Ticks (Acari: Ixodidae) are hematophagous ectoparasites with a significant impact on both human and animal health through their role as vectors of various pathogens. To complete their life cycle, ticks need to feed on the blood of vertebrates, and in doing so, can transmit a variety of pathogens, including bacteria (e.g., Borrelia, Anaplasma), viruses (e.g., tick-borne encephalitis), and parasites (e.g., Babesia, Theileria) (Baneth, 2014; Boulanger et al., 2019; Brites-Neto et al., 2015; Dantas-Torres et al., 2012). In recent decades, climate change and human-induced land use changes have led to an increase in the abundance and geographical expansion of ticks, particularly in the northern hemisphere (Bouchard et al., 2019; Diuk-Wasser et al., 2021; Sonenshine, 2018; Wikel, 2018). As a result, tick-borne diseases have become more prevalent and widespread, posing a growing public health problem (Rochlin and Toledo, 2020). For example, Lyme borreliosis, caused by Borrelia burgdorferi sensu lato, is the most common tick-borne disease in Europe and the USA (Marques et al., 2021). In Western Europe, the incidence of Lyme borreliosis is increasing, particularly in the northern and central regions (Burn et al., 2023; Vandekerckhove et al., 2021). The disease is also a major health issue in North America, with thousands of new infections reported each year (Dumic and Severnini, 2018; Matuschka and Spielman, 1986; Murphree Bacon et al., 1991; Nelson et al., 2015; Schwartz et al., 2017). Therefore, understanding the factors that influence the feeding preferences of ticks is crucial for developing effective strategies to mitigate the risk of tick-borne diseases.

Unlike insects, ticks do not have antennae as a sense organ. Instead, they use the Haller's organ on the tarsus of their first pair of legs to locate possible hosts and conspecifics (Carr and Salgado, 2019; Lees, 1948). The Haller's organ has olfactory (pheromones, kairomones and CO2), mechanosensory, hygrosensory (humidity) and thermosensory (temperature) functions (Carr and Salgado, 2019). Questing behaviour is usually characterized by a tick positioned on vegetation with its front legs extended upwards. This is done not only to grasp a host, but also to pick up cues from hosts with their Haller's organ. For long-range detection, ticks use infrared radiation, vibrations and host odors (Carr and Salgado, 2019; Carroll et al., 1996; Crooks and Randolph, 2006; Mitchell et al., 2017; Wanzala et al., 2004). When a host is closer, ticks also rely on moisture and less volatile chemical cues (Bezerra-Santos et al., 2024; Crooks and Randolph, 2006). Most studies have focused on finding chemical compounds that either repel (Bissinger and Roe, 2010; Faraone et al., 2019; Grenacher et al., 2001; Romashchenko et al., 2012) or attract (Dallas and Foré, 2013; Romashchenko et al., 2012) ticks, to find a way to reduce contact with ticks and tick-borne pathogens. However, little is known about host-associated attractants. Ticks are known to respond positively to CO2 (Steullet and Guerin, 1992; Van Duijvendijk et al., 2017a) and kairomones (Leonovich, 2004; Osterkamp et al., 1999; Soares and Borges, 2012), both of which are used to detect the presence of a host. Some vector-borne pathogens can alter the odor of a host, making it more attractive to vectors, as described for Plasmodium infections and mosquitos (De Moraes et al., 2014). Van Duijvendijk et al. (2017b) found that Ixodes ricinus nymphs were more attracted to the odor of Borrelia afzelii-infected bank voles (Myodes glareolus), but it was not clear whether infection led to a change in odor or an increase in CO2 production in the host.

Changing environmental conditions affect not only the abundance of ticks and the incidence of tick-borne diseases, but also the communities of their hosts. Habitat fragmentation and degradation, for example, have been shown to induce a stress response in free-ranging vertebrates (Wingfield, 2013). This response is characterized by an immediate increase in catecholamine levels, followed by elevated glucocorticoid levels, such as cortisol or corticosterone, depending on the vertebrate species (Karaer et al., 2023; Madliger and Love, 2014). While short-term elevations of these plasma glucocorticoids are considered adaptive, long-term or chronic elevations can negatively affect individual fitness (Bonier et al., 2009; Breuner et al., 2008; Vitousek et al., 2018). These negative effects include delaying or reducing reproduction, suppressing growth, causing oxidative stress or impairing the immune system, and can therefore make individuals more susceptible to diseases (Costantini et al., 2011; Homyack, 2010). The immunosuppressive effects of chronic stress have been repeatedly demonstrated in mice and birds, the two main hosts of the immature stages of I. ricinus (Domínguez-Gerpe and Rey-Méndez, 2001; Frick et al., 2009; Kiank et al., 2006; Nazar and Marin, 2011). However, an effective immune response is crucial for managing and minimizing the impact of tick infestations and fighting potential infections with tick-borne pathogens, especially since tick saliva contains a cocktail of bioactive molecules designed to modulate host immunity and suppress its inflammatory responses (Chmelař et al., 2016; de la Fuente et al., 2016; Wikel, 2013). If questing ticks can detect stress levels before attaching, they could potentially select hosts with impaired immune function, leading to higher feeding success and shorter feeding times. Evidence from other blood-sucking disease vectors supports this idea; for example, Gervasi et al. (2016) showed that zebra finches (Taeniopygia guttata) with experimentally elevated corticosterone levels were approximately twice as likely to be fed on by mosquitoes (Culex quinquefasciatus). Whether ticks operate similarly remains unclear, as this mechanism in ticks is still poorly understood. Interestingly, in the field, it is often observed that while most individuals carry no ticks, a small number are heavily infested (Devevey and Brisson, 2012), suggesting effects of host identity or some form of host selection be might be at play. Although I. ricinus ticks usually have limited opportunities to choose between hosts and may not be able to reject a host once attached, understanding how physiological factors, such as glucocorticoid levels and infection status, influence host selection and feeding success remains relevant.

To fill the knowledge gap on host preference by ticks, we performed a successive series of in vitro experiments, inspired by the work of Žákovská et al. (2018) on tick preferences for human blood groups. In their approach, drops of each blood type (A, B, AB, or O) were applied to sterile filter paper in a Petri dish, followed by the observation of a tick's movements. This in vitro experimental design offers several advantages: it is simple, time-efficient and eliminates the need for animals, thereby excluding host behaviour as a confounding factor. In a first experiment, we test I. ricinus preference for blood from different hosts (mice, birds, and sheep), both with and without the addition of very low concentrations of ethanol. Ethanol is an essential solvent for glucocorticoids prior to blood spiking and therefore needs an evaluation to rule out any potential repellent properties. To examine the role of stress hormones in host selection, in a second experiment, we explore the ticks' response to blood containing different levels of administered glucocorticoids (cortisol and corticosterone), from 0 to 10, 100 and 1000 ng/ml. To investigate the potential role of vector-borne pathogens in tick feeding choices, a third experiment was designed to assess tick preference for blood infected with Borrelia burgdorferi s.l., taking into account both baseline and elevated stress levels in blood. To control for intrinsic preferences of nymphal ticks for different host species, the second and third experiment were performed on both mouse and avian blood, two main host types for I. ricinus nymphs.