Amphibians

Croaking Science: Novel reproductive behaviours in amphibians

April 30, 2019 by editor

There are currently 8,004 species of amphibian (AmphibiaWeb, 2019) of which 88%, (7,064 species) are frogs and toads (anurans). These species exhibit an astonishing range of reproductive modes, of which only a small proportion lay eggs in water which hatch into aquatic larvae. In total, anurans exhibit 40 different modes of reproduction which are based on where the eggs are laid, egg type, and patterns of larval development. Examples of different reproductive modes include: depositing eggs in foam nests which float on water; laying eggs on the back of the female; laying eggs on the ground or in burrows; and depositing eggs on leaves with tadpoles that hatch and fall into water below (Duellman & Trueb, 1986). Of the 40 modes, direct developing embryos are perhaps the most unusual for amphibians. In these species, no water is required for development and instead the tadpoles develop within eggs which then hatch directly into froglets. This type of reproduction has evolved multiple times independently in anurans and currently 1,400 species are recognised to develop in this way (Stuart et al., 2008). Our knowledge of the full range of reproductive modes is still lacking as new patterns of reproduction are still being discovered. For example, some recent discoveries include novel nest building behaviour in the Kumbara night frog (Nyctibatrachus kumbara) from southern India (Gururaja et al., 2014) and unique foot-flagging behaviour has been observed in the Wayanad dancing frog (Micrixalus saxicola) a small torrent frog from India (Preininger et al., 2013). In this article we discuss some further recent discoveries of novel reproductive behaviours in anurans.

The Western Gnats in southern India has been a recent hotspot in discoveries of novel reproductive behaviours in anurans. Due to its glacial history and diversity of habitats, this region supports over 180 species of amphibian which exhibit a range of reproductive patterns. The Chalazodes bubble nest frog (Raorchestes chalazodes) inhabits remote tropical forests in the Western Gnats. This species was presumed to be extinct, until its recent rediscovery in 2011 (Ganesan et al., 2019). Sheshadri et al. (2015), have recently reported a unique breeding behaviour in this frog. Males were found living inside the shafts of bamboo canes where they guarded the eggs laid by the females (Figure 1). It appears that small holes created by insects in the lower shafts of bamboo canes are utilised by this frog, which uses the hollow interior for breeding. Males call to attract females, which lay 5-8 large eggs in clusters inside the bamboo. The male guards these eggs from predators until they hatch into free-living froglets. This is the first time that this type of reproduction has been recorded in any amphibian species and thus represents a unique reproductive mode (Sheshadri et al., 2015). Unfortunately, this frog remains critically endangered, only occurring in five localities, and is highly threatened due to over-harvesting of bamboo (Sheshadri et al., 2015). Unless unregulated overharvest of bamboo is halted, this species may well go extinct due to lack of suitable breeding habitat.Figure 1. Left: Adult Chalazodes bubble nest frog (Raorchestes chalazodes) on top of a bamboo cane; Right: cross section of bamboo cane showing clutch of eggs and an emerging froglet.

***Figure 1.*** *Left: Adult Chalazodes bubble nest frog* (Raorchestes chalazodes) *on top of a bamboo cane; Right: cross section of bamboo cane showing clutch of eggs and an emerging froglet.* [Photo credit: Sheshadri et al., 2015]

Many species of male anuran possess vocal sacs which they use during breeding to vocalise to attract females. In several frog species visual cues of the inflating vocal sac make the acoustic signal more attractive to females or aid to deter competing males. Male African reed frogs (Hyperoliidae) possess a prominent gular patch on the vocal sac which they use to attract females and may also function as a visual cue to competing males (Figure 2). Starnberger et al. (2018) found that in the spotted reed frogs (Hyperolius puncticulatus) males do not respond to the vocal sac of other males but when they heard males calling they performed a previously undescribed gestural display: a tapping behaviour consisting of a series of front and hind limb lifts. It is thought that these gestures serve to deter males and also attract females who may be close by (Starnberger et al., 2018).F

***Figure 2.*** *Male spotted reed frogs (*Hyperolius) often have conspicuous vocal sacs which they use to vocalise and attract females. [Photo credit: Luke & Ursula Verburgt, http://africanamphibians.myspecies.info/file-colorboxed/8072]

Many anurans form amplexus during breeding where by the male takes the female in an embrace. Since fertilization is external in most anurans, the position of the male ensures that he is able to release his sperm to fertilize her eggs as she releases them. The night frogs (genus Nyctibatrachus) from India contains 28 species, many of which have only recently been described. In all Nyctibatrachus species, egg clutches are deposited on rocks or vegetation overhanging water, and tadpoles fall in the water after hatching, where they continue their development. Several novel types of amplexus have been described in this genus (reviewed in Willaert et al., 2016) such as a short axillary amplexus in pairs of the Kumbara night frog (N. kumbara) followed by a handstand, or the complete absence of amplexus in N. petraeus where the female deposits her eggs prior to the male fertilizing them. Willaert et al. (2016) have recently discovered a new breeding behaviour in the Bombay night frog (N. humayuni). During breeding the male forms a loose amplexus on the female, lasting only a few seconds and differs from all previously described amplexus types in anurans. When mounted, the male rests on the female without grabbing her tightly, but instead uses his hands to hold on to a leaf, branch or tree trunk (Willaert et al., 2016). Since the male does not have time to fertilise the eggs whilst engaged with the female, the authors propose that the male releases his sperm onto the back of the female and the eggs are subsequently fertilized by the sperm running down her back and hind legs. This hypothesis is supported by the observation that the female remains motionless after egg deposition for several minutes with her hind legs stretched around the freshly deposited clutch (Willaert et al., 2016) (Figure 3). In addition, females have been observed to make a call which is highly unusual. Female calling behaviour has so far only been reported in only 25 anurans, representing less than 0.5% of the total anuran species that are currently recognized (Willaert et al., 2016). It appears that the females call to aid in mate recognition and location.

***Figure 3.*** *A female Bombay night frog (*N. humayuni) *deposits hers eggs and remains motionless with her hind legs stretched around the eggs. The male is sitting close-by without any physical contact with the* female. [*Photo credit: Willaert et al., 2016]*

This study, along with others recently published, demonstrate the range of reproductive behaviours that are exhibited amongst anurans. The Nyctibatrachus frogs are one of several unique taxa in the Western Ghats biodiversity hotspot and are heavily threatened by human activities. Understanding the ecology of these and other poorly known species is of major importance for planning and successfully implementing conservation strategies.

References

AmphibiaWeb (2019). <https://amphibiaweb.org> University of California, Berkeley, CA, USA. Accessed 18 Apr 2019.

Duellman, W.E. & Trueb, L. (1986) Biology of Amphibians, McGraw-Hill Publishing Company, United States of America.

Ganesan, R., Seshadri, K.S. & Biju, S.D. (2019) Lost! Amphibians of India. Available at: http://www.lostspeciesindia.org/LAI2/new1_rediscovered.php. Accessed 18 April 2019.

Gururaja, K.V., Dinesh, K.P., Priti, H. & Ravikanth, G. (2014) Mud- packing frog: a novel breeding behaviour and parental care in a stream dwelling new species of Nyctibatrachus (Amphibia, Anura, Nyctibatrachus). Zootaxa, 3796: 33-61.

Preininger, D., Boeckle, M., Freudmann, A., Starnberger, I., Sztatecsny, M. & Hödl M. (2013) Multimodal signaling in the Small Torrent Frog (Micrixalus saxicola) in a complex acoustic environment. Behavioral Ecology and Sociobiology, 67: 1449–1456.

Seshadri, K.S., Gururaja, K.V. & Bickford, D.P. (2015) Breeding in bamboo: a novel anuran reproductive strategy discovered in Rhacophorid frogs of the Western Ghats, India. Biological Journal of the Linnean Society, 14 (1): 1-11. https://doi.org/10.1111/bij.12388

Starnberger, I., Maier, P.M., Hödl, W. & Preininger, D. (2018) Multimodal signal testing reveals gestural tapping behavior in spotted reed frogs. Herpetologica, 74 (2): 127–134.

Stuart, S.N., Hoffmann, M., Chanson, J.S., Cox, N.A., Berridge, R.J., Ramani, P. & Young, B.E. (2008) Threatened Amphibians of the World. Barcelona, Spain: Lynx Edicions; Gland, Switzerland: IUCN, Arlington, Virginia, USA: Conservation International.

Willaert, B., Suyesh, R., Garg, S., Giri, V.B., Bee, M.A. & Biju, S.D. (2016) A unique mating strategy without physical contact during fertilization in Bombay Night Frogs (Nyctibatrachus humayuni) with the description of a new form of amplexus and female call. PeerJ 4:e2117; DOI 10.7717/peerj.2117

Croaking Science: Heterospecific attraction: using the sounds of others to find the way

December 19, 2018 by editor

Croaking Science: Heterospecific attraction: using the sounds of others to find the way

Amphibians may use a variety of cues to exploit new habitats including olfactory, visual and celestial cues. Often, individuals may use conspecific cues, i.e. those from individuals of the same species, to locate suitable habitats. For example, many pond-breeding amphibian of the genus Hyla and Rana, have been found to be attracted to calls of the same species (Buxton et al., 2015). However, the use of heterospecific cues, i.e. those from individuals of a different species, is less wide spread and our knowledge and understanding of how species use such cues is generally poorly understood. The heterospecific attraction hypothesis (HAH) has been proposed to describe how individuals may select habitats based on the presence of other species (Mönkkonen et al., 1997). Although initially described for birds, in recent years both field and laboratory experiments have shown an increasing number of amphibian species that use heterospecific cues to locate suitable habitats.

In the tropical forests of Taiwan a number of amphibian species co-exist, with varying degrees of niche overlap. Males of an oriental tree frog species, Rhacophorus prasinatus, form choruses in bushes and produce foam nests on vegetation near or above ephemeral and permanent water (Figure 1). Within the same habitat also lives Babina adenopleura which usually breeds at permanent water bodies and produces an egg mass in the water. This species is therefore a non-competitor as both species have different ecological requirements. However, Polypedates braueri is a resource competitor of R. prasinatus and uses ephemeral and water during the same time period and also produces a foam nest on vegetation. Research by Chang et al. (2018) has found that R. prasinatus frogs initially locate breeding aggregations of males using conspecific cues i.e. the cues of calling males of the same species. However, when these are lacking, they locate suitable habitat using the calls of the non-competitor B. adenopleura, rather than the competitor, P. braueri. This behaviour indicates that the calls of non-competitors could serve as cues for breeding site allocation (Chang et al., 2018). Since breeding sites are limited in this environment, avoiding choruses with heterospecific competitors, such as P. braueri, may reduce competition.

**Figure 1.** The tree frog Rhacophorus prasinatus from Taiwan may use the cues of another species, Babina adenopleura, to locate potential breeding sites. (Photo credit: Evan Pickett, licensed under the Creative Commons Attribution-Share Alike 4.0 International license. https://commons.wikimedia.org/wiki/File:Rhacophorus_prasinatus.jpg )

Many pond-breeding amphibians living in temperate areas often undergo dispersal to seek alternative breeding sites. In particular, in European newt genera such as Triturus, adults may switch breeding ponds in alternative years (Griffiths & Williams, 2000). The dispersal behaviour of individuals is often influenced by environmental factors such as food supply, predation risk, parasite load, and habitat disturbance (context-dependent dispersal) (Cayuela et al., 2018). However, individuals may also make decisions over dispersal direction, distance and habitat selection using the cues of other individuals (social-dependent dispersal). In central Europe great crested newts (Triturus cristatus), palmate newts (Lissotriton helveticus) and alpine newts (Ichthyosaura alpestris) may all co-habit the same ponds. Such newt species have a high degree of niche overlap, with great crested newts being competitively dominant, feeding on similar prey to the other species (Griffiths, 1994). Research by Cayuela et al. (2018) found that great crested newts make decisions on whether to leave a pond or occupy a new one based on the density of palmate newt and alpine newts. In this study, great crested newts were less likely to disperse from ponds which had a high heterospecifc density, i.e. a high density of palmate and alpine newts. In addition, the researchers found that when emigrating from ponds to seek other suitable breeding sites, great crested newts were more likely to select ponds which had a high density of palmate and alpine newts. These findings suggest that great crested newt adults do not avoid ponds with a high density of heterospecifics to limit the negative effects of interspecific competition on larval development and survival (Cayuela et al., 2018), but instead actively choose ponds where the density of other newt species is high. The presence of other newt species may indicate high pond quality with abundant food and egg laying resources, and since great crested newts are competitively dominant over the other newt species, they will gain by breeding in these ponds. The exact mechanism by which great crested newts can detect the density of other species within a pond is unknown, but is likely to be due to chemical cues (Cayuela et al., 2018).

**Figure 2.** Great crested (left), alpine (right) and palmate (below) newts all coexist in the same pond. (Photo credits: 1- Great crested newt: Rainer Theuer, licensed under the Creative Commons Attribution-Share Alike 2.5 Generic license. https://commons.wikimedia.org/wiki/File:Kammmolchmaennchen.jpg. 2- Palmate newt: Froglife. 3- Alpine newt: Anevrisme, Creative Commons Attributio)n-Share Alike 3.0 Unported license. https://commons.wikimedia.org/wiki/File:Triturus_alpestris.jpg.

In Northern Italy, the smooth newt (L. vulgaris) breeds in similar water bodies to both the common toad (Bufo bufo) and green toad (B. viridis). In this region, the common toad breeds in large permanent water bodies and arrives at ponds in early February, whereas the green toad breeds in ephemeral water bodies and has a variable arrival time between February and March, depending on levels of rainfall (Pupin et al., 2007). Since smooth newts will breed in all types of pond, both toad species are therefore potential cues for water presence in a given area and may signal water quality. Research by Pupin et al. (2007) showed that smooth newts positively orientated towards the calls of both species and were using these to indicate location of potential breeding water bodies. Madden & Jehle (2017) carried out similar experiments on the great crested newt and found that individuals oriented to the calls of the common toad, but not common frog (Rana temporaria). They hypothesised that this was because common toads bred later than common frogs, at a more suitable time of year for great crested newts, so orienting towards ponds with common toads was more ecologically adaptive than those of common frogs.

Heterospecific attraction is a relatively newly studied process in amphibians and research has shown a number of examples when individuals use the cues of other species to locate and orientate towards suitable habitats. Such research has conservation significance since it shows the importance of food webs and community cohesion and demonstrates that different species are highly connected. Community level and landscape scale conservation, which targets the conservation of whole groups of species and widespread habitats, may therefore have high benefit to overall biodiversity.

References

Buxton, V.L., Ward, M.P. & Sperry, J.H. (2015) Use of chorus sounds for location of breeding habitat in 2 species of anuran amphibians. Behavioral Ecology, 26: 1111-1118.

Cayuela, H., Grolet, O. & Joly, P. (2018) Context‑dependent dispersal, public information, and heterospecific attraction in newts. Oecologia, 188 (4): 1069 – 1080. https://doi.org/10.1007/s00442-018-4267-3.

Chang, C., Cheng, Y. & Lin, S. (2018) Influence of conspecific and heterospecific cues on phonotaxis behaviour in a polyandrous treefrog. Behavioral Ecology and Sociobiology, 72: 179-190. https://doi.org/10.1007/s00265-018-2593-4.

Griffiths, R.A., Wijer P. de & May, R.T. (1994) Predation and competition within an assemblage of larval newts (Triturus). Ecography, 17 (2): 176-181.

Griffiths, R.A. & Williams, C. (2000) Modelling population dynamics of the great crested newt (Triturus cristatus): a population viability analysis. The Herpetological Journal, 10: 157-163.

Madden, N. & Jehle, R. (2017) Acoustic orientation in the great crested newt (Triturus cristatus). Amphibia-Reptilia, 38 (1): 57-65.

Mönkkonen, M., Helle, P., Niemi, G. & Montgomery, K. (1997) Heterospecific attraction affects community structure and migrant abundances in northern breeding bird communities. Canadian Journal of Zoology, 75: 2077-2083.

Pupin, F., Sacchi, R., Gentilli, A., Galeotti, P. & Fasola, M. (2007) Discrimination of toad calls by smooth newts: support for the heterospecific attraction hypothesis. Animal Behaviour, 74: 1683-1690. doi:10.1016/j.anbehav.2007.03.020.

Croaking Science: Caecilians – unusual reproductive ecology

November 29, 2018 by editor

Croaking Science: Caecilians – unusual reproductive ecology

Caecilians, or blind snakes, as they are also often called, are limbless, elongate amphibians which inhabit tropical regions, with hotspots in South America and Asia (Kupfer et al., 2016; Gomes et al., 2012). Of all the groups of amphibians, caecilians are the most poorly understood, which is in part due to their secretive, fossorial lifestyle (Figure 1). They belong to the order Gymnophiona, one of three orders which comprises the Amphibia. There are currently 209 recognised species, which represents just 3% of the total 7,950 amphibian species worldwide (AmphibiaWeb, 2018). Caecilians are all ground-dwelling tropical amphibians which exhibit a wide range of reproductive modes, from laying eggs which hatch into aquatic larvae to those which hatch into miniature terrestrial juveniles. Little is known of their diverse reproductive modes but in recent years research has elucidated more information on their unusual breeding ecology.

**Figure 1.** Caecilians have an unusual appearance, being primarily fossorial and elongate in form. *[Photo credit: Will Brown – Brown-snouted Blind Snake (Ramphotyphlops wiedii or nirgrogrescens) 3, CC BY 2.0,https://commons.wikimedia.org/w/index.php?curid=71399151]*

Oviparous caecilians lay eggs, which are usually laid in a small terrestrial nest (Figure 2). The females of all these species exhibit extensive parental care, guarding their clutches of eggs against predators (Gomes et al., 2012). Eggs will hatch into either free-swimming larvae which develop in small water bodies, or terrestrial juveniles which are independent of their parents. Ichthyophis kohtaoensis inhabits the tropical forests of northern Thailand and belongs to the second largest caecilian family. In this species, breeding coincides with the rainy season with clutches of eggs laid in damp soil at the onset of the first rains (Kupfer et al., 2004). Females lay their eggs in small chambers near to temporary and permanent ponds, pools and slow flowing brooks and rivers. This allows the free-swimming larvae to readily hatch into the water to complete their aquatic development. Research suggests that females eat little or no food during the three month incubation period since the body condition of females attending young clutches is higher than those with older clutches (Kupfer et al., 2004). This, along with the small clutch size, indicates that there are costs to parental investment and attending eggs. In addition, there is a correlation between female body length and clutch size with older females being longer than young females (Kupfer et al., 2004). This implies that older, more experienced females produce larger clutches and more offspring (Kupfer et al., 2004).

**Figure 2.** A female caecilian guarding her brood of eggs from predators. *[Photo credit: Davidvraju, Creative Commons Attribution-Share Alike 4.0 International, https://commons.wikimedia.org/wiki/File:Caecilian_guarding_its_eggs.jpg]*

The ringed caecilian, Siphonops annulatus, is an unusual oviparous species inhabiting South America. The eggs hatch into poorly developed terrestrial young which feed on the skin of the female. The young possess specialised teeth which allow them to scrape skin from their mother’s back immediately after hatching (Wilkinson et al., 2008). The feeding behaviour of the young is described as being “quite frenetic with the young frequently tearing pieces of skin by spinning along their long axes and sometimes struggling over the same piece of skin” (Wilkinson et al., 2008) (Figure 3). This maternal dermatophagy is also known from a second, distantly related African caecilian species, Boulengerula taitana (Kupfer et al., 2008), which suggests that skin feeding is an ancient form of caecilian parental care and may have persisted for more than 100 million years (Wilkinson et al., 2008). In addition, Wilkinson et al. (2008) report a previously undocumented behaviour which involves the female raising herself upward, exposing her vent and releasing a clear fluid, which the young consume. This appears to be a form of feeding the young, but further studies are required to ascertain the function of this behaviour.

**Figure 3.** A caecilian mother with eggs which will hatch into young which feed off her skin. *[Photo credit: Wilkinson M, Sherratt E, Starace F, Gower DJ (2013)]*

Viviparous caecilians, which give birth to live young, represent another unusual reproductive mode for amphibians. During development inside the female, the foetuses initially obtain their nutrition from a yolk, similar to egg-developing species. After this, the developing young feed on a uterine milk, secreted from the oviducts of the female. The young obtain this through possessing specialised embryonic teeth which they use to scrape at the oviduct wall (Gomes et al., 2012). The evolution of viviparity in caecilians is poorly understood, but since both viviparous and oviparous species possess specialised teeth for scraping (either skin or oviduct milk), this suggests that all these species evolved from a common, oviparous ancestor (Kupfer et al., 2006). Gegeneophis seshachari is the only known species of viviparous caecilian from the Indo-Seychellean region (Gower et al., 2008). In this area the rainy season is short and unpredictable such that laying eggs which hatch into offspring that rely on the presence of water or a damp environment may be highly risky. Water bodies may dry up rapidly before larvae can fully develop. Therefore, Gower et al. (2008) propose that viviparity may have evolved in this species in response to short, unpredictable rainy seasons and the lack of suitable locations to lay and incubate their eggs.

Caecilians possess interesting and unusual reproductive modes, much of which is poorly understood. In addition, many of these species are threatened or Data Deficient and in regions which are undergoing large habitat loss and fragmentation. More primary data are required to fully understand caecilian reproductive ecology and evolution, which will help inform much needed improvements in conservation assessments for these species.

References

AmphibiaWeb (2018) https://amphibiaweb.org. University of California, Berkeley, CA, USA. Accessed 15 Nov 2018.

Gomes, A.D., Moreira, R.G., Navas, C.A., Antoniazzi, M.M. and Jared, C. (2012). Review of the reproductive biology of caecilians (Amphibia, Gymnophiona). South American Journal of Herpetology, 7 (3): 191-202.

Gower, D.J., Giri, V., Dharne, M.S. and Shouche, Y.S. (2008) Frequency of independent origins of viviparity among caecilians (Gymnophiona): evidence from the first ‘live-bearing’ Asian amphibian. Journal of Evolutionary Biology, 21: 1220–1226.

Kupfer, A.K., Nabhitabhata, J. and Himstedt, W. (2004) Reproductive ecology of female caecilian amphibians (genus Ichthyophis): a baseline study. Biological Journal of the Linnean Society, 83: 207-217.

Kupfer, A., Müller, H., Antoniazzi, M.M., Jared, C., Greven, H., Nussbaum, R.A. and Wilkinson, M. (2006) Parental investment by skin feeding in a caecilian amphibian. Nature, 440 (13 April 2006), doi:10.1038/nature04403.

Kupfer, A., Wilkinson, M., Gower, D.J. Müller, H. and Jehle, R. (2008) Care and parentage in a skin-feeding caecilian amphibian. Journal of Experimental Biology, 309A: 460–467.

Kupfer, A., Maxwell, E., Reinhard, S. and Kuehnel, S. (2016) The evolution of parental investment in caecilian amphibians: a comparative approach. Biological Journal of the Linnean Society, 119: 4–14.

Wilkinson, M., Kupfer, A., Marques-Porto, R., Jeffkins, H., Antoniazi, M.M. and Jared, C. (2008) One hundred million years of skin feeding? Extended parental care in a Neotropical caecilian (Amphibia: Gymnophiona). Biological Letters, 4: 358–361.

doi:10.1098/rsbl.2008.0217

Froglife Training 2019

November 19, 2018 by editor

Froglife Ecological Services training programme for 2019 has been released and can be viewed here.

Our expertise in amphibians and reptiles, conservation projects and habitat management enables us to deliver a range of courses to a wide variety of audiences at all levels of experience.

Our current courses include:

Introduction to Amphibians
Great Crested Newt Survey: Working Towards a Licence
Introduction to Reptiles
Reptile Ecology and Survey Techniques
Habitat Creation, Restoration and Enhancement for Amphibians

Our courses are designed to offer something for everyone, from complete beginner to more experienced naturalist or Environmental professional.

Most of our courses involve an element of field work or site visits. Largely the courses run on a single day, however our Great Crested Newt: Working Towards a Licence course does require a site visit the following morning.

In addition to our standard courses we are able to offer bespoke training courses to cater to groups such as NGOs, Charities, Government Departments, volunteer groups, ‘Friends of’ groups and Councils.

Courses are run from our Peterborough location, however if you would like to book a bespoke training course we are able to run this from your group’s own venue so long as there are suitable facilities available. We also occasionally offer courses at our London and Scotland locations. We have excellent facilities and an in-depth knowledge of the sites used offering the opportunity to get a close up view of these species and explore their habitats.

Get in touch with our Science & Research Manager, Laurence Jarvis to discuss and book your training course laurence.jarvis@froglife.org

More information about the services provided by Froglife Ecological Services can be found at www.fles.org.uk

Croaking Science: Toxicity in Amphibians

October 30, 2018 by editor

Croaking Science: Toxicity in amphibians

Frogs, toads and salamanders have long been considered to be noxious and a large range of amphibian species have been found to secrete toxic chemicals from their skin. Over recent decades the type and mode of action of these chemicals has been characterised from many amphibian species. Toads of the genus Bufo, for example the common toad (Bufo bufo), are able to synthesize and store a host of chemicals, some of which are known as steroidal bufadienolides (Daly, 1998). These are a type of cardiac glycoside and specifically block nerve action in the heart, resulting in irregular or slow heartbeat, rapid heartbeat and possibly lethal cardiac arrest in their predators. Leptodactylid frogs, which includes a diverse range of species from the Neotropics, harbour phenolic amines in their skin which are toxic to a range of vertebrate predators. Another class of amphibian toxins are the tetrodotoxins (TTX), which were originally discovered in newts of the family Salamandridae, specifically of the genus Taricha. The rough-skinned newt (Taricha granulosa) of North America is the most toxic salamander in this region and is considered harmful to humans (AmphibiaWeb, 2014) (Figure 1). This species, along with others in the Salamandridae family, such as Triturus newts (e.g. great crested and Italian crested newts) from Europe contain tetrodotoxin in their skin, which is a potent neurotoxin and is a sodium channel blocker. This means that it inhibits the firing of action potentials in neurons and prevents the nervous system from carrying messages and thus muscles from flexing (Bane et al., 2014). Some of the most well documented species of frog containing skin toxins are the poison dart frogs of South America. Unlike Bufo toads or salamanders, which synthesise their own toxins, poison dart frogs accumulate toxic lipophilic alkaloids from their food sources which may be small arthropods, such as mites, ants, springtails, and flies (Daly, 1998).

**Figure 1.** The rough-skinned newt of North America secretes highly toxic tetrodotoxins from its skin which is harmful to humans. [Photo credit: By Chuck Spidell – Imported from 500px (archived version) by the Archive Team. (detail page), CC BY 3.0, https://commons.wikimedia.org/w/index.php?curid=72144165]

The production of skin toxins that have been derived from arthropod prey remains a source of scientific interest and research. The occurrence of alkaloid toxins in different populations and species of poison frogs provides information as to the distribution and availability of the arthropods that provide alkaloids to them. For example, if a species of the bufonid in the genus Melanophryniscus at a particular site do not have alkaloids that are known to be from a certain group of arthropods, then that mite, ant, beetle, or millipede is either not present or not eaten by the toad. Recent research by Daly et al. (2007) investigated the profile of alkaloid skin toxins from different populations of Melanophryniscus toads from Uruguay and Argentina (Figure 2). Daly et al. (2007) found that the nature and availability of dietary sources of alkaloids found in the skin of these toads is strongly dependent on the site of collection and can change with time. This is likely to have a strong impact on the toxicity of the skin toxins produced by individual toads and thus have implications for predator defence. A change or drop in the number of a prey species of alkaloid-producing arthropod is therefore likely to have knock-on impacts for frog chemical defence. Such research highlights the importance of the interconnectedness of species groups and maintaining overall biodiversity in protected areas.

**Figure 2.** Many species of Melanophryniscus toad secrete alkaloid toxins which they obtain from their diet. [Photo credit: Bornschein MR et al., (2015) Three new species of phytotelm-breeding Melanophryniscus from the Atlantic Rainforest of Southern Brazil (Anura: Bufonidae). PLOS ONE 10(12): e0142791. https://doi.org/10.1371/journal.pone.0142791, CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=60310764]

Toxins, such as those secreted from a frog, toad or salamander are only likely to be effective if they have immediate effects on their predator. Small molecules such as alkaloids or steroids are rapidly absorbed across cell membranes even if the predator’s skin is not punctured. This results in immediate effect in the predator such as distastefulness or pain. However, many frog species secrete large molecule toxins such as peptides or small proteins that makes them unsuitable for fast absorption (Nelson et al., 2014). Therefore, the poisons of many frogs seem to be unsuitable for fast toxin uptake by predators and to have any deterrent effect. However, as well as secreting toxins, the skin of many amphibians also contains antimicrobial peptides (AMPs), which are a defence strategy against a range of bacteria and fungi. Until recently, it was thought that AMPs were used only to fight off bacterial infections which would otherwise infect a frog’s skin. However, recent research by Raaymakers et al. (2017) suggests that these AMPs may actually also be used to facilitate the uptake of toxins into predators and increase the speed of action. They found that in frogs which secrete large molecule peptide toxins, the uptake into predators was increased in the presence of AMPs. This highlights the complex role that frog skin chemicals play in antipredator defence.

Most salamander species lack toxic skin secretions, but contain other active compounds such as norepinephrine, steroids, enzymes, and antimicrobial or antifungal substances (Daly, 1998). Many may also produce adhesives for defence (Evans & Brodie, 1994). For example, once attacked by a predatory snake, the California slender salamander (Batrachoseps attenuates) grasps the snake, loops its tail around the snake’s head and coats the predator with a sticky viscous fluid (Arnold, 1982). The adhesives in the secretion harden within seconds upon exposure to air and immobilize the snake immediately (Arnold, 1982). The salamander then escapes and the snake is unable to free itself for up to 48 hours (Arnold, 1982). There is a great deal of interest in natural adhesives for human engineering and nanotechnology (Von Byern et al., 2017) and in creating mimics which can be produced commercially. In a set of experiments using several salamanders, Von Byern et al. (2017) found that some salamanders also produced toxic chemicals which augmented the effect of the adhesives. In addition, two salamander species, the northern slimy salamander (Plethodon glutinosus) and spotted salamander (Ambystoma maculatum) from North America produced adhesives which have the potential to be mimicked commercially and used in nanotechnology products. These findings highlight the importance of understanding skin chemicals in amphibians and the potential use in pharmacology and nanotechnology.

**Figure 3.** The California slender salamander secretes adhesive properties to deter predators. [Photo credit: Greg Schechter from San Francisco, USA – California Slender Salamander, CC BY 2.0, https://commons.wikimedia.org/w/index.php?curid=55625511]

References

AmphibiaWeb (2014) Taricha granulosa: Rough-skinned Newt <http://amphibiaweb.org/species/4288> University of California, Berkeley, CA, USA. Accessed Oct 25, 2018.

Arnold, S.J. (1982). A quantitative approach to antipredator performance: salamander defense against snake attack. Copeia, 1982 (2): 247e253.

Bane, V., Lehane, M., Dikshit, M., O’Riodan, A. & Furey, A. (2014) Tetrodotoxin: chemistry, toxicity, source, distribution and detection. Toxins, 6: 693-755, doi:10.3390/toxins6020693.

Daly, J.W. (1998) Thirty years of discovering arthropod alkaloids in amphibian skin. Journal of Natural Products, 61: 162-172.

Daly, J.W., Wilham, J.M., Spande, T.F., Garraffo, H.M., Gil, R.R., Silva, G.L. & Vaira, M. (2007) Alkaloids in bufonid toads (Melanophryniscus): temporal and geographic determinants for two Argentinian species. Journal of Chemical Ecology, 33: 871–887.

Evans, C.M. & Brodie, E.D. (1994) Adhesive strength of amphibian skin secretions. Journal of Herpetology, 4 (499): 502.

Nelsen, D.R., Nisani, Z., Cooper, A.M., Fox, G.A., Gren, C.K., Corbit, A.G. & Hayes, W.K. (2014) Poisons, toxungens, and venoms: redefining and classifying toxic biological secretions and the organisms that employ them. Biological Reviews, 89: 450–465.

Raaymakers, C., Verbrugghe, E., Hernot, S. Hellebuyck, T., Betti, C. Peleman, C., Myriam, C., Bert, W., Caveliers, V., Baller, S., Martel, A., Pasmans, F. & Roelants, K. (2017) Antimicrobial peptides in frog poisons constitute a molecular toxin delivery system against predators. Nature Communications, DOI: 10.1038/s41467-017-01710-1.

Von Byern, J., Mebs, D., Heiss, E., Dicke, U., Wetjen, O. Bakkegard, K., Grunwald, I., Wolbank, S. & Mühleder, S, Gugerell, A., Fuchs, H. & Nürnberger, S. (2017) Salamanders on the bench: a biocompatibility study of salamander skin secretions in cell cultures. Toxico

Croaking Science: Frogs & toads at the coast

September 28, 2018 by editor

Frogs and toads at the coast: salt tolerance in amphibians

It is generally understood that amphibians breed and associate with freshwater habitats such as ponds, lakes and other small waterbodies. Many scientific studies have demonstrated that amphibians are particularly vulnerable to saline conditions at embryo, larval and adult stages. The most vulnerable life stage is the embryo which often experience high mortality when in salt environments (e.g. Beebee, 1985; Hua & Pierce, 2013). This is because salt water tends to disrupt the ionic and water exchange across permeable membranes. However, an increasing number of studies have found amphibian species that are able to adapt to and tolerate salt water habitats, especially those in coastal marshes. In his observations of the lack of amphibians on islands and in saline habitats, Charles Darwin in 1872 noted that only “one Indian species” was able to tolerate salt water (Darwin, 1872 in Hopkins & Brodie, 2015). Since then a range of scientific observations and studies have vastly increased our knowledge of the number of amphibians that are able to tolerate salt water in the natural environment. In a recent review of all scientific studies relating to amphibians and their ability to resist salt water Hopkins and Brodie (2015) identified 144 amphibian species from 28 amphibian families across the world from anurans (frogs and toads), salamanders and caecilians that are able to inhabit salt habitats. Salt tolerance in amphibians has been recorded from all continents except Antarctica.

The majority of studies which have found amphibians in salt water habitats have recorded them from coastal areas, which get flooded predictably by sea water. For example, mangrove swamps are flooded daily by tidal fluctuations which results in predictable levels of salt water in surrounding standing water. The most well-known frog to inhabit such environments is the crab-eating frog (Fejervarya cancrivora) of southeast Asia (Figure 1). This unique species can live in freshwater as well as 75% seawater and can adapt from one to the other in a matter of hours (Ren et al., 2010). It is able to achieve this by rapidly changing levels of urea in its body tissues which avoids excessive water loss through the skin in salty conditions (Ren et al., 2010). In addition, it has three glands within its skin: mucous glands, mixed glands, and vacuolated gland which help buffer the additional salt which would otherwise enter its body (Amphibiaweb, 2018). Other studies on amphibians which are able to tolerate salt water environments have also noted similar changes in physiology.

Figure 1. Crab-eating frog (Fejervarya cancrivora) from southeast Asia which can tolerate daily influxes of seawater. By Bernard DUPONT from FRANCE – Crab-eating Frog (Fejervarya cancrivora), CC BY-SA 2.0, https://commons.wikimedia.org/w/index.php?curid=40168133

The exact mechanisms behind the evolution of salt tolerance in amphibians is still under debate but may be due to a number of genetic variables. All amphibians have highly permeable skin, which is prone to desiccation. Most amphibians are efficient at water regulation through their skin and retaining salts through effective transport of sodium and chlorine ions. Therefore, there may be a genetic predisposition to adapting to salt water environments through altering the physiology of ion transfer across the skin. Several amphibian species which are tolerant of highly arid conditions are also able to resist salt environments. The green toad (Bufotes viridis) of Central Europe is able to tolerate extremely high temperatures of 40°C and withstand up to 50% body water loss (Amphibiaweb, 2018) (Figure 2). It aestivates (hibernates) during the hottest summer months and has the same adaptations to avoiding water loss as species which inhabit saline environments, i.e. accumulation of urea in body tissues. The same occurs in the tiger salamander (Ambystoma tigrinum) of North America which uses a similar physiological mechanism to inhabit saline habitats. Some biologists have therefore proposed that the ability to tolerate saline environments has evolved through a pre-adaptation to living in arid environments. However, this does not appear to be the case for all amphibians inhabiting salt water. The natterjack toad (Epidalea calamita) is known to inhabit both freshwater and brackish habitats both in the UK and across northern Europe (Beebee, 1985; Gomez-Mestre & Tejedo, 2005) (Figure 3). Through genetic studies Gomez-Mestre & Tejedo (2005) found no support for this hypothesis and suggested that salt and aridity tolerance has evolved independently. They conclude that the mechanisms for resisting salt are fundamentally different in embryos and larvae compared to juveniles and adults. Therefore, amphibians which are able to resist salt whilst in the embryonic or larval stages require unique physiology which has not evolved in arid tolerant species (Gomez-Mestre & Tejedo, 2005). Other theories for the evolution of salt tolerance in amphibians are selection pressures from freshwater predators, forcing amphibians into new habitats or diversification of amphibians into new habitats to allow them to utilise unique prey (Hopkins & Brodie, 2015).

Figure 2. The green toad (Bufotes viridis) of Central Europe is able to tolerate extremely high temperatures using a similar physiological mechanism as species inhabiting salt water. By Umberto Salvagnin – originally posted to Flickr as European Green Toad, CC BY 2.0, https://commons.wikimedia.org/w/index.php?curid=11010130

Figure 3. Typical coastal habitat of the natterjack toad. Breeding ponds in certain areas can easily become saline. As a result, populations can adapt to living in salt water at low concentrations. Inset: male natterjack toad (Epidalea calamita). Natterjack toad habitat: By Gary Rogers, CC BY-SA 2.0, https://commons.wikimedia.org/w/index.php?curid=13571929 Inset image: By Thomas Brown – Natterjack Toad (Epidalea calamita). Uploaded by mgiganteus, CC BY 2.0, https://commons.wikimedia.org/w/index.php?curid=27451130.

In Australia, many amphibian species are threatened with a range of anthropogenic factors including habitat loss, fragmentation and pollution. Salinization of freshwater habitats has been cited as being one of the factors in causing declines of Australian amphibian species but evidence is lacking (Kearney et al., 2012). In experimental studies, Kearney et al. (2012) examined the effects of elevated salinity on growth, metamorphosis and survival of the tadpoles of three species of native Australian frog. Kearney et al. (2012) found that tadpoles of the green and golden bell frog (Litoria aurea) raised in salt conditions had higher survival than those in freshwater. In contrast, a number of studies on other anuran larvae have found that larvae suffered abnormalities when raised in saline conditions. The authors propose that L. aurea has adapted to salt water by rapidly increasing development when in these conditions to avoid the potentially harmful effects of salt. However, other amphibians native to Australia such as the spotted grass frog (Limnodynastes tasmaniensis) and the painted burrowing frog (Neobatrachus sudelli) are unable to exhibit such plasticity in development and consequently are negatively affected by increases in salt water in the environment (Kearney et al., 2012).

Overall, it appears that an increasing number of amphibian species are being recognised as being able to tolerate salt water in the natural environment. However, species vary hugely in their tolerance of salt water habitats and our knowledge of the degree to which amphibians have adapted to salt water is poorly understood, especially those in human dominated habitats. Further in-depth investigations are required to increase our understanding of the mechanisms behind the evolution of salt tolerance in amphibians and their response in a potentially changing environment.

References

AmphibiaWeb (2018) http://amphibiaweb.org. University of California, Berkeley, CA, USA. Accessed 11 Sep 2018.

Beebee, T.J.C. (1985) Salt tolerances of natterjack toad (Bufo calamita) eggs and larvae from coastal and inland populations in Britain. Herpetological Journal, 1: 14-16.

Gomez-Mestre, I. & Tejedo, M. (2005) Adaptation or exaptation? An experimental test of hypotheses on the origin of salinity tolerance in Bufo calamita. Journal of Evolutionary Biology, 18: 847–855.

Hopkins, G.R. & Brodie Jr, E.D. (2015) Occurrence of amphibians in saline habitats: a review and evolutionary perspective. Herpetological Monographs, 29: 1-27.

Hua, J. & Pierce, B.A. (2013) Lethal and sublethal effects of salinity on three common Texas amphibians. Copeia, 2013 (3): 562-566.

Kearney, B.D., Bryrne, P.G. & Reina, R.D. (2012) Larval tolerance to salinity in three species of Australian anuran: an indication of saline specialisation in Litoria aurea. PLOS ONE, 7 (8): e43427.

Ren, Z., Zhu, B., Ma, E., Wen, J., Tu, T., Cao, Y., Hasegawa, M. & Zhong, W. (2015). Complete nucleotide sequence and gene arrangement of the mitochondrial genome of the crab-eating frog Fejervarya cancrivora and evolutionary implications. Gene, 441: 148-155.

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