Salazar Valenzuela, David
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Salazar Valenzuela, David
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Quito
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davidsalazar@uti.edu.ec
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26644312900

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Resistance Is Not Futile: Widespread Convergent Evolution of Resistance to Alpha-Neurotoxic Snake Venoms in Caecilians (Amphibia: Gymnophiona)

2023 , Mancuso M. , Zaman S. , Maddock S.T. , Kamei R.G. , Salazar Valenzuela, David , Wilkinson M. , Roelants K. , Fry B.G.

Predatory innovations impose reciprocal selection pressures upon prey. The evolution of snake venom alpha-neurotoxins has triggered the corresponding evolution of resistance in the post-synaptic nicotinic acetylcholine receptors of prey in a complex chemical arms race. All other things being equal, animals like caecilians (an Order of legless amphibians) are quite vulnerable to predation by fossorial elapid snakes and their powerful alpha-neurotoxic venoms; thus, they are under strong selective pressure. Here, we sequenced the nicotinic acetylcholine receptor alpha-1 subunit of 37 caecilian species, representing all currently known families of caecilians from across the Americas, Africa, and Asia, including species endemic to the Seychelles. Three types of resistance were identified: (1) steric hindrance from N-glycosylated asparagines; (2) secondary structural changes due to the replacement of proline by another amino acid; and (3) electrostatic charge repulsion of the positively charged neurotoxins, through the introduction of a positively charged amino acid into the toxin-binding site. We demonstrated that resistance to alpha-neurotoxins convergently evolved at least fifteen times across the caecilian tree (three times in Africa, seven times in the Americas, and five times in Asia). Additionally, as several species were shown to possess multiple resistance modifications acting synergistically, caecilians must have undergone at least 20 separate events involving the origin of toxin resistance. On the other hand, resistance in non-caecilian amphibians was found to be limited to five origins. Together, the mutations underlying resistance in caecilians constitute a robust signature of positive selection which strongly correlates with elapid presence through both space (sympatry with caecilian-eating elapids) and time (Cenozoic radiation of elapids). Our study demonstrates the extent of convergent evolution that can be expected when a single widespread predatory adaptation triggers parallel evolutionary arms races at a global scale. © 2023 by the authors.

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Disentangling the Anacondas: Revealing a New Green Species and Rethinking Yellows †

2024 , Rivas J.A. , De La Quintana P. , Mancuso M. , Pacheco L.F. , Rivas G.A. , Mariotto S. , Salazar Valenzuela, David , Baihua M.T. , Baihua P. , Burghardt G.M. , Vonk F.J. , Hernandez E. , García-Pérez J.E. , Fry B.G. , Corey-Rivas S.

Anacondas, genus Eunectes, are a group of aquatic snakes with a wide distribution in South America. The taxonomic status of several species has been uncertain and/or controversial. Using genetic data from four recognized anaconda species across nine countries, this study investigates the phylogenetic relationships within the genus Eunectes. A key finding was the identification of two distinct clades within Eunectes murinus, revealing two species as cryptic yet genetically deeply divergent. This has led to the recognition of the Northern Green Anaconda as a separate species (Eunectes akayima sp. nov), distinct from its southern counterpart (E. murinus), the Southern Green Anaconda. Additionally, our data challenge the current understanding of Yellow Anaconda species by proposing the unification of Eunectes deschauenseei and Eunectes beniensis into a single species with Eunectes notaeus. This reclassification is based on comprehensive genetic and phylogeographic analyses, suggesting closer relationships than previously recognized and the realization that our understanding of their geographic ranges is insufficient to justify its use as a separation criterion. We also present a phylogeographic hypothesis that traces the Miocene diversification of anacondas in western South America. Beyond its academic significance, this study has vital implications for the conservation of these iconic reptile species, highlighting our lack of knowledge about the diversity of the South American fauna and the need for revised strategies to conserve the newly identified and reclassified species. © 2024 by the authors.

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Evaluating the utility of camera traps in field studies of predation

2019 , Akcali C.K. , Pérez-Mendoza H.A. , Salazar Valenzuela, David , Kikuchi D.W. , Guayasamin, Juan M. , Pfennig D.W.

Artificial prey techniques-wherein synthetic replicas of real organisms are placed in natural habitats-are widely used to study predation in the field.We investigated the extent to which videography could provide additional information to such studies. As a part of studies on aposematism and mimicry of coral snakes (Micrurus) and their mimics, observational data from 109 artificial snake prey were collected from video-recording camera traps in three locations in the Americas (terra firme forest, Tiputini Biodiversity Station, Ecuador; premontane wet forest, Nahá Reserve, Mexico; longleaf pine forest, Southeastern Coastal Plain, North Carolina, USA). During 1,536 camera days, a total of 268 observations of 20 putative snake predator species were recorded in the vicinity of artificial prey. Predators were observed to detect artificial prey 52 times, but only 21 attacks were recorded. Mammals were the most commonly recorded group of predators near replicas (243) and were responsible for most detections (48) and attacks (20). There was no difference between avian or mammalian predators in their probability of detecting replicas nor in their probability of attacking replicas after detecting them. Bite and beak marks left on clay replicas registered a higher ratio of avian:mammalian attacks than videos registered. Approximately 61.5% of artificial prey monitored with cameras remained undetected by predators throughout the duration of the experiments. Observational data collected from videos could provide more robust inferences on the relative fitness of different prey phenotypes, predator behavior, and the relative contribution of different predator species to selection on prey. However, we estimate that the level of predator activity necessary for the benefit of additional information that videos provide to be worth their financial costs is achieved in fewer than 20% of published artificial prey studies. Although we suggest future predation studies employing artificial prey to consider using videography as a tool to inspire new, more focused inquiry, the investment in camera traps is unlikely to be worth the expense for most artificial prey studies until the cost:benefit ratio decreases. © 2019 PeerJ Inc. All rights reserved.

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Feeding ecology of the Terciopelo pit viper snake (Bothrops asper) in Ecuador

2023 , Loaiza-Lange A. , Székely D , Torres-Carvajal O. , Tinoco N. , Salazar Valenzuela, David , Székely P.

Thoroughly documenting prey items and diet composition is crucial for understanding a predator’s role in the ecosystem. In gape restricted predators, such as snakes, documenting and analyzing the type and size of the prey is important to interpret their ecological role. We describe the diet patterns of a species of venomous snake, the Terciopelo pit viper (Bothrops asper), from its Ecuadorian populations. Examining the gastrointestinal contents of museum specimens collected over an extensive area of the Pacific lowlands of Ecuador, we encountered 69 identifiable prey items from four major taxonomic groups (amphibians, centipedes, mammals, and reptiles). We evaluated the observed composition of prey to check for differences between sexes and size-classes. To complement our observations of the Terciopelo species complex throughout their distribution, we carried out a systematic literature review. Our data show an ontogenetic shift in diet, with a transition from more diverse diet in juveniles towards a mammal-specialized diet in adults, and distinct proportion of prey taxa between the sexes in the juvenile size class. Copyright 2023 Loaiza-Lange et al.

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Bothrops atrox from Ecuadorian Amazon: Initial analyses of venoms from individuals

2021 , Patiño R.S.P. , Salazar Valenzuela, David , Medina-Villamizar E. , Mendes B. , Proaño-Bolaños C. , da Silva S.L. , Almeida J.R.

Bothrops atrox is the most clinically relevant snake species within the Amazon region, which includes Ecuadorian territories. It comprises a large distribution, which could contribute to the genetic and venomic variation identified in the species. The high variability and protein isoform diversity of its venom are of medical interest, since it can influence the clinical manifestations caused by envenomation and its treatment. However, in Ecuador there is insufficient information on the diversity of venomic phenotypes, even of relevant species such as B. atrox. Here, we characterized the biochemical and toxicological profiles of the venom of six B. atrox individuals from the Ecuadorian Amazon. Differences in catalytic activities of toxins, elution profiles in liquid chromatography, electrophoretic patterns, and toxic effects among the analyzed samples were identified. Nonetheless, in the preclinical testing of antivenom, two samples from Mera (Pastaza) required a higher dose to achieve total neutralization of lethality and hemorrhage. Taken together, these data highlight the importance of analyzing individual venoms in studies focused on the outcomes of envenoming. © 2021 Elsevier Ltd

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A retrospective study of clinical and epidemiological characteristics of snakebite in Napo Province, Ecuadorian Amazon

2023 , Patiño R.S.P. , Salazar Valenzuela, David , Robles-Loaiza A.A. , Santacruz-Ortega P , Almeida J.R.

BACKGROUND: Snakebite envenoming remains a relevant public health problem in tropical and subtropical countries. In Ecuador, this is particularly true in an area of great diversity like the Amazon region. Nevertheless, there is scarce information about epidemiological and clinical characteristics of these accidents in this area. METHODS: This was a descriptive and retrospective study of snakebite cases treated at a tertiary hospital in the Napo Province, Ecuadorian Amazon, from 2015 to 2019. We collected sociodemographic and snakebite-related information, clinical aspects and the use of antivenom and antibiotics from medical records. RESULTS: Information from 133 snakebite accidents was reviewed in this time period. Reports of snakebite envenoming decreased over the years. In total, 67% of those bitten were from nearby indigenous communities, which were the most affected groups. When a species was identified, Bothrops atrox was responsible for the highest number of cases registered. Local clinical manifestations were more frequent than systemic signs, in keeping with the typical effects produced by bothropic venoms. Additionally, data showed that more antivenom vials were given than those suggested by the protocol of the Ecuadorian Ministry of Health, in proportion to the grade of severity. Finally, we identified a low incidence of adverse reactions with antivenom administration, as well as a frequent use of antibiotics. CONCLUSIONS: The profile of snakebite accidents in the Napo Province is very similar to that described for other localities in the Amazon region of Ecuador and neighboring countries, with its challenges and limitations. Such aspects underlie the importance of establishing a robust and science-based public health program to respond to this frequent, but neglected, tropical disease. © The Author(s) 2022. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.

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Divergence of tropical pitvipers promoted by independent colonization events of dry montane Andean habitats

2019 , Salazar Valenzuela, David , Kuch U. , Torres-Carvajal O. , Valencia J.H. , Gibbs H.L.

Aim: A poorly explored feature of the origin and maintenance of Neotropical biodiversity is how the evolutionary dynamics of colonization and differentiation in relation to lowland and highland habitats has impacted lineage formation. Most speciation models for this region have focused on vicariant events, whereas the need to assess the influence of demographic processes has been recognized only recently. We evaluate if the origin of Andean montane lineages of terciopelo pitvipers is explained by either of two historical processes that represent distinct phylogeographic mechanisms: differentiation by isolation within the highlands or different dispersal events from the lowlands. Location: Western Ecuador. Taxon: Terciopelo pitvipers (Bothrops asper species complex). Methods: We use genomic data and genetic clustering analyses, evaluation of historical migration between genetic clusters and demographic model selection to investigate recent diversification events in South America using a vertebrate group rarely explored in phylogeographic studies: tropical Andean snakes. Specifically, the origin of two Ecuadorian montane lineages of terciopelo pitvipers was evaluated given ambiguous phylogenetic relationships with the presumably ancestral Pacific lowland lineage. Results: Discrepancies of evolutionary relationships previously obtained with tree-like methods are resolved through the use of modelling approaches. We found strong support for the independent origin of montane lineages based on topologies inferred by maximum-likelihood trees and modelling approaches that take into account possible gene flow. This suggests dispersal rather than in-situ differentiation as the most likely mechanism by which the montane linages originated. Main conclusions: Recent large-scale studies have found support for identifying dispersal events as important drivers of diversification in the Neotropical region. We contribute to these ideas by identifying a fine-scale case in a rarely studied group of animals -Andean snakes- in which river valleys acted as an entrance for the upward colonization of montane dry habitats and subsequent ecological diversification. © 2019 John Wiley & Sons Ltd

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First Insights into the Venom Composition of Two Ecuadorian Coral Snakes

2022 , Hernández-Altamirano, J.A. , Salazar Valenzuela, David , Medina-Villamizar, E.J. , Quirola, D.R. , Patel, K. , Vaiyapuri, S. , Lomonte, B. , Almeida, J.R.

Micrurus is a medically relevant genus of venomous snakes composed of 85 species. Bites caused by coral snakes are rare, but they are usually associated with very severe and life-threatening clinical manifestations. Ecuador is a highly biodiverse country with a complex natural environment, which is home to approximately 20% of identified Micrurus species. Additionally, it is on the list of Latin American countries with the highest number of snakebites. However, there is no local antivenom available against the Ecuadorian snake venoms, and the biochemistry of these venoms has been poorly explored. Only a limited number of samples collected in the country from the Viperidae family were recently characterised. Therefore, this study addressed the compositional patterns of two coral snake venoms from Ecuador, M. helleri and M. mipartitus, using venomics strategies, integrating sample fractionation, gel electrophoresis, and mass spectrometry. Chromatographic and electrophoretic profiles of these snake venoms revealed interspecific variability, which was ascertained by mass spectrometry. The two venoms followed the recently recognised dichotomic toxin expression trends displayed by Micrurus species: M. helleri venom contains a high proportion (72%) of phospholipase A2, whereas M. mipartitus venom is dominated by three-finger toxins (63%). A few additional protein families were also detected in these venoms. Overall, these results provide the first comprehensive views on the composition of two Ecuadorian coral snake venoms and expand the knowledge of Micrurus venom phenotypes. These findings open novel perspectives to further research the functional aspects of these biological cocktails of PLA2s and 3FTxs and stress the need for the preclinical evaluation of the currently used antivenoms for therapeutic purposes in Ecuador. © 2022 by the authors.

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

2020 , Mora-Obando D. , Salazar Valenzuela, David , Pla D. , Lomonte B. , Guerrero-Vargas J.A. , Ayerbe S. , Gibbs H.L. , Calvete J.J.

Bothrops asper is a venomous pitviper that is widely distributed and of clinical importance in Mesoamerica and northern South America, where it is responsible for 50–80% of all envenomations by Viperidae species. Previous work suggests that B. asper has a complex phylogeographic structure, with the existence of multiple evolutionarily distinct lineages, particularly in the inter-Andean valleys of north South America. To explore the impact of the evolutionary history of B. asper on venom composition, we have investigated geographic variation in the venom proteome of this species from the populations from the Pacific side of Ecuador and south-western Colombia. Among the 21 classes of venom components identified, proteins from mainly four major toxin families, snake venom metalloproteases (PI- and PII-SVMP), phospholipases A2 (K49- and D49-PLA2s), serine proteinases (SVSP), and C-type lectins-like (CTL) proteins are major contributors to the geographic variability in venom. Principal component analyses demonstrate significant differences in venom composition between B. asper lineages previously identified through combination of molecular, morphological and geographical data, and provide additional insights into the selection pressures modulating venom phenotypes on a geographic scale. In particular, altitudinal zonation within the Andean mountain range stands out as a key ecological factor promoting diversification in venom. In addition, the pattern of distribution of PLA2 molecules among B. asper venoms complements phylogenetic analysis in the reconstruction of the dispersal events that account for the current biogeographic distribution of the present-day species' phylogroups. Ontogenic variation was also evident among venoms from some Ecuadorian lineages, although this age-related variation was less extreme than reported in B. asper venoms from Costa Rica. The results of our study demonstrate a significant impact of phylogenetic history on venom composition in a pitviper and show how analyses of this variation can illuminate the timing of the cladogenesis and ecological events that shaped the current distribution of B. asper lineages. Biological significance: Bothrops asper, called “the ultimate pitviper” due to its defensive behavior, large body size, and medical importance, represents a species complex that is widely distributed from southern México southwards across north-western South America to north-western Perú. This work reports the characterization of the venom proteomes of B. asper lineages from the Pacific sides of Ecuador and south-western Colombia. Multivariate analyses indicate that variability in venom composition among the B. asper lineages is driven by proteins from four major toxin families, presumably in response to selection pressures created by recent and historical ecological conditions created by geological and climatic events from the Pliocene-Pleistocene to the present along the Central and South American Continental Divide. The emerging biogeographic pattern of venom variation, interpreted in the context of the current phylogenetic hypotheses, support and complement previously proposed evolutionary Plio-Pleistocene dispersal events that shaped the present-day distribution range of B. asper lineages. In addition, our venomics data indicate the occurrence of genetic exchange between Colombian and Pacific Costa Rican populations, which may have occurred during the second wave of B. asper migration into Mesoamerica. Our work represents a foundation for a future broader sampling and more complete “-omics” analyses to deepen our understanding of the patterns and causes of venom variation in this medically important pitviper. © 2020 Elsevier B.V.

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A First Look at the Inhibitory Potential of Urospatha sagittifolia (Araceae) Ethanolic Extract for Bothrops atrox Snakebite Envenomation

2022 , Vera-Palacios, A.L. , Sacoto-Torres, J.D. , Hernández-Altamirano, J.A. , Moreno, A. , Peñuela-Mora, M.C. , Salazar Valenzuela, David , Mogollón, N.G.S. , Almeida, J.R.

Bothrops atrox snakebites are a relevant problem in the Amazon basin. In this biodiverse region, the ethnomedicinal approach plays an important role as an alternative to antivenom therapy. Urospatha sagittifolia (Araceae) is a plant used for this purpose; however, its neutralizing properties have not been scientifically accessed. To fill this gap, we investigated the ability of U. sagittifolia to modulate the catalytic activity of Bothrops atrox venom, and their toxic consequences, such as local damage and lethality. The venom profile of B. atrox was assessed by chromatography and electrophoresis. Inhibition of the three main enzymatic and medically important toxins from the venom was evaluated using synthetic substrates and quantified by chromogenic activity assays. Additionally, the neutralization of lethality, hemorrhage and edema were investigated by in vivo assays. The possible interactions between venom proteins and plant molecules were visualized by polyacrylamide gel electrophoresis. Finally, the phytochemical constituents present in the ethanolic extract were determined by qualitative and quantitative analyses. The ethanolic extract reduced the activity of the three main enzymes of venom target, achieving ranges from 19% to 81% of inhibition. Our in vivo venom neuralizations assays showed a significant inhibition of edema (38.72%) and hemorrhage (42.90%). Additionally, lethality was remarkably counteracted. The highest extract ratio evaluated had a 75% survival rate. Our data support the biomedical value of U. sagittifolia as a source of natural enzyme inhibitors able to neutralize catalytically active B. atrox venom toxins and their toxic effects. © 2022 by the authors.

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