Identification of an Antimicrobial Peptide from the Venom of the Trinidad Thick-Tailed Scorpion Tityus trinitatis with Potent Activity against ESKAPE Pathogens and Clostridioides difficile.

Envenomation by the Trinidad thick-tailed scorpion Tityus trinitatis may result in fatal myocarditis and there is a high incidence of acute pancreatitis among survivors. Peptidomic analysis (reversed-phase HPLC followed by MALDI-TOF mass spectrometry and automated Edman degradation) of T. trinitatis venom led to the isolation and characterization of three peptides with antimicrobial activity. Their primary structures were established asTtAP-1 (FLGSLFSIGSKLLPGVFKLFSRKKQ.NH2), TtAP-2 (IFGMIPGLIGGLISAFK.NH2) and TtAP-3 (FFSLIPSLIGGLVSAIK.NH2). In addition, potassium channel and sodium channel toxins, present in the venom in high abundance, were identified by CID-MS/MS sequence analysis. TtAP-1 was the most potent against a range of clinically relevant Gram-positive and Gram-negative aerobes and against the anaerobe Clostridioides difficile (MIC = 3.1-12.5 µg/mL). At a concentration of 1× MIC, TtAP-1 produced rapid cell death (<15 min against Acinetobacter baumannii and Staphylococcus aureus). The therapeutic potential of TtAP-1 as an anti-infective agent is limited by its high hemolytic activity (LC50 = 18 µg/mL against mouse erythrocytes) but the peptide constitutes a template for the design of analogs that maintain the high bactericidal activity against ESKAPE pathogens but are less toxic to human cells. It is suggested that the antimicrobial peptides in the scorpion venom facilitate the action of the neurotoxins by increasing the membrane permeability of cells from either prey or predator.

Quantification of snake venom proteomes by mass spectrometry-considerations and perspectives.

The advent of soft ionization mass spectrometry-based proteomics in the 1990s led to the development of a new dimension in biology that conceptually allows for the integral analysis of whole proteomes. This transition from a reductionist to a global-integrative approach is conditioned to the capability of proteomic platforms to generate and analyze complete qualitative and quantitative proteomics data. Paradoxically, the underlying analytical technique, molecular mass spectrometry, is inherently nonquantitative. The turn of the century witnessed the development of analytical strategies to endow proteomics with the ability to quantify proteomes of model organisms in the sense of "an organism for which comprehensive molecular (genomic and/or transcriptomic) resources are available." This essay presents an overview of the strategies and the lights and shadows of the most popular quantification methods highlighting the common misuse of label-free approaches developed for model species' when applied to quantify the individual components of proteomes of nonmodel species (In this essay we use the term "non-model" organisms for species lacking comprehensive molecular (genomic and/or transcriptomic) resources, a circumstance that, as we detail in this review-essay, conditions the quantification of their proteomes.). We also point out the opportunity of combining elemental and molecular mass spectrometry systems into a hybrid instrumental configuration for the parallel identification and absolute quantification of venom proteomes. The successful application of this novel mass spectrometry configuration in snake venomics represents a proof-of-concept for a broader and more routine application of hybrid elemental/molecular mass spectrometry setups in other areas of the proteomics field, such as phosphoproteomics, metallomics, and in general in any biological process where a heteroatom (i.e., any atom other than C, H, O, N) forms integral part of its mechanism.

Proteomic analysis of the mandibular glands from the Chinese crocodile lizard, Shinisaurus crocodilurus - Another venomous lizard?

Based on its phylogenetic relationship to monitor lizards (Varanidae), Gila monsters (Heloderma spp.), and the earless monitor Lanthanotus borneesis, the Chinese crocodile lizard, Shinisaurus crocodilurus, has been assigned to the Toxicofera clade, which comprises venomous reptiles. However, no data about composition and biological activities of its oral secretion have been reported. In the present study, a proteomic analysis of the mandibular gland of S. crocodilurus and, for comparison, of the herbivorous Solomon Island skink Corucia zebrata, was performed. Scanning electron microscopy (SEM) of the teeth from S. crocodilurus revealed a sharp ridge on the anterior surface, but no grooves, whereas those of C. zebrata possess a flattened crown with a pointed cusp. Proteomic analysis of their gland extracts provided no evidence of venom-derived peptides or proteins, strongly supporting the non-venomous character of these lizards. Data are available via ProteomeXchange with identifier PXD039424.

Half a century of research on Bothrops asper venom variation: biological and biomedical implications.

Snake venoms are a complex biological mixture of proteins with or without enzymatic activity, peptides, and nucleotides, among other components. It is produced in specialized secretory glands located in the maxillary region, being the result of millions of years of evolution and whose biological functions are defense, immobilization, and digestion of prey. Venoms present intraspecific (i.e., individual, ontogenetic, geographical) and interspecific (i.e., between sympatric and allopatric species) variation, and the study of this variability has become the focus of toxinological research. Bothrops asper is responsible for highest incidence, morbimortality and severe cases of envenoming in Mesoamerica and northern South America. Given its clinical importance, its venom has been characterized and compared qualitatively and quantitatively across the species range. More than 50 years of research show that B. asper venom is endowed with an interesting intraspecific variability. Knowing this variation has allowed advances in the elucidation of the biological role of the venom, a better understanding of the clinical signs and symptoms in patients envenomed by B asper, the immunological implications in the context of antivenoms production, and the generation of new ideas that could be useful to solve different biological and evolutionary questions of one of the venomous snakes with the greatest distribution and strongest public health impact in Latin America.

Interpopulational variation and ontogenetic shift in the venom composition of Lataste's viper (Vipera latastei, Boscá 1878) from northern Portugal.

Lataste's viper (Vipera latastei) is a venomous European viper endemic to the Iberian Peninsula, recognised as medically important by the World Health Organization. To date, no comprehensive characterisation of this species' venom has been reported. Here, we analysed the venoms of juvenile and adult specimens of V. latastei from two environmentally different populations from northern Portugal. Using bottom-up venomics, we produced six venom proteomes (three per population) from vipers belonging to both age classes (i.e., two juveniles and four adults), and RP-HPLC profiles of 54 venoms collected from wild specimens. Venoms from juveniles and adults differed in their chromatographic profiles and relative abundances of their toxins, suggesting the occurrence of ontogenetic changes in venom composition. Specifically, snake venom metalloproteinase (SVMP) was the most abundant toxin family in juvenile venoms, while snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins were the main toxins comprising adult venoms. The RP-HPLC venom profiles were found to vary significantly between the two sampled localities, indicating geographic variability. Furthermore, the presence/absence of certain peaks in the venom chromatographic profiles appeared to be significantly correlated also to factors like body size and sex of the vipers. Our findings show that V. latastei venom is a variable phenotype. The intraspecific differences we detected in its composition likely mirror changes in the feeding ecology of this species, taking place during different life stages and under different environmental pressures. SIGNIFICANCE: Lataste's viper (Vipera latastei) is a medically important viper endemic to the Iberian Peninsula, inhabiting different habitats and undergoing a marked ontogenetic dietary shift. In the current study, we report the first proteomic analysis of V. latastei venom from two environmentally different localities in northern Portugal. Our bottom-up venomic analyses show that snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins are the major components of adult V. latastei venom. The comparative analysis of young and adult venoms suggests the occurrence of ontogenetic shift in toxin abundances, with snake venom metalloproteinases (SVMPs) being the predominant toxins in juvenile venoms. Moreover, geographic venom variation between the two studied populations is also detected, with our statistical analyses suggesting that factors like body size and sex of the vipers are possibly at play in its determination. Our work represents the first assessment of the composition of V. latastei venom, and the first step towards a better understanding of the drivers behind its variability.

Monoclonal-Based Antivenomics Reveals Conserved Neutralizing Epitopes in Type I PLA2 Molecules from Coral Snakes.

For over a century, polyclonal antibodies have been used to treat snakebite envenoming and are still considered by the WHO as the only scientifically validated treatment for snakebites. Nevertheless, moderate innovations have been introduced to this immunotherapy. New strategies and approaches to understanding how antibodies recognize and neutralize snake toxins represent a challenge for next-generation antivenoms. The neurotoxic activity of Micrurus venom is mainly due to two distinct protein families, three-finger toxins (3FTx) and phospholipases A2 (PLA2). Structural conservation among protein family members may represent an opportunity to generate neutralizing monoclonal antibodies (mAbs) against family-conserved epitopes. In this work, we sought to produce a set of monoclonal antibodies against the most toxic components of M. altirostris venom. To this end, the crude venom was fractionated, and its major toxic proteins were identified and used to generate a panel of five mAbs. The specificity of these mAbs was characterized by ELISA and antivenomics approaches. Two of the generated mAbs recognized PLA2 epitopes. They inhibited PLA2 catalytic activity and showed paraspecific neutralization against the myotoxicity from the lethal effect of Micrurus and Naja venoms' PLA2s. Epitope conservation among venom PLA2 molecules suggests the possibility of generating pan-PLA2 neutralizing antibodies.

Combined Molecular and Elemental Mass Spectrometry Approaches for Absolute Quantification of Proteomes: Application to the Venomics Characterization of the Two Species of Desert Black Cobras, Walterinnesia aegyptia and Walterinnesia morgani.

We report a novel hybrid, molecular and elemental mass spectrometry (MS) setup for the absolute quantification of snake venom proteomes shown here for two desert black cobra species within the genus Walterinnesia, Walterinnesia aegyptia and Walterinnesia morgani. The experimental design includes the decomplexation of the venom samples by reverse-phase chromatography independently coupled to four mass spectrometry systems: the combined bottom-up and top-down molecular MS for protein identification and a parallel reverse-phase microbore high-performance liquid chromatograph (RP-µHPLC) on-line to inductively coupled plasma (ICP-MS/MS) elemental mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QToF MS). This allows to continuously record the absolute sulfur concentration throughout the chromatogram and assign it to the parent venom proteins separated in the RP-µHPLC-ESI-QToF parallel run via mass profiling. The results provide a locus-resolved and quantitative insight into the three desert black cobra venom proteome samples. They also validate the units of measure of our snake venomics strategy for the relative quantification of snake venom proteomes as % of total venom peptide bonds as a proxy for the % by weight of the venom toxins/toxin families. In a more general context, our work may pave the way for broader applications of hybrid elemental/molecular MS setups in diverse areas of proteomics.

Venomics of the poorly studied hognosed pitvipers Porthidium arcosae and Porthidium volcanicum.

We report the first proteomics analyses of the venoms of two poorly studied snakes, the Manabi hognosed pitviper Porthidium arcosae endemic to the western coastal province of Manabí (Ecuador), and the Costa Rican hognosed pitviper P. volcanicum with distribution restricted to South Pacific Costa Rica and western Panamá. These venom proteomes share a conserved compositional pattern reported in four other congeneric species within the clade of South American Porthidium species, P. nasutum, P. lansbergii, P. ophryomegas, and P. porrasi. The paraspecific immunorecognition profile of antivenoms produced in Costa Rica (ICP polyvalent), Perú (Instituto Nacional de Salud) and Brazil (soro antibotrópico pentavalente, SAB, from Instituto Butantan) against the venom of P. arcosae was investigated through a third-generation antivenomics approach. The maximal venom-binding capacities of the investigated antivenoms were 97.1 mg, 21.8 mg, and 25.7 mg of P. arcosae venom proteins per gram of SAB, ICP, and INS-PERU antibody molecules, respectively, which translate into 28.4 mg, 13.1 mg, and 15.2 mg of total venom proteins bound per vial of SAB, ICP, and INS-PERU AV. The antivenomics results suggest that 21.8%, 7.8% and 6.1% of the SAB, ICP, and INS-PERU antibody molecules recognized P. arcosae venom toxins. The SAB antivenom neutralized P. arcosae venom's lethality in mice with an ED50 of 31.3 mgV/g SAB AV. This preclinical neutralization paraspecificity points to Brazilian SAB as a promising candidate for the treatment of envenomings by Ecuadorian P. arcosae. BIOLOGICAL SIGNIFICANCE: Assessing the preclinical efficacy profile of antivenoms against homologous and heterologous medically relevant snake venoms represents an important goal towards defining the biogeographic range of their clinical utility. This is particularly relevant in regions, such as Mesoamerica, where a small number of pharmaceutical companies produce antivenoms against the venoms of a small number of species of maximum medical relevance among the local rich herpetofauna, leaving a wide range of snakes of secondary medical relevance, but also causing life-threatening human envenomings without nominal clinical coverage. This work is part of a larger project aiming at mapping the immunological characteristics of antivenoms generated in Latin American countries towards venoms of such poorly studied snakes of the local and neighboring countries' herpetofauna. Here we report the proteomics characterization of the Manabi hognosed pitviper Porthidium arcosae endemic to the western coastal province of Manabí (Ecuador), and the Costa Rican hognosed pitviper P. volcanicum with distribution restricted to southwestern Costa Rica, the antivenomics assessment of three bothropoid commercial antivenoms produced in Costa Rica, Perú, and Brazil against the venom components of P. arcosae, and the in vivo capacity of the Brazilian soro antibotrópico pentavalente (SAB) from Instituto Butantan to neutralize the murine lethality of P. arcosae venom. The preclinical paraspecific ED50 of 31.3 mg of P. arcosae venom per gram of antivenom points to Brazilian SAB as a promising candidate for the treatment of envenomings by the Manabi hognosed pitviper P. arcosae.

Mutual enlightenment: A toolbox of concepts and methods for integrating evolutionary and clinical toxinology via snake venomics and the contextual stance.

Snakebite envenoming is a neglected tropical disease that may claim over 100,000 human lives annually worldwide. Snakebite occurs as the result of an interaction between a human and a snake that elicits either a defensive response from the snake or, more rarely, a feeding response as the result of mistaken identity. Snakebite envenoming is therefore a biological and, more specifically, an ecological problem. Snake venom itself is often described as a "cocktail", as it is a heterogenous mixture of molecules including the toxins (which are typically proteinaceous) responsible for the pathophysiological consequences of envenoming. The primary function of venom in snake ecology is pre-subjugation, with defensive deployment of the secretion typically considered a secondary function. The particular composition of any given venom cocktail is shaped by evolutionary forces that include phylogenetic constraints associated with the snake's lineage and adaptive responses to the snake's ecological context, including the taxa it preys upon and by which it is predated upon. In the present article, we describe how conceptual frameworks from ecology and evolutionary biology can enter into a mutually enlightening relationship with clinical toxinology by enabling the consideration of snakebite envenoming from an "ecological stance". We detail the insights that may emerge from such a perspective and highlight the ways in which the high-fidelity descriptive knowledge emerging from applications of -omics era technologies - "venomics" and "antivenomics" - can combine with evolutionary explanations to deliver a detailed understanding of this multifactorial health crisis.

What's in a mass?

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.

Seminal Plasma: Relevant for Fertility?

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA-the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.

Comparative venomics and preclinical efficacy evaluation of a monospecific Hemachatus antivenom towards sub-Saharan Africa cobra venoms.

Cobras are the most medically important elapid snakes in Africa. The African genera Naja and Hemachatus include snakes with neurotoxic and cytotoxic venoms, with shared biochemical, toxinological and antigenic characteristics. We have studied the antigenic cross-reactivity of four sub-Saharan Africa cobra venoms against an experimental monospecific Hemachatus haemachatus antivenom through comparative proteomics, preclinical assessment of neutralization, and third generation antivenomics. The venoms of H. haemachatus, N. annulifera, N. mossambica and N. nigricollis share an overall qualitative family toxin composition but depart in their proportions of three-finger toxin (3FTxs) classes, phospholipases A2 (PLA2s), snake venom metalloproteinases (SVMPs), and cysteine-rich secretory proteins (CRISPs). A monospecific anti-Hemachatus antivenom produced by Costa Rican Instituto Clodomiro Picado neutralized the lethal activity of the homologous and heterologous neuro/cytotoxic (H. haemachatus) and cyto/cardiotoxic (N. mossambica and N. nigricollis) venoms of the three spitting cobras sampled, while it was ineffective against the lethal and toxic activities of the neurotoxic venom of the non-spitting snouted cobra N. annulifera. The ability of the anti-Hemachatus-ICP antivenom to neutralize toxic (dermonecrotic and anticoagulant) and enzymatic (PLA2) activities of spitting cobra venoms suggested a closer kinship of H. haemachatus and Naja subgenus Afrocobra spitting cobras than to Naja subgenus Uraeus neurotoxic taxa. These results were confirmed by third generation antivenomics. BIOLOGICAL SIGNIFICANCE: African Naja species represent the most widespread medically important elapid snakes across Africa. To gain deeper insight into the spectrum of medically relevant toxins, we compared the proteome of three spitting cobras (Hemachatus haemachatus, Naja mossambica and N. nigricollis) and one non-spitting cobra (N. annulifera). Three finger toxins and phospholipases A2 are the two major protein families among the venoms analyzed. The development of antivenoms of broad species coverage is an urgent need in sub-Saharan Africa. An equine antivenom raised against H. haemachatus venom showed cross-reactivity with the venoms of H. haemachatus, N. mossambica and N. nigricollis, while having poor recognition of the venom of N. annulifera. This immunological information provides clues for the design of optimum venom mixtures for the preparation of broad spectrum antivenoms.

Antivenomics and in vivo preclinical efficacy of six Latin American antivenoms towards south-western Colombian Bothrops asper lineage venoms.

BACKGROUND: Bothrops asper represents the clinically most important snake species in Central America and Northern South America, where it is responsible for an estimated 50-80% of snakebites. Compositional variability among the venom proteomes of B. asper lineages across its wide range mirrors clinical differences in their envenomings. Bothropic antivenoms generated in a number of Latin American countries commonly exhibit a certain degree of paraspecific effectiveness in the neutralization of congeneric venoms. Defining the phylogeographic boundaries of an antivenom's effectivity has implications for optimizing its clinical use. However, the molecular bases and impact of venom compositions on the immune recognition and neutralization of the toxic activities of across geographically disparate populations of B. asper lineages has not been comprehensively studied. METHODOLOGY/PRINCIPAL FINDINGS: Third-generation antivenomics was applied to quantify the cross-immunorecognizing capacity against the individual components of venoms of three B. asper lineages (B. asper (sensu stricto), B. ayerbei and B. rhombeatus) distributed in south-western (SW) Colombia, of six Latin American antivenoms, produced against homologous (Colombia, INS-COL and PROBIOL) and Costa Rica (ICP)), and heterologous (Argentina (BIOL), Perú (INS-PERU) and Venezuela (UCV)) bothropic venoms. In vivo neutralization assays of the lethal, hemorrhagic, coagulant, defibrinogenating, myotoxic, edematogenic, indirect hemolytic, and proteolytic activities of the three SW Colombian B. asper lineage venoms were carried to compare the preclinical efficacy of three (Colombian INS-COL and PROBIOL, and Costa Rican ICP) antivenoms frequently used in Colombia. Antivenomics showed that all the six antivenom affinity matrices efficiently immunoretained most of the B. asper lineages venom proteins and exhibited impaired binding towards the venoms' peptidomes. The neutralization profile of the INS-COL, PROBIOL and ICP antivenoms towards the biological activities of the venoms of SW Colombian B. asper (sensu stricto), B. ayerbei and B. rhombeatus lineages was coherent with the antivenomics outcome. In addition, the combination of in vitro (antivenomics) and in vivo neutralization results allowed us to determine their toxin-specific and venom neutralizing antibody content. Noteworthy, heterologous INS-PERU, BIOL, and UCV bothropic antivenoms had equal or higher binding capacity towards the venoms components of SW Colombian B. asper lineages that the homologous Colombian and Costa Rican antivenoms. CONCLUSIONS/SIGNIFICANCE: The combined in vitro and in vivo preclinical outcome showed that antivenoms manufactured in Colombia and Costa Rica effectively neutralize the major toxic activities of SW Colombian B. asper lineage venoms. The antivenomics profiles of the heterologous antivenoms manufactured in Argentina, Venezuela, and Perú strongly suggests their (pre)clinical adequacy for the treatment of B. asper lineage envenomings in SW Colombia. However, their recommendation in the clinical setting is pending on in vivo neutralization testing and clinical testing in humans. Bothrops asper is a highly adaptable snake species complex, which is considered the most dangerous snake throughout much of its distribution range from the Atlantic lowland of eastern México to northwestern Perú. Antivenoms are the only scientifically validated treatment of snakebite envenomings. Venom variation is particularly common in wide ranging species, such as B. asper, and may result in variable clinical presentations of envenomings, as is the case for the B. asper species complex, potentially undermining the efficacy of snakebite treatments depending on the immunization mixture used in the generation of the antivenom. Conversely, phylogenetic conservation of antigenic determinants confers an unpredictable degree of paraspecificity to homologous antivenoms produced for a geographic area, but also to heterologous congeneric antivenoms, towards the venom components of allopatric conspecific populations. This work aimed at comparing the preclinical profile of a panel of Latin American homologous and heterologous antivenoms against the venoms of B. asper lineages distributed in SW Colombia. The outcome of this study strongly suggests the suitability of considering the heterologous antivenoms BIOL (Argentina), UCV (Venezuela) and INS-PERU (Perú) as alternatives to homologous Colombian INS-COL and PROBIOL and Costa Rican ICP antivenoms for the treatment of envenomings by B. asper (sensu stricto) in W Colombia and Ecuador, B. ayerbei in Cauca and Nariño (Colombia), and B. rhombeatus in Cauca river valley, SW Colombia.

The earless monitor lizard Lanthanotus borneensis - A venomous animal?

Based on its mandibular gland secretion, the earless monitor lizard, Lanthanotus borneensis, has been considered a venomous animal like other members of the Toxicofera group, including Heloderma. In the present study, the gland structure and teeth of L. borneensis were examined by micro-tomography (µCT) and scanning electron microscopy (SEM), respectively, and proteomic analysis of the gland extract was performed. The mandibular gland consists of six compartments with separate ducts. The pleurodont teeth of the lower and upper jaw are not grooved but possess a sharp ridge on the anterior surface. Proteomic analysis of the gland extract confirmed previous studies that kallikrein enzymes are the major biologically active components. In view of the lizard's biology, its mandibular gland secretion is obviously not needed for prey capture or defence. It seems not justified the labelling of L. borneensis as a venomous animal. However, definitively answering this question requires toxinological studies on natural prey.

What's in a mass?

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.

Venom variation in Bothrops asper lineages from North-Western South America.

Bothrops asper is a venomous pitviper that is widely distributed and of clinical importance in Mesoamerica and northern South America, where it is responsible for 50-80% of all envenomations by Viperidae species. Previous work suggests that B. asper has a complex phylogeographic structure, with the existence of multiple evolutionarily distinct lineages, particularly in the inter-Andean valleys of north South America. To explore the impact of the evolutionary history of B. asper on venom composition, we have investigated geographic variation in the venom proteome of this species from the populations from the Pacific side of Ecuador and south-western Colombia. Among the 21 classes of venom components identified, proteins from mainly four major toxin families, snake venom metalloproteases (PI- and PII-SVMP), phospholipases A2 (K49- and D49-PLA2s), serine proteinases (SVSP), and C-type lectins-like (CTL) proteins are major contributors to the geographic variability in venom. Principal component analyses demonstrate significant differences in venom composition between B. asper lineages previously identified through combination of molecular, morphological and geographical data, and provide additional insights into the selection pressures modulating venom phenotypes on a geographic scale. In particular, altitudinal zonation within the Andean mountain range stands out as a key ecological factor promoting diversification in venom. In addition, the pattern of distribution of PLA2 molecules among B. asper venoms complements phylogenetic analysis in the reconstruction of the dispersal events that account for the current biogeographic distribution of the present-day species' phylogroups. Ontogenic variation was also evident among venoms from some Ecuadorian lineages, although this age-related variation was less extreme than reported in B. asper venoms from Costa Rica. The results of our study demonstrate a significant impact of phylogenetic history on venom composition in a pitviper and show how analyses of this variation can illuminate the timing of the cladogenesis and ecological events that shaped the current distribution of B. asper lineages. BIOLOGICAL SIGNIFICANCE: Bothrops asper, called "the ultimate pitviper" due to its defensive behavior, large body size, and medical importance, represents a species complex that is widely distributed from southern México southwards across north-western South America to north-western Perú. This work reports the characterization of the venom proteomes of B. asper lineages from the Pacific sides of Ecuador and south-western Colombia. Multivariate analyses indicate that variability in venom composition among the B. asper lineages is driven by proteins from four major toxin families, presumably in response to selection pressures created by recent and historical ecological conditions created by geological and climatic events from the Pliocene-Pleistocene to the present along the Central and South American Continental Divide. The emerging biogeographic pattern of venom variation, interpreted in the context of the current phylogenetic hypotheses, support and complement previously proposed evolutionary Plio-Pleistocene dispersal events that shaped the present-day distribution range of B. asper lineages. In addition, our venomics data indicate the occurrence of genetic exchange between Colombian and Pacific Costa Rican populations, which may have occurred during the second wave of B. asper migration into Mesoamerica. Our work represents a foundation for a future broader sampling and more complete "-omics" analyses to deepen our understanding of the patterns and causes of venom variation in this medically important pitviper.

Comparative proteomic profiling and functional characterization of venom pooled from captive Crotalus durissus terrificus specimens and the Brazilian crotalic reference venom.

The South American rattlesnake Crotalus durissus spp has a wide geographic distribution in Brazil. Although responsible for only a low proportion of ophidian accidents, it is considered one of the most medically important species of venomous snakes due to the high mortality rate (1.87%). Snake venom is a complex phenotype commonly subjected to individual intraspecific, ontogenetic and geographic variability. Compositional differences in pooled venom used in the immunization process may impact the efficacy of the antivenom. In order to assure standardized high-quality antivenom, the potency of each Brazilian crotalic antivenom batch is determined against the 'Brazilian Crotalic Reference Venom' (BCRV). BCRV is produced by Instituto Butantan using venom obtained from the first milking of recently wild-caught C. d. terrificus specimens brought to the Institute. The decrease in the number of snake donations experienced in recent years can become a threat to the production of future batches of BCRV. To evaluate the feasibility of using venom from long-term captive animals in the formulation of BCRV, we have compared the proteomic, biochemical and biological profiles of C. d. terrificus venom pooled from captive specimens (CVP- captive venom pool) and BCRV. Electrophoretic and venomics analyses revealed a very similar venom composition profile, but also certain differences in toxins abundance, with some low abundant protein families found only in BCRV. Enzymatic (L-amino acid oxidase, phospholipase A2 and proteolytic) and biological (myotoxic and coagulant) activities showed higher values in CVP than in BCRV. CVP also possessed slightly higher lethal effect, although the Instituto Butantan crotalic antivenom showed equivalent potency neutralizing BCRV and CVP. Our results strongly suggest that venom from long-term captive C. d. terrificus might be a valid alternative to generate an immunization mixture of equivalent quality to the currently in use reference venom.

Danger in the Canopy. Comparative Proteomics and Bioactivities of the Venoms of the South American Palm Pit Viper Bothrops bilineatus Subspecies bilineatus and smaragdinus and Antivenomics of B. b. bilineatus (Rondônia) Venom against the Brazilian Pentabothropic Antivenom.

We report a structural and functional proteomics characterization of venoms of the two subspecies (Bothrops bilineatusbilineatus and B. b. smaragdinus) of the South American palm pit viper from the Brazilian state of Rondônia and B. b. smaragdinus from Perú. These poorly known arboreal and mostly nocturnal generalist predators are widely distributed in lowland rainforests throughout the entire Amazon region, where they represent an important cause of snakebites. The three B. bilineatus spp. venom samples exhibit overall conserved proteomic profiles comprising components belonging to 11 venom protein classes, with PIII (34-40% of the total venom proteins) and PI (8-18%) SVMPs and their endogenous tripeptide inhibitors (SVMPi, 8-10%); bradykinin-potentiating-like peptides (BBPs, 10.7-15%); snake venom serine proteinases (SVSP, 5.5-14%); C-type lectin-like proteins (CTL, 3-10%); phospholipases A2 (PLA2, 2.8-7.6%); cysteine-rich secretory proteins (CRISP, 0.9-2.8%); l-amino acid oxidases (LAO, 0.9-5%) representing the major components of their common venom proteomes. Comparative analysis of the venom proteomes of the two geographic variants of B. b. smaragdinus with that of B. b. bilineatus revealed that the two Brazilian taxa share identical molecules between themselves but not with Peruvian B. b. smaragdinus, suggesting hybridization between the geographically close, possibly sympatric, Porto Velho (RO, BR) B. b. smaragdinus and B. b. bilineatus parental populations. However, limited sampling does not allow determining the frequency of this event. The toxin arsenal of the South American palm pit vipers may account for the in vitro recorded collagenolytic, caseinolytic, PLA2, l-amino acid oxidase, thrombin-like and factor X-activating activities, and the clinical features of South American palm pit viper envenomings, i.e., local and progressively ascending pain, shock and loss of consciousness, spontaneous bleeding, and profound coagulopathy. The remarkable cross-reactivity of the Brazilian pentabothropic SAB antivenom toward the heterologous B. b. bilineatus venom suggests that the paraspecific antigenic determinants should have been already present in the venom of the last common ancestor of the Bothrops ″jararaca″ and ″taeniatus″ clades, about 8.5 Mya in the mid-late Miocene epoch of the Cenozoic era. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020043, PXD020026, and PXD020013.

Venomics and biochemical analysis of the black-tailed horned pitviper, Mixcoatlus melanurus, and characterization of Melanurutoxin, a novel crotoxin homolog.

We report a structural and functional venomics characterization of the black-tailed horned pitviper, Mixcoatlus melanurus. The venom phenotype of this small and elusive pitviper endemic to México comprise peptides and proteins of 16 toxin families whose relative abundance mirror those of neurotoxic (type II) venoms described for some species within genera distributed in Central Asia (Gloydius) and the Americas (Sistrurus, Crotalus, Ophryacus, and Bothriechis). A novel ß-neurotoxic heterodimeric PLA2, termed Melanurutoxin was characterized. With a relative abundance of 14.8% of the total M. melanurus venom proteome and a median lethal dose of 0.31 µg/g mouse body weight, Melanurutoxin accounted for 37.8% of the lethality of the whole venom (0.82 µg/g). The low percentage (1.1%) of snake venom metalloproteinases (PIII-SVMPs) and the high content of Melanurutoxin and bradykinin-potentiating peptides (BPP, 16%) found in the type-II venom proteome of M. melanurus correlate with the severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest observed in ex vivo neuromuscular junction experiments and in vivo experimental murine envenoming. Mexican antivenoms manufactured by Birmex and Bioclon showed low neutralization potency per vial (95 LD50s, Birmex; 114 LD50s, Antivipmyn®), and failed to reverse completely the paralysis and the hypotensive effect induced by the black-tailed horned pitviper, Mixcoatlus melanurus. We suggest that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom may be attributed to the use of immunization mixtures that include venom of taxa, C. basiliscus (Birmex) and C. simus (Antivipmyn®), that contain only small amounts of Melanurutoxin-like ß-neurotoxic heterodimeric PLA2s. BIOLOGICAL SIGNIFICANCE: This study represents the first proteomics and funcional investigations conducted on the venom of the black-tailed horned, Mixcoatlus melanurus, a pitviper species endemic to México. The venom's features unveiled through combination of bottom-up venomics and ex vivo and in vivo functional assays provided complementary evidence pointing to severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest as the predominant mechanism of murine prey immobilization and death caused by M. melanurus. A novel ß-neurotoxic heterodimeric PLA2, coined Melanurutoxin, was identified as a major contributor to the lethality of the whole venom. Our study also showed the inefficacy of two commercial Mexican antivenoms to reverse competely the paralytic and hypotensive effects induced by M. melanurus venom in the murine model. We hypothesize that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom should be ascribed to the use as immunogens of venoms that contain only small amounts of Melanurutoxin-like ß-neurotoxic heterodimeric PLA2s.