Discussion
We found that experimental nest parasitism does not elevate circulating
plasma corticosterone in a typically egg-rejecting host of an obligate
avian brood parasite 2 hours following the addition of the model eggs.
However, at the same time point, the expression of the HPA axis-relevant
POMC gene and the stress-related transcription factor ATF3 in the
pituitary was elevated in birds exposed to the non-mimetic model egg
compared to the individuals exposed to the mimetic egg, although these
differences were not significant after false discovery rate correction.
FDR limits the type-I error in datasets with a large number of
comparisons. However, given that we were mainly interested in a few
HPA-axis candidate genes in the pituitary, we also report the
uncorrected p-value, while still acknowledging the possibility of this
result being a false positive. We detected no changes in the immediate
orienting heart rate responses of incubating robins between our model
egg-color treatments.
These results are consistent with a delayed activation of the HPA axis
in response to brood parasitism. In this study, we measured POMC mRNA
levels in the pituitary, not ACTH itself. Although POMC gene expression
can be upregulated within 30 minutes of CRH stimulation (Levin et
al. 1989), translation and posttranslational modification of the POMC
peptide into ACTH likely take time, as POMC needs to be packaged in
vesicles where it is cleaved into daughter peptides (Pritchard & White
2007). Thus, it is possible that a POMC-related corticosterone increase
occurs after our 2-hour mark. Indeed, we found no association between
POMC expression and corticosterone levels at the time of sampling our
subjects. Alternatively, it is possible that at 2 hrs we already missed
a rapid elevation of circulating glucocorticoids following exposure to
the non-mimetic model egg and the p-value based detection of the
increased POMC gene expression data is simply a statistical artifact.
ATF3, which was upregulated in birds exposed to non-mimetic eggs, is
typically upregulated in cells experiencing physiological stress (Haiet al. 1999), although ATF3 expression can also be induced by
psychological restraint stressor (Green et al. 2008). The
upregulation of ATF3 in the pituitary in response to a brood-parasitic
egg thus supports the hypothesis that parasitic eggs are perceived as
stressful by the hosts, although the role of ATF3 expression in the
pituitary is unclear.
In a different study investigating changes in corticosterone in response
to parasitic eggs in a congeneric species (European blackbird) an
increase in baseline corticosterone was detected 24+ hrs following
experimental parasitism with a non-mimetic egg relative to control
treatment (Ruiz-Raya et al. 2018). This suggests that distinctive
parasitic eggs may cause a slow but continuous increase in HPA activity
over multiple hours. Consistent with this interpretation, we found no
difference in the heart rate between the non-mimetic and mimetic egg
treatments immediately following the arrival of the female at the nest.
Heart rate can serve as a rapid, real-time indicator of acute stress
response (Cyr et al. 2009), and the lack of differences in the
heart rate immediately after encountering the egg suggests that
parasitic stimuli do not cause an acute stress response.
A low, but long-term increase in HPA activity may have functional
significance in the context of modulating egg rejection, even if it is
not statistically at a single time point of sampling. For example,
(Abolins-Abols & Hauber 2020a) found that long-term (overnight)
suppression of glucocorticoid synthesis increases the acceptance rate of
parasitic eggs by American robins. If natural glucocorticoid release in
response to parasitic eggs stimulates egg rejection, then minimal but
long-term upregulation of the HPA axis in response to parasitic egg
stimuli is consistent with the timeframe of rejection of parasitic eggs
by American robins. For example, >90% of robins reject
non-mimetic beige cowbird-sized eggs within 5 days (Luro et al.2018), 2 days (Hauber et al. 2019), and even 1 day (Hauberet al. 2020b), but only 33% (7 out of 21) birds rejected the
same non-mimetic egg type within 2 hours in this study (also see Scharf
et al., 2019). However, individual variation in glucocorticoid levels in
this study did not predict egg rejection: circulating corticosterone at
our single time-point of sampling did not differ between females who
accepted or rejected the non-mimetic beige eggs (Fig. 3).
The few steroid-focused endocrine studies on physiological responses to
parasitic egg stimuli so far thus paint a complicated picture. On one
hand, experimental studies now show that glucocorticoids (Abolins-Abols
& Hauber 2020a) can mediate egg rejection. On the other hand, the HPA
axis may only show weak gradual activation in response to exposure to
brood parasitic eggs and may have no discernible effect on egg
rejection. It is possible that egg rejection by brood parasite hosts may
be affected only by pronounced changes in the HPA activity (such as
those due to experimental manipulation of hormone levels or intense
stressors) and not by weak HPA activation in response to brood
parasitism. Furthermore, the relationship between stress physiology and
egg rejection may be species-specific. For example, in incubating
prothonotary warblers (Protonotaria citrea ), a non-rejecter host
species, experimental parasitism with either non-mimetic or mimetic eggs
had no effect on glucocorticoid levels relative to non-parasitized
controls (Scharf et al. 2021). On the other hand, in yellow
warblers (Setophaga petechia ), a species that frequently but
variably abandons its nest if parasitized by brown-headed cowbirds,
individuals with higher circulating corticosterone levels were more
likely to desert an experimentally parasitized nest relative to
non-parasitized controls (Turcotte-van de Rydt et al. 2022).
Methodologically, especially when studying freely behaving wild animals,
we are still limited by our inability to measure the minute-by minute
dynamics in glucocorticoid synthesis and release, as well as our
inability to accurately assess long-term subtle changes in
glucocorticoids (and other hormones) that could cause a behavioral
change. To integrate our findings into a unified framework, future
experiments should characterize the full-time course and magnitude of
HPA axis activation in response to brood parasitism across acceptor and
rejector species. Additionally, dose-response studies, such as those
inducing small and short-term changes in hormone levels (e.g., Vitousek
et al. 2018) are necessary to test the sensitivity of host behavior to
hormones. Finally, future studies must expand beyond the corticosterone
paradigm and test the effect of other hormones, such as prolactin, on
host behaviors (Abolins-Abols & Hauber 2018; Ruiz-Raya et al.2021), as well as stress-response at the cellular level (Ruiz-Rayaet al. 2022). Together, these studies will allow us to more fully
understand the significance of stress signaling in the ecology and
evolution of host defenses.