Trade-off between aerobic performance and egg production in migratory macaroni penguins

Highlights

• Estradiol-mediated physiology is known to have anti-erythropoietic effects in egg-producing birds.

• In migrating female macaroni penguins, high hematocrit is maintained despite active secretion of E2 and yolk precursors.

• As a measure of new red blood cell production, reticulocyte counts were the highest yet measured in any bird species.

• In penguins producing eggs while simultaneously migrating, a trade-off between hematocrit and aerobic performance is apparent.

• Migratory constraints on E2-pathways, well documented in macaroni penguins, also underlie their extreme egg-size dimorphism.

• This study provides an example of physiological carryover effects.

Abstract

When successive stages of an organism's life-history overlap, conflicts and trade-offs can emerge due to competition among physiological pathways. For example, long periods of sustained locomotion in migrating birds are supported by the androgenic up-regulation of aerobic factors, such as new red blood cell production and hematocrit. However, towards the end of migration, many female birds begin up-regulating 17ß-estradiol (E₂) to support vitellogenesis and egg production, but E₂ secretion is known to have suppressive effects on red blood cell production (anti-erythropoiesis). We explored potential trade-offs between factors related to aerobic performance (hematocrit, reticulocyte index) and the expression of factors related to E₂-mediated vitellogenesis (i.e. yolk precursor production) in female macaroni penguins (Eudyptes chrysolophus), a species in which the physiologies controlling egg production and migratory activity run simultaneously (e.g. females experience a migratory conflict). We collected blood samples from penguins immediately upon their return to the colony, prior to egg laying. Hematocrit was elevated when the penguins returned to the colony (50.05% ± 3.40 SD), which is similar to pre-laying values observed in other migratory bird species. Furthermore, mean reticulocyte levels were elevated (34.87% ± 2.34), which is the highest level yet recorded in birds. Similarly, both plasma vitellogenin and yolk-targeted very low density lipoprotein levels were upregulated (2.30 ± 0.06 μg Zn ml⁻¹, and 9.70 ± 0.19 mmol l⁻¹, respectively), indicating that penguins were reproductively active and producing eggs during migration and upon arrival on land. As predicted, a negative relationship between hematocrit and plasma vitellogenin was found, but we found no evidence to suggest that birds were experiencing reproductive anemia. Alternatively, we attribute the negative relationship to a hemodilution effect of yolk precursor secretion into circulation. It appears that female macaroni penguins are able to preserve hematocrit levels and new red blood cell production when migratory activity overlaps with reproductive processes.

Introduction

Aerobic performance, or the ability to deliver oxygen to tissues, is a critical determinant of an animal's physiological endurance during strenuous activity (Joyner, 1991; Calbet et al., 2006). In migratory birds, aerobic performance is needed to sustain work over long distances and time intervals (Morton, 1994; Piersma et al., 1996), particularly when stopover sites are few or rest periods infrequent. To bolster performance, birds can up-regulate aerobic measures prior to and during migrations to distant breeding territories (Holmgren and Hedenström, 1995). One important component of aerobic performance is hematocrit (Hct), which is the relative proportion of the total blood volume occupied by red blood cells (erythrocytes). Hct is often used as a reliable metric of aerobic performance and oxygen transport capacity, and is up-regulated in several avian species at the onset of migrations (Banerjee and Banerjee, 1977; Morton, 1994; Viscor et al., 1985; Piersma et al., 1996; Landys-Ciannelli et al., 2002; Yap et al., 2019; Minias, 2020). Functionally, Hct up-regulation results in an increase in the concentration of circulating hemoglobin, which is central to the binding and delivery of oxygen to muscles and other tissues. The physiological control of erythrocyte production and Hct up-regulation in birds has been linked to increased testosterone secretion via seasonal hypothalamic-pituitary-gonadal axis (HPG) activation (Wingfield et al., 1990; Tonra et al., 2011a).

The seasonal activation of the HPG axis is required for reproductive development in birds, but it also has some influence on aerobic factors. In response to photoperiodic cues, gonadotropin release stimulates testosterone production in both the testes and ovaries, principally for gamete production, but in male birds especially testosterone can have pleiotropic effects on physiological systems related to migratory preparedness (Wingfield et al., 1990; Gwinner, 1996; Dawson et al., 2001; Tonra et al., 2011a; Tonra et al., 2011b). For example, Tonra et al. (2011b) documented a positive correlation between testosterone and Hct in migratory male redstarts (Setophaga ruticilla - a neotropical migrant species), while also demonstrating a positive relationship between testosterone and breeding phenology: birds with high testosterone (and high Hct) arrived earliest at breeding grounds. The effect of testosterone secretion on erythrocyte production thus seems adaptive, as it suggests that an increase in aerobic performance is a key aspect of migratory preparedness, and thus key to migratory success and fitness. An additional way to gauge the aerobic condition of individuals is via the reticulocyte index (RI; e.g. Wagner et al., 2008), which is the relative number of new red blood cells put into circulation. Collectively, Hct and RI provide two means for assessing individual aerobic and/or migratory condition, at least in males. Indeed, most of our working knowledge about the environmental and physiological control of seasonal phenomena in birds comes from studies of males (Williams, 2012); much less is known about females.

In female birds, seasonal activation of the HPG axis also results in the conversion of testosterone to 17ß-estradiol (E₂). It is well known that E₂ and other estrogens can suppress new red blood cell production (anti-erythropoiesis; Williams et al., 2004; Wagner et al., 2008), which in breeding females can lead to reproductive anemia (Crossin et al., 2010; Williams, 2012). Specifically, estrogens inhibit the differentiation, proliferation, and survival of white and red blood cell precursors at their source of production in the bone marrow (Blobel and Orkin, 1996; Perry et al., 2000). In a study of egg-producing zebra finches (Taeniopygia guttata), Wagner and Williams (2007) experimentally prevented the development of anemia via treatment with the estrogen receptor antagonist tamoxifen. This provided mechanistic support to the idea that E₂-dependent suppression of erythropoiesis is responsible for the reduced Hct and reproductive anemia of breeding female birds (Wagner and Williams, 2007). However, for migratory birds, this presents a potential physiological conflict: how can migrant female birds maintain rigorous aerobic activity while simultaneously up-regulating the E₂-mediated pathway necessary for egg production?

To date, most of what we know about physiological conflicts in migrant female birds has been gleaned from studies of Eudyptid penguins, i.e. those from the genus Eudyptes (macaroni penguins – Eudyptes chrysolophus [Crossin et al., 2010; Crossin and Williams, 2016], and rockhopper penguins – E. chyrsocome [Crossin et al., 2012a]; but see also common eiders, Somateria mollissima [Hennin et al., 2016]). Eudyptid penguins are unique among birds by exhibiting a breeding system in which females lay an invariant 2-egg clutch, characterized by extreme egg size dimorphism (ESD) and obligate brood reduction (Stein and Williams, 2013; Crossin and Williams, 2016) which results in a functional clutch size of one. Such extreme ESD, in which the first laid A-egg is 56–84% smaller than the second laid B-egg (Williams, 1995; Crossin et al., 2013), occurs in no other bird species. Experimental studies with penguins have shown that the rapid yolk development phase of egg production takes ~16 days (e.g. Grau, 1982; Astheimer and Grau, 1985), but the females of some Eudyptid species like the macaroni penguin can arrive and begin laying as few as 7 days later (Williams, 1990; Crossin et al., 2010). ESD is therefore putative evidence of a conflict between migration and reproduction that results from a constraint on the E₂-mediated vitellogenic pathway underlying egg production (Crossin et al., 2010; Crossin et al., 2012b; Crossin and Williams, 2016). In the face of this conflict, how or whether the suppression of estrogenic pathways and their associated anti-erythropoietic effects influences red blood cell dynamics and aerobic state of a Eudyptid penguin is currently unknown. A reasonable prediction is that the suppression of vitellogenesis during migration facilitates or permits new red blood cell production during an aerobically demanding time.

The aim of this study is to explore the relationships between E₂-derived yolk precursors (vitellogenin [VTG], yolk-targeted very low density lipoprotein [VLDLy]) and aerobic measures (Hct, RI). We test the hypothesis that a trade-off between aerobic performance and reproductive investment exists in female macaroni penguins, which would favour aerobic performance when migratory activity and egg production occur simultaneously. We therefore predict that 1) Hct levels measured in migrant penguins arriving at a breeding colony will be at levels typical of other migrating birds (~50% packed cell volume (PCV; Piersma et al., 1996; Landys-Ciannelli et al., 2002; Hennin et al., 2016), and 2) that Hct will be negatively correlated with yolk precursor levels – VTG and VLDLy. Because migratory performance is critical to the at-sea survival and timely arrival of penguins to breeding colonies, we would expect female macaroni penguins to have some means for buffering or decoupling red blood cell production from the suppressive, anti-erythropoietic effects of E₂ secretion (Wagner et al., 2008). The known suppression of vitellogenesis underlying ESD in Eudyptid penguins could thus be the key event that preserves aerobic function at a time when migratory and reproductive processes run simultaneously. If a negative correlation between Hct and VTG or VLDLy is observed, we would then predict that 3) the RI will be elevated in these fast- and far-migrating penguins (Bost et al., 2009), as reduced E₂-mediated processes would permit new red blood cell production, resulting in elevated Hct and RI.