Sex differences in behaviour, morphology, and physiology are widespread in nature, with most dioecious animals thought to present at least some degree of sexual dimorphism. The brain – the centre of the nervous system in many organisms – is an example of one of these sexually dimorphic structures as it may differ in size, function and gene expression [1–3]. Sex differences (or sexual dimorphism as is usually termed) in the brain is being increasingly investigated using humans and laboratory animals. Although in wild species these topics have been considerably less explored, recent evidence suggests sex differences in neural organisation and gene expression in the brain [4–9].
With evidence suggesting the importance of functional and structural differences in the male and female brain, it is possible that complementary processes such as disease risk and its defence response may also differ between the sexes. Indeed, sex differences are found in neuronal diseases such as Parkinson disease, where males exhibit a greater reduction in global cognition and language than females, or in Alzheimer disease, presenting women with faster rates of brain atrophy than males [10, 11]. Understanding the causes of these differences are important, since many diseases – including the recent COVID-19 – have different mortality rates in males and females, and ultimately, producing sex-biased mortalities in several organisms [12, 13]. Sex differences in microglial and astrocytic cells, such as their heightened sensitivity to inflammatory stimuli and their anatomical distribution [14–18], have been postulated to mediate sex differences in cognition and memory in rodent models [19, 20]. However, research addressing the main system responsible for inflammatory responses and pathogen defence, i.e. the immune system, in the brain of wild animals is widely lacking, despite showing important differences in various immune parameters between the sexes [21–23].
In the nervous system, immune function seems to be under particularly intense modulation since an insufficient response may result in infection, but an excessive response could result in prolonged inflammation and tissue damage . Also, in tissues like the brain, general immune factors seem to serve a variety of non-immunological functions [25, 26]. Furthermore, many variables may influence the immune response, including biotic and abiotic factors or the combination of both, such as seen in birds in the tropics that seem to upregulate aspects of their immune function in the wet season, presumably as defence mechanism against increased pathogen pressure that emerges from increased rainfall [27, 28].
The Kentish plover (Charadrius alexandrinus) is a small shorebird that is emerging as an ecological model system of sexually dimorphic reproduction and speciation [29, 30]. Kentish plovers are widely distributed along coastal and inland waterbodies across Eurasia and North Africa . Previous studies of Kentish plovers have found that males generally survive better than females [32, 33]. Though the causes of this female-biased mortality in Kentish plover are still unknown, previous studies addressing parasite burden suggest that males and females share comparable infection rates of blood parasites and pathogenic bacteria [34–36].
Here we investigate immune aspects in the male and female brain of Kentish plovers in two contrasting environments in China: the Bohai Bay located on the East coast, and the Qinghai Lake located inland at high elevation in the Qinghai-Tibetan Plateau. We focus on expression patterns of immune system genes, since a healthy immune system in the brain is essential for protecting the animal against infection and maintaining cognitive ability [37–40]. We quantify the expression of genes annotated with immune functions in four brain regions (hypothalamus, medial extended amygdala, nucleus accumbens and septum), with the aim of evaluating sex differences in immune genes . We test two specific hypotheses. First, based on sex differences in gene expression in the brain in various bird species [e.g. 7, 8, 9] and survival demographic analyses of Kentish plover, we expect increased upregulation of immune genes in the male brain. Despite both Bohai Bay and the Qinghai Lake being stopovers of the East Asian-Australasian Flyway and the Central Asian Flyway, respectively, these environments show contrasting differences that could determine patterns of pathogen exposure, namely increased interaction with waterbird and marine diversity in the coastal site, versus the high-altitude inland site. Because local environmental factors such as pathogen pressure, can shape immune function [e.g. 42], we expect differences in immune gene expression between the sites, with a possible upregulation of immune genes in the coastal population.