True toads of the family Bufonidae are amongst the most prominent amphibians on earth. Its members carry specialised poison glands embedded into their skin and possess parotoid macroglands, glandular systems rich in these poison glands, that are visible as parallel bulges at the toads temple (Duellman and Trueb, 1994). From their poison glands, toads are able to secrete strong acting skin poisons that are rich in cardiotoxic steroids (Habermehl, 1994). Some representatives have been introduced to novel geographical areas (e.g. Rhinella marina (Linnaeus 1758) to Australia and Duttaphrynus melanostictus (Schneider 1799) to Madagascar), where they cause serious damage to local faunas (e.g. Hagman et al., 2009; Shine, 2010). Naïve predators, normally preying upon native species, often ingest these invading bufonids and succumb to their skin poison afterwards. Through this, members of Bufonidae emerged as a group of highly infamous anurans and became a concern for conservationists (e.g. Tingley et al., 2017).

Given this outstanding role of bufonids among other anurans, it is not surprising that several scientists recently focused their research on bufonid toxicity as well as on the interaction of these toxins with their potential targets (e.g. Marshall et al., 2018). Contrary to such investigations it is, however, noteworthy that natural history observations on the release of skin poisons remain relatively rare for Bufonidae. This is especially true for non-invasive species. For those, it often remains largely unknown which defensive behaviours are applied and also to what extent skin poisons may be secreted.

The natterjack toad, Epidalea calamita (Laurenti 1768), is such a non-invasive species for which descriptive work on its behavioural and chemical defence has been lacking so far. It is native to Southwestern and Central Europe, including Germany (Sillero et al., 2014). Habitat-wise it prefers well warmed areas with light soils, such as dunes or pine forests and settles in shallow, warm ponds (Brinkmann and Podloucky, 1987; Podloucky, 1994). As typical for bufonids, E. calamita carries a pair of prominent parotoids and a plethora of poison gland openings over its body, which indicates that the species is capable of secreting high amounts of skin poison.

Herein, we report on the observed defensive behaviour of German natterjack toads ranging from body inflation to skin poison secretion. Since this kind of natural history observations for E. calamita is scarce, we evaluate our report as an advance in the understanding of the defensive ecology of this species.

Since 2014, we surveyed amphibian populations at three sites in the area of Gelsenkirchen, North Rhine Westphalia, Germany (Gelsenkirchen-Ückendorf, Gelsenkirchen-Erle and Gelsenkirchen-Bulmke; with permission from the nature conservation authority of Gelsenkirchen) (Stawikowski, 2019). Each amphibian that was found in this context was photographed for documentation purposes. With more than 300 sightings, E. calamita was one of the most abundant species that we observed in our fieldwork sites, especially during the reproduction period in spring (Günther and Meyer, 1996).

In most cases the animals showed no obvious signs of agitation while the photographic documentation was performed, but sporadically a defensive behaviour was displayed (Fig. 1A and B): In that, the toad drastically inflates its body, leading to an artificial increase of its size. Additionally, the animal straightens its legs whereby its body is lifted far from the ground. Together, these mechanisms contribute to the toad appearing a lot

Herpetology Notes, volume 12: 443-445 (2019) (published online on 01 May 2019)

Description of defensive postures of the natterjack toad Epidalea calamita (Laurenti 1768) and notes on the release

of toxic secretions

Rainer Stawikowski1 and Tim Lüddecke2*

1 Siegfriedstr. 14, 45888 Gelsenkirchen, Germany.2 Animal Venomics Research Group, Fraunhofer Institute for

Molecular Biology and Applied Ecology, Winchesterstraße 2, 35394 Gießen, Germany.

* Corresponding author: tim.lueddecke@outlook.com

Rainer Stawikowski & Tim Lüddecke444

larger and therefore more defensive as it actually is. For E. calamita it is anecdotally mentioned that this inflation may be rarely accompanied with vocalization (Günther and Meyer, 1996). We observed this vocalization frequently, often in combination with cloacal discharge (sensu Toledo et al., 2011), when animals were captured. However, we never noticed such a behaviour when inflation was performed. Following inflation and leg extension, the toad lowers its head and presents its prominent pair of parotoids towards the source of agitation. Since these macroglands actually store a vast proportion of the amphibians’ toxin resources, it appears reasonable that this behaviour might protect the toad from some predatory assaults: If a predator would attack the toad frontally in this posture, it would unavoidably be confronted with a high dosage of the toads chemical defence.

This behaviour of presenting body parts with highest loads of toxins towards predators seems to be widespread

in parotoid-bearing amphibians. It was previously described for other bufonid toads such as the common toad Bufo bufo (Linnaeus 1758), a close relative of E. calamita, but was also reported in fire salamanders (Salamandra salamandra (Linnaeus 1758)) (Kowalski et al., 2018; Lüddecke et al., 2018; Lüddecke, 2019). On the other hand, amphibians that lack such macrogland structures usually opt for different behavioural defences, such as tail rising in marbled newts (Triturus marmoratus (Latreille 1800)) or the famous unken reflex in Bombina species, to only name a few (e.g. Haberl and Wilkinson, 1997; Lüddecke et al., 2018).

In E. calamita, the secretion of skin poison is another commonly observed reaction besides the above described defensive postures. The poison appears as white droplets on the skin surface and is often released in small amounts on the toads back, the legs or the parotoids (Fig. 1C and D). Some toads seemingly felt outstandingly threatened from us during their photographic documentation and

Figure 1. Epidalea calamita displaying its defensive posture, illustrated in lateral view (A) and anterior view (B): The animal expands its body by inflation and leg stretching to appear larger, while simultaneously the toxin-rich parotoids are presented. Another strategy to defend themselves is the secretion of skin poison. Often this happens from parotoid glands directly in smaller amounts (C, poison droplets are indicated by an arrow), but can also be performed extensively from poison glands that are distributed over the animals whole body. In this case, the animal is quickly covered in several milky-white droplets (D).

they released their toxic secretions quite extensively across their whole body (Fig. 1D). The secretion of skin poison on the toad’s skin surface and the utilisation of defensive postures can be combined but can also happen independently from each other. It is noteworthy that in some situations the secretion of toxins was performed without actually manipulating the animals. Such a behavior represents a rare observation since usually secretion is performed only after active squeezing of the glandular systems (e.g. when the toad is captured by a predator). Comparative observations of voluntary release of skin poison are rare, but were described from S. salamandra and the bufonid Rhaebo guttatus (Schneider 1799)(Mailho-Fontana et al., 2014; Lüddecke et al., 2018). However our observations in E. calamita indicate that such a behaviour might be more common among amphibians, or at least in bufonid toads, than previously thought.

The skin poison of Bufonidae contains, among other compounds, high amounts of cardiotoxic steroids that cause severe intoxications in vertebrates (Mebs, 2010). The impressive amount of such highly toxic skin poison that is released by E. calamita and on which we report here, represents a good example of the toxic potential of the family.

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Accepted by Wouter Beukema