Introduction
Obligate avian brood parasites lay their eggs in the nests of
heterospecific hosts, imposing substantial fitness costs: parasitized
host broods typically experience a lower hatching rate (Hauber 2003a),
higher host chick mortality (Hauber 2003b), and/or delay in the foster
parents’ future reproduction (Mark & Rubenstein 2013). In turn, many
host species have evolved behavioral defenses against parasitic eggs.
Among the most effective and common host defenses is the rejection of
the foreign egg from the nest before it hatches (Rothstein 1982; Davies
& Brooke 1988; Moksnes et al. 1991). While we know much about
the behavioral and sensory ecology and evolution of egg rejection
(Davies & Brooke 1988; Feeney et al. 2014; Soler 2014, 2017),
the mechanistic basis of this host defense is still poorly understood
(Abolins-Abols & Hauber 2018; Ruiz-Raya 2021). A mechanistic
understanding of a phenotype can offer a unique perspective into the
evolution and ecology of organisms (Ketterson et al. 2009;
Rosvall 2013). In particular, understanding the physiological basis of
egg rejection may explain one of the most puzzling observations in
host-parasite ecology, which is why the propensity to reject parasitic
eggs is highly variable both among species (Stokke et al. 2005;
Krüger 2007) and between individuals of the same species (Luro & Hauber
2017).
While variation in egg rejection across species may be explained by
phylogenetic inertia and other evolutionary constraints (Rothstein 1975;
Medina & Langmore 2016), most of these hypotheses cannot predict why
the likelihood of egg rejection varies between populations (Davies &
Brooke 1989; Soler & Møller 1990; Briskie et al. 1992), among
individuals (Grim et al. 2014; Hauber et al. 2020a, 2020c)
and within individuals over time (Ruiz-Raya and Soler, 2017, 2020).
Intraspecific variation in egg rejection is particularly puzzling
because this behavior has an obvious fitness benefit and typically
incurs comparatively low costs (e.g., the possible rejection of own eggs
(Lotem et al. 1995; Ruiz-Raya & Soler 2017)). Some of the
intraspecific variation in egg rejection can be explained by shifts in
the sensory-perceptual environment of the nest (Honza et al.2011; Rutledge et al. 2021), detecting of an adult brood parasite
adult near the nest (Davies & Brooke 1988; Moksnes & Røskaft 1989), or
previous experience with parasitic eggs (Hauber et al. 2006).
Variation in egg rejection may also be linked to the host life-history
stage (Ruiz-Raya & Soler 2017; Zhang et al. 2021). Many of these
hypotheses treat egg rejection in isolation from the rest of the
phenotype. However, components of egg rejection behavior may share
underlying endocrine mechanisms with related behaviors (e.g.,
aggression, maternal behavior, nest defense (Abolins-Abols & Hauber,
2018)), suggesting that we need to study egg rejection within the rich
context of other physiological and behavioral responses of the
individual to its environment (Ruiz-Raya & Soler 2020; Ruiz-Raya 2021).
One of the recently emerged hypotheses about the mechanistic basis of
egg rejection suggests that it is mediated by the endocrine stress
response (Abolins-Abols & Hauber 2018; Ruiz-Raya et al. 2018;
Abolins-Abols & Hauber 2020a). For example, Ruiz-Raya et al. (2018)
showed that parasitism with non-mimetic model eggs elevated baseline
corticosterone levels in European blackbird (Turdus merula )
females. By experimentally suppressing glucocorticoid synthesis,
Abolins-Abols and Hauber (2020a) demonstrated that corticosterone also
mediates egg rejection in the congeneric American robin (T.
migratorius ). Specifically, female robins with suppressed
glucocorticoid synthesis were less likely to reject non-mimetic model
eggs. Combined, these findings imply that brood parasitic egg stimuli
may induce a stress response which then modulates the egg rejection
decisions and behaviors. Alternatively, the stress response may prime
the host physiologically and cognitively to recognize and reject the
foreign egg, instead of directly inducing the egg rejection response.
The emerging findings that steroid hormones play a role in response to
parasitic eggs in hosts (see also Hahn et al., 2017; Hauber et al.,
2020a) is important not only from a mechanistic perspective but also
from an evolutionary one. Glucocorticoids are hormones that impact
multiple aspects of the phenotype, including stress response,
metabolism, and immune response (Williams 2008; MacDougall-Shackletonet al. 2019). Selection on any of these or other
glucocorticoid-mediated aspects of the phenotype may cause population-
or individual-level variation in glucocorticoid levels (Ketterson &
Nolan 1999), thus also driving variation in egg rejection. In turn, the
glucocorticoid-mediated response to brood-parasitic stimuli may drive
variation in other aspects of the phenotype. For example, stress-induced
corticosterone secretion can suppress maternal investment (Angelier &
Chastel 2009; Angelier et al. 2009). This suggests that
glucocorticoid-mediated responses to parasitic stimuli may induce a
critical trade-off between host defenses and maternal behavior, as
suggested by Abolins-Abols & Hauber (2018).
While the “stress-mediated egg rejection” hypothesis has been
supported by a number of studies (Ruiz-Raya et al. 2018;
Abolins-Abols & Hauber 2020a; Ruiz-Raya et al. 2021), its role
in driving individual variation in egg rejection is yet to be fully
understood. For example, experimental suppression of glucocorticoid
results in a lower propensity for egg rejection (Abolins-Abols & Hauber
2020a); however, contrary to the predictions of the stress-mediated egg
rejection hypothesis, natural circulating corticosterone in the same
species shows a negative association between baseline corticosterone and
the propensity for egg rejection (Abolins-Abols & Hauber 2020b). To
understand if stress response can drive egg rejection decisions in
diverse contexts and across varied timeframes, we need to understand the
multifaceted physiological responses of hosts to parasitic eggs.
For example, response to stressful stimuli is considerably more complex
than a simple read-out of corticosterone levels. Corticosterone is
regulated by adrenocorticotropic hormone (ACTH) secreted from the
anterior pituitary (Romero & Butler 2007), where it is encoded by the
proopiomelanocortin (POMC) gene. The POMC peptide is
post-translationally cleaved to produce a variety of signaling peptides,
including ACTH. ACTH secretion from the anterior pituitary is regulated
by hypothalamic hormones, such as corticotropin releasing hormone (CRH)
and arginine vasotocin (Romero 2006). The stimulation of the pituitary
by CRH results in a rapid release of secretory vesicles containing ACTH
(Deng et al. 2015; Harno et al. 2018), which, in turn,
stimulates rapid glucocorticoid secretion from the adrenal cortex
(Romero & Butler 2007). While it is clear that glucocorticoids play an
important role in regulation behavior and physiology, it is important to
note that other components of the hypothalamic-pituitary-adrenal (HPA)
axis may have glucocorticoid-independent extra-adrenal effects on
behavior (e.g. ACTH: Brain and Evans, 1977; Gallo-Payet, 2016; Miller
and Ogawa, 1962). Furthermore, the activation of the HPA axis is only
one aspects of a multifaceted response to stressful stimuli. Immediate
response to stressors is enabled by catecholamine release (the
fight-or-flight response), which is regulated by the sympathetic and
parasympathetic branches of the autonomic nervous system (Romero &
Gormally 2019). Among effects on behavior, catecholamine release results
in an elevated heart rate (Cyr et al. 2009). Importantly, while
heart rate increases in response to stressors, heart rate may be lowered
during orienteering response (Hauber et al. 2002).
Here we tested the hypothesis that parasitic egg stimuli induce a stress
response in an egg-rejecter host, the American robin. Robins are
parasitized by a generalist brood-parasite, the brown-headed cowbird
(Molothrus ater ). While most robins (>90%) reject
natural or model cowbird eggs (Rothstein 1982; Luro et al. 2018),
some individuals accept natural cowbird eggs and hatch parasitic
offspring (pers. obs.). Furthermore, variation in egg rejection is
repeatable in robins (Luro & Hauber 2017), making this species a great
model system in which to test the mechanisms underlying variation in egg
rejection. We experimentally parasitized robin females with mimetic
(robin-like shell color) or non-mimetic (cowbird like) eggs (Fig. 1) and
measured the three aspects of host physiology in response to the model
eggs: heart rate, corticosterone levels, and POMC expression in the
pituitary. We predicted that, compared to host females receiving a
mimetic egg, females exposed to the parasite-like model egg color
treatment would show lower heart rates (i.e., orienting response),
greater circulating corticosterone, and higher POMC expression.