Introduction

Chemical defence is one of the mechanisms that organisms use to enhance their survival prospects. Several different animal taxa from arthropods [1]–[3] to amphibians [4] defend themselves against predators, parasites and/or competitors by producing deterrent or noxious substances [5]. Within amphibians, poison frogs that sequester alkaloids from their prey to be chemically protected are the best known example [4]. In other groups, such as endothermal tetrapods, however, examples of chemically defended animals are limited to a small number of lineages [6]. Indeed, birds have not been included in lists of chemically-defended animals until the end of the twentieth century [5], when species of the genus Pitohui were found to deter predators and/or parasites by means of the neurotoxin homobatrachotoxin that is contained in their tissues [7]. Apart from this extreme example of poisonous Pitohuis, several other bird species also contain toxic or unpalatable compounds that may help to defend them (see reviews in [5], [8]). For instance, hoopoes Upupa epops and green woodhoopoes Phoeniculus purpureus, when disturbed at the holes where they roost, secrete drops of a fetid substance from their uropygial glands [9].

The full understanding of the functioning of a chemical defence needs to address the three following general issues [5]: a) the origin of defensive chemicals, which can either be produced de novo by the defended organism or be obtained from other organisms, usually through consumed food; b) the composition of the defensive substances; and, c) the effects of the chemicals on generalist predators because chemical defences are effective against generalist consumers but can be circumvented by specialists [10]. This research pathway has been widely adopted to investigate the evolution of chemical defences in several taxa [4], [11], [12]. However, many of the examples of avian chemical defence are based on anecdotal reports [5]. Therefore, the ecological and evolutionary relevance of avian chemical defences still needs to be thoroughly investigated.

Here, we aim to investigate in depth the possibility that the odorous orange substance that Eurasian roller (Coracias garrulus) nestlings regurgitate when disturbed [13] acts as a defence. Vomiting is a particular behaviour of the nestlings of rollers, and, as far as we know among birds, only described in chicks of some Procellariiform species [14]. Roller parents returning from a foraging trip approach to their nests more cautiously when they smell nestling vomit, suggesting they have interpreted this smell as a signal of offspring fear [13]. However, the primary function of this substance is unknown. The expelling of vomit must be costly for nestlings due to the loss of body fluids; thus, it seems unlikely that the secretion has no function at all. Indeed, as indicated above, this vomiting behaviour is not a common feature of nestling birds but limited to a small number of species. Consequently, we hypothesize that the vomit might have a defensive function in rollers by making nestlings distasteful to predators.

Many herbivorous insects such as grasshoppers regurgitate when disturbed [15]. The defensive role of the expelled fluid has been attributed primarily to ingested plant secondary compounds [16]–[18]. Grasshoppers are the main prey that rollers hunt to feed their nestlings ([19], see below. Furthermore, rollers feed their offspring with a large share of poisonous arthropods [19] that are avoided by most of the other sympatric insectivorous birds [20]. This suggests that rollers are resistant to these toxic substances and could have the ability to sequester chemicals from their protected prey to defend themselves, like phytophagous insects do with plants secondary compounds [15], [21], [22]. Therefore, we expected that vomit expelled by nestling rollers contained the defensive substances used by plants as anti-herbivore defence, but accumulated in some specialized herbivores that are able to circumvent this defensive strategy as the phytophagous arthropods consumed by rollers. The effect that it produces on potential predators is crucial for assigning a defensive role to the substance. Indeed, if vomit has a defensive function, it should be produced in response to a threat and elicit rejection or avoidance by predators, parasites and/or microbes.

In this context, we combine detailed diet and behavioural analysis of roller nestlings, experimental approaches and chemical analyses with high performance liquid chromatography–mass spectrometry (LC-MS) to address the following four objectives: 1) whether the substance that nestling rollers expel has a dietary origin; 2) the type of stimulus (mobile, visual, auditive or tactile) that triggers its expelling; 3) the composition of nestling oral secretion; and, finally 4) whether it is effective eliciting aversion by generalist predators.

Methods

Results

Nestling Diet

We identified at least one prey item provided by parents in 34 video recordings (36.2% of total recordings) from 32 different nests (50% of the observed nests). From these 34 video recordings, we identified 112 items, all of them arthropods, mainly belonging to the order Orthoptera (N = 103, 92%), but also some Coleoptera (N = 2, 1.8%), Lepidoptera (N = 3, 2.7%) and centipedes Scolopendromorpha (N = 4, 3.6%) (Table 1).

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Table 1. Number of prey by taxa and percentage of frequency of each order (in brackets) in the diet of nestling rollers estimated from video recordings (N = 32 nests) or by the application of collars to nestlings’ necks (N = 14 nest).

https://doi.org/10.1371/journal.pone.0068862.t001

We also collected some prey items from neck collars sporadically applied to nestlings in 2008 and in the experiment of food deprivation in 2012. Specifically, we collected 21 arthropods from 14 different nests, 19 belonged to the order Orthoptera (90.5%), 1 to the order Coleoptera (4.8%) and 1 to the order Scolopendromorpha (4.8%) (Table 1).

Discussion

In this paper we first show that arthropods from the order Orthoptera are the main prey of roller nestlings in the study area. We also demonstrate that the vomit expelled by roller nestlings depends on food provided by parents and that vomiting is triggered by grasping and moving of nestlings. In addition, we have found that vomit samples contain variable concentrations of hydroxycinnamic and hidroxybenzoic acids, two phenolic acids, and that some of the vomit samples also have traces of psoralen, a furanocoumarin. Finally, we have shown that vomit of nestling rollers alone makes chicken meat unappealing for dogs. Below, we will critically assess these findings in the light of the hypothesis that nestling rollers regurgitate when disturbed, expelling an orange and odorous substance [13], which could result from the sequestration of chemicals from their prey for their own use, as their main prey (grasshoppers and beetles) do from plants. As this behaviour is produced in response to a threat, it could have a defensive function during the nesting period in which nestlings are not able to escape from predators.

We have found that the movement of nestlings by the investigator seemed to trigger vomit ejection. This fact suggests that the vomit might be produced in response to some kind of predators that actively grasp and move prey during the predation event such as snakes, rats and mustelids, which are common predators of hole-nesting species [36], [37] as rollers. Holding nestlings was always the last tested stimulus in our experiment which raises the possibility that was order, rather than stimulus per se, which was determining the found pattern. However, this is unlikely because we previously knew from our long-term monitoring of rollers that chicks vomited when straightly handled (Parejo and Avilés unpublished data). Anyway, which is important here is that disturbance causes vomiting.

Our results also indicate that the production of vomit depends directly on recently consumed food because when nestlings were food-deprived for 1 hour they reduced vomit production. This result suggests that the vomit has not an endogenous (i.e. glandular) but a dietary origin. The oral emissions of arthropods contain a blend of digestive enzymes, salivary secretions, and partially digested food as plant secondary compounds [15]. Therefore, in rollers a similar mechanism of secretion production seems to be feasible. Moreover, toxins seem not to be produced de novo by any vertebrate group [38], which leads us to think that all the chemicals found in vomit samples have a dietary origin. What is clear is that nestling rollers need food either as a source of chemicals or as a source of energy to produce the vomit.

In the study area roller nestlings are mainly fed with Orthoptera, which are relatively polyphagous species [39], [40]. Therefore, we expected to find a tritrophic effect of plant secondary compounds, from plant to insects and then to rollers. In agreement with our expectation, we found that all vomit samples contained hydroxycinnamic and hydroxybenzoic acids, that are phenolic acids usually found in leaves of many Gramineae and cell walls of most higher plants [18], [28], [41] and that deter insect feeding [29], [42]. Furthermore, some samples also contained traces of psoralen that is a furacoumarin produced by a wide variety of plants in response to pathogens and/or herbivore attacks [17]. Despite the occurrence of these substances in several plants, most phytophagous insects develop the ability to cope in greater or lesser extent with these unpalatable substances (see [30] for a revision). Therefore, phytophagous insects may first feed on defended plants and, second, use opportunistically plant secondary compounds for their own defence [16], [21]. Indeed, oral secretions produced by several species of grasshoppers, such as Romalea microptera, R. guttata and Taeniopoda eques, are dominated by phenolics and quinones [31], [32]. These armed insects, hunted by adult rollers to feed their offspring, would be the putative source of phenolic acids contained in the vomit of roller. Previous work has demonstrated that oral secretion of different grasshopper species can deter predators [16]–[18], [31]. Here, for the first time, we show the deterrent effect of the oral secretion of a vertebrate, the avoidance of the oral secretion of nestling rollers by domestic dogs. These results suggest that vomit can be used by rollers as a way to be chemically-defended, which would improve brood survival and, consequently, parental fitness. As nestlings only vomit after being grasped and moved, some of the common predators of hole-nesting birds, as rats and mustelids, must perhaps bite a nestling roller before realising that the prey is unpleasant. Thus, one could wonder about the nestling advantage of this defence. Kin selection is a possible answer to that question because a predator that finds the first nestling of a brood of five to be distasteful may leave alive the others [5]. Alternatively, the advantage might be found in parental fitness because parents would benefit from an incomplete predation event at their nest. For other predators as snakes, however, the advantage of the defence is easier to understand. Snakes would first try to immobilize nestlings by constriction while holding them with the mouth, which would induce nestling vomiting and hence the immediate savouring of the unpleasant prey through the snake olfactory tongue [37], thus avoiding chick death.

It should be acknowledged here that despite the initial avoidance that dogs showed against meat with vomit, many dogs finally ate it. However, they did that after some minutes, perhaps after the volatilization of much of the smell of the vomit [13]. This fact probably means that vomiting only serve in the short time against predators because of the volatile nature of the expelled substance. Nevertheless, it is interesting to highlight that 30% of the tested dogs avoided consumption of the meat experimentally smeared with vomit even as a second option. This result clearly shows that roller vomit can be effective in avoiding nestling predation. A direct test of predator avoidance function with natural predators would require experimental manipulation of vomit production in nests in the field and estimating its effects on predation rates. Such a protocol, however, needs the development of a method to inhibit vomiting.

To summarize, several lines of evidence support the idea that the vomit of nestling rollers might have a defensive function against predation: 1) It is expelled in response to a threat, our handling, at nests. 2) Vomit seems not to be produced de novo by nestlings but has a dietary origin, which suggests that vomiting might be a costly behaviour that should have an adaptive function. 3) Vomit contains deterrent chemicals used by plants against herbivores and by phytophagous insects against their predators. Therefore, these substances could be acquired by rollers from plants through prey insects to deter predation at nests. 4) The fact that the vomit makes meat unpalatable to mammalian generalist predators supports the idea that secondary compounds of plants present in the vomit could be used by rollers as a chemical defence. However, we have no data yet to show that individuals with less protection experience reduced fitness [5].

Supporting Information

Acknowledgments

We are grateful to the staff of the Centro Zoosanitario Municipal in Almería, who kindly provided help with dogs, to the Instrumentation Service of the EEZ (CSIC) in Granada, where chemical analyses were done, and to Juan Rodríguez for his help in the field.