Implications for the field of environmental toxic ecology through studying Lithobates palustis

Pickerel frog
Lithobates palustris

Pickerel frogs, Lithobates palustris, are the only toxic frog in North America and range throughout the eastern half of the United States. Their brown bodies, speckled with black spots, are well camouflaged to their environment. Researchers collecting frogs in the field reported pickerel frogs cause other captured frogs in the collection bag to die, leading them to hypothesize pickerel frogs are poisonous (Duellman and Trueb 1994). This conclusion has been backed up by studies identifying toxic peptides in L. palustris excretions as a response to stress. However, little has been done to investigate the frog’s toxic ecology and even general ecology. “Pickerel frogs may produce toxic skin secretions in some parts of their range but not others, or may be toxic only at certain life stages. A third possibility is that pickerel frogs produce toxic secretions only in some habitats or under certain diet regimes….Some aspects of the general ecology and life history, including size of the home range, whether they establish territories, and life span, are completely unknown” (Dorcas and Gibbons 2008).

Given that pickerel frogs are severely understudied, inferences about their ecology and toxins must be made from studies conducted with poison frogs. Poison frogs are much more intensively studied than the pickerel frog. This could be due to the charismatic coloration of poison frogs, their high potency of toxin, and the dozens of species that make up their taxonomic family, all of which the pickerel frog lacks. Mud-colored pickerel frogs are not very toxic and can be handled bare-handed, causing a rash at worst. Could it be because their toxicity is so low, they need to blend in with their environment to avoid predation? Might their toxicity be affected by their diet? If their diet is a factor, then their levels of toxicity would vary throughout their range and habitats. At this point we can only look to dart frogs for answers to questions about their ecology.

Toxicology is an expanding field especially since some toxins have the potential to help cure ailments. Initially, ethnic folk medicine provided the basis for choosing which toxins to investigate.  In traditional Chinese medicine, for example, dried and powered toad secretions were used as a pharmaceutical agent to treat a number of illnesses. It has since been proven some secretions, like that from Bufo bufo gargarizans, can induce apoptosis in bladder carcinoma cells (Gomes et al. 2010). There is a decent amount of funding available to researchers who choose to identify proteins in toxins and venoms present in animals and plants around the world, but toxicity can be affected by an animal’s ecology, especially if their toxin is derived environmentally. To fully understand toxins, ecological studies must be conducted as well.

Six-novel families of similarly structured peptides are present in the Pickerel frog’s skin secretions (Basir and Conlon 2003). Some of these peptides are antimicrobial, discouraging bacterial and fungal growth on the frog, and are thought to be a natural defense against the chytrid fungus which is responsible for severe declines in amphibian populations (Ali et al 2002). Most frogs secrete antimicrobial peptides, but toxic frogs secrete an extra family of proteins called bradykinins. Bradykinins coevolved with vertebrate predators to be structural homologues to regulatory peptides and bind to target receptors (Chen et al 2002). They are known to cause vasodilation, hypotension, smooth muscle contraction, pain, and inflammation in mammals (Mccrudden et al 2007). L. palustris bradykinins were injected into hamster smooth muscle tissue, but a reaction to the toxin did not occur, leading researchers to conclude the “biological functions, if any, remain to be established” (Basir and Conlon 2003).

Toxic species are typically associated with aposematic coloration, warning predators of their unpalatability with vibrant hues and patterns. Visual predators learn to avoid species with aposematic traits and prey upon members of a toxic species whose patterns differ from the traditional signals, stabilizing an aposematic trait and promoting mimicry in similar species (Gamberale-Stille and Tullberg 1999; Darst et al 2006). Decreased predation pressures and female poison frog preference for bright coloration result in a colorful, toxic frog species (Maan and Cummings 2008). This leads to the assumption that the brighter a species is the more toxic it is as well. Two popular theories speculate the relationship between toxicity and aposematic traits: 1) coloration evolved to compensate for lack of toxin and 2) coloration evolved with increasing toxicity. L. palustris does not fit into these theories with its camouflaged, brown skin. Pickerel frog’s may not have a sexual selection pressure for increased coloration, but, with decreased predation, some color and pattern variation would be expected to occur. The mimicry theory does not hold water either as there are multiple camouflaged, brown frogs almost indistinguishable from pickerel frogs, whose habitat they share, and all these stream-dwelling frogs, including L. palustris, tend to have a long list of similar predators, suggesting other frogs are not benefiting from mimicry. A recent paper with poison frogs found coloration and toxicity did not coevolve with each other, and stated differences in coloration and toxin potency do not significantly influence predation (Wang 2011).  A new way of thinking about coloration and toxicity is needed for species with environmentally derived toxins, and the pickerel frog may be an important species to help unlock that way of thinking.

Examining a common predator among stream-dwelling frogs, the garter snake (Thamnophis sirtalis), may shed light on L. paulustris’s role in the food chain. Garter snakes do not seem to be adversely affected by bradykinins despite a previous study’s findings regarding bradykinins’ effects on serpentine cardiovascular systems (Wand et al 2000). The garter snake and the hog-nosed snake (Heterodon platirhinos) participate in an evolutionary arms race with newt toxin, adapting to changing tetrodotoxins to keep newts on the menu (Feldman et al 2010; Feldman et al 2015). The pickerel frog may be evolving different bradykinins to discourage snake predation, but local snakes are experts at adapting to amphibian toxins. This suggests the presence of a unique, under-studied food chain in eastern North America, resulting from the evolutionary ecology of amphibian toxin and predator adaptation.

If bradykinins in pickerel frogs function in the same manner as bradykinins in Central American poison frogs, then their toxins would be derived from alkaloid containing arthropods in their diet. The pickerel frog diet has not been adequately described but is generally believed to consist of small invertebrates, presumably including arthropods (Dorcas and Gibbons 2008). Toxin composition of poison frogs varies geographically and temporally depending on availability and presence of arthropod prey items (Saporito et al 2007). If diet is the source of pickerel frog toxin, potency of the toxin would vary depending on availability of their alkaloid-containing prey. Availability is affected by habitat, season, and prey range. Variation in diet during life stages of frogs influence toxicity as well (Flores et al 2015). Poison frog tadpoles are unable to eat arthropods and are non-toxic as a result. Newly metamorphosed froglets are less toxic than adults, because they are transitioning to their adult diet. It would not be farfetched to propose predation on frog populations may be higher in areas, seasons, or life stages where certain arthropods are not present or available, causing low toxicity.

L. palustris is an untapped gold mine of evolutionary toxic ecology data. Though poison frogs are generally the only toxic frogs studied, the sole existing toxic frog in North America deserves attention too. Their large range may signify the presence of alkaloid rich arthropods, and there may be populations of non-toxic pickerel frogs. If there are non-toxic populations, how does their mortality compare to toxic populations? If there is an abundance of alkaloids in the environment, why doesn’t the pickerel frog’s sister species, L. areolatus, excrete bradykinins? These questions open the pathway to diet, food web, and molecular cell biology studies. The possibilities of L. palustris experiments are almost endless because hardly anyone studies this species, and the majority of studies that do exist do not account for the frog’s ecology.

One of the few toxic analyses of L. palustris published used hamster muscle tissue to test for responses to pickerel frog toxin, but did not have conclusive findings. Lack of a reaction to the toxins is most likely a result of hamsters not being a predator of L. palustris or inhabiting the same continent, so the bradykinins would not have coevolved to be homologous to hamster regulatory peptides or capable of producing a negative reaction in hamster anatomy. A different paper stated in its discussion that L. palustris bradykinins have no effect on hamster gastric or vascular smooth muscle tissue (Basir et al 2007). Another study tested the potency of toxins from a poison frog on mice, admitting mice were not ideal, because they are not natural predators (Wang 2011). Natural predators of toxic frogs may be difficult to obtain and approve for use in experimentation. Accounting for the evolutionary ecology of the pickerel frog in toxin studies by using an animal – such as a mink – with a natural history of preying upon pickerel frogs would establish the biological function of L. palustris bradykinins.

Toxins may have a non-predatory purpose. Aquatic animals sensitive to changes in water quality, such as other amphibians, could avoid pickerel frog toxin. If a frog excretes these toxins and clears the area of other amphibians, the frog would have an advantage over non-toxic amphibian species when competing for local resources. Another hypothesis for avoidance behavior, if it exists, is other species, rather than being intolerant of toxins, learn bradykinins signify the presence of predators and consequently avoid areas with toxins. Avoidance behavior studies are required to answer these questions. Studying evolutionary toxic ecology in a local stream environment doesn’t have to include field work in Central America, making experiments much more cost effective.

Pickerel frogs have much to offer the field of environmental toxic ecology. Studying them could help unlock the relationship between toxins and coloration in species with environmentally derived toxins, having huge implications for how evolution is understood. In order to reveal ecological or evolutionary relationships, experiments must be designed with a toxic species’ evolutionary ecology in mind to yield accurate results. Filling gaps in the literature will provide a more complete ecological picture of this species and how its toxin interacts with its environment.

Literature Cited

Ali, M. F., Lips, K. R., Knoop, F. C., Fritzsch, B., Miller, C., & Conlon, J. (2002). Antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, Rana areolata. Biochimica Et Biophysica Acta (BBA) – Proteins and Proteomics, 1601(1), 55-63.

Basir, Y. Knoop, F. Dulka, J. Conlon, J. (2000). Multiple antimicrobial peptides and peptides related to bradykinin and neuromedin N isolated from skin secretions of the pickerel frog, Rana palustris. Biochimica et Biophysica Acta, 1543, 95-105.

Basir, Y. J., & Conlon, J. (2003). Peptidomic analysis of the skin secretions of the pickerel frog Rana palustris identifies six novel families of structurally-related peptides. Peptides, 24(3), 379-383.

Chen, T., Orr, D. F., Bjourson, A. J., Mcclean, S., O’Rourke, M., Hirst, D. G., . . . Shaw, C. (2002). Bradykinins and their precursor cDNAs from the skin of the fire-bellied toad (Bombina orientalis). Peptides, 23(9), 1547-1555.

Darst, C. R., M.E. Cummings, and D.C. Cannatella. (2006). A mechanism for diversity in warning signals: conspicuousness versus toxicity in poison-dart frogs. Proc. Nat. Acad. Sci. USA, 103, 5852-5857.

Dorcas, M. E., & Gibbons, W. (2008). Frogs & toads of the southeast. Athens: University of Georgia Press.

Duellman, W. E., Trueb, L. (1994) Biology of Amphibians, Baltimore/London: John Hopkins University Press.

Feldman, C. R., Brodie, E. D., Brodie, E. D., & Pfrender, M. E. (2010). Genetic architecture of a feeding adaptation: Garter snake (Thamnophis) resistance to tetrodotoxin bearing prey. Proceedings of the Royal Society B: Biological Sciences, 277(1698), 3317-3325.

Feldman, C. R., Durso, A. M., Hanifin, C. T., Pfrender, M. E., Ducey, P. K., Stokes, A. N., . . . Jr, E. D. (2015). Is there more than one way to skin a newt? Convergent toxin resistance in snakes is not due to a common genetic mechanism. Heredity, 116(1), 84-91.

Flores, E. E., Stevens, M., Moore, A. J., Rowland, H. M., & Blount, J. D. (2015). Body size but not warning signal luminance influences predation risk in recently metamorphosed poison frogs. Ecol Evol Ecology and Evolution, 5(20), 4603-4616.

Gamberale-Stille, G., and B.S. Tullberg. (1999). Experienced chicks show biased avoidance of stronger signals: an experiment with natural colour variation in live aposematic prey. Evolutionary Ecology, 13, 579-589.

Gomes, A., Bhattacharjee, P., Mishra, R., Biswas, A., Dasgupta, S., & Biri, B. (2010). Anticancer potential of animal venoms and toxins. Indian Journal of Experimental Biology, 48, 93-103.

Maan, M. E., & Cummings, M. E. (2008). Female Preferences For Aposematic Signal Components In A Polymorphic Poison Frog. Evolution, 62(9), 2334-2345.

Mccrudden, C. M., Zhou, M., Chen, T., O’Rourke, M., Walker, B., Hirst, D., & Shaw, C. (2007). The complex array of bradykinin-related peptides (BRPs) in the peptidome of pickerel frog (Rana palustris) skin secretion is the product of transcriptional economy. Peptides, 28(6), 1275-1281.

Saporito, R. A., M. A. Donnelly, P. Jain, M. H. Garraffo, T. F. Spande, and J. W. Daly. (2007). Spatial and temporal patterns of alkaloid variation in the poison frog Oophaga pumilio in Costa Rica and Panama over 30 years. Toxicon, 50, 757-778.

Wand, T., Axelsson, M., Jenson, J., Conlon, J.M. (2000). Cardiovascular actions of python bradykinin and substance P in the anesthetized python, Python regius, Am. J. Physiol., 279, 531-538.

Wang, I. J. (2011). Inversely Related Aposematic Traits: Reduced Conspicuousness Evolves With Increased Toxicity In A Polymorphic Poison-Dart Frog. Evolution, 65(6), 1637-1649.

Author’s note

This paper was written for my evolutionary ecology class. Follow my twitter for updates on my works!

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