Pheromones for amphibian conservation – considering new approaches in conservation breeding programmes

Abstract

The sixth mass extinction, leading to massive biodiversity declines, is currently particularly affecting vertebrates (Ceballos et al., 2015). Amphibians are the most vulnerable vertebrate class, with 41% of the species threatened with extinction (IUCN, 2022). The reasons for amphibian species loss are diverse, including habitat loss, chytridiomycosis, global warming, overexploitation, invasive species, chemical pollution, illegal trafficking and others (for review see Allentoft & O'Brien, 2010). Most of these factors are difficult to overcome in the short term (if at all). To protect species at imminent extinction risk, numerous ex-situ conservation breeding programmes have been established in recent decades, with the larger goal of future reintroduction (Wren et al., 2015). One example of such breeding programmes are zoos (Murphy & Gratwicke, 2017), such as the Frankfurt Zoo in Germany. Here, a number of endangered amphibians are kept and bred, with the potential for future reintroduction. However, while conservation breeding has proven successful for many species, others do not thrive in human care, and breeding, so far, remains unsuccessful, leading to a conservation breeding crisis (Kouba, Vance, & Willis, 2009; Tapley et al., 2015). Hormone treatments have been established to overcome reproduction problems in particular groups (Silla & Byrne, 2019), but their success varies substantially due to the extreme diversity of courtship, mating and reproductive behaviour, including the way of mate attraction, type of amplexus, fertilization mode, oviposition site, presence of parental care, etc. (Wells, 2007; Carvajal-Castro et al., 2020; Schulte et al., 2020). A factor that strongly influences mate attraction and courtship behaviour in many animal species is olfactory signals, that is, pheromones (Wyatt, 2014). Because this also applies to amphibians, this is a plea to consider the involvement of pheromones and pheromone-producing structures in amphibian species in ex-situ programmes as an additional approach to help with breeding-problematic species, as well as future focal species with low population densities unable to recover naturally. The majority of pheromones described in amphibians are proteins directly transferred to the mating partner (over the nose or through the skin) and categorized as courtship pheromones (Arnold & Houck, 1982). In salamanders, it has been shown that such courtship pheromones positively influence female receptivity for reproduction (for reviews see Bossuyt et al., 2019a; Woodley & Staub, 2021). Courtship pheromones are produced by the males in large glandular clusters (i.e. breeding glands; Houck & Sever, 1994; Brizzi, Delfino, & Jantra, 2003). Such breeding glands can be found in countless amphibian species, including many frogs which produce proteins closely related to salamander courtship pheromones (Bossuyt et al., 2019b; Schulte et al., 2021, 2022). The ubiquitous nature of these glands in amphibians suggests a significant role of the herein produced proteins for mating success, and calls for a collective effort to try to incorporate such potential courtship pheromones into conservation breeding programmes. One possibility is to choose males with well-developed glands. At Frankfurt Zoo, we closely monitored the breeding glands of the critically endangered (in Germany) yellow-bellied toad (Bombina variegata; Kühnel et al., 2009). During the breeding season, we monitored the thumb pads of the males on a weekly basis and, where possible, tried to track which of the males had the best breeding success. While a rather skinny male barely developed breeding glands (and was, therefore, eventually taken out of the breeding colony), those males that actually went into amplexus with the females had very large, dark glandular tissue (Fig. 1). Due to this finding, we encourage animal caretakers and researchers actively involved in ex-situ programmes, as well as in in-situ species assessments (e.g. choosing future candidates for breeding programmes), to look out for the quality of male breeding glands. Another possibility, to further amphibian reproductive success, may be synthetic pheromones. Frankfurt Zoo and Goethe University Frankfurt are currently collaborating in first efforts to synthesize potential anuran courtship pheromones. The intention is to apply such synthetic pheromones to the females using minimally invasive methods, that is, by dripping them onto the nostrils or topically applying them to the skin (as shown in some salamanders, see Houck et al., 2007, 2008). These synthetic pheromones could be applied before or during courtship/amplexus and used instead or in addition to hormone treatments. For example, in species where males are actively calling but females remain uninterested, a few drops of male courtship pheromones may have the potential to change her mind (without undergoing stressful hormone injections). However, similar to hormone treatments, pheromone treatments would have to be considered and tested for each species individually. We, therefore, encourage amphibian conservationists to observe, whenever possible, how, where and when the male breeding glands are brought into contact with the females – and thereby get a better picture of the importance of these glands on a species-specific basis. Admittedly, research on protein courtship pheromones in amphibians (especially in frogs) is still in its early stages and many questions still need to be tackled. However, because of their potential for conservation breeding programmes, not just the research on the pheromones themselves, but also the assessment of breeding gland quality in ex-situ breeding programmes and the collection of more natural history data regarding chemical communication in threatened species are immensely important. Even though the involvement of pheromones in conservation breeding programmes might seem like a drop in the ocean, it could be an important new opportunity to help at least some species to breed under human care and, thereby, escape extinction. We are grateful to the Frankfurt Zoo for financially supporting the work of the Wildlife-/Zoo-Animal-Biology and Systematics Laboratory, and thereby our efforts to further the knowledge about amphibian pheromones. We furthermore thank the animal caretaker team of the Exotarium at Frankfurt Zoo for their help during the Bombina-project.