Discussion
Our results suggest that P. vaillanti and P. bicolor show conspicuous colorations in some areas of the body are unpalatable, have toxins and prefer to remain on the surface of the water column. These strategies may be evolving in tandem, and represent an adaptive syndrome that reduces the risk of predation (Relyea 2001). Most of the larvae were depredated; the mortality of the naiads that consumed the P. vaillanti larvae is an indication of the presence of toxicity from tadpole’s skin. However, there is a great variation in the effect that a toxin can cause to different predators and in how a predator can more easily recognize a non-palatable prey than another (Adams et al. 2011). For example, fish and amphibians can recognize and identify a toxic prey faster than aquatic insects (Kats et al. 1988). This is because vertebrates consume whole prey including skin, while insects use buccal parts to suck the prey content (Brodie et al. 1978; Gunzburger and Travis 2005). Therefore, it is necessary to corroborate the lack of palatability using other predators that can coexist with the larvae.
Toxicity assays helped us to reinforce the hypothesis about the presence of alkaloids in the skin extracts of these larvae. The presence of these toxins as an aposematic signal in anuran larvae is a rare characteristic in which few studies have focused (Toledo and Haddad 2009). Some investigations have shown that various larvae of the genusAtelopus, Rhinella and Oophaga are fed with unfertilized eggs the female deposits and they possess chemical defenses for the tadpoles. Also, some eggs of these same species hatch with chemical defenses the female has provided during oviposition as protection against predators (Pavelka et al. 1977; Hayes et al. 2009; Stinosky et al. 2014). In the Phyllomedusine larvae, the acquisition of chemical defenses has not been reported; however, we can be sure the toxicity assays are reliable to demonstrate these tadpoles possess toxic substances, although we cannot prove their origin. The presence of glands that secrete opioid peptides such as dermorphins, bradykinin and delthorphine by some adult Phyllomedusa are highly unpleasant to predators because they can modify cardiac function, induce regurgitation, or produce catalepsy (Sazima 1974; Negri et al. 1992). These same substances may be appearing along larval growth, as well as serous glands, which could function as regulatory and defense mechanisms in the aquatic environment (Delfino et al. 1998).
About the presence of conspicuous colorations in our model, we observed that there is a bimodal pattern of coloration. On the dorsal area, the tadpoles have conspicuous colorations, while ventrally the tadpoles can be considered inconspicuous. This would suggest that dorsal coloration would be targeted at aerial or terrestrial predators, whereas the coloration of the ventral area makes them cryptic for predators living underwater (Thibaudeau and Altig 2012). P. vaillanti was the most conspicuous species in our experiment. It also had a very significant metalized interocular patch. It is possible this conspicuous patch not only gives it advantages to warning predators, but it can also serve as a disruptive coloration, thus increasing their opportunity to avoid predators (Eterovick et al. 2010). On the other hand, C. tomopterna , presents a black spot at the end of the tail, which could be used as a strategy to redirect the predator’s attack. Such an attack would be directed toward a less vulnerable area and compensate the absence of some conspicuous coloration (Caldwell 1982).
On the other hand, conspicuousness did not to have a correlation with the toxicity in P. bicolor. This correlation was only observed in the P. vaillanti larvae and only associated to the coloration of the interocular spot, but not all tadpole coloration. The reason can be that the conspicuousness and presence of toxins could operate decoupled. For example, some species of dendrobatids may differ significantly in toxicity, but their coloration is the same. Other individuals may exhibit similar colorations, but their toxicity varies significantly (Darst et al. 2006).
Conspicuous dorsal colorations of these larvae may work together with schools towards the surface of the water. This would suggest a positive effect on predator learning. Other phyllomedusine larvae (P. gutatta, P. jandaia and P.vaillanti ), mainly present congregation behaviors supposedly as an antipredatory mechanism. They are predominately grouped on the water surface forming schools with individuals of the same size during the day while at night they are dispersed and located along the water column (Branch 1983; Caramaschi and Jim 1983). This behavior towards the water surface could be strongly related to environmental factors such as light and the different sensory systems of the larvae, which would explain the behavior of P. vaillanti and P. bicolor (Katz et al. 1981).
On the other hand, it has been evidenced the effectiveness of this conspicuous coloration and the toxicity present in both P. vaillanti and P. bicolor increases as the individuals’ size, which produces greater aversion by predators (Hagman and Forsman 2003). In addition, the palatability experiments support this hypothesis, since it was observed that the larger larvae were not depredated. This has suggested this aposematic signal may evolve favoring larger prey (Lever 2001, Gunzburger and Travis 2005). Depending on the stage of larval development, it is possible for toxins or alkaloids to change their composition and abundance, making the animals less palatable to predators (Brodie and Formanowicz 1987; Peterson and Blaustein 1991; Hayes et al. 2009; Stynoski et al., 2014). For example, there has been an apparent change in the toxicity of Bufo during ontogeny. Large mortality of giant water bugs (Belostomatidae) was observed when the insects predated tadpoles in late stages of development (Crossland 1998). In other experiments, were offered to chickens and predatory rejection of larger insects was observed (Gamberale and Tulberg 1998).
In conclusion, P. vaillanti and P. bicolor larvae may present an antipredatory syndrome using various mechanisms to avoid predators. These combined strategies can reduce the probability of attack, confuse the predator, reinforce the alarm signal, and warn of the presence of toxins in the skin of the tadpoles (Aronsson and Gamberale 2012). Finally, the high cost of having a conspicuous coloration associated with chemical defense and the formation of congregations is a point that merits greater attention in future investigations, specifically at the time the larvae complete their cycle or survival rates associated with the use of these strategies.