Spatial versus spatio-temporal approaches for studying metacommunities: a multi-taxon analysis in Mediterranean and tropical temporary ponds.

Prior research on metacommunities has largely focused on snapshot surveys, often overlooking temporal dynamics. In this study, our aim was to compare the insights obtained from metacommunity analyses based on a spatial approach repeated over time, with a spatio-temporal approach that consolidates all data into a single model. We empirically assessed the influence of temporal variation in the environment and spatial connectivity on the structure of metacommunities in tropical and Mediterranean temporary ponds. Employing a standardized methodology across both regions, we surveyed multiple freshwater taxa in three time periods within the same hydrological year from multiple temporary ponds in each region. To evaluate how environmental, spatial and temporal influences vary between the two approaches, we used nonlinear variation partitioning analyses based on generalized additive models. Overall, this study underscores the importance of adopting spatio-temporal analytics to better understand the processes shaping metacommunities. While the spatial approach suggested that environmental factors had a greater influence, our spatio-temporal analysis revealed that spatial connectivity was the primary driver influencing metacommunity structure in both regions. Temporal effects were equally important as environmental effects, suggesting a significant role of ecological succession in metacommunity structure.

Proteomic profile of the venom of three dark-colored Tityus (Scorpiones: Buthidae) from the tropical rainforests of Costa Rica.

OBJECTIVE: We aimed to elucidate the potential differences in the venom peptide sequences of three Tityus species from Costa Rican rainforests: T. jaimei, T. championi and T. dedoslargos, compared to T. cf. asthenes from Colombia, which could explain the low level of scorpionism in Costa Rica, evidenced by the lack of epidemiological data. METHODOLOGY: We applied venom proteomics of peptides purified by RP-HPLC and compared the obtained sequences from venoms of these Tityus species to the sequences previously identified from Tityus inhabiting other Central and South American regions. RESULTS: Venom proteome analysis evidences that most of the putative peptide toxins identified in Costa Rican dark-colored Tityus are very similar to those present in other T. (Atreus) from the region. CONCLUSIONS: Our study suggests that, in the case of potential envenomation by Tityus in Costa Rica, the same level of toxicity should be observed, compared to other cases caused by members of the subgenus from other geographical localities. On the other hand, compared to countries with more accelerated urban expansion, Costa Rican Tityus still inhabit secondary rainforests and do not commonly share the same spaces with humans, so the lack of epidemiological evidence of medical emergencies caused by envenoming by this scorpion group could be more related to ecological and demographic factors and less attributed to the characteristics of the venom.

Inconsistent response of taxonomic groups to space and environment in mediterranean and tropical pond metacommunities.

The metacommunity concept provides a theoretical framework that aims at explaining organism distributions by a combination of environmental filtering, dispersal, and drift. However, few works have attempted a multitaxon approach and even fewer have compared two distant biogeographical regions using the same methodology. We tested the expectation that temperate (mediterranean-climate) pond metacommunities would be more influenced by environmental and spatial processes than tropical ones, because of stronger environmental gradients and a greater isolation of waterbodies. However, the pattern should be different among groups of organisms depending on their dispersal abilities. We surveyed 30 tropical and 32 mediterranean temporary ponds from Costa Rica and Spain, respectively, and obtained data on 49 environmental variables. We characterized the biological communities of bacteria and archaea (from the water column and the sediments), phytoplankton, zooplankton, benthic invertebrates, amphibians and birds, and estimated the relative role of space and environment on metacommunity organization for each group and region, by means of variation partitioning using generalized additive models. Purely environmental effects were important in both tropical and mediterranean ponds, but stronger in the latter, probably due to their larger limnological heterogeneity. Spatially correlated environment and pure spatial effects were greater in the tropics, related to higher climatic heterogeneity and dispersal processes (e.g., restriction, surplus) acting at different scales. The variability between taxonomic groups in the contribution of spatial and environmental factors to metacommunity variation was very wide, but higher in active, compared with passive, dispersers. Higher environmental effects were observed in mediterranean passive dispersers, and higher spatial effects in tropical passive dispersers. The unexplained variation was larger in the tropical setting, suggesting a higher role for stochastic processes, unmeasured environmental factors, or biotic interactions in the tropics, although this difference affected some actively dispersing groups (insects and birds) more than passive dispersers. These results, despite our limitations in comparing only two regions, provide support, for a wide variety of aquatic organisms, for the classic view of stronger abiotic niche constraints in temperate areas compared with the tropics. The heterogeneous response of taxonomic groups between regions also points to a stronger influence of regional context than organism adaptations on metacommunity organization.

Bases conceptuales para la compensación ambiental bajo el enfoque ecológico

Introducción: La generación de impactos negativos producto de proyectos de infraestructura o actividades de desarrollo generan pérdida de biodiversidad y reducción de la calidad de los servicios que el ambiente provee a sociedades humanas. Afortunadamente, muchos de esos impactos pueden ser reducidos, controlados o resarcidos por medio de medidas de mitigación, rehabilitación y compensación, que suelen sustentarse en el marco jurídico ambiental de los países o estados donde son implementados. Esta revisión provee una visión teórica del uso de compensación ambiental como instrumento jurídico/técnico integrado al manejo del entorno natural. Metodología: Realizamos una revisión de la literatura global sobre el tema mediante los buscadores de referencias GoogleScholar y SciELO empleando varios indicadores en inglés y español. Resultados: Un total de 244 referencias sobre compensación ambiental fueron encontradas, catalogadas y evaluadas, mostrándose un claro patrón de incremento del número de ellas en la última década. La compensación ambiental constituye un instrumento jurídico/técnico que permite resarcir por pérdidas ambientales y suele ser un componente de los Estudios de Impacto Ambiental regulado por el estado. Varias estrategias metodológicas son empleadas para establecer acciones compensatorias, cada una de ellas sustentada en alguno de cuatro enfoques posibles: económico, ecológico, político y cultural/social. Dentro del enfoque ecológico, se busca la equivalencia ecológica entre los elementos ambientales impactados y la compensación, procurando que no exista pérdida neta. Así, este enfoque tiene la ventaja de posibilitar la incorporación de múltiples criterios para la evaluación del daño y de las acciones de indemnización, por lo que es posible de aplicar en situaciones diversas. A pesar de su uso, la aplicación de medidas compensatorias no está exenta de crítica y hay situaciones en que la singularidad de los elementos impactados imposibilita un resarcimiento efectivo de las pérdidas. Conclusiones: La compensación tiene gran relevancia actualmente en la gestión ambiental y debe favorecer el derecho universal a un ambiente sano. Sin embargo, su aplicación efectiva requiere claridad en los procedimientos seguidos y una muy estrecha vigilancia prevenir pérdidas o incluso una ganancia neta ambiental.

Introduction: Infrastructure projects or development activities often generate a loss of biodiversity and reduce the quality of the environment's services to human societies. Fortunately, many of these impacts can be reduced, controlled, or compensated through mitigation, rehabilitation, and compensation measures, which usually rely on the environmental legal framework of the countries or states where they are implemented. This review provides a theoretical vision of using environmental compensation as a legal/technical instrument integrated into natural environment management. Methodology: We reviewed the global literature on the subject using the GoogleScholar and SciELO reference search engines using various indicators in English and Spanish. Results: We found 244 references on environmental compensation, showing a clear increasing pattern in the last decade. Environmental compensation constitutes a legal/technical instrument that allows offsets for environmental losses and often is part of the Environmental Impact Studies regulated by the state. Several methodological strategies are used to establish compensatory actions, each of them based on one of four possible approaches: economic, ecological, political, and cultural. The ecological equivalence between the impacted environmental elements and compensation is sought within the ecological approach, ensuring no net loss. Thus, this approach allows the incorporation of multiple criteria for the evaluation of damage and compensation actions: it can be applied in different situations. Nevertheless, compensatory measures are not exempt from criticism. There are situations in which the singularity of the impacted elements makes an adequate compensation for losses impossible. Conclusions: Compensation is highly relevant in environmental management and must favor the universal right to a healthy environment. However, its practical application requires clear procedures and close vigilance to prevent losses or even a net environmental gain.

Compensación Ambiental en Costa Rica: Legislación y alcances

Introducción: La compensación ambiental es un instrumento técnico-jurídico de gestión ambiental utilizado para compensar el daño ambiental debido a los impactos irreversibles e inevitables de proyectos de desarrollo. Por lo general, las regulaciones de compensación se incluyen en la legislación ambiental nacional. Este artículo resume la legislación de compensación ambiental en Costa Rica, un país con una reconocida tradición de conservación y conciencia ambiental. Nuestro objetivo es describir cómo se formula la compensación en ese país e identificar posibles vacíos regulatorios. Métodos: Realizamos una revisión sistemática de la legislación ambiental a través del Sistema Costarricense de Información Jurídica (http://www.pgrweb.go.cr/scij/avanzada_pgr.aspx), considerando tanto normativa como pronunciamientos sobre medidas de compensación y mitigación. Resultados: La Constitución Política de Costa Rica reconoce el medio ambiente sano como un derecho fundamental de todos los ciudadanos. Actualmente se encuentran en vigencia al menos siete leyes que tratan de temas ambientales y sus respectivos reglamentos. Esta voluminosa legislación tiene como objetivo proteger los recursos naturales regulando su uso y creando un conjunto de áreas silvestres protegidas. Sin embargo, la legislación es algo repetitiva y abierta a interpretación, lo que facilita la confusión. Las acciones que requieren compensación están reguladas de manera general, lo que dificulta establecer cuándo y cómo aplicarlas. Además, la compensación se considera en función de cuándo se identifica el daño y no debido a su nivel de impacto ambiental. Asimismo, la normativa no hace explícitos los principios y objetivos que sustentan las medidas compensatorias ni cómo deben llevarse a cabo, mencionando brevemente algunas acciones que no aseguran que la pérdida ambiental neta sea cero. Conclusiones: Esta revisión revela la necesidad de estandarizar los mecanismos para determinar las medidas compensatorias a nivel regulatorio, analizando además las estrategias que se seguirán para asegurar la proporcionalidad y la equivalencia ecológica. La legislación debe enfatizar la importancia de la compensación como práctica positiva, la cual debe ser incorporada dentro de los lineamientos del Estudio de Impacto Ambiental.

Introduction: Environmental compensation is a technical-legal instrument of environmental management used to offset damage due to non-reversible and unavoidable impacts. Usually, offset regulations are included in national environmental legislations. This paper summarizes environmental compensation legislation in Costa Rica, a country with a recognized conservation tradition and environmental awareness. Our goal is to describe how compensation is formulated in that country and identify possible regulations gaps. Methods: We carried out a systematic review of environmental legislation through the Costa Rican Legal Information System (http://www.pgrweb.go.cr/scij/avanzada_pgr.aspx), considering both regulations and pronouncements on compensation measures and mitigation. Results: The Political Constitution of Costa Rica recognizes a healthy environment as a fundamental right for all citizens. At least seven laws dealing with environmental issues and their respective regulations are currently enforced. This bulky legislation aims to protect natural resources by regulating their use and creating an assemblage of protected wild areas. However, the legislation is somewhat repetitive and open to interpretation, facilitating confusion. Actions requiring compensation are regulated in a general way, making it difficult to establish when and how to apply them. In addition, compensation is considered based on when the damage is identified and not on its level of environmental impact. Furthermore, the regulations do not make explicit the principles and goals that support the compensatory measures or how they should be carried out, briefly mentioning some actions that do not ensure that the net environmental loss is zero. Conclusions: This review reveals the need to standardize the mechanisms to determine compensatory measures at the regulatory level, also analyzing the strategies that will be followed to ensure proportionality and ecological equivalence. The legislation must emphasize the importance of compensation as a positive practice, which must be incorporated within the guidelines of the Environmental Impact Study.

Aplicación del Método Hectárea de Hábitat en compensación ambiental: El caso del Embalse Río Piedras, Costa Rica

Introducción: La compensación ambiental es la alternativa final para enfrentar los impactos de proyectos de desarrollo que no pueden ser evitados, reducidos o mitigados. La compensación de hábitats pretende sustituir recursos ecológicamente equivalentes. El Método Hectárea de Hábitat se diseñó inicialmente para evaluar la equivalencia ecológica de vegetación nativa y emplea puntajes de indicadores relativos a un hábitat de referencia. La suma de estos puntajes mide la condición estructural del ambiente de manera relativa a la referencia, cuantificando el número de hectáreas necesarias para compensar la pérdida de un hábitat similar. Aunque el método no está exento de limitaciones, los valores se pueden estimar a partir de datos de campo recogidos sin ambigüedad, de forma reproducible, con menos sesgos que métodos más cualitativos. En este artículo, usamos una modificación del Método Hectárea de Hábitat para determinar el área equivalente necesaria para compensar la pérdida de hábitat en un área silvestre protegida en Costa Rica. Metodología: Trabajamos en la Reserva Biológica Lomas de Barbudal, Pacífico norte de Costa Rica, un área silvestre protegida donde se inundarán cerca de 113 hectáreas para el Embalse de Río Piedras. En una propiedad previamente identificada como un sitio potencial de compensación, evaluamos indicadores de paisaje, suelo, estructura de la vegetación y servicios ecosistémicos; asignamos los puntajes en relación con el sitio a inundar. Resultados: El puntaje de la condición ambiental en el sitio de compensación fue de 44,7% (±15,9%) del sitio inundado. En consecuencia, considerando la incertidumbre de nuestras mediciones, compensar la pérdida de cada hectárea requeriría entre 2.23 y 3.49 hectáreas de un ambiente similar. Conclusión: Se necesitan entre 2.23 y 3.49 hectáreas de reemplazo, para compensar la pérdida de hábitat en el Embalse de Río Piedras.

Introduction: Environmental compensation is the final alternative to face the impacts of development projects that cannot be avoided, reduced, or mitigated. The offset of affected habitats or environmental elements usually substitutes ecologically equivalent resources. The Habitat-Hectare Method was initially designed to assess the ecological equivalence of native vegetation, and employs indicator scores relative to a reference habitat. The sum of these scores measures the structural condition of the environment against the reference, quantifying the number of hectares needed to compensate for the loss of similar habitat. Although it is not exempt from limitations, the values can be estimated from field data collected without ambiguity, in a reproducible way, with less bias than more qualitative methods. In this paper, we use a modification of the Habitat-Hectare Method to determine the equivalent area needed to compensate for habitat loss in a protected wilderness in Costa Rica. Methods: We worked in the Lomas de Barbudal Biological Reserve, north Pacific of Costa Rica, a protected wilderness where nearly 113 hectares will be flooded for the Río Piedras Reservoir. In a property previously identified as a potential compensation site, we assessed indicators for landscape, soil, vegetation structure, and ecosystem services; the scores were assigned in relation to the environment found at the flood site. Results: The environmental condition score at the compensation site was 44.7% (±15.9%) of the impact site. Consequently, considering the uncertainty in our measurements, compensating for the loss of each hectare would require between 2.23 and 3.49 hectares of a similar environment. Conclusion: Between 2.23 and 3.49 hectares of are needed to compensate for the loss of habitat in the Río Piedras Reservoir.

Uso de la diversidad biológica de grupos indicadores para evaluar la compensación ecológica: el caso del Embalse Río Piedras, Costa Rica

Introducción: Cerca de 113 hectáreas dentro de la Reserva Biológica Lomas de Barbudal (RBLB) se inundarían si se llegara a establecer el embalse de riego Río Piedras en el noroeste de Costa Rica. Ante ese impacto inevitable, la legislación costarricense requiere evaluar la pérdida de diversidad en el sitio y compensarla siguiendo el principio de equivalencia ecológica. Objetivo: Nuestro objetivo fue evaluar la pérdida de diversidad en el sitio y, valorar esta condición en una propiedad privada adyacente considerada como sitio potencial para compensar por esas pérdidas. Metodología: La diversidad, composición y prioridad de conservación de plantas vasculares, fauna vertebrada y artrópodos del sotobosque fueron evaluadas en el sitio de inundación en RBLB y en una propiedad privada adyacente identificada como sitio potencial de compensación. La equivalencia en diversidad entre ambos sitios se valoró mediante un procedimiento que asigna puntajes a los diferentes indicadores del sitio de compensación respecto a los observados en RBLB. La suma de esos puntajes representa la condición de diversidad del sitio de compensación y puede utilizarse como criterio para fundamentar acciones resarcitorias. Resultados: Todos los grupos sustitutos registraron un alto número de especies, aunque la información aportada por las plantas herbáceas, los mamíferos no voladores y los reptiles fue limitada. En contraste, los otros grupos indican una mayor diversidad en el sitio potencial de inundación, atribuida a sutiles diferencias ambientales y a su mejor conservación. En consecuencia, se valoró sustituir el área afectada por un área mayor del sitio de compensación para resarcir las perdidas en diversidad estimadas. Conclusión: Nuestro análisis muestra que grupos sustitutos pueden ser empleados en estudios ambientales a corto plazo. Sin embargo, los grupos a utilizar deben seleccionarse cuidadosamente, considerando los objetivos y el alcance del proyecto. Cuantificar la condición de diversidad del sitio de compensación respecto a la que se encuentra en un sitio de referencia parece ser un procedimiento válido, repetible y evaluable que permite establecer criterios sobre los cuales basar medidas resarcitorias.

Introduction: About 113 hectares within the Lomas de Barbudal Biological Reserve (RBLB) would be flooded if the Río Piedras irrigation reservoir were established in northwestern Costa Rica. Given this inevitable impact, Costa Rican legislation requires evaluating the loss of diversity in the site and compensating for it following the principle of ecological equivalence. Objetive: Our goal was to assess the loss of diversity at that site and evaluate the condition of an adjacent private property as a potential site to compensate for those losses. Methodology: The diversity, composition, and conservation priority of vascular plants, vertebrate fauna, and understory arthropods were assessed at the RBLB flood site and on an adjacent private property identified as a potential offset site. The equivalence in diversity between both sites was evaluated by assigning scores to the different indicators at the compensation site concerning those observed in RBLB. The sum of these scores represents the diversity condition of the compensation site and can be used as a criterion to support compensatory actions. Results: All surrogate groups recorded a high number of species, although the information provided by herbaceous plants, non-flying mammals, and reptiles was limited. In contrast, the other groups indicate a greater diversity in the potential flood site, attributed to subtle environmental differences and the better conservation at this site. Consequently, it was assessed to replace the affected area with a larger offset site area to compensate for the estimated losses in diversity. Conclusion: Our analysis shows that surrogate groups can be used in short-term environmental studies. However, the groups to be used must be carefully selected, considering the objectives and scope of the project. Quantifying the diversity condition of the compensation site concerning that found in a reference site seems to be a valid, repeatable, and evaluable procedure that allows establishing criteria on which to base compensatory measures.

Compensación ambiental por la segregación y uso de un área dentro de un refugio de vida silvestre: un caso de estudio empleando coste de oportunidad

Introducción: El establecimiento del Puesto Fronterizo Las Tablillas implica la segregación de 12.12 ha del Refugio Nacional de Vida Silvestre Corredor Fronterizo (RNVSCF), en el norte de Costa Rica. El sitio tiene una larga historia de uso de la tierra y su entorno natural está muy degradado. Este es un caso que se puede abordar desde el coste de oportunidad que se refiere al valor de la alternativa a la que se renuncia al tomar una decisión económica. Objetivo: En este artículo, usamos este precepto para evaluar la compensación por pérdidas en la estructura del hábitat y la biodiversidad si se interrumpiera la dinámica de sucesión dentro de un área silvestre protegida. Métodos: Para encontrar la ganancia neta en compensación requerida por dicha segregación, analizamos la diversidad y composición de la cubertura forestal cercana para pronosticar el ambiente que se perdería si se interrumpieran sus objetivos de conservación (costo de oportunidad). Compensar estas pérdidas requiere un ambiente equivalente, por lo que seleccionamos dos propiedades, entre 27 investigadas adyacentes al RNVSCF, que comparten cobertura forestal, están registradas legalmente y poseen atributos ambientales favorables para el diseño de reservas. Las dos propiedades seleccionadas y el sitio Las Tablillas fueron caracterizados a nivel de atributos geológicos e hidrológicos y de composición biológica de grupos indicadores. Resultados: Ambas propiedades son física y biológicamente favorables para el intercambio. La escogencia de cualquiera aumentaría el área del RNVSCF en más de 100 ha de un ambiente con mejor cobertura, más diversidad y mejor conexión con otras masas forestales que el observado en el sitio de segregación. Esto es una ventaja para el objetivo de conservación del refugio. Conclusión: En este caso, el coste de oportunidad favoreció la ganancia ambiental para la propuesta de compensación y podría ser utilizado para evaluar otras compensaciones que involucren ambientes sensibles o protegidos.

Introduction: The establishment of the Las Tablillas Border Post implies the segregation of 12.12 ha within the National Wildlife Refuge Border Corridor (RNVSCF) in northern Costa Rica. The site has a long land use history, so its environment is highly degraded. This case can be studied from the opportunity cost, which refers to the amount of the alternative given up when making an economic decision. Objective: In this paper, we use this precept to assess the compensation for losses in habitat structure and biodiversity if succession dynamics within a protected wilderness area were interrupted. Methods: To find the net gain in compensation required by this segregation, we analyzed the diversity and composition of nearby forest cover to forecast the environment that would be lost if its conservation objectives were interrupted (opportunity cost). Compensating for these losses requires an equivalent environment, so we selected two properties, among the 27 investigated, that shared forest cover, are located adjacent to the RNVSCF, are registered, and possess other attributes favorable to the design of reserves. The two selected properties and the Las Tablillas site were characterized at the level of geological and hydrological attributes and the composition of indicator groups. Results: Both properties are physically and biologically favorable for the exchange. It would increase the area of the RNVSCF by more than 100 ha of an environment with better coverage, more diversity, and a better connection to other forest masses than the one observed in the segregation site, which is an advantage to the conservation objective of the refuge. Conclusion: In this case, the opportunity cost favored the environmental gain for the offset proposal and could be used to evaluate other compensation involving sensitive or protected environments.

Proteomic and toxicological analysis of the venom of Micrurus yatesi and its neutralization by an antivenom.

Coralsnakes belong to the family Elapidae and possess venoms which are lethal to humans and can be grouped based on the predominance of either three finger toxins (3FTxs) or phospholipases A2 (PLA2s). A proteomic and toxicological analysis of the venom of the coralsnake Micrurus yatesi was performed. This species, distributed in southeastern Costa Rica, was formerly considered a subspecies of M. alleni. Results showed that this venom is PLA2-rich, in contrast with the previously studied venom of Micrurus alleni. Toxicological evaluation of the venom, in accordance with proteomic data, revealed that it has a markedly higher in vitro PLA2 activity upon a synthetic substrate than M. alleni. The evaluation of in vivo myotoxicity in CD-1 mice using histological evaluation and plasma creatine kinase release also showed that M. yatesi venom caused muscle damage. A commercial equine antivenom prepared using the venom of Micrurus nigrocinctus displayed a similar recognition of the venoms of M. yatesi and M. nigrocinctus by enzyme immunoassay. This antivenom also immunorecognized the main fractions of the venom of M. yatesi and was able to neutralize its lethal effect in a murine model.

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.

The Genetic Diversity of Cranberry Crop Wild Relatives, Vaccinium macrocarpon Aiton and V. oxycoccos L., in the US, with Special Emphasis on National Forests.

Knowledge of the genetic diversity in populations of crop wild relatives (CWR) can inform effective strategies for their conservation and facilitate utilization to solve agricultural challenges. Two crop wild relatives of the cultivated cranberry are widely distributed in the US. We studied 21 populations of Vaccinium macrocarpon Aiton and 24 populations of Vaccinium oxycoccos L. across much of their native ranges in the US using 32 simple sequence repeat (SSR) markers. We observed high levels of heterozygosity for both species across populations with private alleles ranging from 0 to 26. For V. macrocarpon, we found a total of 613 alleles and high levels of heterozygosity (HO = 0.99, HT = 0.75). We also observed high numbers of alleles (881) and levels of heterozygosity (HO = 0.71, HT = 0.80) in V. oxycoccos (4x). Our genetic analyses confirmed the field identification of a native population of V. macrocarpon on the Okanogan-Wenatchee National Forest in the state of Washington, far outside the previously reported range for the species. Our results will help to inform efforts of the United States Department of Agriculture Agricultural Research Service (USDA-ARS) and the United States Forest Service (USFS) to conserve the most diverse and unique wild cranberry populations through ex situ preservation of germplasm and in situ conservation in designated sites on National Forests.

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. BIOLOGICAL SIGNIFICANCE: With more than 2200 species in family Colubridae (sensu lato), rear-fanged snakes comprise approximately two-thirds of the extant species of advanced snakes. Snakebites from venomous snakes that are of medical concern are predominantly from front-fanged snakes of families Viperidae and Elapidae. On the other hand, rear-fanged snakes have been conventionally considered non-venomous, and thus their venoms have remained a largely untapped area of venomics. However, increasing documentation of life-threadening, even fatal, envenomings from rear-fanged snakes has sparked interest in their venoms. Appying bottom-up venomics we have revealed that the Duvernoy's gland secretory proteome of R. bovallii comprises a handful of toxins belonging to only three protein families, with slow-acting three-finger toxins (3FTx) that are non-toxic towards mammalian prey and show preference towards diapsid taxa representing the dominant structural and functional proteins. Our work documents for the first time 3FTxs exerting different effect in an avian model than in a reptile model. Besides, the 3FTx fractions that faithfully mimicked the irreversible iguana-immobilizing effect of the whole venom were identified through toxicovenomic analysis of R. bovallii venom on Iguana iguana. Our work underscores the importance of using biologically-relevant animal toxicity models for investigating the biological roles of venoms in an evolutionary-ecological context.

Venomics of the Central American Lyre Snake Trimorphodon quadruplex (Colubridae: Smith, 1941) from Costa Rica.

Rear-fanged colubrid snakes include hundreds of species globally that possess a Duvernoy's venom gland and often one-several enlarged rear maxillary teeth. We investigated the venom proteome of the Central American Lyre Snake (Trimorphodon quadruplex), a moderate-sized rear-fanged colubrid snake and the southernmost Trimorphodon, using a bottom-up proteomic approach coupled with enzyme and inhibitor assays, cytotoxicity assays and lethal toxicity assays. Several enzymes uncommonly observed in colubrid venoms were purified and characterized further. Trimorphodon quadruplex has a rather low complexity venome, typical of many rear-fanged snakes, but its venom contains L-amino acid oxidase, phospholipase A2, and a dimeric 3FTx, and 3FTxs dominate the proteome. Its PLA2 is catalytically quite active, but it lacks myotoxicity or acute toxicity; LAAO exhibits conserved structure and appears to be highly labile. Several P-III metalloproteinases are present and hydrolyze azocasein and the α-subunit of fibrinogen but lack hemorrhagic activity. Trimorphodon quadruplex produces venom and retains constriction, utilizing both chemically-mediated and mechanical feeding modes. SIGNIFICANCE: We demonstrate that T. quadruplex venom proteins are similar to those found in front-fanged snake species are present but show different biological activities. Our results underscore the importance of considering the biological roles of venoms from more than a mammal-centric perspective.

Venom diversity in the Neotropical scorpion genus Tityus: Implications for antivenom design emerging from molecular and immunochemical analyses across endemic areas of scorpionism.

Scorpions of the Neotropical genus Tityus are responsible for most severe envenomations in the Caribbean, South America, and Lower Central America (LCA). Although Tityus is taxonomically complex, contains high toxin polymorphism, and produces variable clinical manifestations, treatment is limited to antivenoms produced against species with restricted distributions. In this study, we explored the compositional and antigenic diversity of Tityus venoms to provide improved guidelines for the use of available antivenoms at a broader geographic scale. We used immunoblotting, competitive ELISA, and in vivo studies to compare reactivity against commercial antivenoms from Brazil, Venezuela, and Mexico, as well as MALDI-TOF mass spectrometry, cDNA sequencing, and phylogenetic analyses to assess venom sodium channel-active toxin (NaTx) content from medically important Tityus populations inhabiting Brazil, Colombia, Costa Rica, Ecuador, Panama, Trinidad and Tobago, and Venezuela. Additionally, we raised rabbit antibodies against Tityus venoms from LCA to test for cross-reactivity with congeneric species. The results suggest that Tityus spp. possess high venom antigenic diversity, underlying the existence of four toxinological regions in Tropical America, based on venom composition and immunochemical criteria: LCA/Colombia/Amazonia (Region I), Venezuela (Region II), southeast South America (Region III), and a fourth region encompassing species related to toxinologically divergent Tityus cerroazul. Importantly, our molecular and cross-reactivity results highlight the need for new antivenoms against species inhabiting Region I, where scorpions may produce venoms that are not significantly reactive against available antivenoms.

Venom characterization of the bark scorpion Centruroides edwardsii (Gervais 1843): Composition, biochemical activities and in vivo toxicity for potential prey.

In this study, we characterize the venom of Centruroides edwardsii, one of the most abundant scorpions in urban and rural areas of Costa Rica, in terms of its biochemical constituents and their biological activities. C. edwardsii venom is rich in peptides but also contains some higher molecular weight protein components. No phospholipase A2, hemolytic or fibrinogenolytic activities were found, but the presence of proteolytic and hyaluronidase enzymes was evidenced by zymography. Venom proteomic analysis indicates the presence of a hyaluronidase, several cysteine-rich secretory proteins, metalloproteinases and a peptidylglycine α-hydroxylating monooxygenase like-enzyme. It also includes peptides similar to the K+-channel blocker margatoxin, a dominant toxin in the venom of the related scorpion C. margaritatus. MS and N-terminal sequencing analysis also reveals the presence of Na+-channel-modulating peptides with sequence similarity to orthologs present in other scorpion species of the genera Centruroides and Tityus. We purified the hyaluronidase (which co-eluted with an allergen 5-like CRiSP) and sequenced ~60% of this enzyme. We also sequenced some venom gland transcripts that include other cysteine-containing peptides and a Non-Disulfide Bridged Peptide (NDBP). Our in vivo experiments characterizing the effects on potential predators and prey show that C. edwardsii venom induces paralysis in several species of arthropods and geckos; crickets being the most sensitive and cockroaches and scorpions the most resistant organisms tested. Envenomation signs were also observed in mice, but no lethality was reached by intraperitoneal administration of this venom up to 120 µg/g body weight.

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.

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

Venoms of medically important scorpions from Buthidae family have been intensively studied, in contrast to 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 ß-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 time.

Hydrology Affects Environmental and Spatial Structuring of Microalgal Metacommunities in Tropical Pacific Coast Wetlands.

The alternating climate between wet and dry periods has important effects on the hydrology and therefore on niche-based processes of water bodies in tropical areas. Additionally, assemblages of microorganism can show spatial patterns, in the form of a distance decay relationship due to their size or life form. We aimed to test spatial and environmental effects, modulated by a seasonal flooding climatic pattern, on the distribution of microalgae in 30 wetlands of a tropical dry forest region: the Pacific coast of Costa Rica and Nicaragua. Three surveys were conducted corresponding to the beginning, the highest peak, and the end of the hydrological year during the wet season, and species abundance and composition of planktonic and benthic microalgae was determined. Variation partitioning analysis (as explained by spatial distance or environmental factors) was applied to each seasonal dataset by means of partial redundancy analysis. Our results show that microalgal assemblages were structured by spatial and environmental factors depending on the hydrological period of the year. At the onset of hydroperiod and during flooding, neutral effects dominated community dynamics, but niche-based local effects resulted in more structured algal communities at the final periods of desiccating water bodies. Results suggest that climate-mediated effects on hydrology can influence the relative role of spatial and environmental factors on metacommunities of microalgae. Such variability needs to be accounted in order to describe accurately community dynamics in tropical coastal wetlands.