Substrate vibrations in the scorpion Centruroides margaritatus (Scorpiones: Buthidae) during courtship

articulo (arbitrado)--Universidad de Costa Rica. Escuela de Biología, 2009Mating behavior in Centruroides margaritatus, as in other scorpion species, includes a series of rapid rocking or shaking movements of the male (“juddering”). It has been suggested that substrate vibrations are generated by juddering and that females respond to them by approaching the male, but its functional significance remains little studied. For the first time, substrate vibrations produced by males during courtship in Centruroides margaritatus are documented. The male started juddering after his first physical contact with the female and only one type of male vibratory signal was registered. The signal is produced during a series of rapid shaking of the male’s body from front to rear and consists of multiple short pulses. Each pulse is called a judder and several judders “a series”. The average duration of each judder was 0.018±0.009s (n=50) with an interval of 0.028±0.013s (n=50); the average duration of each series of judders was 4.2±3.5s with an interval between series of 3.5±6.3s and a rate of 0.21±0.17 series per second. The females responded in 72% of the time to the males juddering.Universidad de Costa RicaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

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.Ministerio de Ciencia e Innovacion/[BMC 2004-01432]//EspañaMinisterio de Ciencia e Innovacion/[BFU 2007-61563]//EspañaMinisterio de Ciencia e Innovacion/[BFU 2010-173730]//EspañaMinisterio de Ciencia e Innovacion/[BFU 2013-42833-P]//EspañaMinisterio de Ciencia e Innovacion/[BFU 2017-89103-P]//EspañaNorwegian Research Council/[No.287462.]/NFR/NoruegaNational Health and Medical Research Council/[Grant 13/093/002 AVRU]/AustraliaDBT/Wellcome Trust India Alliance/[IA/I/19/2/504647]//IndiaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Integrated “omics” profiling indicates that miRNAs are modulators of the ontogenetic venom composition shift in the Central American rattlesnake, Crotalus simus simus

Background Understanding the processes that drive the evolution of snake venom is a topic of great research interest in molecular and evolutionary toxinology. Recent studies suggest that ontogenetic changes in venom composition are genetically controlled rather than environmentally induced. However, the molecular mechanisms underlying these changes remain elusive. Here we have explored the basis and level of regulation of the ontogenetic shift in the venom composition of the Central American rattlesnake, Crotalus s. simus using a combined proteomics and transcriptomics approach. Results Proteomic analysis showed that the ontogenetic shift in the venom composition of C. s. simus is essentially characterized by a gradual reduction in the expression of serine proteinases and PLA2 molecules, particularly crotoxin, a β-neurotoxic heterodimeric PLA2, concominantly with an increment of PI and PIII metalloproteinases at age 9–18 months. Comparison of the transcriptional activity of the venom glands of neonate and adult C. s. simus specimens indicated that their transcriptomes exhibit indistinguisable toxin family profiles, suggesting that the elusive mechanism by which shared transcriptomes generate divergent venom phenotypes may operate post-transcriptionally. Specifically, miRNAs with frequency count of 1000 or greater exhibited an uneven distribution between the newborn and adult datasets. Of note, 590 copies of a miRNA targeting crotoxin B-subunit was exclusively found in the transcriptome of the adult snake, whereas 1185 copies of a miRNA complementary to a PIII-SVMP mRNA was uniquely present in the newborn dataset. These results support the view that age-dependent changes in the concentration of miRNA modulating the transition from a crotoxin-rich to a SVMP-rich venom from birth through adulhood can potentially explain what is observed in the proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus. Conclusions Existing snake venom toxins are the result of early recruitment events in the Toxicofera clade of reptiles by which ordinary genes were duplicated, and the new genes selectively expressed in the venom gland and amplified to multigene families with extensive neofunctionalization throughout the approximately 112–125 million years of ophidian evolution. Our findings support the view that understanding the phenotypic diversity of snake venoms requires a deep knowledge of the mechanisms regulating the transcriptional and translational activity of the venom gland. Our results suggest a functional role for miRNAs. The impact of specific miRNAs in the modulation of venom composition, and the integration of the mechanisms responsible for the generation of these miRNAs in the evolutionary landscape of the snake's venom gland, are further challenges for future research.Ministerio de Economía y Competitividad/[BFU2010-17373]//EspañaGeneralitat Valenciana/[PROMETEO/2010/005]//EspañaUniversidad de Costa Rica/[741-B2-093]/UCR/Costa Rica/2009CR0021/CRUSA-CSIC/EspáñaPrograma Iberoamericano de Ciencia y Tecnología para el Desarrollo/206AC0281/CYTED/EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Anfibios y reptiles de los pantanos dominados por la palma de yolillo Raphia taedigera (Arecaceae) en el noreste de Costa Rica

The herpetofauna that inhabits Caribbean Costa Rica has received considerable attention in the last two decades. This assemblage includes a total of 141 species of reptiles and 95 amphibians mostly distributed in tropical wet and moist lowland forests. While most information available came from primary and secondary forest sites, little is known about the amphibians and reptiles that inhabit more open habitats, such as wetlands and swamps. For instances, swaps dominated by the yolillo palm Raphia taedigera extend through much of the northeastern Caribbean coast of Costa Rica and eastern Nicaragua, but information about the herpetological community that uses such environments remains practically unknown. This situation reflects the little research conducted in such inhospitable environments. Here, we report the results of an intensive survey conducted to assess the herpetological community that inhabit R. taedigera palm-swamps. A total of 14 species of amphibians and 17 of reptiles have been recorded from these swamps. Amphibians and reptiles that inhabit yolillo swamps have wide distributions along much of Middle America and are considered common species throughout their range. In general, yolillo swamps are poor environments for herpetofauna: richness of reptiles and amphibians is almost two times higher in the adjacent forest than in the palm dominated swamps. Furthermore, most species observed in this swamps can be considered habitat generalists that are well adapted to the extreme conditions imposed by the changes in hydroperiods, reduce understory cover, low tree diversity and simple forest architecture of these environments. Despite similarities in the herpetofauna, it is clear that not all forest species use yolillo habitat, a characteristic that is discussed in terms of physical stress driven by the prolonged hydroperiod and reduced leaflitter in the ground, as these features drive habitat structure and herpetofaunal complexity. Our list of species using yolillo swamps is not definitive, and is likely to grow as more studies are conducted in this unexplored environment. La herpetofauna que habita el Caribe y norte de Costa Rica constituye una unidad ecológica que ha recibido considerable atención en las últimas dos décadas. Esta unidad incluye cerca de 141 reptiles y 95 anfibios distribuidos principalmente en los bosques tropicales húmedos de bajura. La mayor información disponible sobre esa agregación proviene de estudios en bosques primarios y secundarios, pero poco se conoce de la herpetofauna que habita ambientes más abiertos de la región, tales como humedales y pantanos. En particular no existe información sobre la comunidad herpetológica que utiliza los pantanos dominados por la palma de yolillo R. taedigera y/o la palma real M. saccifera, pese a las enormes extensiones cubiertas por esos ambientes en la región. Esta situación es un reflejo de la poca investigación que se ha llevado a cabo en estos inhóspitos ambientes, caracterizados por largos periodos de inundación. En este trabajo, reportamos los resultados de un estudio llevado a cabo para evaluar la comunidad herpetológica que utiliza los yolillales en el noreste de Costa Rica. A partir de muestreos en ambientes de yolillo y bosques adyacentes en cinco sitios, encontramos un total de 14 especies de anfibios y 183 de reptiles en pantanos dominados por palmas, por lo que la herpetofauna en esos ambientes es más bien pobre. En contraste, la riqueza de reptiles y anfibios es casi dos veces mayor en el bosque adyacente. Anfibios y reptiles que habitan en el yolillal tienen amplia distribución a lo largo de gran parte de Mesoamérica y se incluyen entre las especies más comunes de la región. Además, la mayoría de las especies observadas son generalistas y están bien adaptadas a las condiciones extremas impuestas por los cambios en hidroperiodos, cobertura del sotobosque reducida, baja diversidad arbórea y a la arquitectura simple de estos entornos. Es evidente que no todas las especies forestales utilizan el ambiente de yolillo, una característica que se discute en términos de estrés físico inducido por la inundación prolongada y la reducida capa de hojarasca en el suelo del pantano, hidroperiodo prolongado y reducido mantillo en el suelo, factores que inciden en la estructura del hábitat y la complejidad de herpetofauna. Nuestra lista de especies que utilizan los yolillales no es definitiva, y es probable que crezca a medida que más estudios se realicen en estos ambientes tan poco explorados. 

Distribución, superficie y área protegida de humedales dominados por pantanos de palmas (Arecaceae) en Costa Rica y Nicaragua

In Central America, palm swamps are known collectively as yolillales. These wetlands are usually dominated by the raffia palm Raphia taedigera, but also by the royal palm Manicaria saccifera and –in lower extensions– by the American oil palm Elaeis oleifera. The yolillales tend to be poor in woody species and are characteristic of regions with high rainfall and extensive hydroperiods, so they remain flooded most of the year. The dominance of large raffia palm leaves in the canopy, allow these environments to be distinguishable in aerial photographs, which consequently has helped to map them along most of their distribution. However, while maps depicting yolillales are available, the extent of their surface area, perimeter and connectivity remains poorly understood. This is particularly true for yolillales in Costa Rica and Nicaragua, countries that share a good proportion of palm dominated swaps in the Rio San Juan Basin. In addition, it is not known the actual area of these environments that is under any category of protection according to the conservation systems of both countries. As a first step to catalog yolillal wetlands in Costa Rica and Nicaragua, this paper evaluates cartographic maps to delineate yolillales in the region. A subsample of yolillales mapped in this study were visited and we geo-referenced them and evaluate the extent and condition of the swamp. A total of 110 883.2ha are classified as yolillales in Nicaragua, equivalent to 22% of wetland surface area recorded for that country (excluding the Cocibolca and Xolotán Lakes). In Costa Rica, 53 931.3ha are covered by these palm dominated swamps, which represent 16.24% of the total surface area covered by wetlands. About 47% of the area covered by yolillales in Nicaragua is under some category of protection, the largest extensions protected by Cerro Silva, Laguna Tale Sulumas and Indio Maíz Nature Reserves. In Costa Rica, 55.5% of the area covered by yolillal is located within protected areas, mainly the Tortuguero National Park, Barra del Colorado Wildlife Refuge and the Sierpe-Térraba National Wetland. Therefore, in both countries, about half the area covered by these wetlands is not protected by their systems of protection of wilderness areas.En Centroamérica, los pantanos dominados por palmas son conocidos como yolillales. Estos humedales suelen ser dominados por la palma de yolillo R. taedigera, la palma real M. saccifera y en algunas ocasiones, por la palma aceitera americana E. oleifera. Los yolillales suelen ser pobres en especies leñosas y son característicos de regiones con alta precipitación e hidroperiodos extensos, de modo que permanecen anegados gran parte del año. Si bien su cartografía está disponible en Costa Rica y Nicaragua, la extensión de yolillales a lo largo de su distribución permanece pobremente conocida y no existe una evaluación del área superficial cubierta por esta asociación vegetal; ni de la extensión que se encuentra bajo alguna categoría de protección de áreas. Como un primer paso para catalogar los humedales de yolillo de Costa Rica y Nicaragua, en este trabajo evaluamos imágenes aéreas y cartografía para delinear y caracterizar yolillales en la región. Una muestra de los yolillales mapeados en este trabajo fueron visitados, georeferenciados y se evaluó la extensión y estado del humedal. Un total de 110 883.2ha son catalogadas como yolillales en Nicaragua, lo que equivale a 22% del área superficial de humedales registrados para ese país (excluyendo los lagos Cocibolca y Xolotlán). En Costa Rica, el área correspondiente a ambientes de yolillo es menor: 53 931.3ha, las que representan 16.24% de la superficie del territorio considerada humedales. Cerca del 47% del área cubierta por yolillales en Nicaragua se encuentra bajo alguna categoría de protección, cuyas mayores extensiones corresponden a las Reservas Naturales Cerro Silva, Laguna Tel Suluma e Indio Maíz. En Costa Rica, un 55.5% del área de yolillos se ubica dentro de áreas protegidas, especialmente en el Parque Nacional Tortuguero, Refugio de Vida Silvestre Barra del Colorado y Humedal Nacional Sierpe-Térraba. Por lo tanto, en ambos países, cerca de la mitad del área cubierta por estos pantanos no está resguardada por sus sistemas de protección de áreas silvestres

Uso y conservación de humedales de yolillo Raphia taedigera (Arecaceae) en el Área de Conservación Tortuguero, Costa Rica

The swamps dominated by raffia palm Raphia taedigera are conspicuous environments in the Tortuguero floodplains and in other wet regions along the Caribbean and Pacific coasts of Costa Rica. However, these environments have been little studied and are exposed to numerous threats, most importantly their replacement by agricultural activities or pastureland. In this paper, we describe some applications and uses of the raffia palms and other palms that are common in these flooded swamps. We also describe the efforts that have been made in Costa Rica for the protection or raffia-dominated swamps, through the environmental law frame of the country and the establishment of a protection system based on wilderness areas under different categories of protection. We discuss issues relevant to the future of these environments in the regions where they are distributed. Los humedales dominados por las palmas de yolillo Raphia taedigera son ambientes conspicuos en las llanuras de inundación de Tortuguero, así como en otras regiones húmedas de la costa Caribe y Pacífica de Costa Rica. Estos ambientes sin embargo han sido muy poco estudiados y están expuestos a numerosas amenazas, la más importante su drenado y substitución por actividades agrícolas o pecuarias. En este ensayo, describimos algunos usos que se le han dado a las palmas de yolillo y otras que son frecuentes en pantanos anegados. Además describimos los esfuerzos que se han realizado en Costa Rica para su protección, a partir de su legislación ambiental y la instauración de un sistema de protección basado en áreas silvestres bajo distintas categorías de protección. Por último, discutimos aspectos relevantes del futuro de estos ambientes en las regiones donde se distribuyen.

Proteomic profiling, functional characterization, and immunoneutralization of the venom of Porthidium porrasi, a pitviper endemic to Costa Rica

The genus Porthidium includes nine pitviper species inhabiting Mexico, Central America, and northern South America. Porthidium porrasi is a species endemic to the Southwest of Costa Rica, for which no information on its venom was available. In this study, the proteomic composition and functional activities of P. porrasi venom are described. The most abundant venom proteins were identified as metalloproteinases (36.5%). In descending order of abundance, proteins belonging to the disintegrin, phospholipase A2, serine proteinase, C-type lectin/ lectin-like, vascular endothelial growth factor, Cysteine-rich secretory protein, L-amino acid oxidase, phospholipase B, and phosphodiesterase families were also identified. P. porrasi venom showed a weak lethal potency in mice (10 μg/g body weight by intraperitoneal route), induced marked hemorrhage and edema, and weak myotoxic effect. These in vivo activities, as well as those assayed in vitro (proteolytic and phospholipase A2 activities) correlated with compositional data. A comparison of P. porrasi venom with those of three other Porthidium species studied to date reveals a generally conserved compositional and functional pattern in this pitviper genus. Importantly, the lethal effect of P. porrasi venom in mice was adequately cross-neutralized by a heterospecific polyvalent antivenom, supporting its use in the treatment of eventual envenomings by this species.Universidad de Costa Rica/[741-B7-608]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Venomics of the Duvernoy's gland secretion of the false coral snake Rhinobothryum bovallii (Andersson, 1916) and assessment of venom lethality towards synapsid and diapsid animal models

The Duvernoy's gland secretory proteome of the false coral snake Rhinobothryum bovallii (Costa Rica), unveiled applying bottom-up venomics, comprises a handful of toxins belonging to only three protein families, three-finger toxin (3FTx), cysteine-rich secretory protein (CRISP), and snake venom metalloprotease (PIII-SVMP). Except for small differences in the relative abundance of the PIII-SVMPs, which may be due to individual variability, no evidence of geographic variability or ontogenetic changes was found among the venom proteomes of the juvenile and adult R. bovallii snakes sampled. Major monomeric (86.5%mol) and minor dimeric (2.8%mol) 3FTxs dominate the toxin arsenal of the Costa Rican false coral snake. The remaining 10.7% of the venom proteome comprises CRISP (8.2%) and PIII-SVMP (2.4%) molecules. In vivo lethality assays showed that R. bovallii produces venom that is non-toxic towards mammalian prey, and which exerts a different toxic effect on domestic chicken chicks and baby green iguana. Toxicovenomic analysis of R. bovallii venom in the iguana model identified two 3FTx RP-HPLC fractions that faithfully mimicked the irreversible immobilizing effect of the whole venom.Ministerio de Ciencia, Innovación y Universidades/[BFU2017-89103-P]/MICIU/EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Biochemical characterization of the venom of Central American scorpion Didymocentrus krausi Francke, 1978 (Diplocentridae) and its toxic effects in vivo and in vitro

non-buthid venoms, for which knowledge is scarce. In this work, we characterized the venom of a Diplocentridae species, Didymocentrus krausi, a small fossorial scorpion that inhabits the Tropical Dry Forest of Central America. D. krausi venom soluble fraction contains proteases with enzymatic activity on gelatin and casein. Mass spectrometry and venomic analysis confirmed the presence of elastase-like, cathepsin-O-like proteases and a neprilysin- like metalloproteinase. We did not detect phospholipase A2, C or D, nor hyaluronidase activity in the venom. By homology-based venom gland transcriptomic analysis, NDBPs, a β-KTx-like peptide, and other putative toxin transcripts were found, which, together with a p-benzoquinone compound present in the venom, could potentially explain its direct hemolytic and cytotoxic effects in several mammalian cell lines. Cytotoxicity of D. krausi venom was higher than the effect of venoms from two buthid scorpion species distributed in Costa Rica, Centruroides edwardsii and Tityus pachyurus. Even though D. krausi venom was not lethal to mice or crickets, when injected in mouse gastrocnemius muscle at high doses it induced pathological effects at 24 h, which include myonecrosis, weak hemorrhage, and inflammatory infiltration. We observed an apparent thrombotic effect in the skin blood vessels, but no in vitro fibrinogenolytic activity was detected. In crickets, D. krausi venom induced toxicity and paralysis in short periods of timeUniversidad de Costa Rica/[741-B8-133]/UCR/Costa RicaUniversidad de Costa Rica/[741-A5-048]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de MedicinaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de BiologíaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA

Venomics of the scorpion Tityus ocelote (Scorpiones, Buthidae): Understanding venom evolution in the subgenus Archaeotityus

The subgenus Tityus (Archaeotityus) includes small variegated species considered until recently, a basal group within Tityus, one of the most diverse and medically relevant genera of scorpions in the New World. Archaeotityus species are distributed in the humid forests of Lower Central America and the Choco, Guiana Shield, and Amazonian regions. Due to their size and habits, Archaeotityus species are not usually considered dangerous, however, there are some clinical reports that show otherwise. To contribute to the toxinological knowledge of these poorly explored species, we characterized the venom of Tityus ocelote from three localities in Costa Rica. In addition, we assessed the evolutionary relationships of putative sodium channel-modulating peptides found in this species with those reported for other members of the genus, through a maximum likelihood phylogenetic analysis based on their amino acid sequences. We observed the presence of homologs of previously identified NaTx from the species Tityus (Archaeotityus) clathratus and some other putative Na+ and K+ channel modulating peptides related to the T. bahiensis group. In addition, we sequenced some peptides related to toxins present in the venom of the subgenus Atreus, such as those reported for T. obscurus, T. pachyurus, and the Costa Rican endemic species T. dedoslargos. Our phylogenetic analysis suggests that the venom of this Archaeotityus species is very complex and that some of the ion channel toxins expressed in it are related to distinct lineages within the genus Tityus, which could represent a plesiomorphic condition conserved in this group of scorpions of the New World.Universidad de Costa Rica/[741-C2-076]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biodiversidad y Ecología Tropical (CIBET)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin