Toxicological, enzymatic, and immunochemical characterization of Bothrops asper (Serpentes: Viperidae) reference venom from Panama / Caracterización toxicológica, enzimática e inmunoquímica del veneno de referencia de Bothrops asper (Serpentes: Viperidae) de Panamá

Introduction: It is estimated that 2 000 snakebites occur in Panama every year, 70 % of which are inflicted by Bothrops asper. Objective: To determine the biochemical and toxicologic effects and to assess the immunochemical characteristics of a reference pool of B. asper venom representative of Panama. Methods: The reference venom was prepared as a homogeneous mixture of the venoms obtained from 78 adult snakes collected in four geographic areas of Panama. Enzymatic and toxicological activities were assessed. The electrophoretic pattern was studied by SDS-PAGE. Immunoreactivity of various antivenoms was analyzed by Western blot. Results: B. asper reference venom has lethal, hemorrhagic, myotoxic, edema-forming, coagulant, defibrinating, proteinase and phospholipase A2 activities. SDS-PAGE showed the presence of protein bands with molecular weights ranging from 8 to 70 kDa, with the presence of predominant bands at ≈ 15 kDa and ≈ 30 to 66 kDa, which likely correspond to phospholipases A2 and metalloproteinases, respectively. Immunoblotting showed a high degree of recognition by various antivenoms, especially by antivenoms from Colombia and Costa Rica. Conclusions: Following recommendations by the World Health Organization, this reference venom of B. asper of Panama will become a useful tool for the preclinical evaluation of antivenoms distributed in this country.

Introducción: Se estima que 2 000 mordeduras de serpiente ocurren en Panamá cada año, el 70 % de las cuales son infligidas por Bothrops asper. Objetivo: Determinar los efectos bioquímicos y toxicológicos y evaluar las características inmunoquímicas del veneno de referencia de B. asper representativo de Panamá. Métodos: El veneno de referencia se preparó como una mezcla homogénea de los venenos obtenidos de 78 serpientes adultas recolectadas en cuatro áreas geográficas de Panamá. Se evaluaron las actividades enzimáticas y toxicológicas. El patrón electroforético se estudió mediante SDS-PAGE. La inmunoreactividad de varios antivenenos se analizó mediante transferencia de Western. Resultados: El veneno de referencia de B. asper tiene actividades letales, hemorrágicas, miotóxicas, formadoras de edema, coagulantes, desfibrinante, proteolítica y de fosfolipasa A2. El análisis de SDS-PAGE mostró la presencia de bandas de proteínas con pesos moleculares que varían de 8 a 70 kDa, con la presencia de bandas predominantes a ≈ 15 kDa y ≈ 30 a 66 kDa, que probablemente corresponden a fosfolipasas A2 y metaloproteinasas, respectivamente. La inmunotransferencia mostró un alto grado de reconocimiento por varios antivenenos, especialmente por antivenenos de Colombia y de Costa Rica. Conclusiones: Siguiendo las recomendaciones de la Organización Mundial de la Salud, este veneno de referencia de B. asper de Panamá se convertirá en una herramienta útil para la evaluación preclínica de antivenenos distribuidos en este país.

Envenenamiento ofídico en Costa Rica: logros y tareas pendientes / Snakebite envenoming in Costa Rica: achievements and pending tasks

Resumen Objetivo: Efectuar un análisis de los logros en Costa Rica en cuanto al problema de los envenenamientos por mordeduras de serpientes, y señalar tareas pendientes para reducir aún más el impacto de esta patología. Metodología: Se efectuó una revisión de bibliografía relacionada con el estudio del envenenamiento ofídico en Costa Rica y con los avances efectuados en el tema, en el país. Paralelamente, se identificaron aspectos que requieren atención en el manejo de esta enfermedad tropical desatendida. Conclusiones: Desde las primeras décadas del siglo XX, se han realizado avances significativos en la comprensión y manejo del problema de los envenenamientos por mordedura de serpiente en Costa Rica. Se ha trabajado desde una visión integral que incluye: investigación científico-tecnológica, producción y distribución de antivenenos, esfuerzos en prevención, capacitación de las personas profesionales de la salud en el diagnóstico y tratamiento, y docencia de grado y posgrado en el tema. El país ha asumido un papel de liderazgo a nivel internacional, tanto en el plano académico como en la provisión de antivenenos a muchos países. No obstante, aún quedan aspectos del problema que requieren nuevos esfuerzos en nuestro medio, sobre todo en lo referente a la atención de las consecuencias biomédicas, psicológicas, sociales y económicas que sufren las personas afectadas.

Abstract Aim: To analyze the achievements made in Costa Rica in confronting the problem of snakebite envenomings, and to identify pending tasks to further reduce the impact of this pathology. Methods: A review of the literature on snakebite envenomings in Costa Rica was carried out, identifying the main achievements reached in the country. In parallel, issues that require renewed attention in the management of this neglected tropical disease were identified. Conclusions: Since the first decades of the 20th century, significant advances have been made in Costa Rica for understanding and confronting the problem of snakebite envenomings. An integrative perspective has been implemented, which includes scientific and technological research, production and distribution of antivenoms, prevention campaigns, training of health professionals in the diagnosis and treatment of envenomings, and teaching at graduate and undergraduate levels. Costa Rica has had a leading international role in this topic at the academic level, and by providing antivenoms to many countries. Nevertheless, there are issues that require further efforts, especially regarding the attention to the biomedical, psychological, social and economic consequences suffered by people affected by snakebites.

Antibiotic therapy for snakebite envenoming

There are numerous conflicting recommendations available on the use of antibiotics following snakebite. The present letter to the editor presents some recommendations based on recent studies, and aims to stimulate debate on this topic.(AU)

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Background 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. Methods 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. Results 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). Conclusion 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.(AU)

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Abstract Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patients 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. Methods The articular inflammation was induced by MT-II (10 g/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. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 g/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 TNF, IL-1, 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). Conclusion 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.

The need for an integrated approach in confronting snakebite envenoming in Latin America: the relevance of endogenous scientific and technological research / Hacia un abordaje integral del envenenamiento ofídico en américa latina: la importancia de la generación de conocimiento científico-tecnológico endógeno

Snakebite envenomings constitute a serious public health problem on a global level, especially in Africa, Asia and Latin America [1, 2]. In our Latin American region, it is estimated that at least 70,000 snakebite cases occur every year, although the actual number is likely to be higher [3]. This pathology is one of the so-called 'neglected tropical diseases', i.e. a group of diseases that affect primarily poor people in poor settings and, therefore, does not receive the necessary attention from research agencies, pharmaceutical companies, and health authorities. Consequently, it has been described as a 'disease of poverty' [3, 4]. In our continent it largely affects impoverished populations living in rural areas, including indigenous groups [3]

Current challenges for confronting the public health problem of snakebite envenoming in Central America

Snakebite envenoming is a serious public health problem in Central America, where approximately 5,500 casesoccur every year. Panama has the highest incidence and El Salvador the lowest. The majority, and most severe,cases are inflicted by the pit viper Bothrops asper (family Viperidae), locally known as ‘terciopelo’, ‘barba amarilla’ or‘equis’. About 1% of the bites are caused by coral snakes of the genus Micrurus (family Elapidae). Despite significantand successful efforts in Central America regarding snakebite envenomings in the areas of research, antivenommanufacture and quality control, training of health professionals in the diagnosis and clinical management of bites,and prevention of snakebites, much remains to be done in order to further reduce the impact of this medicalcondition. This essay presents seven challenges for improving the confrontation of snakebite envenoming inCentral America. Overcoming these challenges demands a coordinated partnership of highly diverse stakeholdersthough inter-sectorial and inter-programmatic interventions.

Current challenges for confronting the public health problem of snakebite envenoming in Central America

Snakebite envenoming is a serious public health problem in Central America, where approximately 5,500 casesoccur every year. Panama has the highest incidence and El Salvador the lowest. The majority, and most severe,cases are inflicted by the pit viper Bothrops asper (family Viperidae), locally known as ‘terciopelo’, ‘barba amarilla’ or‘equis’. About 1% of the bites are caused by coral snakes of the genus Micrurus (family Elapidae). Despite significantand successful efforts in Central America regarding snakebite envenomings in the areas of research, antivenommanufacture and quality control, training of health professionals in the diagnosis and clinical management of bites,and prevention of snakebites, much remains to be done in order to further reduce the impact of this medicalcondition. This essay presents seven challenges for improving the confrontation of snakebite envenoming inCentral America. Overcoming these challenges demands a coordinated partnership of highly diverse stakeholdersthough inter-sectorial and inter-programmatic interventions.

New approaches & technologies of venomics to meet the challenge of human envenoming by snakebites in India.

The direct estimate of 46,000 snakebite deaths in India in 2005 (1 for every 2 HIV/AIDS deaths), based on verbal autopsies, renders unrealistic the total of only 47,000 snakebite deaths in the whole world in 2010, obtained indirectly as part of the “Global Burden of Disease 2010” study. Persistent underestimation of its true morbidity and mortality has made snakebite the most neglected of all the WHO’s “neglected tropical diseases”, downgrading its public health importance. Strategies to address this neglect should include the improvement of antivenom, the only specific antidote to envenoming. To accommodate increased understanding of geographical intraspecific variation in venom composition and the range of snake species that are medically important in India, the design of antivenoms (choice of venom sources and species coverage) should be reconsidered. Methods of preclinical and clinical testing should be improved. The relatively new science of venomics involves techniques and strategies for assessing the toxin composition of snake venoms directly through proteomics-centred approaches or indirectly via high-throughput venom gland transcriptomics and bioinformatic analysis. Antivenomics is translational venomics: a proteomics-based protocol to quantify the extent of cross-reactivity of antivenoms against homologous and heterologous venoms. These approaches could revolutionize the preclinical assessment of antivenom efficacy, leading to a new generation of antivenoms that are clinically more effective.

Las publicaciones de investigacion biomedica en la Revista de Biologia Tropical / Biomedical research in Revista de Biologia Tropical

The contributions published in Revista de Biología Tropical in the area of Biomedical Sciences are reviewed in terms of number of contributions and scope of research subjects. Biomedical Sciences, particularly Parasitology and Microbiology, constituted the predominant subject in the Revista during the first decade, reflecting the intense research environment at the School of Microbiology of the University of Costa Rica and at Hospital San Juan de Dios. The relative weight of Biomedicine in the following decades diminished, due to the outstanding increment in publications in Biological Sciences; however, the absolute number of contributions in Biomedical Sciences remained constant throughout the last decades, with around 80 contributions per decade. In spite of the predominance of Parasitology as the main biomedical subject, the last decades have witnessed the emergence of new areas of interest in the Revista, such as Pharmacology of natural products, Toxinology, especially related to snake venoms, and Human Genetics. This retrospective analysis evidences that Biomedical Sciences, particularly those related to Tropical Medicine, were a fundamental component during the first years of Revista de Biología Tropical, and have maintained a significant presence in the scientific output of this journal, the most relevant scientific publication in biological sciences in Central America

Comprendiendo los venenos de serpientes: 50 años de investigaciones en América Latina / Understanding snake venoms: 50 years of research in Latin America

As a tribute to Revista de BiologÝa Tropical in its 50th anniversary, this review describes some of the main research efforts carried out in the study of the chemical composition and the mechanism of action of toxins present in the venoms of snakes distributed in Latin America. Venom proteins involved in neurotoxicity, coagulopathies, hemorrhage and muscle necrosis are discussed, together with a description of the inflammatory reactions elicited by these venoms and toxins. In addition, the search for inhibitory substances present in plants and animals that may be utilized in the neutralization of venoms is analyzed. Some of the clinical studies performed on snakebite envenomations in Latin America are also reviewed, together with the development of technologies aimed at improving the quality of antivenoms produced in the region. Toxinology has become a fruitful and stimulating research field in Latin America which has contributed to a better understanding of snake venoms as well as to an improved management of snake bitten patients.

Geographic and ontogenic variability in the venom of the neotropical rattlesnake Crotalus durissus: pathophysiological and therapeutic implications

A comparative study was performed on the venoms of adult specimens of the neotropical rattlesnake, Crotalus durissus, from Guatemala, Costa Rica, Venezuela and Brazil, together with the venom of newborn specimens of C. d. durissus from Costa Rica. Venoms from Brazil (C. d. terrificus) and from newborn specimens of C. d. durissus presented an electrophoretic pattern characterized by the predominance of bands with molecular mass of 36 and 15 kDa, whereas those of adult specimens of C. d. durissus from Guatemala and Costa Rica, and C. d. cumanensis from Venezuela, showed a conspicuous band of 62 kDa, and additional bands of 36, 29 and 15 kDa. Moreover, venoms from C. d. terrificus and C. d. cumanensis showed a prominent band of < 10 kDa that probably corresponds to crotamine, since a 'crotamine-like' activity was detected in these venoms upon intraperitoneal injection in mice. Venoms of C. d. terrificus, C. d cumanensis and newborn C. d. durissus induced higher lethal and myotoxic effects than those of adult C. d. durissus. In contrast, adult C. d. durissus and C. d. cumanensis venoms induced hemorrhage, whereas venoms of C. d. terrificus and newborn C. d. durissus lacked this effect. All venoms showed coagulant effect in plasma, the highest activity being observed in the venom of newborn C. d. durissus. An anti-crotalic antivenom produced by Instituto Butantan (Brazil), using C. d. terrificus venom as antigen, was effective in the neutralization of lethal, myotoxic and coagulant effects of all venoms studied, being ineffective in the neutralization of hemorrhagic activity of the venoms of C. d. cumanensis and C. d. durissus. On the other hand, a polyvalent antivenom produced by Instituto Clodomiro Picado (Costa Rica), using the venoms of C. d. durissus. Bothrops asper and Lachesis stenophrys as antigens, was able to neutralize lethal, myotoxic, coagulant and hemorrhagic effects of C. d. durissus venom, but was ineffective in the neutralization of lethality and myotoxicity of C. d. terrificus, C. d. cumanensis and newborn C. d. durissus venom. The high toxicity of South American and newborn C. d. durissus venoms is related to the presence of high concentrations of the neurotoxic phospholipase A2 complex 'crotoxin'. Accordingly, antivenom from Instituto Butantan has a much higher titer of anti-crotoxin antibodies than antivenom from Instituto Clodomiro Picado. Crotalus durissus represents an example of intraspecies variation in venom composition and pharm

Neutralización del efecto hemorrágico inducido por veneno de Bothrops asper (Serpientes : Viperidae) por extractos de plantas tropicales / Neutralization of hemorrhagic effect inducid by venom of Bothrops asper (Serpentes: Viperidae) for extracts of tropical plants

Se evaluó la capacidad de extractos orgánicos de 48 especies de plantas costarricense para neutralizar la actividad hemorrágica del veneno de la serpiente Bothrops asper (terciopelo). Los extractos se evaluaron mediante un bioensayo basado en inyecciones intradérmicas de veneno en ratones, o de mezclas veneno-extracto, seguidas de la cuantificación macroscópica de la hemorragia. Se observó una inhibición total de la hemorragia con los extractos etanólicos, de acetato de etilo y acuoso de Bursera simaruba, Clusia torresii, C. palmana, Croton draco, Persea americana, Phoebe brenesii, Pimenta dioica, Sapindus saponaria, Smilax cuculmeca y Virola koschnyi. El análisis químico de estos extractos permitió identificar catequinas, flavonas, antocianinas y taninos condensados, los cuales podrían jugar un papel en la inhibición del efecto hemorrágico debido a la capacidad de quelar el ion zinc requerido por las metaloproteínas hemorrágicas para su acción

Actividades hemorrágicas y coagulante del veneno de Bothrops alternatus de Argentina / Haemorrhagic and clotting activities of Argentina B. alternatus venon

Se trabajó con veneno crudo de Bothrops alternatus para determinar sus actividades hemorrágica y coagulante. Para hallar la actividad hemorrágica se inocularon ratones, en forma intradérmica, con diluciones seriadas de veneno, en un volumen de 0,1 ml de solución salina amortiguada con fosfato, pH 7,2. Luego de 2 horas, se sacrificaron con éter y se midió el área hemorrágica. La Dosis Hemorrágica Mínima (DHM) es la cantidad de veneno que produce un área hemorrágica mínima de 10 mm de diámetro, para el veneno de B. alternatus fue de 3,6 µg. El estudio de la Dosis Coagulante Mínima (DCM) se realizó confrontando diluciones seriadas de veneno con plasma humano. La DMC, capaz de coagular el plasma humano en 60 seg, fue de 14,5 µg/ml. Al igual que otras especies del género Bothrops de Latinoamérica, el veneno de B. alternatus de Argentina posee marcada actividad hemorrágica y coagulante

Valores hematológicos y de enzimas séricas en caballos inoculados con venenos de serpientes para la produción de antivenenos en Costa Rica / Hematologic values and of serum enzymes in inoculated horses with snake venoms for the production of antivenins in Costa Rica

Se estudiaron algunos componentes sanguíneos en un grupo de seis caballos inmunizados por primera vez con venenos de serpientes para la producción de suero antiofídico polivalente en Costa Rica. No hubo cambios significativos en los valores de hematocrito y hemoglobina, aunque las proteínas totales sufrieron un pequeno pero significativo aumento en la segunda mitad del esquema de inmunización, probablemente relacionado con un aumento en la producción de globulinas. No hubo cambios significativos en los niveles de CK, mientras que las enzimas TGO y TGP aumentaron levemente, sin sobrepasar el límite superior normal. Estos resultados sugieren la ausencia de lesiones tisulares importantes en músculo esquelético, músculo cardíaco e hígado. Los caballos presentan únicamente alteraciones locales en el sitio de inoculación del veneno, caracterizadas por edema, abscesos y fístulas. No se observó ningún tipo de alteración sistémica. Se observó una gran variabilidad individual en el desarrollo de la respuesta de anticuerpos anti-fosfolipasa A2

Desarrollo de la respuesta de anticuerpos anti-fosfolipasa A2 en caballos inoculados con veneno para la producción de suero antiofídico polivalente en Costa Rica / Development of antibody anti-phospholipase A2 response in inoculated horses with venom for the productions of polivalent antiofidico serum in Costa Rica

Se estudió el desarrollo de la respuesta inmune y las alteraciones en el estado general en un grupo de ocho caballos adultos sanos utilizados en la producción del suero antiofídico polivalente en el Instituto Clodomiro Picado. Se observó una gran variación individual en la elevación del título de anticuerpos aunque, en términos generales, los máximos títulos se alcanzaron luego de que se inocularon las dosis de 30 mg y 50 mg de la mezcla de venenos. Con respecto a caballos que habían sido previamente utilizados en la producción de suero y que recibieron una dosis de refuerzo, el máximo título se obtuvo entre los 16 y 22 días. Las inoculaciones de la mezcla de venenos indujeron alteraciones leves en el estado general de los cabllos. Sin embargo, la mayoría de ellos desarrollaron abscesos, fístulas y fibrosis en el sitio de inoculación del veneno. Se concluye que, dada la gran variabilidad individual de los caballos en lo que respecta a la respuesta inmune, se hace necesaria su evaluación individual, con el fin de seleccionar los animales que produzcan una respuesta satisfactoria

Descripción de un programa para la determinación de capacidad de los antivenenos para neutralizar la actividad hemolítica de los venenos / Description of a program to determinate neutralizing antihemolitic capacity of antivenems

Se describe un programa en lenguaje Pascal para calcular la capacidad de los anntivenenos para neutralizar la actividad hemolítica indirecta de los venenos de serpientes, cuando se emplea la técnica de hemólisis radial descrita por Gutiérrez et al. el operador debe suministrar el número de niveles, asi como los niveles mayor y menor, el diámetro de los halos, de hemólisis y el diámetro del orificio que se hace en el gel para aplicar las muestras. El resultado se expresa como dosis efectiva 50%(DE50), definida como la razón Ul de antiveneno/mg de veneno en la que se neutraliza en un 50%la actividad hemolítica del veneno.

Bothrops ophryomegas Bocourt (Serpentes: Viperidae) en Costa Rica: distribución, lepidosis, variación sexual y cariotipo / Bothrops ophryomegas Bocourt (Serpentes: Viperidae) in Costa Rica: distribution, lepidosis, sexual variation and karyotype

The distributions, karyotype and morphological characteristics of Bothrops ophryomegas from Costa Rica are discribed. This species presents a conspicuous sexual dimorphism and dichromatism. It is distributed in the dry forest areas of Guanacaste and Puntarenas. Its karyotype is indistnguisable from those discribed for other crotaline species.