Alien Vs Predator Reaction

[Earleen Muffley]

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Alien predators are considered to be one of the major threats to global biodiversity loss, causing declines and extinction of species worldwide1,2,3. Introduced predators can create novel ecological contexts, posing new threats to which antipredatory responses of native prey may lack adaptive value4,5,6. Native prey are usually equipped with adaptive morphologies and/or behaviours to cope with local, coexisting predators. However, prey are often nave to the hunting tactics of novel predators with whom they lack a shared evolutionary past1, 7. Because of this, alien predators frequently cause more severe impacts to prey populations than native predators8.

Amphibians are the most threatened group of vertebrates with ca. 41% of the species endangered23, 24. Over the last decades, numerous studies have linked the introduction of alien predators with global amphibian declines, and even local extinctions25,26,27,28,29,30,31,32,33. Amphibian eggs and tadpoles are particularly vulnerable to alien aquatic predators, which can consume them intensively28,29,30,31. Nave tadpoles, like many freshwater organisms, typically respond to the presence of water-borne cues from local predators by developing defensive morphologies and adjusting their behaviour34, 35 reviewed in36, 37. In contrast, tadpoles usually fail at inducing adaptive responses against alien predators, since activation of plastic defences necessarily requires predator recognition, and nave tadpoles may be unable to recognize introduced predators with which they lack evolutionary history38,39,40,41,42. However, experimental studies have shown that many aquatic organisms including tadpoles can learn to recognize new predators as a threat by associating the unknown predator stimulus with conspecific alarm cues41, 43,44,45,46. Alarm cues are released when prey skin is damaged during a predatory attack, warning nearby individuals of imminent risk of predation and being crucial tools in associative learning40, 47, 48.

Hence, learning might be key to enabling the use of inducible defences against introduced predators, which can prove critical for amphibian populations until innate recognition (i.e. genetic adaptation) evolves. In this regard, tadpoles in some amphibian populations have been reported to recognize introduced predators39, 49,50,51,52, suggesting that, given enough time, native amphibians may evolve the ability to innately detect and avoid novel predators over generations, presumably assisted by behavioural plasticity through recurrent individual learning during the initial exposure. Geographic variation in plastic or innate responses against novel predators are likely to arise among prey populations, mostly due to variation in local predator abundance and history of exposure to the novel predators9, 53. Moreover, among-population variation in their behavioural plasticity and learning capacity may be influenced by available genetic diversity and gene flow with other populations with varying degrees of exposure to the novel predators. Thus, given sufficient genetic variation and selection from the novel predator, adaptive plasticity and even innate recognition can evolve locally, and even be exported to neighbouring populations with less predation pressure54, 55. Conversely, the evolution of such adaptations may be hindered by incoming gene flow from non-adapted populations54, 56, 57.

Tadpole alarm cues were prepared from three conspecific donor tadpoles. Tadpoles were euthanized by immersion in a highly concentrated solution of MS-222 and homogenized with a bench top homogenizer (Miccra D-1, Germany). We then diluted the homogenate in 600 mL of carbon-filtered, dechlorinated tap water and filtrated it with filter paper to remove solid particles. The water containing the alarm cues was immediately frozen in 10 mL aliquots until use75.

Different histories of coexistence with predators may also lead to dissimilar evolved responses to invasive crayfish among populations. In fact, generalization of predator recognition (i.e. when prey lacking innate recognition of novel predators have the ability to label them as predatory if they are phylogenetically closely related to already known predators92) has been demonstrated in larval amphibians93,94,95. In the Iberian Peninsula, the native crayfish Austropotamobius pallipes has been historically absent from Doana area and almost from the entire Madrid region, but this indigenous species was present in the area of STOME population (in the limits between Segovia and Madrid provinces) about 50 years ago, before Iberian crayfish populations were drastically decimated by the crayfish plague caused by the pathogen Aphanomyces astaci96,97,98. Interestingly, we observed a significant reduction in activity of nave tadpoles in response to water-borne cues from P. clarkii in the STOME population (Fig. 1). This suggests that a certain degree of generalized predator recognition of the alien crayfish linked to the presence of a native crayfish in the recent past might be taking place in this non-invaded population. However, an innate response to crayfish cues was observed also in the MAN population (Fig. 1), where A. pallipes has been historically absent. Although the arrival of P. clarkii to central Spain is fairly recent (ca. 35 years), we cannot completely discard that innate recognition of crayfish cues could have evolved in this population under the predatory pressure posed by the alien crayfish during the last decades.

Predator diet seems to be a critical factor in the recognition of alien predators by nave amphibian larvae, and the consumption of conspecific tadpoles is precisely the ecological scenario in which learned recognition would take place. Even though innate responses may fail to be triggered against novel predators, prey can still rely on learning to reduce their impact. Learning of new threats through cognitive association with alarm cues has been observed in a variety of aquatic prey (see47 for an extended review), including amphibians44,45,46, 95. In a previous study, we demonstrated that learning via association with conspecific alarm cues allows successful activation of antipredatory responses by spadefoot toad tadpoles against P. clarkii, increasing larval survival in predatory assays41. Here we show that such learning ability is common to multiple populations of P. cultripes regardless of the intensity of the invasion by crayfish. This widespread learned predator recognition suggests that amphibian larvae might broadly benefit from associative learning to recognize and avoid new predatory threats, thus modulating the initial advantage of alien predators in amphibian assemblages.

Our results emphasize the importance of integrating phenotypic plasticity and behavior of native organisms in the management of biological invasions, in order to achieve the conservation goal of long-term persistence. Indeed, the capacity to plastically modify behavior in response to new or unusual challenges (i.e., the cognitive buffer104) is an essential mechanism for animals to cope with rapid environmental changes such as the introduction of novel predators. By enabling tadpoles to adjust defensive behaviour and activate inducible defences, widespread learned predator recognition may be decisive for amphibian populations to persist in the new ecological context posed by aliens, soothing the impact of invasions and buying time for innate recognition to evolve17, 18.

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The experiment involved four olfactory treatments: (1) aged tap water (serving as the control); (2) cues (kairomones) from fasting dragonfly larvae; (3) fasting pond sliders; and (4) fasting red swamp crayfish. To collect dragonfly cues, 100 ml of water was gathered each test day from five randomly chosen rearing tubs and combined in a separate container. For turtle cues, 250 ml of water were extracted from each container just before the experiment and mixed. The same protocol was applied to collect crayfish cues, taking 150 ml of water from each container. The day before collecting the olfactory cues, water was totally changed in each predator container, and no food was provided, ensuring a 24-h fasting period (Polo-Cavia et al., 2010).

All videos were analyzed using ToxTrac, a source executable software designed for image-based tracking (Rodriguez et al., 2018). This software provided detailed locomotor information by recording the x and y coordinates of the central point of each tadpole at intervals of 0.04 s (Fig. 1). To prevent the risk of bias, a blind approach was employed; the operator analyzing the recordings was unaware of the specific chemical stimulus received by each tadpole within the experimental arena.

Since the 1950s, the rate of species introductions on a global scale has increased exponentially (Seebens et al., 2017), and the number of invasive alien species is expected to double in the next decade (Mormul et al., 2022). In this scenario, understanding the potential impact of alien predators on native assemblages is crucial for conservation. Despite its importance, there is currently limited knowledge about the ability of aquatic anuran larvae, including endemic and threatened species, to detect and cope with unfamiliar predators. We investigated the behavioral responses of three sympatric, closely related Rana species. Two of these species, Rana dalmatina and Rana latastei, coexist in the residual forested areas of the intensively cultivated Po River plain, while the third species, Rana temporaria, is widespread in hilly and mountainous regions.

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