The Venom Composition of the Snake Tribe Philodryadini: 'Omic' Techniques Reveal Intergeneric Variability among South American Racers.

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

The venom composition of the snake tribe Philodryadini: ‘Omic’ techniques reveal intergeneric variability among South American racers

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Proteomic comparison of adult and juvenile Santa Catalina rattlesnake (Crotalus catalinensis) venom.

Rattlesnake's venom constitutes an important ecological trait that dynamically changes over time. Venoms of adult and juvenile rattleless rattlesnakes, Crotalus catalinensis, an endemic insular species from the Gulf of California, were compared by electrophoretic profile, fibrinogenolytic activity, and proteomic composition to assess ontogenetic variability. The SDS-PAGE profiles show important differences at 12, 22, and 45 kDa, which were prominent in adult samples and absent in juvenile samples, while bands around 20, 25, and 70 kDa are almost absent in adults. Both venoms hydrolyze Aa and Bb chains of fibrinogen generating different patterns of degradation products. This activity was partially inhibited by EDTA and PMSF and completely abolished only in the presence of both inhibitors. More than 260 proteins were identified and quantified in both venoms by proteomic analysis. Metalloproteinases (more than 60%), serine proteinases (14.5% in adult venom and 17.7% in juvenile venom), and C-type lectins (7.1 and 5.9%) represent the three most abundant toxin-related protein families. Bradykinin inhibitor peptides and L-amino acid oxidases were not detected in juvenile venom. A protein-specific comparison shows that adult and juvenile venom share about 30.5% of total toxin-related proteins, while 32% and 35% are exclusively present in adult and juvenile venoms, respectively. This work represents one of the first efforts to understand phenotypic diversity in the venom composition of insular rattlesnake species from Mexico.

Geographic variation of individual venom profile of Crotalus durissus snakes.

BACKGROUND: South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. METHODS: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. RESULTS: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. CONCLUSIONS: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.

Geographic variation of individual venom profile of Crotalus durissus snakes

South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.(AU)

Geographic variation of individual venom profile of Crotalus durissus snakes

Background: South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.

Geographic variation of individual venom profile of Crotalus durissus snakes

South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.(AU)

Molecular composition of the paralyzing venom of three solitary wasps (Hymenoptera: Pompilidae) collected in southeast Mexico.

The chemical and biological characterization of peptide and protein components of the paralyzing venom from three Pompilidae solitary spider wasps (Pepsis mexicana, Pepsis terminata, and Anoplius nigritus) is described for the first time. The molecular masses of the most abundant peptides were determined. The N-terminal sequences of two cysteine-rich peptides were obtained from Pepsis. Metalloproteinase and hyaluronidase activities were identified in the venom of P. mexicana. A novel non-lethal method to collect venom is described.

Compositional and functional investigation of individual and pooled venoms from long-term captive and recently wild-caught Bothrops jararaca snakes.

Intraspecific venom variability has been extensively reported in a number of species and is documented to be the result of several factors. However, current evidence for snake venom variability related to captivity maintenance is controversial. Here we report a compositional and functional investigation of individual and pooled venoms from long-term captive (LTC) and recently wild-caught (RWC) B. jararaca snakes. The composition of individual venoms showed a remarkable variability in terms of relative abundance of toxins (evidenced by 1-DE and RP-HPLC), enzymatic activities (proteolytic, PLA2, and LAAO) and coagulant activity, even among captive specimens. Thus, no compositional and functional pattern could be established to assign each individual venom to a specific group. Conversely, pooled venom from LTC and RWC snakes showed no significant differences regarding protein composition (characterized by 1-DE and shotgun proteomics), enzymatic activities (proteolytic, PLA2 and LAAO) and biological function (coagulant, hemorrhagic and lethal activities), except for edematogenic activity, which was more prominent in RWC venom pool. Additionally, both pooled venoms displayed similar immunoreactivity with the bothropic antivenom produced by Instituto Butantan. Taken together, our results highlight the complexity and the high intraspecific variation of B. jararaca venom, that is not influenced at a discernible extent by captivity maintenance. BIOLOGICAL SIGNIFICANCE: Bothrops jararaca snakes are one of the main causes of snakebites in Southeastern Brazil. Due to its medical interest, the venom of this species is the most studied and characterized among Brazilian snakes and captive B. jararaca specimens are maintained for long periods of time in our venom production facility. However, knowledge on the influence of captivity maintenance on B. jararaca venom variability is scarce. In this report, we described a high compositional and functional variability of individual venoms from LTC and RWC B. jararaca snakes, which are not observed between LTC and RWC pooled venoms. This intraspecific variability is more likely to be due to genetic/populational differences rather than "captivity vs wild" conditions. In this regard, data generated by the present work support the use of venom from captive and wild snakes for antivenom production and scientific research. Moreover, the data generated by this study highlight the importance of analyzing individual venom samples in studies involving intraspecific venom variability.

Functional and proteomic comparison of Bothrops jararaca venom from captive specimens and the Brazilian Bothropic Reference Venom.

Snake venom is a variable phenotypic trait, whose plasticity and evolution are critical for effective antivenom production. A significant reduction of the number of snake donations to Butantan Institute (São Paulo, Brazil) occurred in recent years, and this fact may impair the production of the Brazilian Bothropic Reference Venom (BBRV). Nevertheless, in the last decades a high number of Bothrops jararaca specimens have been raised in captivity in the Laboratory of Herpetology of Butantan Institute. Considering these facts, we compared the biochemical and biological profiles of B. jararaca venom from captive specimens and BBRV in order to understand the potential effects of snake captivity upon the venom composition. Electrophoretic analysis and proteomic profiling revealed few differences in venom protein bands and some differentially abundant toxins. Comparison of enzymatic activities showed minor differences between the two venoms. Similar cross-reactivity recognition pattern of both venoms by the antibothropic antivenom produced by Butantan Institute was observed. Lethality and neutralization of lethality for B. jararaca venom from captive specimens and BBRV showed similar values. Considering these results we suggest that the inclusion of B. jararaca venom from captive specimens in the composition of BBRV would not interfere with the quality of this reference venom. BIOLOGICAL SIGNIFICANCE: Snakebite envenomation is a neglected tropical pathology whose treatment is based on the use of specific antivenoms. Bothrops jararaca is responsible for the majority of snakebites in South and Southeastern Brazil. Its venom shows individual, sexual, and ontogenetic variability, however, the effect of animal captivity upon venom composition is unknown. Considering the reduced number of wild-caught snakes donated to Butantan Institute in the last decades, and the increased life expectancy of the snakes raised in captivity in the Laboratory of Herpetology, this work focused on the comparative profiling of B. jararaca venom from captive snakes and the Brazilian Bothropic Reference Venom (BBRV). BBRV is composed of venom obtained upon the first milking of wild-caught B. jararaca specimens, and used to assess the potency of all bothropic antivenoms produced by Brazilian suppliers. The use of proteomic strategies, added to biochemical and neutralization tests, allowed to conclude that, despite some subtle differences detected between these two venoms, venom from captive specimens could be used in the BBRV composition without affecting its quality in antivenom potency assays.

Compositional and functional investigation of individual and pooled venoms from long-term captive and recently wild-caught Bothrops jararaca snakes

Intraspecific venom variability has been extensively reported in a number of species and is documented to be the result of several factors. However, current evidence for snake venom variability related to captivity maintenance is controversial. Here we report a compositional and functional investigation of individual and pooled venoms from long-term captive (LTC) and recently wild-caught (RWC) B. jararaca snakes. The composition of individual venoms showed a remarkable variability in terms of relative abundance of toxins (evidenced by 1-DE and RP-HPLC), enzymatic activities (proteolytic, PLA2, and LAAO) and coagulant activity, even among captive specimens. Thus, no compositional and functional pattern could be established to assign each individual venom to a specific group. Conversely, pooled venom from LTC and RWC snakes showed no significant differences regarding protein composition (characterized by 1-DE and shotgun proteomics), enzymatic activities (proteolytic, PLA2 and LAAO) and biological function (coagulant, hemorrhagic and lethal activities), except for edematogenic activity, which was more prominent in RWC venom pool. Additionally, both pooled venoms displayed similar immunoreactivity with the bothropic antivenom produced by Instituto Butantan. Taken together, our results highlight the complexity and the high intraspecific variation of B. jararaca venom, that is not influenced at a discernible extent by captivity maintenance. Biological significance: Bothrops jararaca snakes are one of the main causes of snakebites in Southeastern Brazil. Due to its medical interest, the venom of this species is the most studied and characterized among Brazilian snakes and captive B. jararaca specimens are maintained for long periods of time in our venom production facility. However, knowledge on the influence of captivity maintenance on B. jararaca venom variability is scarce. In this report, we described a high compositional and functional variability of individual venoms from LTC and RWC B. jararaca snakes, which are not observed between LTC and RWC pooled venoms. This intraspecific variability is more likely to be due to genetic/populational differences rather than "captivity vs wild" conditions. In this regard, data generated by the present work support the use of venom from captive and wild snakes for antivenom production and scientific research. Moreover, the data generated by this study highlight the importance of analyzing individual venom samples in studies involving intraspecific venom variability.

Examination of biochemical and biological activities of Bothrops jararaca (Serpentes: Viperidae; Wied-Neuwied 1824) snake venom after up to 54 years of storage

The number of snakes donated to the Brazilian Instituto Butantan has been decreasing in the past 10 years. This circumstance motivated us to compare the properties of five venom pools of Bothrops jararaca snake stored for up to 54 years. Results showed differences among venom pools regarding enzymatic and other biological activities, such as caseinolytic, phospholipase A(2,) hemorrhagic and coagulant activities, as well as antigenicity. Protein content, reverse-phase chromatographic profile, and immunorecognition by commercial Bothrops antivenom were comparable for all venom pools, although lethality of the most recent preparations was higher. Since the lowest functional activities did not always correspond to older venoms, differences among venom pools used for antivenom production during the period 1963-2008 may correlate with the different proportions of venoms from different localities used in their generation, rather than to long-term storage. We conclude that B. jararaca venoms properly stored for long periods of time retain their structural and pharmacological activities, thus representing useful materials for scientific research and antivenom production.

Functional and proteomic comparison of Bothrops jararaca venom from captive specimens and the Brazilian Bothropic reference venom

Snake venom is a variable phenotypic trait, whose plasticity and evolution are critical for effective antivenom production. A significant reduction of the number of snake donations to Butantan Institute (Sao Paulo, Brazil) occurred in recent years, and this fact may impair the production of the Brazilian Bothropic Reference Venom (BBRV). Nevertheless, in the last decades a high number of Bothrops jararaca specimens have been raised in captivity in the Laboratory of Herpetology of Butantan Institute. Considering these facts, we compared the biochemical and biological profiles of B. jararaca venom from captive specimens and BBRV in order to understand the potential effects of snake captivity upon the venom composition. Electrophoretic analysis and proteomic profiling revealed few differences in venom protein bands and some differentially abundant toxins. Comparison of enzymatic activities showed minor differences between the two venoms. Similar cross-reactivity recognition pattern of both venoms by the antibothropic antivenom produced by Butantan Institute was observed. Lethality and neutralization of lethality for B. jararaca venom from captive specimens and BBRV showed similar values. Considering these results we suggest that the inclusion of B. jararaca venom from captive specimens in the composition of BBRV would not interfere with the quality of this reference venom.

Does the administration of pilocarpine prior to venom milking influence the composition of Micrurus corallinus venom?

Considering that the scarcity of venom represents a huge challenge for biochemical and functional studies of Micrurus species (coral snakes), in this report we describe for the first time the influence of pilocarpine administration prior to venom milking on the yield and protein composition of Micrurus corallinus venom. The administration of pilocarpine resulted in an increase of about 127% in the volume of venom milked, with similar protein content. Venoms showed similar protein bands distribution and intensity by SDS-PAGE and equivalents RP-HPLC profiles. Our proteomic analysis showed that venoms milked in the presence and absence of pilocarpine presented comparable protein profiles, in terms of protein composition and relative abundance. The toxins identified were assigned to 13 protein families and represent the most complete M. corallinus venom proteome described so far, in terms of number of protein families identified. Our data indicate that the administration of pilocarpine prior to venom milking increases the venom yield and does not change significantly the venom composition of M. corallinus. The employment of pilocarpine represents a useful approach to increase the yield of venom not only for Micrurus species, but also for other genera of snakes with limitations regarding the amount of venom available.

Maturity-related changes in venom toxicity of the freshwater stingray Potamotrygon leopoldi.

Aquatic venomous animals such as stingrays represent a largely untapped source for venom-based drug development. However, the major challenge for a potential drug development pipeline is the high inter- and intraspecific variability in toxicity and venom composition. As of today, little is known about maturity-driven changes in these traits in stingrays. The present study investigates the differences in toxicity and venom composition in different maturity stages of the freshwater stingray Potamotrygon leopoldi. This species can be found in the Xingú River basin (Brazil), where it mainly feeds on invertebrates, while being predated by other stingrays or large catfishes. P. leopoldi, as commonly known for stingrays, can cause severe injuries with the venomous dentine spine located at its tails. The toxicity of tissue extracts of juvenile and mature specimens was recorded on a myoblast cell culture bioassay. Venom composition and bioactivity of compounds were analyzed with planar chromatography linked to an Aliivibrio fischeri bioassay. Results revealed a decrease in venom toxicity during maturation, but no changes in venom composition. These findings may indicate that toxicity in mature specimens becomes evolutionary less important, probably due to a decrease in predation pressure.

Influence of post-starvation extraction time and prey-specific diet in Tityus serrulatus scorpion venom composition and hyaluronidase activity.

The role of diet in venom composition has been a topic of intense research interest. This work presents evidence that the variation in the venom composition from the scorpion Tityus serrulatus (Ts) is closely associated with post-starvation extraction time and prey-specific diet. The scorpions were fed with cockroach, cricket, peanut beetle or giant Tenebrio. The venoms demonstrated a pronounced difference in the total protein and toxins composition, which was evaluated by electrophoresis, reversed-phase chromatography, densitometry, hyaluronidase activity and N-terminal sequencing. Indeed, many toxins and peptides, such as Ts1, Ts2, Ts4, Ts5, Ts6, Ts15, Ts19 frag. II, hypotensins 1 and 3, PAPE peptide and peptide 9797 (first described in Ts venom), were all identified in different proportions in the analyzed Ts venoms. This study is pioneer on assessing the influence of the starvation time and the prey diet on hyaluronidase activity as well as to describe a modification of Tricine-gel-electrophoresis to evaluate this enzyme activity. Altogether, this study reveal a large contribution of the extraction time and diet on Ts venom variability as well as present a background to recommend the cockroach diet to obtain higher protein content and the cricket diet to obtain higher hyaluronidase specific activity.