Among amphibians, conspecific chemical communication has been widely studied in Caudata. Adult anurans, by contrast, have received less attention. Recently, it was shown that chemical scents are also relevant for adult anuran intraspecific communication. In this context, we evaluate whether females of the four-eyed frog (Pleurodema thaul) respond to conspecific male scents. We carried out a double choice experiment in a Y-maze. Females were repeatedly presented with the scents of several males versus distilled water. To extract the scent from males, we acoustically stimulated males and then used the water from their aquaria for the experiments. Our data suggest that females are capable of responding behaviourally to male scents, since they spent longer periods in the zones with male scent, rather than in zones with water. We propose that under natural breeding conditions, females of P. thaul may use either their chemical sense or chemical cues to facilitate their encounters with males.

anuran communication, chemical communication, female choice, male scent

Animal communication is a process by which information is transferred from a sender to a receiver, provoking in the receiver a behavioural or physiological reaction (Dusenbery 1992). This communication process is essential for social interactions (e.g. agonistic interactions, courtship rituals), and can depend on different channels (e.g. acoustic, visual, vibrational, chemical), which might be utilised independently or in association (Partan 2004; Bradbury and Vehrencamp 2011).

Chemical communication is one of the oldest systems of communication in the animal kingdom, and it has been studied across ontogenetic stages and in different social situations across several taxa (Gorman and Mills 1984; Roberts et al. 2003; Wyatt 2014). Particularly in amphibians, most knowledge regarding conspecific chemical communication has been developed for Caudata, where scents have a significant role in different aspects of their social life (Park and Sung 2006; Houck et al. 2007, 2008). For instance, salamanders of the family Plethodontidae have been widely studied for the use of chemical signals in their social interactions (Guillaume 2000; Rollinson and Hackett 2015). It was observed that males of Plethodon shermani produce a sexual pheromone denominated Plethodon Receptivity Factor (PRF) that increases the female`s receptivity (Rollmann et al. 1999). In addition, courtship behaviour involving pheromone delivery (Houck 2009), the genetic components for sex pheromone productions and their evolution across amphibian groups, have been widely described (Watts et al. 2004). The genus Cynops, has received strong attention for their intersexual interactions mediated by chemosignals (Toyoda et al. 2004). Kikuyama et al. (1995), discovered that males of the fire belly salamander (C. phyrrogaster) release a female-attracting pheromone (denominated Sodefrina) and males of C. ensicauda release a peptide from their abdominal gland called Silefrin that attracted females (Yamamoto et al. 2000). The fire belly salamander and their Sodefrin pheromone have become a model for the study of sex pheromones in aquatic salamanders, resulting in the discovery of a multi-component pheromone system (Van Bocxlaer et al. 2016).

Anurans have been described as acoustically dependent; several studies have, however, by now demonstrated that scents can mediate conspecific recognition (Kiseleva 1996; Carlson et al. 2015), provide information about kinship identity (Waldman 1985) and play a role in predator-prey interactions in the larval stages of anurans (Pearl et al. 2003; Chivers and Ferrari 2013; Troyer and Turner 2015). Even learning of chemical cues at embryonic stages has been described (Hepper and Waldman 1992). Additionally, chemical signals are also important in different social contexts in adult anurans (Kam 2002; Still et al. 2019). For instance, Leiopelma hamiltoni releases a chemical compound in the faeces that provides information to conspecific individuals about the size of the sender (Lee and Waldman 2002). In courtship situations, it has been observed that males of Litoria splendida release a sexual pheromone called Splendipherin that elicits changes in postural and permanence times in females (Wabnitz 1999). A recent study has contributed knowledge about anuran chemical communication through the study of compound secretions (Brunetti et al. 2019) and also several investigations provided evidence for their role in social interactions (Pearl et al. 2000; Asay et al. 2005; King et al. 2005). For example, Byrne and Keogh (2007) showed that the Australian terrestrial Pseudophryne bibronii toadlets use scents to identify and locate potential mates. In addition, the frog Lithobates pipiens shows differences in their behavioural responses to adult male and female chemical cues (Graham et al. 2020). Compared with other amphibian groups, however, more investigation describing chemical communication in adult anurans is still needed.

Adult males of the four-eyed frog, Pleurodema thaul, emit advertisement calls during the reproductive season, inflating their vocal sacs while floating on the surface of slow-flowing streams or pools (Penna and Veloso 1990; Velásquez et al. 2014). Research has shown that females do not exhibit a preference for males based on their calls (Velásquez et al. 2015). Alternatively, their preference may be guided by male scent. Currently, the only information available on chemosensory recognition in this species focuses on tadpoles using chemicals under predation risk (Pueta et al. 2016; Pueta and Perotti 2016), but the question of whether adults utilise scent recognition has not been studied yet. In this context, and considering the available information on sexual scents involved in mate attraction in other anurans (Wabnitz et al. 1999), we aimed to determine whether female Pleurodema thaul responded behaviourally to male scents.

A total of 60 trials were done, and, in 80% of these cases, females showed a positive response toward the scent of males by entering the corresponding arm. Despite this observation, females chose the arm with the conspecific male scent and the arm with the control solution indiscriminately (One-tailed Binomial test: p = 0.385, Fig. 2A), but they spent more time in the male scent solution than in the control solution arm (Sign test: Z = 2.33; p < 0.05, Figs 2B, 3). The analysis of permanence time indexes showed that females were consistent between replicates (U-Mann Whitney test: Z = -0.483; p = 0.629), and the identity of the male did not affect the response of the females (Kruskal-Wallis test: H = 2.736, p = 0.741).

Figure 2. 

Total number of choices (A), and permanence time (B) shown by females of Pleurodema thaul in the Y-maze experiment. The asterisk indicates significant differences (p < 0.05).

Figure 3. 

Heat-map showing the permanence time of females of Pleurodema thaul in the Y-maze. A) represent arm containing male scents and B) represent water-control arm. The lateral bar indicates the relation between colour and the permanence time (s). Heat-map is the outcome of overlaying track routine videos.

Females of Pleurodema thaul exhibit a behavioural response towards the scents of conspecific males by remaining in the arm containing the conspecific scent for a longer period compared to the arm containing water, suggesting that females of P. thaul are capable of detecting chemical cues originating from conspecific males. This, however, was not reflected in the first-choice behaviour of the females. Females seemed to choose both arms at similar rates.

The lack of initial choice for the arm dowsed with a male’s scent may be explained by females depending on water-soluble compounds during mate choice. During the breeding season, male P. thaul congregate in pools to call and attract females (Penna and Veloso 1990; Velásquez et al. 2015). Females might need to be in close contact with water so as to show a behavioural response, similar to what was observed in Litoria splendida (Wabnitz et al. 2000). The water-soluble sexual pheromone Splendipherin produced by L. splendida males was effective in eliciting female attraction when the aquarium had a water column depth of 2 cm (Wabnitz et al. 2000). The lack of female choice for the conspecific scents in P. thaul, together with the longer period they spent in the arm with the conspecifics scent, suggests that females depend on water-soluble molecules to elicit specific behavioural responses, as it has been previously proposed (Wyatt 2010). Therefore, it is likely that similar experiments that include more water (e.g., Pearl et al. 2000; Asay et al. 2005) may trigger more robust behavioural responses in females of P. thaul.

Despite the lack of first choice preference, females remained for longer periods in zones containing the conspecific scent solution, hinting that females reacted to the scents produced by males. This result might indicate that chemical compounds promote intersexual interactions in P. thaul. Females may alter their locomotion or activity levels to move in the direction of the scent source in order to find a mate (Norris and Lopez 2011). Similar results were obtained in the dwarf African clawed frog (Hymenochirus sp.) in which females were attracted to water containing male scent in a two-choice aquatic Y-maze and then remained for longer periods closer to the chemical scent source, suggesting an intersexual recognition mediated by scent (Pearl et al. 2000). Permanence time has been used to determinate the existence of conspecific chemical communication across different anuran species (Lee and Waldman 2002; Graham et al. 2020). Thus, considering the time spent in male scented zones and that conspecific male solution used in this experiment could resemble pool water during a breeding season, our results are in concordance with previous studies that demonstrate how chemical communication plays an important role in conspecific interactions. A previous study showed that females of P. thaul do not exhibit a preference for males based on their calls (Velásquez et al. 2015). Potentially, females of P. thaul need both acoustic and chemical information to approach males, where acoustic signals can be used to find breeding ponds, and chemical signals to pick within a chorus (e.g., Rendall et al. 2009; Buxton et al. 2015). The consistency showed by females toward scents obtained from conspecific males (N=3; mean ± SD; weight=4.32 ± 0.67 g; snout-vent length= 31.98 ± 0.53 mm) could subtly suggest that: i) there were no differences between the males’ characteristics or ii) in P. thaul frog, chemical and acoustic signals promote reproduction without involving sexual selection. Future studies must set up experiments that explore all factors influencing chemical behaviour, such as the breeding season and non-breeding season, largest sample size and intra and/or intersexual interactions. In this way, it would be possible to identify the exact role of the chemical compounds in male-female interactions and female preferences.

Our study demonstrates the capability of female Pleurodema thaul to discriminate conspecific male scents from a water control solution, since they remained for longer periods in zones with the conspecific solution compared to the control. Considering this result, we suggest that chemical compounds are involved in the intersexual interactions in this species helping females to find a mate. This study expands our knowledge on chemical communication, which is relevant to understand the factors and mechanism that take part in and modulate reproductive events.

We are grateful to Carlos López and Javier Rivas for assisting us with the experiments. This study was supported by FONDECYT 11140752 assigned to N. A. Velásquez, and Iniciativa Cientifica Milenio, Biomedical Neuroscience Institute, ICM P09-015-F assigned to J. L. Valdés. We also thank Alejandra Fabres who helped with comments on the final version of this manuscript.