RESUMEN

Snake venoms are primarily composed of proteins and peptides, which selectively interact with specific molecular targets, disrupting prey homeostasis. Identifying toxins and the mechanisms involved in envenoming can lead to the discovery of new drugs based on natural peptide scaffolds. In this study, we used mass spectrometry-based peptidomics to sequence 197 peptides in the venom of Bothrops cotiara, including a novel 7-residue peptide derived from a snake venom metalloproteinase. This peptide, named Bc-7a, features a pyroglutamic acid at the N-terminal and a PFR motif at the C-terminal, homologous to bradykinin. Using FRET (fluorescence resonance energy transfer) substrate assays, we demonstrated that Bc-7a strongly inhibits the two domains of angiotensin converting enzyme (Ki < 1 µM). Our findings contribute to the repertoire of biologically active peptides from snake venoms capable of inhibiting angiotensin-converting enzyme (ACE), beyond current known structural motifs and precursors. In summary, we report a novel snake venom peptide with ACE inhibitory activity, suggesting its potential contribution to the hypotensive effect observed in envenomation.

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The genus Bothrops are considered Category 1 of medical importance by the World Health Organization, responsible for approximately 85 % of snakebites occurring throughout Brazil. Main factors determining snake venom variations can be genetics, diet, gender, geographic distribution, age, or even seasonality. In this study, we compared the composition of protein profile, biochemical activities, and immunorecognition of toxins present in the venom of eight adults of Bothrops species (B. alternatus, B. atrox, B. jararaca, B. jararacussu, B. leucurus, B. moojeni, B. neuwiedi and B. pauloensis). The following methods were used to analyze the venoms: protein dosage; electrophoresis in polyacrylamide gel containing SDS; High Performance Liquid Chromatography – Reverse Phase; enzymatic activities, western blotting and Enzyme Linked Immuno Sorbent Assay. The results show inter and intraspecific differences in the electrophoretic profile. LAAO and PLA2 activities, in general, were higher in males than females and proteolytic activity was higher in females than males. The bothropic antivenom produced by Instituto Butantan recognized most of the protein bands in all Bothrops species analyzed, with only the regions between 37 and 25 kDa presenting lower intensity. A notable variability in the chromatograms was observed. Bothrops venom demonstrated inter-intraspecific disparities in protein composition and biochemical activity.

RESUMEN

Knowledge on hormonal regulation of reproductive cycles in viperid snakes is still incipient, especially when it comes to females and tropical species. There is an urgent need to understand the reproduction of venomous snakes to improve assisted reproduction techniques and optimize the maintenance of these animals in captivity. With this in mind, we monitored Northern pit viper females year-round throughout different seasons via serum levels of progesterone (P4) and estradiol (E2) in conjunction with ultrasound examinations. Ovarian follicles were classified according to their size and stage of vitellogenesis in F-I and F-II (non-vitellogenic phase) or in F-III and F-IV (vitellogenic phase). During autumn and winter, five adult males were rotated among these females for reproductive pairing, which resulted in 17 copulations and 2 pregnancies in the first year and 12 copulations and 5 pregnancies in the second year. Then, we assessed changes in P4 and E2 levels according to seasons, predominant ovarian structures and the presence of embryos or eggs in the oviduct. Our findings showed high levels of E2 when a greater number of vitellogenic follicles were detected, indicating a possible influence of E2 on vitellogenesis and higher levels of P4 whenever eggs and embryos were visualized in the oviduct, implying its role in maintaining pregnancy. Descriptive analysis of the vipers’ ovarian cycles revealed a greater number of vitellogenic follicles during winter, probably as a result of increases in E2; whereas pregnancies occurred predominantly in spring, under the influence of P4. The use of ultrasound images, as a minimally invasive methodology, associated with serum steroid levels has proven to be an efficient approach in the reproductive monitoring of Northern pit vipers in vivo. In addition, these data suggest that female pit vipers under human care display a seasonal reproductive cycle, despite earlier studies involving captive males of the species indicating a lack of seasonality in sperm production and quality.

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Snakes in the family Elapidae largely produce venoms rich in three-fnger toxins (3FTx) that bind to the α1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specifc mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specifcity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan→serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

RESUMEN

Snakes in the family Elapidae largely produce venoms rich in three-fnger toxins (3FTx) that bind to the α1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specifc mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specifcity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan→serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

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Variability in snake venom composition is well-documented and crucial for understanding snake ecology and predicting snakebites. In this study, we characterize the venom composition and biological activities of newborn female and male Bothrops moojeni and their mother. Our results reveal significant differences between the venom of newborn females and males, demonstrating a broad and diverse range of proteins. The venoms of newborn females showed higher serine protease effects, increased hemorrhagic activity, and greater lethality compared to the venom of newborn males. However, no differences were observed in phospholipase A2 and coagulant activity. The differences in protein composition and toxic activities between maternal and neonatal venom, as well as between the venoms of newborn females and males, contribute to understanding the diverse outcomes of snakebites. These results underscore the importance of considering sex and ontogeny in understanding venom composition in snakes.

A variabilidade na composição dos venenos de serpentes é bem documentada e crucial para a compreensão da ecologia das serpentes ou do prognóstico dos envenenamentos ofídicos. Nesse estudo, caracterizamos a composição e a atividade biológica do veneno de fêmeas e machos recém-nascidos e sua mãe. Nossos resultados revelaram diferenças significativas entre o veneno de fêmeas e machos recém-nascidos, demonstrando uma ampla e diversificada gama de proteínas. Os venenos de fêmeas recém-nascidas apresentaram mais efeitos de serina protease, aumento da atividade hemorrágica e maior letalidade em comparação ao veneno dos machos recém-nascidos. No entanto, nenhuma diferença foi observada na fosfolipase A2 e na atividade coagulante. As diferenças na composição proteica e nas atividades tóxicas entre o veneno materno e neonatal, bem como entre os venenos de fêmeas e machos recém-nascidos contribuem para a compreensão dos diversos resultados dos acidentes ofídicos. Esses resultados ressaltam a importância de considerar o sexo e a ontogenia na compreensão da composição do veneno desses animais.

RESUMEN

Snake venoms are primarily composed of proteins and peptides, which selectively interact with specific molecular targets, disrupting prey homeostasis. Identifying toxins and the mechanisms involved in envenoming can lead to the discovery of new drugs based on natural peptide scaffolds. In this study, we used mass spectrometry-based peptidomics to sequence 197 peptides in the venom of Bothrops cotiara, including a novel 7-residue peptide derived from a snake venom metalloproteinase. This peptide, named Bc-7a, features a pyroglutamic acid at the N-terminal and a PFR motif at the C-terminal, homologous to bradykinin. Using FRET (fluorescence resonance energy transfer) substrate assays, we demonstrated that Bc-7a strongly inhibits the two domains of angiotensin converting enzyme (Ki < 1 μM). Our findings contribute to the repertoire of biologically active peptides from snake venoms capable of inhibiting angiotensin-converting enzyme (ACE), beyond current known structural motifs and precursors. In summary, we report a novel snake venom peptide with ACE inhibitory activity, suggesting its potential contribution to the hypotensive effect observed in envenomation.

RESUMEN

Snake venoms are known to be major sources of peptides with different pharmacological properties. In this study, we comprehensively explored the venom peptidomes of three specimens of Lachesis muta, the largest venomous snake in South America, using mass spectrometry techniques. The analysis revealed 19 main chromatographic peaks common to all specimens. A total of 151 peptides were identified, including 69 from a metalloproteinase, 58 from the BPP-CNP precursor, and 24 from a l-amino acid oxidase. To our knowledge, 126 of these peptides were reported for the first time in this work, including a new SVMP-derived peptide fragment, Lm-10a. Our findings highlight the dynamic nature of toxin maturation in snake venoms, driven by proteolytic processing, post-translational modifications, and cryptide formation.

RESUMEN

Considerable heterogeneity and ontogenetic changes in venom composition have already been observed in different species of snakes within the Viperidae family. Since the venom of young and adult can cause distinct pathological effects and because the antivenom may be less effective in neutralizing envenoming by young snakes compared to adults, it is of paramount importance to understand the ontogenetic variation of snake venom. Thus, the present study aimed to analyze and compare the venom of Bothrops pauloensis snakes, searching for possible influences of ontogeny and sex in their biochemical and biological aspects. The venom of younger individuals was more complex in relation to high molecular mass proteins, with a greater abundance of metalloproteinases, while adults showed a greater abundance of medium and low molecular mass proteins, such as phospholipases A2 (PLA2), C-type lectins and serine proteases. The antivenom showed better immunorecognition towards the venom of adult snakes than younger ones, in addition to a deficiency in the recognition of medium molecular mass proteins, suggesting the need for an improvement in the antivenom. Younger snakes showed higher coagulant, caseinolytic, and hemorrhagic activity, while adult snakes showed higher L-amino acid oxidase (LAAO) activity and acted faster in lethality. Differences between males and females were observed mainly in the rate of loss of coagulant activity, change in PLA2 activity and lethality action time. Furthermore, considering only the adult groups, males showed a higher LAAO and thrombin-like activity, while females showed a higher caseinolytic and hyaluronidase activity. With the results obtained in this work, it was possible to conclude that there is an ontogenetic variation in the composition and some activities of the B. pauloensis snake venom, in addition to differences between the venom of males and females, reinforcing that there is an intraspecific variation that may result in different symptoms in their envenoming and, consequently, differences in the response to treatment with the antivenom.

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Due to their major medical importance in Latin America, lancehead pitvipers are frequently kept and bred in captivity for venom extraction to the production of antivenom serums. Nevertheless, despite the great contribution given to captive breeding, much of the knowledge of Bothrops' reproductive biology derived from sporadic and insufficient data provided by zoological collections. Thus, we aimed to investigate seasonal changes in gonadosomatic index (GSI) and seminal parameters (e.g., volume, concentration, motility, viability, and acrosome integrity) of five species of lancehead pitvipers from different biomes and phylogenetic groups, maintained in the indoors serpentarium at Butantan Institute (Brazil). Patterns of variation in GSI and semen parameters differed from one species to another, suggesting that captive populations should perhaps be managed distinctly to maximize reproductive success. Furthermore, in none of the studied species did changes in GSI occur concomitantly with seminal variations. GSI remained unaltered year-round for Jararaca (Bothrops jararaca) and Brazilian lancehead (Bothrops moojeni), whereas it peaked in the autumn for Common lancehead (Bothrops atrox), Jararacussu (Bothrops jararacussu), and Whitetail lancehead (Bothrops leucurus). But surprisingly, the scenario was inverted when we estimated the total number of motile spermatozoa per season, as Jararaca and Brazilian lancehead displayed seasonal differences and the other species did not vary throughout the year. Potential ecological and evolutionary factors underlying these differences were also discussed in the present article. Together, these findings can help to better define breeding management strategies for each species in captivity, in addition to optimizing the future use of artificial insemination and semen cryopreservation.

RESUMEN

Considerable heterogeneity and ontogenetic changes in venom composition have already been observed in different species of snakes within the Viperidae family. Since the venom of young and adult can cause distinct pathological effects and because the antivenom may be less effective in neutralizing envenoming by young snakes compared to adults, it is of paramount importance to understand the ontogenetic variation of snake venom. Thus, the present study aimed to analyze and compare the venom of Bothrops pauloensis snakes, searching for possible influences of ontogeny and sex in their biochemical and biological aspects. The venom of younger individuals was more complex in relation to high molecular mass proteins, with a greater abundance of metalloproteinases, while adults showed a greater abundance of medium and low molecular mass proteins, such as phospholipases A2 (PLA2), C-type lectins and serine proteases. The antivenom showed better immunorecognition towards the venom of adult snakes than younger ones, in addition to a deficiency in the recognition of medium molecular mass proteins, suggesting the need for an improvement in the antivenom. Younger snakes showed higher coagulant, caseinolytic, and hemorrhagic activity, while adult snakes showed higher L-amino acid oxidase (LAAO) activity and acted faster in lethality. Differences between males and females were observed mainly in the rate of loss of coagulant activity, change in PLA2 activity and lethality action time. Furthermore, considering only the adult groups, males showed a higher LAAO and thrombin-like activity, while females showed a higher caseinolytic and hyaluronidase activity. With the results obtained in this work, it was possible to conclude that there is an ontogenetic variation in the composition and some activities of the B. pauloensis snake venom, in addition to differences between the venom of males and females, reinforcing that there is an intraspecific variation that may result in different symptoms in their envenoming and, consequently, differences in the response to treatment with the antivenom.

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Despite coagulotoxicity being a primary weapon for prey capture by Bothrops species (lancehead pit vipers) and coagulopathy being a major lethal clinical effect, a genus-wide comparison has not been undertaken. To fill this knowledge gap, we used thromboelastography to compare 37 venoms, from across the full range of geography, taxonomy, and ecology, for their action upon whole plasma and isolated fibrinogen. Potent procoagulant toxicity was shown to be the main venom effect of most of the species tested. However, the most basal species (B. pictus) was strongly anticoagulant; this is consistent with procoagulant toxicity being a novel trait that evolved within Bothrops subsequent to their split from anticoagulant American pit vipers. Intriguingly, two of the arboreal species studied (B. bilineatus and B. taeniatus) lacked procoagulant venom, suggesting differential evolutionary selection pressures. Notably, some terrestrial species have secondarily lost the procoagulant venom trait: the Mogi Mirim, Brazil locality of B. alternatus; San Andres, Mexico locality of B. asper; B. diporus; and the São Roque of B. jararaca. Direct action on fibrinogen was extremely variable; this is consistent with previous hypotheses regarding it being evolutionary decoupled due to procoagulant toxicity being the primary prey-capture weapon. However, human patients live long enough for fibrinogen depletion to be clinically significant. The extreme variability may be reflective of antivenom variability, with these results thereby providing a foundation for such future work of clinical relevance. Similarly, the venom diversification trends relative to ecological niche will also be useful for integration with natural history data, to reconstruct the evolutionary pressures shaping the venoms of these fascinating snakes.

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Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane – EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.

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The breeding of medically important snakes is of great value, not only for the production of serums, but also for scientific studies aimed at the maintenance and well-being of animals in captivity. The present study aimed to establish the correlation between body size, sex and age in the venom production of Bothrops leucurus snakes, when manually milked under intensive captivity conditions, for one year. Were used 31 specimens kept in the Herpetology laboratory of Instituto Butantan, grouped by sex and age (young, adult and old). Biometric data (weight and length) and solid and liquid weight of venoms extracted from each fang were recorded, totaling 106 extractions in the period. We evaluated the electrophoretic profile (SDS-PAGE) under reducing conditions, the 50% lethal dose (LD50) and the minimum coagulant dose (DMC) of the extracted venoms. The body size was positively correlated with venom production in B. leucurus snakes. Regardless of sex and age, the venom showed no differences between liquid and solid composition or between right and left fang, however, the production of venom in females was twice the one found in males and more lethal. The clotting ability was lost as the animals aged, indicating that older snakes are not the best choice for venom pools in the production of antivenoms. These results are important to choose of serum production animals, and to understand the composition of snake venoms under captive conditions.

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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.

RESUMEN

The venom color variation of Crotalus durissus terrificus (Cdt) is attributed to the presence of the toxin L-amino acid oxidase (LAAO). During the venom milking routine of Instituto Butantan, we have noticed that most venoms of captive Cdt specimens show a yellowish color, while most venoms of wild specimens are white. Here we describe a comparative analysis of long-term captive (LTC) and recently wild-caught (RWC) Cdt, focusing on LAAO variation. For the identification of LAAO in individual venoms, four different approaches were employed: evaluation of the enzymatic activity, SDS-PAGE, Western blotting, and ELISA. In addition, mass spectrometry analysis was performed using pooled samples. Although some variation among these methodologies was observed, it was possible to notice that the presence of LAAO was significantly higher in the venom of LTC individuals. LAAO was identified in 60-80% LTC specimens and in only 10-12% of RWC specimens. Furthermore, this enzyme accounts for 5.6% of total venom proteins of LTC Cdt pooled venom, while it corresponds to only 0.7% of RWC Cdt pooled venom. These findings strongly suggest that captive maintenance increases the expression of LAAO in Cdt venom.

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The Brazilian lancehead (Bothrops moojeni) has a wide distribution in Brazil and represents a serious public health hazard. Previous works reported that the symptoms of snakebites caused by B. moojeni juveniles’ bites were mainly related to coagulation, while those caused by adults’ bites had a more prominent local damage. In this work, we analyzed the venoms of B. moojeni at different life stages to better understand the ontogeny shift in this species. Snakes were grouped by age and sex, and venom pools were formed accordingly. Compositional analyses by one-dimensional electrophoresis (1-DE), chromatography, and mass spectrometry revealed that ontogenetic changes might be mostly related to phospholipase A2 (PLA2) and metalloproteases. Regarding the venoms functional aspect, proteolytic, L-amino acid oxidase, PLA2, and coagulant in vitro activities were assayed, but only the first and the last ones showed age-related changes, with the venom of snakes up to 1 year-old displaying lower proteolytic and higher coagulant activities, while those from 2 years-old onward presented the opposite relation. The venoms of 3 years-old snakes were exceptions to the compositional and functional pattern of adults as both venoms presented profiles similar to neonates. Sex-related differences were observed in specific groups and were not age-related. In vivo experiments (median lethal dose and hemorrhagic activity) were statistically similar between neonates and adults, however we verified that the adult venom killed mice faster comparing to the neonates. All venoms were mostly recognized by the antibothropic serum and displayed similar profiles to 1-DE in western blotting. In conclusion, the Brazilian lancehead venom showed ontogenetic shift in its composition and activities. Furthermore, this change occurred in snakes from 1 to 2 years-old, and interestingly the venom pools from 3 years-old snakes had particular characteristics, which highlights the importance of comprehensive studies to better understand venom variability.

RESUMEN

This short essay pretends to make the reader reflect on the concept of biological mass and on the added value that the determination of this molecular property of a protein brings to the interpretation of evolutionary and translational snake venomics research. Starting from the premise that the amino acid sequence is the most distinctive primary molecular characteristics of any protein, the thesis underlying the first part of this essay is that the isotopic distribution of a protein's molecular mass serves to unambiguously differentiate it from any other of an organism's proteome. In the second part of the essay, we discuss examples of collaborative projects among our laboratories, where mass profiling of snake venom PLA2 across conspecific populations played a key role revealing dispersal routes that determined the current phylogeographic pattern of the species.