Venom Production Research Articles

Hematological parameters and globulin profile of Bothrops asper and Crotalus simus snakes (Serpentes: Viperidae), after the main venom gland removal

Snakebite accidents are a neglected tropical disease worldwide recognized, and in Costa Rica the snakes Bothrops asper and Crotalus simus causes more than the 60% of the snakebite accidents reported. To fight this neglected disease, institutions need to establish and keep a venomous snake collection in order to extract the venom and produce the antivenom. The welfare of the snakes in captivity must be guaranteed, specially to old animals with defective venom production, which is the case of the Clodomiro Picado Institute. A surgical removal of the main venom gland was performed in five animals of B . asper and C . simus with none or low venom production, and the hematological parameters and plasma proteins were analyzed pre-surgery and for 90 days post-surgery. As result of the surgical procedure, the animals did not show anemia post-surgery, a heterophilia was found in respond to the invasive method, a relative monocytosis and lymphopenia at day one to three was also found, meaning that the surgical removal of the main venom gland is a safe procedure and did not affect the snake’s health. Additionally, the electrophoretic determination of the albumin:globulin ratio, is a good test to assess the body health condition in the snakes studied. Finally, the relative monocytosis and lymphopenia seems to be directly linked to a continuously handling of the snakes during consecutive days. with monocytes playing a key role in a stress respond, therefore, these relative monocytosis and lymphopenia values should be considered in the interpretation of stress leukogram reports.

Reversible plasticity in brain size, behaviour and physiology characterizes caste transitions in a socially flexible ant (Harpegnathos saltator).

Phenotypic plasticity allows organisms to respond to changing environments throughout their lifetime, but these changes are rarely reversible. Exceptions occur in relatively long-lived vertebrate species that exhibit seasonal plasticity in brain size, although similar changes have not been identified in short-lived species, such as insects. Here, we investigate brain plasticity in reproductive workers of the ant Harpegnathos saltator. Unlike most ant species, workers of H. saltator are capable of sexual reproduction, and they compete in a dominance tournament to establish a group of reproductive workers, termed 'gamergates'. We demonstrated that, compared to foragers, gamergates exhibited a 19% reduction in brain volume in addition to significant differences in behaviour, ovarian status, venom production, cuticular hydrocarbon profile, and expression profiles of related genes. In experimentally manipulated gamergates, 6-8 weeks after being reverted back to non-reproductive status their phenotypes shifted to the forager phenotype across all traits we measured, including brain volume, a trait in which changes were previously shown to be irreversible in honeybees and Drosophila. Brain plasticity in H. saltator is therefore more similar to that found in some long-lived vertebrates that display reversible changes in brain volume throughout their lifetimes.

Derivation of snake venom gland organoids for in vitro venom production

More than 400,000 people each year suffer adverse effects following bites from venomous snakes. However, snake venom is also a rich source of bioactive molecules with known or potential therapeutic applications. Manually 'milking' snakes is the most common method to obtain venom. Safer alternative methods to produce venom would facilitate the production of both antivenom and novel therapeutics. This protocol describes the generation, maintenance and selected applications of snake venom gland organoids. Snake venom gland organoids are 3D culture models that can be derived within days from embryonic or adult venom gland tissues from several snake species and can be maintained long-term (we have cultured some organoids for more than 2 years). We have successfully used the protocol with glands from late-stage embryos and recently deceased adult snakes. The cellular heterogeneity of the venom gland is maintained in the organoids, and cell type composition can be controlled through changes in media composition. We describe in detail how to derive and grow the organoids, how to dissociate them into single cells, and how to cryopreserve and differentiate them into toxin-producing organoids. We also provide guidance on useful downstream assays, specifically quantitative real-time PCR, bulk and single-cell RNA sequencing, immunofluorescence, immunohistochemistry, fluorescence in situ hybridization, scanning and transmission electron microscopy and genetic engineering. This stepwise protocol can be performed in any laboratory with tissue culture equipment and enables studies of venom production, differentiation and cellular heterogeneity.

MicroRNAs from Snellenius manilae bracovirus regulate innate and cellular immune responses of its host Spodoptera litura

To avoid inducing immune and physiological responses in insect hosts, parasitoid wasps have developed several mechanisms to inhibit them during parasitism, including the production of venom, specialized wasp cells, and symbioses with polydnaviruses (PDVs). These mechanisms alter the host physiology to give the wasp offspring a greater chance of survival. However, the molecular mechanisms for most of these alterations remain unclear. In the present study, we applied next-generation sequencing analysis and identified several miRNAs that were encoded in the genome of Snellenius manilae bracovirus (SmBV), and expressed in the host larvae, Spodoptera litura, during parasitism. Among these miRNAs, SmBV-miR-199b-5p and SmBV-miR-2989 were found to target domeless and toll-7 in the host, which are involved in the host innate immune responses. Microinjecting the inhibitors of these two miRNAs into parasitized S. litura larvae not only severely decreased the pupation rate of Snellenius manilae, but also restored the phagocytosis and encapsulation activity of the hemocytes. The results demonstrate that these two SmBV-encoded miRNAs play an important role in suppressing the immune responses of parasitized hosts. Overall, our study uncovers the functions of two SmBV-encoded miRNAs in regulating the host innate immune responses upon wasp parasitism.

Maintenance of venomous snakes in captivity for venom production at Butantan Institute from 1908 to the present: a scoping history.

Maintenance of snakes at Butantan Institute started in the last century, intending to produce a different antivenom serum to reduce death caused by snakebites. Through a successful campaign coordinated by Vital Brazil, farmers sent venomous snakes to Butantan Institute by the railway lines with no cost. From 1908 to 1962, the snakes were kept in an outdoor serpentarium, where venom extraction was performed every 15 days. During this period, the snake average survival was 15 days. In 1963, the snakes were transferred to an adapted building, currently called Laboratory of Herpetology (LH), to be maintained in an intensive system. Although the periodicity of venom extraction remained the same, animal average survival increased to two months. With the severe serum crisis in 1983, the Ministry of Health financed remodeling for the three public antivenom producers, and with this support, the LH could be improved. Air conditioning and exhausting systems were installed in the rooms, besides the settlement of critical hygienic-sanitary managements to increase the welfare of snakes. In the early 1990s, snake survival was ten months. Over the years to the present day, several improvements have been made in the intensive serpentarium, as the establishment of two quarantines, feeding with thawed rodents, an interval of two months between venom extraction routines, and monitoring of snake health through laboratory tests. With these new protocols, average snake survival increased significantly, being eight years for the genus Bothrops, ten years for genus Crotalus and Lachesis, and four years for the genus Micrurus. Aiming the production of venoms of good quality, respect for good management practices is essential for the maintenance of snakes in captivity. New techniques and efficient management must always be sought to improve animal welfare, the quality of the venom produced, and the safety of those working directly with the venomous snakes.

Therapeutic Potential of Peptides Derived from Animal Venoms: Current Views and Emerging Drugs for Diabetes.

The therapeutic potential of venom-derived drugs is evident today. Currently, several significant drugs are FDA approved for human use that descend directly from animal venom products, with others having undergone, or progressing through, clinical trials. In addition, there is growing awareness of the important cosmeceutical application of venom-derived products. The success of venom-derived compounds is linked to their increased bioactivity, specificity and stability when compared to synthetically engineered compounds. This review highlights advancements in venom-derived compounds for the treatment of diabetes and related disorders. Exendin-4, originating from the saliva of Gila monster lizard, represents proof-of-concept for this drug discovery pathway in diabetes. More recent evidence emphasises the potential of venom-derived compounds from bees, cone snails, sea anemones, scorpions, snakes and spiders to effectively manage glycaemic control. Such compounds could represent exciting exploitable scaffolds for future drug discovery in diabetes, as well as providing tools to allow for a better understanding of cell signalling pathways linked to insulin secretion and metabolism.

An optimized method of IgG’s precipitation with ammonium sulphate from hyper immune horse plasma for snake anti venom production

An optimized method of IgG’s precipitation with ammonium sulphate from hyper immune horse plasma for snake anti venom production

Physiological demands and signaling associated with snake venom production and storage illustrated by transcriptional analyses of venom glands

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.

Some like it hot: population-specific adaptations in venom production to abiotic stressors in a widely distributed cnidarian

BackgroundIn cnidarians, antagonistic interactions with predators and prey are mediated by their venom, whose synthesis may be metabolically expensive. The potentially high cost of venom production has been hypothesized to drive population-specific variation in venom expression due to differences in abiotic conditions. However, the effects of environmental factors on venom production have been rarely demonstrated in animals. Here, we explore the impact of specific abiotic stresses on venom production of distinct populations of the sea anemone Nematostella vectensis (Actiniaria, Cnidaria) inhabiting estuaries over a broad geographic range where environmental conditions such as temperatures and salinity vary widely.ResultsWe challenged Nematostella polyps with heat, salinity, UV light stressors, and a combination of all three factors to determine how abiotic stressors impact toxin expression for individuals collected across this species’ range. Transcriptomics and proteomics revealed that the highly abundant toxin Nv1 was the most downregulated gene under heat stress conditions in multiple populations. Physiological measurements demonstrated that venom is metabolically costly to produce. Strikingly, under a range of abiotic stressors, individuals from different geographic locations along this latitudinal cline modulate differently their venom production levels.ConclusionsWe demonstrate that abiotic stress results in venom regulation in Nematostella. Together with anecdotal observations from other cnidarian species, our results suggest this might be a universal phenomenon in Cnidaria. The decrease in venom production under stress conditions across species coupled with the evidence for its high metabolic cost in Nematostella suggests downregulation of venom production under certain conditions may be highly advantageous and adaptive. Furthermore, our results point towards local adaptation of this mechanism in Nematostella populations along a latitudinal cline, possibly resulting from distinct genetics and significant environmental differences between their habitats.

Kinetic analysis of effects of temperature and time on the regulation of venom expression in Bungarus multicinctus

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.

Actividad neurotóxica del veneno de serpientes del género Micrurus y métodos para su análisis. Revisión de la literatura

Introducción. Las serpientes del género Micrurus son animales de hábitos fosoriales, de temperamento pasivo y escasa producción de un potente veneno con características neurotóxicas que bloquean la transmisión sináptica en la placa neuromuscular.Objetivo. Presentar un panorama general de la neurotoxicidad del veneno de las serpientes Micrurus y su caracterización funcional mediante métodos de análisis ex vivo.Materiales y métodos. Se realizó una revisión de la literatura en MedLine y ScienceDirect usando términos específicos y sus combinaciones. Estrategia de búsqueda: tipo de estudios: artículos sobre la neurotoxicidad del veneno de serpientes Micrurus y técnicas para determinar su actividad neurotóxica mediante modelos in vitro, in vivo y ex vivo; periodo de publicación: sin limite inicial a junio de 2018; idiomas: inglés y español.Resultados. De los 88 estudios identificados en la búsqueda inicial, se excluyeron 28 por no cumplir los criterios de inclusión (basándose en la lectura de títulos y resúmenes); además, se incluyeron 8 documentos adicionales (libros e informes), que, a criterio de los autores, complementaban la información reportada por las referencias seleccionadas. Los estudios incluidos en la revisión (n=68) correspondieron a las siguientes tipologías: investigaciones originales (n=44), artículos de revisión (n=16) y capítulos de libros, informes, guías y consultas en internet (n=8).Conclusiones. Los estudios que describen el uso de preparaciones ex vivo de músculo y nervio para evaluar el efecto de neurotoxinas ofrecen un buen modelo para la caracterización del efecto presináptico y postsináptico del veneno producido por las serpientes Micrurus.

The Genome of Shaw's Sea Snake (Hydrophis curtus) Reveals Secondary Adaptation to Its Marine Environment.

The transition of terrestrial snakes to marine life ∼10Ma is ideal for exploring adaptive evolution. Sea snakes possess phenotype specializations including laterally compressed bodies, paddle-shaped tails, valvular nostrils, cutaneous respiration, elongated lungs, and salt glands, yet, knowledge on the genetic underpinnings of the transition remains limited. Herein, we report the first genome of Shaw's sea snake (Hydrophis curtus) and use it to investigate sea snake secondary marine adaptation. A hybrid assembly strategy obtains a high-quality genome. Gene family analyses date a pulsed coding-gene expansion to ∼20Ma, and these genes associate strongly with adaptations to marine environments. Analyses of selection pressure and convergent evolution discover the rapid evolution of protein-coding genes, and some convergent features. Additionally, 108 conserved noncoding elements appear to have evolved quickly, and these may underpin the phenotypic changes. Transposon elements may contribute to adaptive specializations by inserting into genomic regions around functionally related coding genes. The integration of genomic and transcriptomic analyses indicates independent origins and different components in sea snake and terrestrial snake venom; the venom gland of the sea snake harbors the highest PLA2 (17.23%) expression in selected elapids and these genes may organize tandemly in the genome. These analyses provide insights into the genetic mechanisms that underlay the secondary adaptation to marine and venom production of this sea snake.

Producción y caracterización del veneno de abejas en Chulucanas, Piura

Producción y caracterización del veneno de abejas en Chulucanas, Piura

Snake venomics at the crossroads between ecological and clinical toxinology

Venoms are complex secretions used for predatory and defensive purposes by a wide range of organisms. Venoms and venom production represent fascinating systems to study fundamental evolutionary processes. Understanding the evolution of venom generation demands the integration of the selective interactions and mechanisms, which transformed ordinary genes into deadly toxins, in the context of the natural history of the producing organism. Humans are not prey for any extant venomous creature on Earth, and thus human envenomings result from unexpected encounters with venomous animals, e.g., snakes. Research on snake venoms conducted on mammalian prey from an ecologically informed perspective is conceptually transferable to the clinic, highlighting the mutually enlightening relationship between evolutionary and translational venomics.

Venom Collection from Spiders and Snakes: Voluntary and Involuntary Extractions ("Milking") and Venom Gland Extractions.

Venom collection (often called "milking") provides the toxic secretions essential for studying animal venoms and/or generating venom products. Methods of venom collection vary widely, falling into three broad categories: voluntary venom extraction (inducing the animal to willingly release its venom), involuntary venom extraction (glandular massage, electrical stimulation, or administration of induction chemicals to promote venom expulsion), and venom gland extraction (surgical aspiration or trituration of homogenized gland tissue). Choice of method requires consideration of animal species, animal welfare, human safety (avoiding envenomation), venom yield and composition desired, and level of toxin purity required. Here, we summarize the materials and methods used to obtain venom by each of these approaches from spiders and snakes.

Biochemical Modulation of Venom by Spiders is Achieved Via Compartmentalized Toxin Production and Storage

Animal venoms are complex chemical arsenals containing hundreds or even thousands of diverse small molecules, peptides, and proteins that affect a myriad of molecular targets. Because this biochemical arsenal is metabolically expensive, venom must be used sparingly and therefore one might predict that venom evolution would result in retention of only the most essential toxins for predation and/or defence. However, this presents a strong dichotomy in the functional requirements of venoms that are used for both predation and defence, which results in extensive functional redundancy. Here we show that spiders can overcome this potential metabolic burden by modulating the biochemical composition of their venom. We demonstrate that the venom peptidome of the lethal Australian funnel-web spider Hadronyche infensa changes both qualitatively and quantitatively throughout a series of defensive secretions — secretion of the most potent insecticidal toxins is preceded by secretion of non-insecticidal, presumably defensive, toxins. Using mass spectrometry imaging, we show that modulation of toxin secretion is facilitated by differential storage of toxins in the venom gland. We propose that the establishment of distinct venom-gland zipcodes for toxin production is an adaptation that reduces the metabolic expense of venom production and perhaps also serves to minimize effects from development of toxin resistance during predator-prey co-evolution. Our results highlight the importance of considering behavioral aspects of natural venom secretions in understanding toxin function and evolution.

The Ecological Importance of Toxicity: Sea Anemones Maintain Toxic Defence When Bleached.

Cnidarians are amongst the most venomous animals on the planet. They are also under significant threat due to the impacts of climate change. Corals and anemones undergo climate-induced bleaching during extreme environmental conditions, where a loss of symbiotic photosynthetic algae (zooxanthellae) causes whitening in colour, loss of internal food supply, and reduction in health, which can ultimately lead to death. What has yet to be determined is whether bleaching causes a reduction in the production or quality of venom. In this study, the sea anemone Entacmaea quadricolor was exposed to long-term light-induced bleaching to examine the effect that bleaching has on venom. Venom quality and quantity, as determined through lethality and haemolysis measures and nematocyst production was highly preserved over the five-month imposed bleaching event. Maintenance of venom and nematocyst production, despite a loss of an internal food source provided by endosymbiotic algae, indicates both the ecological importance of maintaining toxicity and a remarkable resilience that anemones have to major environmental stressors.

Comparative analysis of intestinal bacteria among venom secretion and non-secrection snakes

To further investigate the bacterial community and identify the bacterial biomarkers between venom secretion and non-venom secretion snakes, 50 intestinal samples (25 large intestine, 25 small intestine) were obtained from 29 snakes (13 gut samples from Deinagkistrodon, 26 from Naja and 11 from Ptyas mucosa). 16S rDNA high-throughput sequencing results showed that 29 bacterial phyla, 545 bacterial genera, and 1,725 OTUs (operational taxonomic units) were identified in these samples. OTU numbers and the Ace, Chao, Shannon, and Simpson indexes were very similar among the three breeds of snakes included in this study. The Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria were predominant bacterial phyla. The relative abundance at the phylum level among these samples was similar, and the difference between small and large intestinal samples was not obvious. However, at the genus level, venom secretion snakes Deinagkistrodon and Naja clustered together according to different breeds. 27, 24, and 16 genera were identified as core microbes for Deinagkistrodon, Naja, and Ptyas mucosa, respectively. Interestingly, the relative abundances of genera Hafnia_Obesumbacterium, Providencia, and Ureaplasma were found to be significantly higher in non-venom secretion snakes, and the genera Achromobacter, Cetobacterium, Clostridium innocuum group, Fusobacterium, Lachnoclostridium, Parabacteroides, and Romboutsia were only detected in venom secretion snakes. The function of these bacteria in venom secretion needs to be further studied, and these venom secretion related genera may be the promising target to improve venom production.

The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes.

Here we use a chromosome-level genome assembly of a prairie rattlesnake (Crotalus viridis), together with Hi-C, RNA-seq, and whole-genome resequencing data, to study key features of genome biology and evolution in reptiles. We identify the rattlesnake Z Chromosome, including the recombining pseudoautosomal region, and find evidence for partial dosage compensation driven by an evolutionary accumulation of a female-biased up-regulation mechanism. Comparative analyses with other amniotes provide new insight into the origins, structure, and function of reptile microchromosomes, which we demonstrate have markedly different structure and function compared to macrochromosomes. Snake microchromosomes are also enriched for venom genes, which we show have evolved through multiple tandem duplication events in multiple gene families. By overlaying chromatin structure information and gene expression data, we find evidence for venom gene-specific chromatin contact domains and identify how chromatin structure guides precise expression of multiple venom gene families. Further, we find evidence for venom gland-specific transcription factor activity and characterize a complement of mechanisms underlying venom production and regulation. Our findings reveal novel and fundamental features of reptile genome biology, provide insight into the regulation of snake venom, and broadly highlight the biological insight enabled by chromosome-level genome assemblies.

Effect of the Honeybee Hybrid and Geographic Region on the Honey Bee Venom Production

The present study was carried out at the apiary of Plant Protection Department, Faculty of Agriculture, Al-Azhar University at Nasr city, Cairo Governorate, and Motobes region Kafr El-Sheikh Governorate. The study was designed to investigate the effect of the Carniolan and Italian hybrids and geographic regions on the honey bee venom production during different seasons, at the beginning from September 2017 to the end of August 2018.Data showed that the average amounts of dry bee venom collected from Carniolan and Italian colonies were 51.3 and 44.8 mg/colony, respectively, and there were no significant differences observed between dry bee venom amounts collected from the two honey bee hybrids.Results indicated that the highest amount of dry bee venom (99.9 mg/colony) was recorded in May, and the least amount (11.1 mg/colony) was recorded at October, under Nasr city conditions. While under Motobes region conditions, the highest amount of dry bee venom (102.5 mg/colony) was recorded in June, and the least amount of dry bee venom (60.2mg/colony) was obtained at the end of November.