From bugs to behavior: the science of LOBSU
The guiding mission driving research in the Laboratory of Organismal Biology is to understand evolutionary adaptations that enable new behavioral and physiological functions across different scales of biology - from small molecular and cellular level changes all the way up to ecological and evolutionary processes. Most of us use poison frogs as our study organisms to ask these biological questions, although we also have some cane toads and other critters in the lab as well. Poison frogs have many novel characteristics that make them quite interesting to study, including a wide variety of parental care strategies, impressive spatial navigation skills, and an arsenal of protective toxins. Like the frogs themselves, the research conducted in our lab is very diverse. We study many aspects of poison frogs: the bugs they eat, how they are able to be toxic, the impacts of toxicity on microbiomes, and the neural circuitry and evolution of their complex behaviors.
Chemical Ecology
Chemical ecology is the study of how organisms communicate with one another using chemically-mediated signals, and how these chemicals in turn structure populations, communities, ecosystems, and evolutionary processes. In the context of poison frogs, we are interested in the ecological role that the alkaloid toxins play in shaping communities and driving evolutionary processes. Alkaloids are a numerous and diverse class of organic compounds that often have strong physiological responses in animals. They can be found in drugs such as morphine, and in poisons such as the pesticide strychnine, but can also be found in some less potent substances like the caffeine in coffee. We know that poison frogs obtain alkaloid toxins from their diet of arthropod prey and that these alkaloids in turn provide defense against potential predators; however, there are still so many unanswered questions about this toxicity, including how it is evolutionarily and physiologically derived and what it means in the context of a broader ecosystem. Our chemical ecology work is focused on three primary topics: how diet contributes to toxicity, the physiology and metabolism of toxins, and the microbiome.
Diet
While it is known that poison frogs obtain their toxins from their diet, we still don’t know what exactly is in their diet. Some of the alkaloids found on the frogs’ skins have been tied back to either ants or mites, but the specific source of many of the alkaloids remains unknown. There is also a larger question that remains unexplored: Do poison frogs exhibit a preference for toxin-containing prey? One hypothesis is that poison frogs preferentially eat bugs that contain toxins, which makes toxin uptake from their diet more efficient. Alternatively, poison frogs may eat indiscriminately, and show no preference for specific toxic prey. This would suggest that poison frogs have a well-tuned ability to sequester toxins from the few alkaloid-containing prey items that they do consume. We are actively trying to characterize the diet of the frogs, and through that, we are hoping to better understand whether dietary preference contributes to toxicity in poison frogs. The primary person in the lab working on this area of research is Nora Moskowitz, who is a third-year graduate student.
Physiology and metabolism
The physiological mechanisms by which toxins are extracted from prey items and transported through the body to the skin remain unknown. In the lab, we are looking at how gene expression and protein abundances differ across toxic and non-toxic frogs to try to identify proteins that may be involved in this process. Additionally, we are using a comparative approach across many species to try to better understand how toxicity evolved in poison frogs. In addition to understanding the general mechanisms of uptake and their evolutionary origins, we are also interested in understanding how these toxins are metabolized, if at all. In most other organisms, potentially harmful foreign compounds like toxins are metabolized by the liver or kidneys and excreted from the body as waste. However, poison frogs not only keep these toxins for their own use, but they can actually modify them in order to make them more toxic. In particular, pumiliotoxin 251D is modified to the more potent allopumiliotoxin 267A, but this mechanism remains unknown. It has been hypothesized that this conversion is performed by a cytochrome P450, but little else is known. We are working to identify potential cytochrome P450s that may be involved in this process of metabolism, as well as identifying whether other toxins may undergo similar metabolic changes on their journey to the skin. This physiology work is being done by Aurora Alvarez-Buylla, a fourth year graduate student in the lab.
Microbiome
Alkaloid toxins are often thought of in a larger, ecological context in terms of how they may protect a frog from predation. However, they may also play an ecological role in shaping frog-microbe associations. Poison frog alkaloids were demonstrated to have some antimicrobial effects against multiple species of bacteria and fungi. But the influence of toxins on microbial colonization (either beneficial or pathogenic) of frogs remains unknown. We are hoping to develop a better understanding of how poison frog toxins shape the microbiome and how these shifts in the microbial community may contribute to changes in frog health, including the ability to ward off infections, such as the chytrid fungus. This microbial work is led by Stephanie Caty, a third year graduate student.
Other chemical ecology projects
In addition to our frog-focused chemical ecology work, we also have two additional chemical ecology projects. These projects aim to identify the sources of hallucinogenic compounds in Hawaiian dreamfish and Sonoran toads. In both the Hawaiian dreamfish system and in Sonoran toads, the source of the hallucinogenic compounds is unknown and could be dietary, microbial, or a direct metabolite produced by the animal. This work is led by Marina Luccioni and Jules Wyman, two undergraduate researchers in the lab, who are hoping to determine the identity and/or sources of these hallucinogens.
Neuroethology
Poison frogs have unique and complex social behaviors that make them an excellent system to study the neural circuits involved in social communication and behavior. Many poison frogs display parental care by transporting their tadpoles to nurseries, where they occasionally check on and feed their tadpoles. In some species, this is only done by the dad. In others, it’s only by the mom. In others, this task is shared by both parents. This creates an excellent opportunity for studying the neural circuitry of parental care behaviors and spatial cognition. In order to communicate with mom and dad, some species of tadpoles make a vigorous begging display towards their parents. Tadpole begging behavior involves rapid body vibrations and is thought to be an honest indicator of need, demonstrating to the mom that the tadpole needs to be fed. Moms will then deposit unfertilized eggs as meals for the tadpoles to consume. This behavior is analogous to lactation in mammals. Because these tadpoles are translucent, we can perform imaging studies of the brain in live animals. Calcium imaging is a technique that allows us to visualize the neural activity in real time. Essentially, this makes the neurons glow, and the more they glow, the more that neuron was activated. The complex social behaviors and translucent physiology of tadpoles makes them a great system for neuroethology research.
Parent/offspring communication
We are investigating the sensory and neural control of parent-offspring communication using Mimetic poison frogs, Ranitomeya imitator. In this species, males carry hatched tadpoles to individual nurseries. Males check occasionally on tadpoles, and if tadpoles display begging behavior for food, males will produce an acoustic signal to call females to come feed the tadpoles. This complex system involves communication both between the parents and between the parents and tadpoles. PhD student Billie Goolsby is studying this interaction from the perspective of the parents, while Postdoc Julie Butler is studying it from the perspective of the tadpole. Together, their research examines the sensory modalities that poison frog parents and tadpoles use to communicate with each other and the neural circuitry associated with parental behaviors and tadpole caregiver recognition.
Tadpole Social Decision Making
Tadpoles must assess each visitor to their nursery and determine if they are a potential caregiver or threat. Incorrectly displaying begging behavior is energetically costly and increases predation risk, making this a life-or-death decision. We are using a combination of techniques to examine how tadpoles make this complex social decision. We first expose tadpoles to different scenarios and watch the resulting behavior. We use a technique called phosphoTRAP, which combines RNA sequencing with immunoprecipitation of active neurons. By comparing levels of RNA transcripts between the total RNA sample and from the active neurons, we can determine what transcripts are involved in different types of behavior. We then use staining techniques to map this neural circuitry. Based on these data, we hope to implement new techniques that will allow us to functionally control these neurons to test their role in tadpole social behaviors. We are also looking at how infants convey nutritional need through brain-specific knockdowns of the FOXP2 protein. This work is primarily being completed by two undergraduates in the lab, Jordan McKinney and Sarah Ludington, with the help of Dr. O’Connell and Julie Butler.
Space use
Poison frogs show great ability to navigate their surroundings. One extreme example of navigation and space use in poison frogs is tadpole transport and care. Once hatched, tadpoles are transported from the leaf litter to pools of water. In some species, mothers place tadpoles individually in small plants and then return to feed each tadpole every few days for several months. These behaviors are energetically expensive and cognitively demanding, as not only do frog parents need to remember where these pools are located, but some moms frequently return to feed their tadpoles. We are investigating species differences in spatial cognition as a function of sex in parental behavior in poison frogs. In addition, we are studying spatial behavior in cane toads, who are large enough to carry neural recording devices on their heads and have some applied invasion ecology aspects to their behavior. By implanting electrodes into the brain, we can record from neurons as these toads navigate around an arena. This work is led by postdoctoral researcher Andrius Pasukonis and third year graduate student Daniel Shaykevich. Two co-term masters students, Andrew Hayden and Chris Jackson, are also working on this project.
Evolution of parental care and pair bonding
Other research in the lab has focused on the evolution of pair bonding and parental care neural circuitry. Dr. Jessica Nowicki is taking a comparative approach by examining the neural transcripts and brain regions associated with pair bonding in quails, lizards, frogs, and fish. She is using a combination of phosphoTRAP to identify transcripts that differ between paired and unpaired males and immunohistochemistry with the neural activation marker pS6 to identify brain regions activated differentially in paired animals. A similar approach was taken by former postdoc Eva Fischer in her comparative study of parental care neural circuits in poison frogs. Her work, which identified molecules and brain regions associated with parental care and tadpole transport, can be found here.