability to ferritin. Reduction of the lumen size following enzyme discharge seemed to be achieved by withdrawal of lumen membrane in the form of small smooth vesicles which appeared mostly in the apical part of the cell. At the same time, the cell retracted towards the lumen, the whole process being completed within 2 hr from onset of secretion. Disappearance of the smooth vesicle followed, concomitant with formation of many condensing vacuoles and appearance of mature zymogen granules. The fate of the zymogen granule membrane, including its fusion with the lumen membrane, resorption in the form of small smooth vesicles, and its eventual reutilization mediated by the Golgi system, is discussed.
It has been established by electron microscopic radioautography of guinea pig pancreatic exocrine cells (Caro and Palade, 1964) that secretory proteins are transported from the elements of the rough-surfaced endoplasmic reticulum (ER) to condensing vacuoles of the Golgi complex possibly via small vesicles located in the periphery of the complex. To define more clearly the role of these vesicles in the intracellular transport of secretory proteins, we have investigated the secretory cycle of the guinea pig pancreas by cell fractionation procedures applied to pancreatic slices incubated in vitro. Such slices remain viable for 3 hr and incur minimal structural damage in this time. Their secretory proteins can be labeled with radioactive amino acids in short, well defined pulses which, followed by cell fractionation, makes possible a kinetic analysis of transport. To determine the kinetics of transport, we pulse-labeled sets of slices for 3 min with leucine-(14)C and incubated them for further +7, +17, and +57 min in chase medium. At each time, smooth microsomes ( = peripheral elements of the Golgi complex) and rough microsomes ( = elements of the rough ER) were isolated from the slices by density gradient centrifugation of the total microsomal fraction. Labeled proteins appeared initially (end of pulse) in the rough microsomes and were subsequently transferred during incubation in chase medium to the smooth microsomes, reaching a maximal concentration in this fraction after +7 min chase incubation. Later, labeled proteins left the smooth microsomes to appear in the zymogen granule fraction. These data provide direct evidence that secretory proteins are transported from the cisternae of the rough ER to condensing vacuoles via the small vesicles of the Golgi complex.
The sedimentation rates of ribosomal RNA from Escherichia coli, Bacillus subtilis, etiolated pea stem, white potato tuber, rabbit liver, rabbit reticulocytes, rat liver and rat fat-pads were compared by mixing any two of the above and analyzing them independently (32P and absorbancy at 260 mμ) on sucrose gradients. By this criterion rabbit liver, rabbit reticulocytes, rat liver and rat fat-pad ribosomal RNA's were indistinguishable. Pea stem and potato tuber as well as E. coli and B. subtilis ribosomal RNA's were also indistinguishable; in other words, within each small group of organisms studied, there was no detectable difference in sedimentation rate of the ribosomal RNA's.
1. A neurotoxic protein similar to erabutoxins a and b of Laticauda semifasciata was isolated in crystalline form from the venoms of Laticauda laticaudata and Laticauda colubrina. The name ;laticotoxin a' is proposed. 2. Laticotoxin a is homogeneous by CM-cellulose column chromatography, disc electrophoresis and ultracentrifugation and by terminal amino acid analyses. 3. Laticotoxin a consists of 62 amino acid residues. The molecular weight by ultracentrifuging is 6520. 4. The minimal 50% lethal dose of laticotoxin a by intramuscular injection to mice is 0.13mug./g. body wt. The toxin attacks the postsynaptic membrane, competing with acetylcholine. 5. Radioactive amino acids are incorporated into laticotoxin a in vivo. The incorporation is inhibited by puromycin, suggesting that the biosynthesis of the toxin follows the mechanism of protein biosynthesis, although the toxin molecule is rather small as a protein.
Synchronization of the secretory cycle in vivo was obtained by injecting isoprenaline as an inducer of secretion. A quantitative correlation between enzyme release, its subsequent reaccumulation, and the sequence of ultrastructural changes was found. At the ultrastructural level secretion was paralleled by depletion of zymogen granules through fusion of the granule membrane with the lumen membrane and discharge of the content. Each zymogen granule membrane, once connected with the lumen, acted as a lumen membrane. Fusion was thus sequential and resulted in a dramatic enlargement of the lumen space. During the entire process the passage between the lumen and the intercellular space remained blocked by the tight junctions, as shown by their impenetrability to ferritin. Reduction of the lumen size following enzyme discharge seemed to be achieved by withdrawal of lumen membrane in the form of small smooth vesicles which appeared mostly in the apical part of the cell. At the same time, the cell retracted towards the lumen, the whole process being completed within 2 hr from onset of secretion. Disappearance of the smooth vesicle followed, concomitant with formation of many condensing vacuoles and appearance of mature zymogen granules. The fate of the zymogen granule membrane, including its fusion with the lumen membrane, resorption in the form of small smooth vesicles, and its eventual reutilization mediated by the Golgi system, is discussed.
A technique is described for the isolation of tobacco mosaic virus (TMV) ribonucleic acid (RNA) of higher purity and, as will be shown in the subsequent paper, of increased stability. The method relies on the use of bentonite as an adsorbent for nucleases during the phenol degradation of the virus.If S35-labeled virus is used as starting material, the S35 content, indicative of the presence of residual protein, is markedly lower in the resultant RNA than it is in preparations made without bentonite. The upper limit for the presence of TMV protein based on these analyses is 0.04%, or one peptide chain per 23 moles RNA. The lower limit is zero. Amino acid analyses also suggest the presence of very little proteinaceous material. The possibility of traces of TMV protein contributing any biological role to the activity of the RNA is excluded on the basis of such data in conjunction with biological grounds which are discussed.
Observations on the secretion of venom by 150 captive Vipera palaestinae indicate that snakes separately caged and properly cared for may be milked regularly for 3 years or longer, and large quantities of venom can thus be obtained. With artificial heating, the milking can be continued throughout the year. No influence of ecdysis, pregnancy, or food consumption on the yield of venom was observed. Taking into account the amounts and concentration of the venom obtained as well as the physical well being of the snakes, a period of rest of one month between milkings is suggested. Temperature was found to be one of the factors governing the venom yield. A rise in temperature increased both the amount and the concentration of venom. The freshly secreted venom is rather dilute initially, and is subsequently concentrated by the reabsorption of water.
The amount of venom injected in a single bite was estimated by allowing the vipers to strike dead mice. The amount injected was found to range from nil to 190 mg of fresh venom; in most bites less than 50 mg amounts (mean, 32 ± 3.1 mg) were injected. The mean proportionate amount injected was approximately 11% of the venom available in the glands; in most strikes less than 15% of the available venom was injected. Results obtained in the laboratory when considered in relation to the severity of viper bites in human beings suggest that the minimal lethal dose of Vipera palaestinae venom for man is approximately 75 mg.
A method has been developed for the isolation of RNA from E. coli “protoplasts”, by extraction with a phenol-water mixture. The RNA preparation contained no detectable DNA and only traces of proteins and polysaccharides. Sedimentation in the ultracentrifuge yielded three boundaries. The two faster moving components were separated from the slower one by (NH4)2SO4 precipitation from a phenol-saturated water solution. From sedimentation, viscosity and light-scattering data the molecular weight was estimated to be of the order of one million. The RNA preparations in their viscosity behavior closely resemble coiling synthetic polyelectrolytes, and behave quite unlike DNA. The viscosity behavior, birefringence of flow, and potentiometric titration data are discussed in terms of a single contractile coil model.The ϵ(P)260 mμ values of the isolated material in 1 M phosphate (pH 7.1) were about 7400. Alkaline hydrolysis or polyribonucleotide phosphorylase action brought about an increase in absorption ranging from 56 to 59% while pancreatic ribonuclease caused a 28–31% increment. At very low concentrations of RNAase the change in optical density remained constant, while viscosity decreased rapidly with time.
- E Kochva
- C Gans
Kochva, E. & Gans, C. (1970). Clin. Toxicol. 3, 363.
- E Bamberger
- D Rotenberg
- Y Sharf
- E Kochva
Bamberger, E., Rotenberg, D., Sharf, Y. & Kochva, E.
(1967). I8rael J. Chem. 5, 116p.
In Toxin8 of Animal and Plant Origin
- Y Ben-Shaul
- Sh Lifshitz
- E Kochva
Ben-Shaul, Y., Lifshitz, Sh. & Kochva, E. (1971). In
Toxin8 of Animal and Plant Origin. Ed. by de Vries,
A. & Kochva, E. New York and London: Gordon and
Breach (in the Press).
In Animal Toxin8, p. 195
- E Kochva
- C Gans
Kochva, E. & Gans, C. (1967). In Animal Toxin8, p. 195.
Ed. by Russell, F. E. & Saunders, P. R. Oxford:
Pergamon Press Ltd.
- A Amsterdam
- I Ohad
- M Schramm
Amsterdam, A., Ohad, I. & Schramm, M. (1969). J. Cell
Biol. 41, 753.