It’s common knowledge that some frogs secrete toxins from special glands in their skin. But according to a paper published today in Current Biology, an international team of researchers report for the first time ever that two frog species are actually venomous. To be considered venomous, an animal must possess a toxin and must have some sort of mechanism, such as fangs, to deliver that toxin into another animal. And these frogs’ delivery mechanism of choice? Sharp spines on their faces.



Although these Brazilian frog species, Aparasphenodon brunoi and Corythomantis greeningi, both have been long known to science, almost nothing is known about their lives in the wild. Thus, this revelation is important because it increases our understanding of the biology of amphibians and provides a glimpse into some of their interactions with predators in the wild.

Two Brazilian frog species have bony spines growing on their faces

People sometimes confuse the terms “poison”, “toxin” and “venom”. Basically, toxins are a subgroup of poisons that are produced by living organisms, and venoms are toxins that are injected into another animal, typically either by a bite or sting. Thus, to demonstrate that an organism is venomous, it is essential to establish that an animal possesses toxins and that they have some sort of toxin delivery mechanism.

Today, an international team of researchers report their discovery that two frog species are not only toxic, but venomous. In their study, the researchers show that the skin secretions from both the head and the body of Aparasphenodon brunoi (Figure 1A) and Corythomantis greeningi (Figure 1B) are highly toxic. When injected into mice in microgram quantities, these secretions proved lethal. They also reveal that both species possess an efficient delivery mechanism. Both A. brunoi (Figures 1A, 1C, and 1E) and C. greeningi (Figures 1B, 1D, and 1F) have skulls covered with bony spines associated with toxin-producing skin glands.

Figure 1. Head Spines of Aparasphenodon brunoi and Corythomantis greeningi (A and B) Adult frogs A. brunoi (A) and C. greeningi (B). (C and D) Co-ossified skulls of A. brunoi (C) and C. greeningi (D); arrowheads point to occipital region. (E and F) Higher magnification of the rostral margin of the skull of A. brunoi (E) and C. greeningi (F). Photograph: Carlos Jared et al./Current Biology 2015

These frogs’ secretions are more toxic than pit viper venom

Many animal-made toxins are enzymes, which are catalytic proteins. Since proteins often carry an overall electrical charge, they can be separated into distinct groups of proteins based upon both their size and electrical charge by placing them into a matrix of some sort and running an electrical current through the apparatus. Examining the resulting patterns of “bands” can provide recognisable toxin profiles for secretions collected from a frog’s head and body.

This electrophoresis study revealed a lot of similarities between the protein profiles for each frog’s head and body secretions, but each species showed some unique bands, too. Further, these secretions showed an assortment of destructive activities, indicating the presence of a variety of different enzymes, as is typical for animal venoms (doi:10.1146/annurev.genom.9.081307.164356).

Surprisingly, the toxin profiles also indicated that these two frog species may not obtain their toxins from a diet of noxious insects, as is typical for most frogs, such as the iconic poison dart frogs (Dendrobatidae) of Central and South America.

“[W]e strongly suspect the frogs produce their own toxin”, said evolutionary biologist Edmund Brodie, a professor in the biology department at Utah State University, who is a co-author of the paper.

“The presence of alkaloids would suggest toxins were coming from a food source”, explained Professor Brodie in email.

“[But] we don’t see alkaloids.”

Further studies quickly made it clear that both frogs’ toxins are potent and lethal. When the researchers injected small amounts of either head or body secretions from each frog species into lab mice, they found that the LD 50 (the median lethal dose that can kill 50 per cent of a test population) for A. brunoi secretions were just 3.12 µg for head secretion and 4.36 µg for body secretion. The LD 50 for C. greeningi was roughly an order of magnitude greater: 51.94 µg for head secretion and 49.34 µg for body secretion (Figures 2A & B):

Figure 2. Edematogenic and Nociceptive Activity of Venoms (A–C) Edematogenic activity of A. brunoi (A) and C. greeningi (B) venoms. Nociceptive activity of venoms of both species (C). Two-way ANOVA followed by Tukey post-test. Differences between results were considered statistically significant when p ≤ 0.05. All experiments utilized six Swiss male mice per dose per group, weighing 18–20 g. Bars represent means, and vertical lines show SEM. Significant statistical difference versus control (*) versus same dose of venom (#). Illustration: Carlos Jared et al./Current Biology 2015

According to the researchers’ calculations, a single gram (roughly the same mass as a large paper clip) of the toxic secretion from A. brunoi could kill more than 300,000 mice or approximately 80 humans. In other words, A. brunoi secretion is 25 times more potent than pit viper (Bothrops species) venom, whereas C. greeningi secretion is twice as potent.

“The high levels of toxicity aren’t too surprising, as quite a lot of venomous animals go in for what’s been called ‘overkill’”, said geneticist John Mulley, a Lecturer at Bangor University, who was not part of this study.

Although neither of these frogs have any known predators, something clearly does prey on them: their high toxicity suggests that selection has driven the evolution of more toxic individuals.

“The question of why so strong a toxin is the same one we asked with our garter snake-newt studies [doi:10.1007/s00049-010-0057-z]. The answer there was the presence of a predator with resistance to the toxin. That may be the case also with these frogs”, said Professor Brodie.

Although gram-for-gram these two frogs’ venoms are more toxic than pit viper venoms, snakes have a much more effective venom delivery system -- long hollow fangs.

“Another possibility is that very small amounts [of toxin] would be transmitted by the head spines. These are not the hollow fangs of vipers and the toxin is stronger, but much less is transmitted” by the frogs’ spines, said Professor Brodie.

“My guess is that [toxin potency] comes down to being unable to predict how much venom/toxin will actually make it into the prey, or in this case, predator. To be on the safe side (especially if it’s life or death) it’s best to go overboard”, explained Dr Mulley in email.

These studies indicate that death-by-frog would probably be excruciating, especially if you happen to be the size of a mouse. The research team found that skin secretions of both frog species caused swelling in mice at all tested doses, reaching the highest values at a dose of 8 µg after 30 min for A. brunoi and after 60 min for C. greeningi (Figures 2A and 2B). Swelling persisted for 72 hours, particularly at doses of 32 µg and 8 µg in both species, and for C. greeningi, even at a dose of just 0.5 µg. Further, secretions from both frogs also caused pain in mice, regardless of dose (Figure 2C).

The frogs’ venoms are injected via bony spines on their faces

The researchers went on to show that the bony spines on these frogs’ heads are associated with toxin-producing skin glands. The skulls of both A. brunoi (Figures 1A, 1C, and 1E) and C. greeningi (Figures 1B, 1D, and 1F) are flattened and rough, with numerous enlarged bony spines in the nasal, jaw, and occipital regions (Figures 1C– 1F). Both frogs -- especially C. greeningi -- also have a conspicuous protrusion that resembles an almost cartoonishly large upper lip. This upper lip protrusion has prominent bony spines that are associated with concentrations of mucous and granular glands, which is where frogs manufacture and store their toxins (Figures 3B, 3C, and 4B–4D). Further, in both species, these bony spines pierce the skin in areas that have a lot of granular glands (Figures 3A–3D, 4C, and 4D):

Figure 3. Spines Pierce the Skin in A. brunoi and C. greeningi. (A) Live specimen of C. greeningi. (B–E) Scanning electron microscopy (SEM) of the rostral area and skin glands Q8 of A. brunoi (B & C) and C. greeningi (D & E). Spines (*) penetrate the skin through regions with high number of granular gland pores (arrows) on skin surface. (D) Tangential and superficial section through the dorso-lateral region of the head, near the upper jaw, showing spines (*) surrounded by granular glands (g). (E) Higher magnification of region equivalent to (D) showing connective tissue surrounding each gland. Photograph: Carlos Jared et al./Current Biology 2015

As first author, Carlos Jared, a herpetologist at the Instituto Butantan in Brazil, discovered first-hand whilst collecting C. greeningi for research, when restrained, the struggling frogs release a sticky secretion onto their skins. At the same time, the frog flexes its head -- an unusual ability for frogs -- ensuring that the animal jabs and rubs its venom-coated spines into its captor’s hand. Dr Jared’s injury caused intense, radiating pain for a period of about 5 hours -- pain that he had to endure since the team was far from any medical facilities at the time. Fortunately, he was wounded by the less toxic of the two species.

Figure 4. Head Spine & Skin Gland Histology. (A) Median sagittal section through the head of A. brunoi; arrows: major concentrations of glands. (B) Median sagittal section through the head of C. greeningi showing a high number of large granular glands. Asterisk: a spine almost reaching skin surface. High magnification of an area of co-ossification in the top of the head of A. brunoi (C) & in the upper lip of C. greeningi (D). (A-D) Stained with hematoxylin-eosin. Granular glands of A. brunoi (E) and C. greeningi (F) are rich in protein content (stained with bromophenol blue). Photograph: Carlos Jared/Butantan Institute/Current Biology 2015

Needless to say, that agonising injury captured the researchers’ attention: “This action should be even more effective on the mouth lining of an attacking predator,” they note, somewhat dryly, in their paper.



Frogs are understudied and poorly known animals

To my knowledge, this study is the most thoroughly documented description of a venomous amphibian published so far. The authors use histology, morphology, toxicology and behavioural observations to demonstrate for the first time that frogs can be venomous.

“This is very very cool”, said evolutionary biologist Bryan Fry, from the Venom Evolution Lab and an associate professor at the University of Queensland in Australia, who was not part of the study.

“Unprecedented would actually be an understatement”, said Professor Fry in email. “The authors provide compelling evidence for the evolution of venom in frogs. This is quite an amazing discovery.”

“The frogs’ venom delivery system is quite similar to a convergently evolved structure in the Iberian ribbed newt, the only venomous type of salamander”, Professor Fry pointed out.

The large mud-coloured Iberian ribbed newt, Pleurodeles waltl, has sharp ribs that can puncture through its sides, becoming coated with toxins secreted from special glands in its skin. Its sharp, toxin-coated ribs are thus transformed into a defensive stinging mechanism that effectively injects toxins directly through the thin skin in a predator’s mouth. However, this newt’s use of venom is purely defensive, which differs from most venomous animals, such as snakes, spiders and scorpions, that rely on venom to subdue their prey.

Photograph: Carlos Jared et al./Current Biology 2015

“[T]his is the first study on amphibians that I am aware of where they have actually functionally tested the ‘venom’ secretions in terms of toxicity”, said Nicholas Casewell, a Lecturer and NERC Research Fellow at the Liverpool School of Tropical Medicine, who was not part of this study.

“What I do find interesting is that the toxicity of the secretions collected from the head and the body was essentially the same”, said Dr Casewell in email.

“So whilst the granular glands had increased in size in one of the species and there was apparent ‘head butting’ behaviour to aid ‘envenoming’, in reality these frogs could well be technically classified as venomous and poisonous, although the distinction may simply be the product of where on the frog a predator bites (head - toxin injection = venom; body - toxin ingestion = poison)”, said Dr Casewell.

This research serves as a provocative reminder that frogs are still a very understudied and poorly known group. It demonstrates how much we still have to learn about their natural history and indicates that there are many fundamental discoveries about these animals that still await us. And of course, this study raises many important questions, such as whether there are more venomous frogs lurking out there somewhere.

“There are a number of frogs, both hylids and other families that have spines on the head. These have not been studied from this point of view, specifically for the toxins and proximity of granular (poison) glands to spines”, said Professor Brodie. “We have some of these specimens and are working on them now.”

The location of the toxic spines appear to be the best possible location for making a strong defence: in the wild, these frogs also exhibit phragmotic behaviour, which is where the animal hides its body inside some sort of shelter, such as a bromeliad, or a hole or burrow, and sits facing outward with its head filling up the entrance. This behaviour also helps reduce water loss.

Many highly toxic poison dart frogs have striking aposematic colouration, which is a visual signal indicating their toxicity to potential predators. So why aren’t these venomous frogs more colourful?

“[A]posematic species are usually diurnal, as is the case of most aposematic frog species, including the well-known toxic poison frogs in the family Dendrobatidae. There is evidence that the presence of aposematic coloration in frogs is the result of the selective action of visually oriented predators, which can perceive colors, such as birds”, explained evolutionary ecologist, Ivan Prates, a doctoral candidate at the City University of New York (CUNY), who was not part of this study. Mr Prates, who earned his masters degree at Butantan Institute in Brazil, has extensive research experience studying the ecology and evolution of poisonous Brazilian frogs.

But unlike the poison dart frogs, which obtain their toxins from their diet, A. brunoi and C. greeningi probably produce their own toxins. Thus, they may be balancing the energetic costs associated with producing brilliant warning colouration with those of toxin production. It is also possible their relatively subdued colouring is the result of an evolutionary trade off between the frog displaying itself as toxic and exposing itself to potential predators; the possibility that bright colouration may reduce the possibility of catching prey; how toxic the frog’s secretions should be to deter its predators; and the potential confounding factor that some predators may be resistant to the frog’s toxins.

Venomous frog, Corythomantis greeningi, perches peacefully on Carlos Jared’s hand. Photograph: Carlos Jared/Current Biology 2015

I am especially intrigued by the less toxic of the two frog species, C. greeningi. This frog appears to be camouflaged, yet it is venomous and very toxic -- an unusual combination. What might this indicate about its natural history and ecology?

“It suggests that there may be a predator that is resistant to lower doses of the venom causing an arms-race between toxicity and resistance”, said Professor Brodie. “Perhaps a snake or an opossum?”

This frog’s natural history and ecology likely play other roles in toxicity, too.

“Data on the natural history of C. greeningi indicates that this species has nocturnal habits; eating, calling and reproducing during the night, while hiding in rock crevices and tree holes during the day. At night, lack of light limits the perception of colors by predators; moreover, being active at night, C. greeningi is exposed to nocturnal predators having limited color vision, such as snakes and other frogs. As a result, nocturnal frogs rarely show aposematic coloration, even if they may be defended chemically and show high toxicity,” explained Mr Prates in email.

The field site for C. greeningi is in the Caatinga, an arid region in Brazil. Photograph: Carlos Jared/Current Biology 2015

C. greeningi lives in the interior of northeastern Brazil in an ecoregion known as the Caatinga. This beautiful, but forbidding, habitat is arid with a brief rainy season; accordingly, the Caatinga hosts a variety of drought-tolerant annual plants, cactus, thorny brush, and small, thorny trees that shed their leaves seasonally.

“As you can see, everything has spines, including the frogs. This is frog habitat”, noted Professor Brodie dryly.

According to Professor Brodie, the research team is planning to better characterise the venom and the skin glands that produce the venom for both A. brunoi and C. greeningi. And already, they are studying several other frog species from around the world that they suspect may also be venomous.

Sources:

Carlos Jared, Pedro Luiz Mailho-Fontana, Marta Maria Antoniazzi, Vanessa Aparecida Mendes, Katia Cristina Barbaro, Miguel Trefaut Rodrigues, and Edmund D. Brodie, Jr. (2015). Venomous Frogs Use Heads as Weapons, Current Biology, published online on 6 August 2015 ahead of print | doi:10.1016/j.cub.2015.06.061

Also cited:

Bryan G. Fry, Kim Roelants, Donald E. Champagne, Holger Scheib, Joel D.A. Tyndall, Glenn F. King, Timo J. Nevalainen, Janette A. Norman, Richard J. Lewis, Raymond S. Norton, Camila Renjifo, and Ricardo C. Rodríguez de la Vega (2009). The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms, Annual Review of Genomics and Human Genetics 10:483–511 | doi:10.1146/annurev.genom.9.081307.164356 (OA)

Becky L. Williams, Charles T. Hanifin, Edmund D. Brodie Jr., and Edmund D. Brodie III (2010). Tetrodotoxin affects survival probability of rough-skinned newts (Taricha granulosa) faced with TTX-resistant garter snake predators (Thamnophis sirtalis), Chemoecology, 20:285–290 | doi:10.1007/s00049-010-0057-z (OA)

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