We found that the presence of the great tits drives a hormonal, as well as behavioural, response in gregarious desert locusts. The desert locusts spent twice as much time under the shelter and limited the time of feeding by 60% when a real bird and its alarm call were presented to them, compared to controls. Levels of stress hormones in the CNS and haemolymph were significantly higher in the presence of the real bird compared to the birds call and control treatments. Our experiment thus provides additional information, based on the neurochemical analysis, on the interaction among the level of stress hormones and behaviour in insects, despite the casual mechanism for behaviour is not studied. It is also in line with our expectations and would mean that fear of birds might result in lower feeding rate, slower growth, and maybe lower reproduction thus shaping evolution of interactions between insects and plants (Fournier et al. 2013).
Against expectations (Minoli et al. 2012) the alarm call of great tits was not an appropriate signal to induce hormonal and behavioural change in gregarious desert locusts. The time devoted to hiding and feeding during the continuous alarm calling of great tits was not significantly different from the control treatments. Several alternative explanations are possible. The birds singing and calling are nearly ubiquitous in nature and the cost of hiding permanently would be high. The production of stress hormones is known to change the prey's behaviour and prevent its predation (Adamo et al. 2013), but a permanently occurring level of stress hormones could be debilitating to the animal (Clinchy et al. 2013) It might be also speculated that a warning call is usually used by birds in response to bird’s predators, which might mean that the bird is less likely to continue the search for prey and can be evaluated as less dangerous by the insect. Unlike bat vocalisation used to locate prey, the bird calls may not represent an immediate risk of attack (Cinel and Taylor 2019). Further, it might be because desert locusts did not perceive the great tit as a potentially risky predator, but they were afraid of silhouette or movement as birds are generally predators of locusts. This would however suggest that locusts recognize the specific call of great tit but generalise the silhouette. Our additional experiments which we present in the Supplementary material (as Additional experiment IV) then showed, that locusts reacted less fearfully to a warning Call of kestrel than to a live Bird (i.e., Great tit), while their reaction to a Call of great tit and a Call of kestrel did not differ. Thus, despite the kestrel might represent a more dangerous predator to them, the presence of an actual bird might be needed for the development of a strong stressful reaction.
The final explanation might lay in the locust's ability to hear. Locusts have a well-developed auditory system that is able to determine a wide range of pitch. Desert locusts show extreme phenotypic plasticity forming, solitary and gregarious phases, which differ extensively in behaviour, physiology including their sensory abilities, and morphology (Simpson et al. 1999; Gordon et al. 2014). In previous experiments, gregarious forms of desert locusts responded in 6 out of 12 cases to locust swarm sounds by an escape (Haskell 1957) and took evasive action after hearing ultrasound calls of hunting bats (Weber et al. 1981). Yet, Gordon et al (2014) found differences in the audition of both forms of locusts. Solitarious locusts fly at night (Ould Ely et al. 2011), and hence are potentially at much greater risk from predation by bats (Haskell 1957; Robert 1989) which seems to be in line with their sensitivity to the higher frequencies used by bats in their echolocation calls (Gordon et al. 2014). On the other hand, gregarious locusts are more active during the day and so they should need a stronger detection of birds. However, our current observation that the desert locusts did not react to bird alarm calls cannot support this hypothesis. Our additional experiment which we present in Supplementary material (as Additional experiment IV) then showed, that locusts reacted the same way to quiet Control, as well as to the Call of great tit and Rattling of keys. However, they reacted a bit more fearfully to the Call of kestrel. This proves that they can distinguish individual types of sounds but evaluated the Call of great tit as harmless as the Rattling of keys.
Existing studies have revealed that various arthropods have variable responses to different types of predator-induced stimuli. Lohrey et al. (2009) found that spiders were freezing when a seismic or acoustic stimulus was presented, and they increased movement when they were exposed to a visual stimulus. Desert grass spider Agelenopsis aperta exhibited antipredator behaviour to puffs of air simulating bird wing beats (Riechert and Hedrick 1990). Some crickets were shown to respond to species-specific vibrations that lizards made when walking (Adamo et al. 2013). Even the placement of a dummy predator (robotic hamster) in the terrarium resulted in changes in behaviour of the crickets (Adamo et al. 2013). In another experiment, the mere presence of spiders with their mouths glued shut changed the behaviour of the grasshoppers (similarly to predation treatment, where the spiders were allowed to eat them), which resulted in the grasshoppers acquiring less food, which in turn decreased grasshopper populations (Schmitz et al. 1997). The indirect effects of insectivorous arthropods on arthropods have been further proved through experiments in several systems: mantids and their predation on herbivorous insects (Moran and Hurd 1997), the effect of large predatory mosquitoes on smaller mosquitoes (Chandrasegaran and Juliano 2019), the effect of Anolis lizards on Homoptera and Araneae (Spiller and Schoener 1990a; b), the effect of beetle larvae on ants (Letourneau and Dyer 1998), the effect of the sound of wasps on Lepidoptera larvae (Lee et al. 2021). Orthoptera specifically was shown to be able to detect risk by the air particle movement generated by the predator wing beat due to the filiform hair sensilla (Gnatzy and Kämper 1990).
However, birds have been rarely used as the source of fear in terrestrial experiments with insects, although they are one of their main predators. Signals of bird presence are visual (whole animal, part of an animal, or just its shadow), localized disturbance (e. g. vibration, movement of leaves, air movement), or different types of vocalizations. The perceived signals might be dependent on the distance and duration in which the arthropod and predatory bird interact. The dimensions of our birdcage in the indoor conditions were only 0.7 x 0.4 x 0.5 m, so the laboratory experiment was intense, as the locusts were in close contact with the bird, and they could see its movement. In contrast, the locusts in outdoor aviaries were not in close contact with the birds, as the birds typically moved 1–2 m far from the locusts. Pitt (1999) used 5 x 5 m large aviaries protecting grasshoppers from birds and observed that grasshoppers were significantly more often lower in vegetation but did not change their mobility and feeding behaviour. In other avian-free treatments, free-living birds were not able to access the locusts closer than 2 metres, while they had free access to the locusts in the control treatment. The activity of locusts tended to be lower in the control plots; however, the pattern varied over the summer season. Similarly, to the earlier study, the locusts tended to forage deeper in the grass and did not climb so high, when birds were present (Belovsky et al. 2011).
Another crucial feature in the experiment could be the duration of the exposure of the prey to predators. Our 30-minute laboratory experiment (with a predator being close) and 3 hours-long outdoor experiment (with a predator) resulted in similar levels of stress hormones. In general, a 30 min long exposure to predators was sufficient to detect the hormonal stress response. This would be in line with an earlier study, which found that the concentration of AKH in the haemolymph of S. gregaria changes and increases with the time of exposure to the stress factor but appears as quickly as in 2 minutes and peaks after 60 minutes (Candy 2002).
The link between various components of the stress response system and behaviour controlled by the nervous and endocrine systems is far from a proper understanding (Storey 2004; Johnstone et al. 2012). Nevertheless, in arthropods, biogenic amines and certain neurohormones seem to play a crucial role in the control of stress response (Kodrik 2008; Nelson et al. 2021). It is obvious that these amines are involved directly in the defence reactions, e.g., octopamine modulates anti-predator behaviour in beetles (Tribolium castaneum) (Nishi et al. 2010) and an orb-weaving spider (Jones et al. 2011). On the other hand, biogenic amines are thought to be involved in regulation of AKH production (Van der Horst et al. 2001) or the AKH release from the corpora cardiaca into haemolymph (Orchard et al. 1993). There are many examples in the literature describing fluctuations in AKH levels in the insect CNS and haemolymph after a stressor application. For example, 20 minutes of forced running (in a horizontal laboratory shaker) resulted in a slight increase of AKH titre in the CNS of the firebug, Pyrrhocoris apterus, but in a strong increase of the titre in the haemolymph (Kodrík and Socha 2005). A similar picture can be seen when applying various pathogens (Ibrahim et al. 2017; Ibrahim et al. 2018; Gautam et al. 2020a; Gautam et al. 2020b) or toxins including insecticides (Candy 2002; Kodrík et al. 2015). Changes in the AKH levels in the haemolymph are often faster, while in the CNS are slower but more profound. Nevertheless, in this study, all changes in Schgr-AKH-II titre after the bird treatment were similar except for the no change of Schgr-AKH-II in the haemolymph within the external experiment. To explain this, we can only speculate that due to the large distance of the locusts from the birds, the stress was not strong enough, as mentioned above.
In conclusion, we report evidence that insects in the presence of birds decreased their foraging activity, reducing their food income roughly by 60%, and we relate the results of behavioural tests to a physiological stress response. Our experimental results are in line with the risk allocation hypothesis (Lima and Dill 1990; Lima and Bednekoff 1999) predicting that animals should increase their foraging effort in the low-risk environment and decrease foraging in high-risk environments. We argue that the methodological approach combining behavioural experimentation with the physiological assessment of animal responses should be crucial for insight into fear perception in animals and for understanding their fitness optimization strategies. Last, future tests should focus on the effect of bird calls on the behaviour of other insect species to extend the information gained by our study.