Croaking Science

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

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.

Croaking Science: The toad fly Lucilia bufonivora in common toads

August 29, 2018 by editor

Croaking Science: The toad fly Lucilia bufonivora in common toads

Blow-flies are dipteran flies that evolved 105 million years ago and there are now over 1,000 species occurring in 150 genera in a range of countries worldwide (Figure 1) (McDonagh, 2009). Within the family Calliphoridae there are 80 species which are known to cause myiasis, which is the infestation of live human and vertebrate animals, where the fly larvae feed on the host’s dead or living tissue, liquid body substances, or ingested food (Zumpt, 1967). There are three main types of blow-fly that cause myiasis. The first are known as saprophages and simply infect animal carcasses that are already dead and decaying. These species do not initiate myiasis but rather take advantage of animals that are already decaying. The second group of blow-flies are generally ectoparasites (i.e. parasites that attach to the skin or fur of an animal) that occasionally initiate myiasis by feeding on damaged tissue of their host. These flies sometimes feed on dead decaying matter, like the saprophages. The last group of blow-flies are known as obligate parasites, only feeding on the living tissue of their host and initiating myiasis as a result (Stevens & Wall, 1997). It has been proposed that the obligate myiasis-causing blow-fly parasites have evolved from an ancestral saprophageous stage, with flies occasionally being attracted to dead or decaying tissue or wounded animals. This later involved into flies which relied more on living tissue until obligate parasites evolved (Zumpt, 1967; McDonagh, 2009).

*Figure 1. Blow-flies belonging to the genus Lucilia range from feeding on flower nectar to liquefied dead vertebrate tissues.*

The blow-fly genus Lucilia comprises approximately 27 species, all of which are similar in appearance. The larvae of the majority of these species are saprophageous, feeding on dead matter. However, there are several species which are occasionally ectoparasites on large mammal species and which may cause myiasis, particularly in sheep (Stevens & Wall, 1997). One species, Lucilia silvarum, sometimes feeds on dead amphibian species, but the toad fly, Lucilia bufonivora, is a highly specialised blow-fly and is an obligate parasite of live prey. Adult L. bufonivora make their first emergence in May to July and seek out living common toads (Bufo bufo) and their lays eggs on the back and flanks of an amphibian host. The larvae then hatch and migrate to the head and nasal cavities of the toad where they continue to feed on the live host (Figure 2). Infected toads tend to change their behaviour and instead of hiding away in sheltered areas, move out into exposed locations where they are susceptible to further infestation by blow-flies. The nasal cavities and head are gradually consumed until eventually the infected toad dies. Once the toad has died, the larvae continue to feed on the flesh of the toad before dropping off to pupate in the soil. Here they undergo metamorphosis and hatch into new adult flies a few weeks later. Toad flies are capable of having three generations each year, depending on the weather and availability of toads.

*Figure 2. A common toad (Bufo bufo) infected with blow-fly larvae from Lucilia bufonivora. The larvae have migrated to the nostrils where they will continue to consume the living flesh.*

Toad-flies have a wide distribution across Europe, North Africa and Asia and are particularly common in the Netherlands where between 15 and 70% of common toads may be affected each year, with adults being most commonly affected (Weddeling & Kordges, 2008). Despite this high level of infection, there is no evidence of toad flies causing decline in the common toad. In the UK, toad flies are relatively uncommon and the number of reports each year is low.

L. bufonivora and another blow-fly species L. silvarum are highly similar in appearance. In Europe, L. silvarum tends to only feed on carrion with a preference for dead toads. However, in North America, the species has been recorded as infecting living toads with larvae found in the neck, legs and parotid glands (Eaton et al., 2008). Research by the University of Bristol and Exeter, in collaboration with RAVON, has looked at how closely related L. bufonivora and L. silvarum are to each other. The two species are so similar in appearance it is possible that the eggs found on toads in Europe are from one or both species. Using genetic analysis, the researchers found that the two are sister species, being genetically distinct, but are very closely related and have only recently diverged as separate species. In addition, the researchers found that L. silvarum blow-flies in North America are more closely related to toad flies L. bufonivora, than they are to their own L. silvarum species in Europe. This suggests that obligate parasitism in Lucilia blow-flies may have evolved independently several times and originally diverged from L. silvarum (Arias-Robledo et al., 2008). The obligate parasite traits of L. bufonivora may have evolved as the two species diverged. The findings from this research also show that in Europe and the UK common toads are only infected by L. bufonivora and L. silvarum has yet to become an obligate parasite in these countries (Arias-Robledo et al., 2008). Further research is required to determine the evolutionary status of other closely related blow-fly species such as L. elongata, which is relatively poorly understood.

References

Arias-Robledo, G., Stark, T., Wall, R.L. & Steven, J. R. (2018) The toad fly Lucilia bufonivora: its evolutionary status and molecular identification. Medical and Veterinary Entomology, doi: 10.1111/mve.12328.

Eaton, B. R., Moenting, A. R., Paszkowski, C. A. & Shpeley, D. (2008) Myiasis by Lucilia silvarum (Calliphoridae) in Amphibian Species in Boreal Alberta, Canada. Journal of Parasitology, 94 (4): 949 – 952.

McDonagh, L. M. (2009) Assessing patterns of genetic and antigenic diversity in Calliphoridae (blowflies). PhD thesis, University of Exeter.

Stevens, J. & Wall, R. (1997) The evolution of ectoparasitism in the genus Lucilia (Diptera: Calliphoridae). International Journal of Parasitology, 27 (1): 51-59.

Wellling, K. & Kordges, T. (2008) Lucilia bufonivora-Befall (Myiasis) bei Amphibien in

Nordrhein-Westfalen – Verbreitung, Wirtsarten, Ökologie und Phänologie. Zeitschrift für Feldherpetologie, 15: 183–202.

Zumpt F. & Ledger J. (1967) A malign case of mylasts caused by Hemipyrellia fernandica (Macquart) (Diptera Calliphoridae) in a cape hedgehog (Erinaceus frontalis A. Smith). Acta Zoologica et Pathologica Antverpiensia, 43: 85-91.

Croaking Science: Overwintering in Frog Tadpoles

July 27, 2018 by editor

Overwintering in frog tadpoles

During June and July in the UK the majority of Common Frog (Rana temporaria) tadpoles metamorphose into juveniles which leave ponds for terrestrial habitats. They will spend the rest of the summer and autumn foraging and feeding on small invertebrates in preparation for the winter. However, a number of common frog tadpoles each year will remain in ponds and spend the winter in the water, metamorphosing into juveniles the following spring (Figure 1). What causes some tadpoles to metamorphose and others to remain in the water over the winter? Often, ponds drying out or freezing over can limit the capacity for frog tadpoles to overwinter. However, there are many ponds in the UK where this does not occur, which allows for overwintering of tadpoles. Our understanding of the exact factors which trigger overwintering in tadpoles are not fully understood, but research by Walsh et al. (2016) found that temperature and food availability were key factors. In addition, the decision on whether to over‐winter as a tadpole appears to be made relatively early in the season (Walsh et al., 2008). Under laboratory conditions, lower temperatures and reduced food availability during the summer resulted in a higher proportion of individuals remaining as tadpoles during the winter (Walsh et al., 2016). These findings suggest that cold weather conditions in the early autumn may affect tadpole development, perhaps by affecting endocrine function. However, temperature alone does not appear to be enough to trigger overwintering in tadpoles; food availability also appears to be important. Although these two factors play an important role in triggering overwintering in common frog tadpoles, there are other factors, not fully understood, that appear to be involved.

***Figure 1.*** *Common Frog Rana temporaria tadpoles may spend the winter in their natal ponds under certain environmental conditions. (Photo credit: John Roston)*

Living in areas with variations in altitude provides challenges for the development of common frog tadpoles. Higher altitude ponds experience lower temperatures and food availability and these may promote a higher incidence of overwintering in tadpoles. However, Muir at al. (2014) found that in tadpoles living at high altitude in Scotland, individuals had a lower resting metabolic rate which allowed for more energy to be allocated to growth. This is likely to allow individuals to grow faster under cooler environmental conditions and allow tadpoles to metamorphose earlier than expected and prevent the necessity to overwinter. However, if tadpoles developing at high altitudes do need to overwinter, they are able to better tolerate the colder winter temperatures. Muir et al. (2014) found that the tadpoles of Common Frogs at high altitude were able to tolerate freezing for short periods of time. This adaptation provides common frog tadpoles with the ability to withstand the cooler pond conditions at high altitude over the winter and therefore lead to greater survival.

Across Central Europe the incidence of overwintering tadpoles varies by species and is more typical in later breeding species with large tadpoles such as the Midwife Toad Alytes obstetricans, Spadefoot Toad Pelobates fuscus, and water frogs (Pelophylax species) (Gilbert & Harmsel, 2016). The incidence of overwintering in Common Frogs Rana temporaria is rare and was reported for the first time in the Netherlands in 2013 (Gilbert, 2016). This was likely to be due to the unusual weather conditions of 2013/14 along with the sheltered site where this was observed. Overwintering in the Edible Frog (Pelophylax esculentus complex) is a relatively rare phenomenon across Central Europe (Figure 2). Research by Péntek et al. (2018) found that overwintering by tadpoles only seems to occur in unusual weather conditions. In one particular incidence cold temperatures over the winter resulted in late breeding by adults which led to tadpoles getting a late start to their growth and development. Cool conditions early in the autumn (October) appeared to trigger halting of development and the subsequent mild winter promoted successful overwintering by tadpoles of this species (Péntek et al., 2018). Although still relatively rare, overwintering may become more common across Central Europe with changing environmental conditions causing increased variability in seasonal temperatures.

**Figure 2.** Overwintering by the tadpoles of the Edible Frog is relatively rare across Central Europe.

In the United States, 15 species of Ranid frogs occur but only five of these species are known to overwinter as larvae (North American Bullfrog Rana catesbeiana, Pig Frog R. grylio, River Frog R. heckscheri, Red-legged Frog R. aurora, and Southern Mountain Yellow-legged Frog R. muscosa). It appears that pond desiccation is one of the main factors limiting Ranid frog species in North America from overwintering as tadpoles. The Southern Leopard Frog Rana sphenocephala is a common frog inhabiting freshwater ponds across the Northern United States (Figure 3). Tadpoles of this species will remain in the larval stage until reaching a critical metamorphic size and if this is not reached by the end of the autumn, then the tadpoles will overwinter (Pintar & Resetarits, 2018). If winter conditions are suitable, tadpoles will remain in the pond for several years until the critical size for metamorphosis has been reached. This is different to Common Frogs in the UK which do not need a critical size to metamorphose but do require specific environmental conditions. These results highlight the variations that occur across Ranid species in their requirements for metamorphosis.

**Figure 3.** Tadpoles of the Southern Leopard Frog from the United States have to attain a critical body size before they can metamorphose. This can result in the tadpoles spending several years in one pond.

References

Gilbert, M.J. & Harmsel, R. (2016) Hibernating larvae of the common frog (Rana temporaria) in the Netherlands. Herpetology Notes, 9: 27-30.

Muir, A.P., Biek, R., & Mabel, B.K. (2014) Behavioural and physiological adaptations to low-temperature environments in the common frog, Rana temporaria. BMC Evolutionary Biology, 14: 110.

Péntek, A.L., Sárospataki, M., & Zsuga, K. (2018) Larval overwintering of the Pelophylax esculentus complex in a sodic bomb crater pond near Apaj, Hungary. North-western Journal of Zoology, 2018: e1775023/9.

Pintar, M.R. & Resetarits Jr., W.J. (2018) Variation in pond hydroperiod affects larval growth in Southern Leopard Frogs, Lithobates sphenocephalus. Copeia, 106 (1): 70–76.

Walsh, P.T., Downie, J.R. & Monaghan, P. (2016) Factors affecting the overwintering of tadpoles in a temperate amphibian. Journal of Zoology, 298 (3): 183-190.

Walsh, P.T., Downie, J.R. & Monaghan, P. (2008) Larval over‐wintering: plasticity in the timing of life‐history events in the common frog. Journal of Zoology, 276 (4): 394-401.

Croaking Science: Kin Recognition

May 31, 2018 by editor

Kin recognition – when recognising relatives is important

During the spring in temperate countries tadpoles of frogs and toads often develop in a range of water bodies from small ponds to lakes. Swimming around in the open water, tadpoles are highly vulnerable to predation so in many species such as the common toad (Bufo bufo), tadpoles swim in large groups or shoals (Figure 1). By living in groups the tadpoles gain advantages such as decreased risks of predation and increased access to food. However, there are costs to group living such as an increase in the risk of transmitting infectious disease and intraspecific competition. Research has shown that tadpoles further increase the benefits of group living by associating with relatives, or close kin. If forming shoals reduces the risk of predation, then swimming with relatives who possess similar genes will increase the chances that these genes will survive to the next generation. The exact mechanism of kin recognition in anuran tadpoles is not clear but studies suggest that frog and toad tadpoles recognise each other through chemical cues (Eluvathingal et al., 2009). The persistent dense swimming shoals of tadpoles of many amphibians from the genera Rana and Bufo (e.g. wood frog (Rana sylvatica); common toad (Bufo bufo); and boreal toad (Bufo boreas)) have been shown to consist primarily of associations of closely related kin (Blaustein & Waldman, 1982). However, in the red-legged frog (Rana aurora), Schneider’s toad (Duttaphrynus scaber) and Günther’s golden-backed frog (Indosylvirana temporalis), tadpoles only exhibit kin recognition early in their development when they form dense shoals. As they mature, tadpoles disperse and kin recognition drops (Rajput et al., 2014). During late development the risks of pond desiccation are high and individual tadpoles seek isolated patches of water. Under these conditions the risks of suffocation through desiccating water is higher than the benefits of associating with relatives. Further to this, recent research has shown that the persistence of associating with kin varies considerably even within a single species. For example, larvae of the wood frog (Rana sylvatica) from North America exhibits kin recognition and individuals often choose to swim with relatives. However, Halverston et al. (2006) have shown that the level and occurrence of swimming with relatives is not consistent but depends on a range of factors including levels of competition, predation and parasitism. Tadpoles from two adjacent ponds exhibited different levels of association with kin due to localised differences in these factors (Halverston et al., 2006). This demonstrates that associating and swimming with relatives only occurs under certain environmental conditions and groups of tadpoles may show variations in their degree of kin association.

*Figure 1. Several species of Bufo tadpole form shoals which may consist of close relatives.*

Many amphibians within temperate zones are herbivorous, however there are a number of tropical species of tadpole which are carnivorous. This provides the opportunity for cannibalism to evolve where larger tadpoles may predate smaller ones within the same species. In these species it may be advantageous for tadpoles to recognise kin to avoid potential predation on relatives who carry the same genes. Research has shown that cannibalism generally occurs when the nutritional benefits gained from eating relatives outweigh the disadvantages such as risks of injury, transmitting infectious diseases and losing potential members of the same species who carry similar genes. As a result, cannibalism has evolved in some carnivorous amphibian larval species, but not others, depending on the environmental conditions and the relative advantages and disadvantages of consuming close kin.

Many tropical frogs in the genus Dendrobates lay their eggs in terrestrial habitats that are then transported by males to small water-filled water bodies formed in the axils of tree leaves (phytotelmata). Females of the poison dart frog Dendrobates auratus only lays a few eggs per year, so the number of tadpoles developing within any given water body is low. In some species of Dendrobates frogs, the female lays non-fertile eggs into the water to feed the tadpoles. However, female D. auratus does not perform this behaviour so the tadpoles rely completely on external food sources e.g. fallen insects, for survival. Starvation within such small water bodies is very high and therefore cannibalism has evolved to allow some of the tadpoles to survive. In this species the larger tadpoles predate the smaller ones which provides additional nutrition which is crucial for survival. However, male D. auratus may bring tadpoles from a number of clutches so there is also the potential for kin recognition i.e. larger tadpoles may choose to consume non-relatives over relatives since the latter may share the same genes. However, experiments carried out by Gray et al. (2009) suggest this is not the case and that larger tadpoles indiscriminately predate kin from non-kin. The authors hypothesise that since food sources are so scarce for developing tadpoles then the benefits of individual survival outweigh the costs of losing genetic relatives.

Additional research by Poelman & Dicke (2007) has shown that in the poison dart frog Ranitomeya ventrimaculata, females exhibit a plasticity in where she lays her eggs, which depends on environmental conditions (Figure 2). In this species, like D. auratus, tadpoles are highly cannibalistic, consuming both kin and non-kin. At the beginning of the breeding season, when water-filled phytotelmata do not contain many tadpoles, the female will spread her eggs widely amongst different water bodies, avoiding those which already contain a tadpole. This reduces the chances that her eggs will get consumed by another tadpole (which may or may not be her own). However, nearer to the end of the breeding season, the female changes behaviour and actively starts laying more eggs in phytotelmata which already contain large tadpoles. Poelman & Dicke (2009) hypothesise that this is because at the end of the breeding season there are few free phytotelmata left and by provisioning those that already contain tadpoles with fertilised eggs, she is providing food to the tadpoles which may be her own. This sacrifice of eggs will indirectly improve the survival of her existing tadpoles, the eggs of which she laid early in the breeding season. This plasticity in behavioural response allows a greater number of tadpoles to survive when the risk of desiccation of water bodies is high.

Figure 2. The poison dart frog Ranitomeya ventrimaculata exhibits different egg-laying strategies depending on the environmental conditions.

The green and golden bell frog (Litoria aurea) was once a common species widely distributed in south eastern Australia (Figure 3). It has also been introduced into New Zealand and surrounding islands. However, the extreme sensitivity of these species to the infectious chytrid fungus has been the major cause of the species’ decline in its native range and the species is currently restricted to small isolated populations. The tadpoles form groups where individuals gain protection from predators, increased foraging efficiency and better growth. Associating with relatives is advantageous since individuals would be indirectly protecting those carrying similar genes. Pizzatto et al. (2016) examined the behaviour of tadpoles of the green and golden bell frog in relation to kin recognition. The authors found that in the bell frog L. aurea tadpoles did indeed distinguish kin from non-kin and thus formed shoals containing mainly relatives. The authors suggest the advantages to this species include increased growth and increased resistance to disease since related members are likely to carry the same disease resistance genes.

Figure 3. Tadpoles of the green and golden bell frog (Litoria aurea) from Australia congregate in groups containing related individuals.

References

Blaustein, A.R. & Waldman, B. (1982) Kin recognition in anuran amphibians. Animal Behaviour, 44: 207 -221.

Eluvathingal, L.M., Shanbhag, B.A. & Saidapur, S.K. (2009) Association preference and mechanism of kin recognition in tadpoles of the toad Bufo melanostictus. Journal of Bioscience, 34 (3): 435–444.

Gray, H.M., Summers, K. & Ibáñez, R.D. (2009) Kin discrimination in cannibalistic tadpoles of the Green Poison Frog, Dendrobates auratus (Anura: Dendrobatidae). Phyllomedusa, 81 (1): 41-50.

Halverson, M.A., Skelly, D.K. & Caccone, A. (2006) Kin distribution of amphibian larvae in the wild. Molecular Ecology, 15: 1139–1145.

Poelman, E.H. & Dicke, M. (2007) Offering offspring as food to cannibals: oviposition strategies of Amazonian poison frogs (Dendrobates ventrimaculatus). Evolution and Ecology, 21: 215–227.

Pizzatto, L., Stockwell, M., Clulow, S., Clulow, J. & Mahony, M. (2016) How to form a group: effects of heterospecifics, kinship and familiarity in the grouping preference of green and golden bell frog tadpoles. Journal of Herpetology, 26: 157–164.

Rajput, A.P., Saidapur, S.K. & Shanbhag, B.A. (2014) Kin discrimination in tadpoles of Hylarana

temporalis (Anura: Ranidae) and Sphaerotheca breviceps (Anura: Dicroglossidae): influence of hydroperiod and social habits. Phyllomedusa, 13 (2): 119–131.

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