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Bothrops asper is the medically most important venomous snake in Central America. In Panama, the country having the highest incidence of snakebites in Latin America, B. asper is widely distributed throughout the country and is responsible for the vast majority of snakebites. This study was performed to analyze whether there are variations in the toxicological profile and in some biochemical parameters between the venoms of B. asper from four different regions in Panama. The venoms showed a similar profile of lethal, hemorrhagic, in vitro coagulant, defibrinogenating, edema-forming, myotoxic and indirect hemolytic activities, with subtle quantitative variations between samples of some regions. The venoms also had similar SDS-PAGE patterns and reverse phase HPLC profiles. A polyvalent antivenom manufactured in Costa Rica, and regularly used in Panama, was effective in the neutralization of lethal activity of the venoms of the four populations, with Mean Effective Doses (ED50) ranging from 5.98 to 9.72 mg venom/mL antivenom. In agreement, a widespread pattern of cross-reactivity between this antivenom and the four venoms was observed by immunoblotting. Overall, results highlight the lack of marked differences between the venoms of the various populations of B. asper in Panama, and that the antivenom from Costa Rica is effective in neutralizing lethality. Secretaría Nacional de Ciencia y Tecnología/[ INF10-051]/SENACYT/Panamá Secretaría Nacional de Ciencia y Tecnología/[COL06-017]/SENACYT/Panamá Universidad de Panamá/[VIP-UP CUFI-2015-CS-P-010]/UP/Panamá Universidad de Panamá/[VIP-01-04-15-2016-01]/UP/Panamá Universidad de Panamá/[CE-PT-327-15-02-15-33]/UP/Panamá UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Jicaro (Crescentia alata) is a tropical tree native to Mesoamerica well adapted to severe drought conditions. The seeds of the fruits were analyzed for protein, fatty acid, dietary fiber, phytate, polyphenol, tannin, tocopherol, sugar, mineral, amino acid and trypsin inhibitor contents. The jicaro cotyledons contained 43.6 1.15 g protein/100 g and 38.0 0.20 g fat/100 g (d.w.), which is comparable to most protein-rich and oleaginous seeds. Among the lipids, 77.6% were unsaturated fatty acids, particularly oleic acid, and essential amino acids represented 16.0 0.9% (d.w.) of the protein fraction, which is similar to soybean amino acid contents. A proteomic analysis and SDS-PAGE electrophoresis revealed that the proteins are mostly of low molecular weight ( 10 kDa), and the storage protein 2S albumin dominated. Jicaro seed trypsin inhibitory activity was low (0.1 trypsin inhibitor units TIU/mg), which enhances the digestibility of its proteins. The jicaro seed cotyledon represents an autochthonous and high-quality food source. Programa Regional de Seguridad Alimentaria y Nutricional/[C-15-2012]/PRESANCA/ French Cooperation in Central America (Institut Français pour l’Amérique Centrale-IFAC)///Francia UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro Nacional de Ciencia y Tecnología de Alimentos (CITA) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Bothriechis is a genus of eleven currently recognized slender and arboreal venomous snakes, commonly called palm-pitvipers that range from southern Mexico to northern South America. Despite dietary studies suggesting that palm-pitvipers are generalists with an ontogenetic shift toward endothermic prey, venom proteomic analyses have revealed remarkable divergence between the venoms of the Costa Rican species, B. lateralis, B. schlegelii, B. supraciliaris, and B. nigroviridis. To achieve a more complete picture of the venomic landscape across Bothriechis, the venom proteomes of biodiversity of the northern Middle American highland palm-pitvipers, B. thalassinus, B. aurifer, and B. bicolor from Guatemala, B. marchi from Honduras, and neonate Costa Rican B. lateralis and B. schlegelii, were investigated. B. thalassinus and B. aurifer venoms are comprised by similar toxin arsenals dominated by SVMPs (33–39% of the venom proteome), CTLs (11–16%), BPP-like molecules (10–13%), and CRISPs (5–10%), and are characterized by the absence of PLA2 proteins. Conversely, the predominant (35%) components of B. bicolor are D49-PLA2 molecules. The venom proteome of B. marchi is similar to B. aurifer and B. thalassinus in that it is rich in SVMPs and BPPs, but also contains appreciable amounts (14.3%) of PLA2s. The major toxin family found in the venoms of both neonate B. lateralis and B. schlegelii, is serine proteinase (SVSP), comprising about 20% of their toxin arsenals. The venom of neonate B. schlegelii is the only palm-pitviper venom where relative high amounts of Kunitz-type (6.3%) and γPLA2 (5.2%) inhibitors have been identified. Despite notable differences between their proteomes, neonate venoms are more similar to each other than to adults of their respective species. However, the ontogenetic changes taking place in the venom of B. lateralis strongly differ from those that occur in the venom of B. schlegelii. Thus, the ontogenetic change in B. lateralis produces a SVMP-rich venom, whereas in B. schlegelii the age-dependent compositional shift generates a PLA2-rich venom. Overall, genus-wide venomics illustrate the high evolvability of palm-pitviper venoms. The integration of the pattern of venom variation across Bothriechis into a phylogenetic and biogeographic framework may lay the foundation for assessing, in future studies, the evolutionary path that led to the present-day variability of the venoms of palm-pitvipers. Significance Bothriechis represents a monophyletic basal genus of eleven arboreal palm-pitvipers that range from southern Mexico to northern South America. Despite palm-pitvipers' putative status as diet generalists, previous proteomic analyses have revealed remarkable divergence between the venoms of Costa Rican species, B. lateralis, B. schlegelii, B. supraciliaris, and B. nigroviridis. Our current proteomic study of Guatemalan species, B. thalassinus, B. aurifer, and B. bicolor, Honduran B. marchi, and neonate B. lateralis and B. schlegelii from Costa Rica was undertaken to deepen our understanding of the evolutionary pattern of venom proteome diversity across Bothriechis. Ancestral characters are often, but not always, preserved in an organism's development. Venoms of neonate B. lateralis and B. schlegelii are more similar to each other than to adults of their respective species, suggesting that the high evolvability of palm-pitviper venoms may represent an inherent feature of Bothriechis common ancestor. Our genus-wide data identified four nodes of venom phenotype differentiation across the phylogeny of Bothriechis. Integrated into a phylogenetic and biogeographic framework, the pattern of venom variation across Bothriechis may lay the groundwork to establish whether divergence was driven by selection for efficient resource exploitation in arboreal ‘islands’, thereby contributing to the ecological speciation of the genus.

Snake venom phospholipases A2 (PLA2) share high sequence identities and a conserved structural scaffold, but show important functional differences. Only a few PLA2s have been purified and characterized from coral snake (Micrurus spp.) venoms, and their role in envenomation remains largely unknown. In this report, we describe the isolation, sequencing and partial functional characterization of two Micrurus PLA2s: MmipPLA2 from Micrurus mipartitus and MdumPLA2 from Micrurus dumerilii, two species of clinical importance in Colombia. MmipPLA2 consisted of 119 amino acid residues with a predicted pI of 8.4, whereas MdumPLA2 consisted of 117 residues with a pI of 5.6. Both PLA2s showed the conserved 'group I' cysteine pattern and were enzymatically active, although MdumPLA2 had higher activity. The two enzymes differed notably in their toxicity, with MmipPLA2 being highly lethal to mice and mildly myotoxic, whereas MdumPLA2 was not lethal (up to 3 μg/g body weight) but strongly myotoxic. MdumPLA2 displayed higher anticoagulant activity than MmipPLA2 in vitro and caused more sustained edema in the mouse footpad assay. Neither of these enzymes was cytolytic to cultured skeletal muscle C2C12 myotubes. Based on their structural differences, the two enzymes were placed in separate lineages in a partial phylogeny of Micrurus venom PLA2s and this classification agreed with their divergent biological activities. Overall, these findings highlight the structural and functional diversity of Micrurus venom PLA2s. Universidad de Costa Rica/[741-B3-760]/UCR/Costa Rica Universidad de Antioquia/[111556933661]/UdeA/Colombia Universidad de Antioquia/[617]/UdeA/Colombia UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Pterois volitans/miles lionfish (adult and juvenile) dorsal spines and caudal fin extracts were compared in their general composition, enzymatic activities and hemolytic and cytotoxic effects on bovine aortic endothelial cells and murine myoblasts, to distinguish between the activities present in the venom and epidermal mucus. Intradermal and intramuscular injections were also administered in mice to determine in vivo effects. This work shows that crude venom of Caribbean species of lionfish, present in dorsal spines, induces several in vitro effects including hemolysis, weak cytotoxicity, proteolytic and hyaluronidase activities, whereas in vivo, it is not hemorrhagic nor myotoxic, but causes edema, plasma extravasation and a thrombotic-associated lesion on the skin. Some small differences were observed between adult and juvenile venomous secretions. Gelatinolytic activity of the epidermal mucus, the only activity found in caudal fin extracts, could contribute to the in vivo toxicity of the venom. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicina

Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient’s lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation Centro para el Estudio de Venenos y Animales Venenosos/[063-2010]/CEVAP/Brasil UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

This work offers a general overview on the evolving strategies for the proteomic analysis of snake venoms, and discusses how these may be combined through diverse experimental approaches with the goal of achieving a more comprehensive knowledge on the compositional, toxic, and immunological characteristics of venoms. Some recent developments in this field are summarized, highlighting how strategies have evolved from the mere cataloguing of venom components (proteomics/venomics), to a broader exploration of their immunological (antivenomics) and functional (toxicovenomics) characteristics. Altogether, the combination of these complementary strategies is helping to build a wider, more integrative view of the life-threatening protein cocktails produced by venomous snakes, responsible for thousands of deaths every year., Research at IBV-CSIC was supported by grant BFU2013-42833-P from the Ministerio de Economía y Competitividad, Madrid, Spain. Research at ICP-UCR was supported by Vicerrectoría de Investigación, Universidad de Costa Rica.

Phospholipase A2 are esterolytic enzymes that catalyze the hydrolysis of 1,2-diacyl-3-sn-glycerophospholipids at the C2 position, resulting in the formation of lysophospholipids and free fatty acid. They constitute one of the largest families of lipid hydrolyzing enzymes. PLA2s are known to be one of the most potent components found in snake venoms, where they exhibit a diverse array of pharmacological activities. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

Envenoming following snakebite is largely a neglected threat to public health in tropical and subtropical regions of Africa, Asia, Latin America and Oceania addicting some of the world’s poorest rural communities. An estimated 5.5. Million people are bitten by sneakers each year, resulting in about 400 000 amputations, and between 20 000 and 125 000 deaths; however, the true scale of this “disease of poverty” may be much greater than these hospital-based statistics. Ministerio de economía y competitividad/[BFU2010-17373]//España Generalitat Valenciana/[PROMETEO 2010-005]//España Fundación Costa Rica- Estados Unidos para la cooperación/[2009CR0021]/CRUSA-CSIC/Costa Rica Programa Iberoamericano de ciencia y tecnología para el desarrollo/[BIOTOXP211RT0412]/CYTED/España Universidad de Costa Rica/[741-B2-652]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

Los envenenamientos por mordedura de serpiente constituyen un importante problema de salud pública en América Latina. La gran mayoría de estos envenenamientos son causados por mordeduras de serpientes de la familia Viperidae, en tanto un pequeño porcentajes de casos son consecuencia de mordeduras por serpiente de coral. Por otra parte, también ocurren accidentes por serpientes de la familia Colubridae, los cuales no cursan con envenenamiento, con algunas excepciones como el caso de la especie Philodryas olferssi, cuya mordedura produce algunas efectos locales. Universidad de Costa Rica/[]/UCR/Costa Rica International Foundation for Science/[]/IFS/Suecia Agency for International Development/[]/AID/Estados Unidos Swedish Agency for Research Cooperation with Developing Countries/[]/SAREC/Europa UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

De nombreuses envenimations ophidiennes en Amérique centrale sont dues aux crotales (famille des Viperidae) parmi lesquels Bothrops asper est le principal responsable. Quelques accidents sont provoqués par les serpents corail (famille des Elapidés, genre Micrurus). Des sérums antivenimeux (SAV) polyvalents et anti-serpent corail sont produits depuis 1967 á I´Institut Clodomiro Picado, Costa Rica. Les envenimations par crotales sont sévères et complexes, et caractérisée par des lésions locales et des désordres systémiques. L´envenimation par Micrurus sp. est caractérisée par un syndrome neurotoxique paralytique. Une série de tests de laboratoire a été adaptée á I´Institut Clodomiro Picado pour évaluer le pouvoir neutralisant des sérums antivenimeux de L´Amérique Centrale. Ces tests portent sur le pouvoir neutralisant des effets létaux, hémorragiques, oedémateux, myotoxiques, coagulants, défibrinants, neurotoxiques, protéolytiques, et des activités phospholipase A2 et hyaluronidase des venins. Ils permettent donc une évaluation précise des sérums antivenimeux fabriqués distribués en Amérique Centrale. Les résultats indiquent que les sérums antivenimeux fabriqués au Costa Rica sont efficaces contre les venins des espèces d´Amérique Centrale testées, tandis que d´autres produits disponibles dans la région ont un pouvoir neutralisant moins efficace. Ces observations montrent la nécessité d´un contróle de qualité rigoureux des sérums antivenimeux. Most snake-bite envenomings in Central America are inflicted by pit vipers (family Viperidae, subfamily Crotalinae); Bothrops asper is responsible for most accidents. A few envenomings are caused by coral snakes (family Elapidae, genus Micrurus). Polyvalent and Micrurus antivenoms have been produced in Costa Rica since 1967. Envenomings induced by pit vipers are severe and complex and are characterized by local and systemic pathophysiological alterations. Neurotoxic paralysis is the most severe effect in Micrurus envenomigs. A series of laboratory assays has been adapted at the Instituto Clodomiro Picado for evaluating the neutralizing ability of antivenoms in Central America. It includes tests for the neutralization of lethal, haemorrhagic, oedema-forming, myotoxic, coagulant, defibrinating, neurotoxic, proteolytic, phospholipase A2 and hyaluronidase activities. Thus, these assays allow for a complete, detailed evaluation of antivenoms distributed in Central America. Our studies indicate that antivenoms produced in Costa Rica are effective against the Central American venoms tested, whereas other products that are commercially available in the region are less effective in neutralizing the activities of several venoms. Our observations stress the need for rigorous quality control of antivenoms. Universidad de Costa Rica/[]/UCR/Costa Rica International Foundation for Science/[F/1388-1]/IFS/Suecia International Foundation for Science/[F/1388-2]/IFS/Suecia International Foundation for Science/[F/1388-3]/IFS/Suecia International Foundation for Science/[F/0883-1]/IFS/Suecia International Foundation for Science/[F/0883-2]/IFS/Suecia International Foundation for Science/[F/0883-3]/IFS/Suecia International Foundation for Science/[F/0883-4]/IFS/Suecia UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología