**1. Introduction**

Free-living wild animals are repeatedly exposed to different parasites. These parasites can be found on plants and other animals, in soil and water, and generally throughout the animal's environment [1,2]. When an animal interacts with one or more parasites, that animal's immune system can respond in different ways [3]. For example, some hosts engage in strategies of resistance, while others rely on tolerance [4]. These general strategies combined with myriad immunological defenses form a host's immunological phenotype [4,5]. Qualitative and quantitative differences in aspects of immunological phenotypes have been documented among individuals, populations, and species of animals through space and time [6–10]. As with other plastic phenotypic traits, immunological phenotypes and variation therein are shaped by evolutionary (i.e., genetic) and ecological (i.e., environmental) factors.

Two influential factors are parasite pressure and parasite load [1]. Parasite pressure is largely a characteristic of the broader environment, the specific habitat in which animals live, or both [1]. Exposure to parasite pressure across generations is thought to drive selection and shape immune system evolution, but this has mostly been tested indirectly [1,11]. Parasite load is a trait associated with individual animals [1,12]. The magnitude of parasite load likely depends on parasite pressure and other environmental characteristics, but critically, it also integrates host ecology and immunology [1,11]. Parasite load can be decomposed into several parameters, including the infection status (i.e., presence/absence) and infection intensity. In field studies of wild animals, parasite pressure, parasite load, and host immune defenses are rarely all characterized simultaneously in the same study population.

Knowledge is limited regarding the relative contribution of parasite pressure and parasite load to processes shaping immunological phenotypes in wild animals. Yet understanding these relationships is increasingly important in the light of emerging pathogens that can cause disease in humans [13]. These so-called zoonotic pathogens are often maintained in enzootic cycles by wildlife populations [14], and several zoonotic pathogens have increased in occurrence in recent decades [13]. Of the few studies that have begun to explore ecological immunology in the context of specific pathogens, even fewer study multiple zoonotic pathogens (e.g., [8]). Evaluating relationships among zoonotic pathogens and immune defenses helps in understanding these natural systems and the risks they pose to humans.

We quantified immunological and physiological indices and parasite load parameters in two common species of small rodent from wooded areas in the Netherlands (Supplementary Figure S1) that were previously shown to differ in their parasite pressure (Supplementary Table S1 [15]). Moreover, the bank vole (*Myodes glareolus*) and the wood mouse (*Apodemus sylvaticus*), our two study species, differ in their ability to *host* ticks and to *infect* ticks with zoonotic pathogens [6,16]. Bank voles, but not wood mice, acquire resistance to some ectoparasites (e.g., the tick *Ixodes ricinus* [16]), while wood mice mount a stronger antibody-mediated response against zoonotic pathogens than bank voles [6]. Given our interest in both parasite pressure and parasite load and given that infection with one parasite can mediate infection with another (i.e., via mechanisms of co-infection [17]), we took a holistic "parasite assemblage approach"; however, we also maintained a strong focus on vector-borne microparasites. To this end, we screened rodents for an array of ectoparasites, gastrointestinal parasites, and microparasites. (For a full list, see Supplementary Table S2.) We also characterized the immunological phenotypes of the same individuals via six indices of the immune system and other allied physiological systems.

We formulated two parasite-related hypotheses. First, if parasite pressure drives immunological phenotype, then populations from different sites are expected to express different immunological phenotypes. In general, higher parasite pressure is thought to select for stronger immune systems [1]. Second, if parasite load drives immunological phenotype, then individuals carrying higher parasite loads are expected to express immunological phenotypes that differ from those carrying lower loads, irrespective of population. The direction of this relationship likely depends on the parasite load parameter under consideration, since some members of the parasite assemblage can be immunostimulatory and others immunosuppressive [17]. Additionally, we expected intrinsic host factors to shape immunological phenotype. In the light of the differences between our study species described above, immunological indices are expected to correlate more strongly with microparasite infection status in wood mice compared to bank voles. Furthermore, immunological indices are expected to correlate positively with body mass (a proxy for age in rodents [18]), a result of immune system development.

#### **2. Materials and Methods**
