Guatemala and the entire Mesoamerican region are sites teeming with a rich diversity of fauna and flora. Numerous studies related to these subjects have been conducted in these areas by outstanding Guatemalan scientists. These investigations contribute significantly, not only to science but also to expanding our understanding of the fascinating species surrounding us in Mesoamerica. The crux of any research lies in its dissemination and the public disclosure of its findings. The publication of research plays a pivotal role in disseminating scientific knowledge and promoting conservation in the Mesoamerican region. Hence, FLAAR Mesoamerica has pioneered a new approach, where valuable research on Mesoamerican fauna and flora will be published, aiming to showcase the results of these endeavors and spotlight the scientists and researchers behind them. By sharing the discoveries of studies and research, we aim to connect with a broader audience, disseminating valuable information about the biodiversity and incredible fauna species present in this region. Our goal is to inspire greater interest and awareness regarding the importance of preserving the Mesoamerican ecosystem and species. Through this initiative, we aspire not only to inform but also to motivate everyone to deepen their commitment to conservation and foster a greater respect for nature.
“Caracterización toxicológica del veneno de la serpiente mano de piedra (Metlapilcoatlus occiduus: Viperidae) y su variación ontológica”
Autora: Licenciada Mariana Rivas Galvez (2022)
What is an Ontogenetic Change and How Does it Influence Snakes?
An ontogenetic change is a common characteristic in the production and composition of venom in many reptiles, especially snakes. Studies report that both the elapid family (Elapidae) and vipers (Viperidae) exhibit this characteristic. “Ontogenetic change” refers to a variation in the venom composition of the same species, which arises from different factors such as distribution, sex, age, and, most importantly, the snake’s diet. All snakes are carnivores, and one of their most successful mechanisms for hunting is the injection of a protein cocktail that paralyzes and digests the prey. The venom will be composed of all the proteins necessary to perform its function on the type of prey, so the secretion’s composition will be specialized to exert harmful effects on the specific type of prey that the snake consumes. Thus, a snake that feeds on reptiles and amphibians will have a different venom composition than a snake that preys on mammals.
Snakes exhibit variations in their diets throughout their development. Neonates prefer to feed on small invertebrates such as insects, while juvenile snakes also feed on small prey, but at this stage de feeding is characterized mostly by herpetofauna or small rodents. In contrast, adult snakes choose larger prey, primarily mammals or birds, although it may also include other reptiles like snakes or lizards in their diet.
Note: This diagram does not necessarily show the biology of B. asper, it is only used for exemplification and illustration motives (Diagram by Mariana Rivas Gálvez).
Diagram 2. Differences in the diets of adult and juvenile/neonate snakes. Juvenile specimens feed on small-sized, mostly ectothermic prey, including rodent offspring. Adult specimens feed on larger-sized, mostly endothermic prey, but they can also consume reptiles such as other snakes or lizards. Note: For illustrative purposes, photographs of N. atara and B. atrox were included in the reports, highlighting ontogenetic variation in both species (Diagram by Mariana Rivas Gálvez)
The “Mano de piedra” snake (Metlapilcoatlus occiduus) shows ontogenetic changes in its venom
Metlapilcoatlus occiduus, commonly known as one of the “Mano de piedra” snake, belongs to the Viperidae family and is part of the group of the pit vipers. Its distribution extends from southeastern Chiapas, Mexico, to western El Salvador. Snakes of the same genus and family have shown ontogenetic changes in their venom based on age and diet. Considering that the diet of M. occiduus is very similar to that of its “sister” species during its growth stages (Juveniles feed on invertebrates and small vertebrates and adults on mammals mainly), it was hypothesized that there would be variation in the venom of the species based on the reptile’s diet in its juvenile and adult stages. The hypothesis suggests that there is an ontogenetic change in the venom of juveniles and adults of the species Metlapilcoatlus occiduus, determined by the difference in diet during these stages of the snake’s growth. Accordingly, it is anticipated that the venom of juveniles would be more harmful to invertebrates due to the presence of specific proteins designed to paralyze in contrast to the venom from adults.
Figure 1. Adult specimen of Metlapilcoatlus occiduus. Medium size. A: Photograph: Maquin, 2021; B: Photograph: Herrera, 2015; C: Photograph: Herrera, 2015. All photographs have been modified by Mariana Rivas Galvez.
How was the research conducted?
From snake venom extractions, SDS-PAGE bioassay, Bradford assay, and venom inoculation laboratories on alive invertebrate models (Common cricket (Acheta domesticus)) authorized, endorsed, and reviewed by the Ethics Committee of the Universidad del Valle de Guatemala) to determine lethality and LD50, it was found that there is an ontogenetic change in the venom of both adult and juvenile M.occiduus snakes. However, contrary to the study hypothesis, it was reported that the lethality and toxicity of the venom in invertebrates were higher for the venom of adult snakes. This can be attributed to the variety and complexity of the protein cocktail present in the venom of adult snakes compared to juvenile snakes. In addition, the proteins found in the venom of adult M.occiduus snakes were shown to be selective for both ectothermic and endothermic animals. This indicated the presence of a possible varied and generalist diet during the adult stage, not limited only to mammals as in other species of the genus or family. This makes sense when considering that the snake’s habits during its adult stage are entirely terrestrial, under leaf litter, old logs, or any area that can offer varied prey, from insects and other arthropods to reptiles, amphibians, and mammals.
Figure 2. Laboratory work for lethality and LD50 bioassays. Inoculation of samples into a live invertebrate model (Photograph by Mariana Rivas Galvez, 2022, during thesis work).
What is suggested and recommended?
As a recommendation, it is suggested to repeat the bioassays with snakes in the “neonate” stage, as the juvenile snakes in this study were close to their adult stage, which could explain the similarity in proteins with the other sample along with the differences in lethality. Similarly, it is crucial to continue the study with other vertebrate models (Under all ethical authorizations and reviews), such as mammals, birds, and reptiles, to have a more comprehensive comparison of the different effects of protein types in the venom of M.occiduus and thus be able to associate it with its diet and growth stages.
Figure 3. Process of venom extraction and investigation of the protein composition of different samples of M.occiduus venom for laboratory tests (Photograph by Rocío León, 2022 during the thesis work of Mariana Rivas Galvez).
This study provides an approach to the biology and toxicology of the snake M.occiduus, about which little is known despite its medical importance and its “common” occurrence in the Mesoamerican region. In addition to being the first toxicological study for the species, it offers new ways and proposals for laboratory methodologies that can be replicated in similar studies. The information from this study is extremely valuable to promote the conservation of the species, raise awareness about it, and encourage more scientists to work in this field, continuing toxicological and biological research for this fascinating species.
Figure 4.Juvenile M.occiduus feeding on rodent offspring as part of its diet (Photographs by Mariana Rivas Galvez, 2022, during thesis work).
Acknowledgments by Mariana Rivas Gálvez
“I thank God for the opportunity to undertake this project. I express my gratitude to my thesis advisors, PhD Daniel Ariano, PhD Alejandra Zamora, and MSc Miguel Morales, for their ideas, corrections, suggestions, and involvement in this project. I appreciate my family and friends for accompanying me during the process of this work. Thanks to PhD. Rowland Griffin, Dr. Alejandro Striedinger, and Erick Hernández, who participated in the extractions and obtained permissions to work with specimens of M.occiduus at La Aurora Zoo. Thanks to the Universidad del Valle de Guatemala (UVG) Biology department and its executives for the support in using materials and laboratory space. I extend my gratitude to the UVG Bioethics Committee for reviewing and authorizing this work. I am eternally grateful to the crickets that gave their lives for science and the M. occiduus snakes that donated their venom for this thesis. I dedicate this work to myself, as a demonstration of self-love and everything I can achieve when I set my mind to it with leadership, effort, perseverance, and above all, doing it with love.
A special thanks to FLAAR Mesoamerica for providing the platform to publish this work, reaching more people with the aim of disseminating science, promoting conservation, and informing individuals about the wonderful fauna species in the Mesoamerican region.”
Written by Licenciada Mariana Rivas Gálvez
Found out more about this incredible research! Read this thesis in this link:
Notes and concepts:
Ectothermic animals: Ectothermic animals, also known as cold-blooded animals, depend on external sources to regulate their body temperature. Unlike endothermic or warm-blooded animals, they lack the ability to generate enough metabolic heat for a consistent internal body temperature. Instead, ectothermic animals adjust their body temperature by seeking or avoiding external heat sources, like sunlight or shade.
Endothermic animals: Endothermic animals, also known as warm-blooded animals, have the ability to regulate their body temperature internally. Unlike ectothermic or cold-blooded animals, they generate sufficient metabolic heat to maintain a relatively constant internal body temperature, independent of external environmental conditions.
LD50: Refers to “median lethal dose”. Measure used in toxicology to indicate the amount of a substance expected to cause death in 50% of a specific test population after a set exposure period. It is commonly expressed in terms of substance quantity per unit of body weight (e.g., milligrams per kilogram) and is used to assess the acute toxicity of chemical substances. A lower LD50 value indicates higher toxicity.
Lethality: Refers to the degree of capability a substance or agent has to cause death. It is a measure of how harmful or deadly a particular factor, such as a chemical, substance or condition, can be to living organisms.
Toxicity: Refers to the degree to which a substance, usually a chemical or environmental factor, can cause harm or damage to living organisms. It is a measure of how poisonous or harmful a particular substance is and can vary depending on the concentration and duration of exposure.
Cocktail of proteins/Cocktail: Mixture of various proteins present in the venomous secretion of certain animals, such as snakes. This concoction typically includes proteins with specific functions, such as enzymes that aid in prey digestion, toxins that immobilize or kill the prey, and other components that contribute to the venom’s overall effectiveness.
Bioassays: Experimental procedures that use living organisms or biological systems to assess the effects of substances, such as drugs, venom or toxins.
SDS PAGE assay: “Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis”, is a widely used laboratory technique in biochemistry and molecular biology. It is employed to separate proteins based on their size.
Bradford assay: A commonly used biochemical technique for quantifying the concentration of proteins in a solution.