A Comparison of Techniques Measuring Stress in Birds

Abstract

Free-living birds are subjected to both external and internal stresses which can affect their health, activity, and reproductive success. To study stress in free living birds, they must be captured in nets and handled by the researcher to take blood samples for commonly used measures of stress, an activity which itself can induce stress and confound results. This study compares the effects of handling time on three different measures of stress: levels of the stress hormone corticosterone (CORT), levels of Heat Shock Protein 60 (HSP 60) and the ratio of heterophils to lymphocytes (H/L ratio) in tufted titmouse (Baeolophus bicolor) captured at feeders between December and January (2011-2013) in Fredericksburg, VA. Blood samples collected between two and 15 minutes from 12 birds were assayed for levels of CORT and HSP and from 24 birds for H/L ratios. Relationships were examined between these stress indicators and handling time, body mass and body condition. CORT was significantly correlated with handling time (p<0.01), which reinforces existing evidence of CORT’s sensitivity to the way subjects are handled immediately prior to blood collection. HSP or H/L ratios were not affected by handling time, suggesting that they may be preferable indicators of stress in free living birds under some circumstances. INTRODUCTION Free-living birds face a variety of internal and external sources of stress, which may affect physiological function and reduce fitness. Acute stress results from a specific stressful event, such as an attack by a predator or sudden storms, whereas chronic stress results from prolonged exposure to biologically challenging conditions, such as exposure to extreme temperatures (Vleck et al. 2000), periods of limited food availability (Herring et al. 2011), and anthropogenic pressures such as pollution, habitat disturbance (Arriero et al. 2008; Busch and Hayward 2009), and from prolonged psychosocial stressors (Cyr et al. 2007; Cyr and Romano 2007; Landys et al. 2011). To Corresponding author: dodell@umw.edu 1 Virginia Journal of Science, Vol. 65, No. 3, 2014 http://digitalcommons.odu.edu/vjs/vol65/iss3 134 VIRGINIA JOURNAL OF SCIENCE cope with such stressors, birds and other animals have a protective physiological stress response that allows them to withstand immediate threats to their homeostatic balance. When this response is elevated chronically, however, it can become biologically costly and have negative impacts on birds’ fitness by weakening the immune system (Dabbert et al. 1997), which could increase susceptibility to disease, and compromising growth and reproduction (Sapolsky et al. 2000). Thus, stress levels can indicate the general physiological condition of birds and point to possible environmental perturbations. Biomarkers such as the glucocorticoid corticosterone (CORT), heterophil/lymphocyte ratios (H/L), and heat shock proteins (HSPs) have all been used as tools to assess chronic or long-term stress in wild populations. These markers may be predictably regulated according to environmental conditions and various biological challenges (Gross and Siegel 1983; Sapolsky et al. 2000; Vleck et al. 2000; Moreno et al. 2002; Martinez-Padilla et al. 2004; Tomas et al. 2004; Davis 2005; Herring and Gawlik 2007; Busch and Hayward 2009; Cockrem et al. 2009; Krams et al. 2010; Herring et al. 2011), and thus can provide researchers with consistent methods of detecting stress experienced by birds in their natural habitats. However, they may also be affected to varying degrees by acute stress caused by capture and handling leading up to blood sample collection. Therefore, drawing inferences about birds’ long-term stress status prior to their capture by researchers may be problematic since the relationships between the different measures used have not been adequately studied. Quantifying glucocorticoids, such as CORT, is presently the most frequently applied method of assessing individual stress in birds (Sapolsky et al. 2000; Tomas et al. 2004; Herring and Gawlik 2007). However, the release of CORT is highly influenced by acute stress associated with capture and handling during field research which may confound results. Capture and handling can rapidly mobilize CORT, which can make the interpretation of CORT measurements difficult under some circumstances (Sapolsky et al. 2000; Romero and Reed 2005; Fridinger et al. 2007; Herring and Gawlik 2007; Busch and Hayward 2009; Cockrem et al. 2009). When handling time before sampling lasts for more than 2 or 3 minutes, CORT levels may no longer accurately reflect birds’ physiological status before their capture (Romero and Reed 2005; Cockrem et al. 2009). In studies involving free-living birds, field conditions may prevent sufficiently prompt blood collection, leading to less reliable measures of chronic stress. Additionally, fecal CORT, can degrade over time in frozen samples (Herring et al. 2007) which reduces reliability of measurements. Herring and Gawlick (2007) compared the use of CORT with that of HSPs as ways of measuring stress associated with allostatic overload (when the energy requirements needed to maintain homeostasis in the body exceed the capacity of the animal) and concluded that HSPs have some advantages over CORT and may represent a viable supplementary or even alternative indicator of chronic stress. Circulating H/L ratios have also been used to measure chronic stress in birds. These ratios are used because the avian immune response to stress takes significantly longer to initiate, by hours to days, than the rapid CORT response, and changes in leukocyte numbers last longer than changes in CORT levels (Davis et al. 2008). Their slower response to stress and longer endurance indicate that H/L ratios may be informative, especially in obtaining baseline stress measurements. However H/L levels are impacted by disease and infection and may not reflect true levels of stress to external stressors. While studies on Adélie penguins (Vleck et al. 2000) and house finches (Davis 2005) Virginia Journal of Science, Vol. 65, No. 3, 2014 http://digitalcommons.odu.edu/vjs/vol65/iss3 MEASURING STRESS IN BIRDS 135 indicate that H/L ratios are resistant to handling up to 1 hour, a study investigating wintering male great tits (Cirule et al. 2012) found that acute stress due to capture and handling caused an increase in heterophil counts between 30 and 60 minutes and a decline in lymphocyte counts between 60 and 120 minutes after capture. Therefore, H/L changes may be species specific and may change more rapidly than originally thought, which may limit the reliability of results in a way similar to CORT. A different cellular response to stress is mediated by HSPs. HSPs are a family of proteins whose expression is increased when cells are exposed to both cellular stressors such as parasites (Merino et al. 1998; Martinez-Padilla et al. 2004; Arriero et al. 2008; del Cerro et al. 2010), limited food availability (Zulkifli et al. 2002; Herring et al. 2011), and sibling competition (Martinez-Padilla et al. 2004; Merino et al. 2006) as well as psychosocial stressors such as crating in birds (Zulkifli et al. 2009), fear (AlAqil et al. 2013) and social interactions in fish (Currie et al. 2009). They have been found to exist in almost all organisms, including bacteria, plants, and animals (Feder and Hofmann 1999). HSPs are a special class of proteins referred to as molecular chaperones which protect proteins from degradation and correct damage caused by stress-induced instability (Merino et al. 1998; Feder and Hofmann 1999; Tomas et al. 2004; del Cerro et al. 2010). Essentially, they serve to restore and maintain cellular homeostasis during times of increased stress (Tomas et al. 2004; del Cerro et al. 2010). These molecules may be reliable indicators of chronic stress because they are maintained at high levels for longer periods after stressors are applied, and some research has shown that handling stress does not cause their rapid up-regulation (Martinez-Padilla et al. 2004; Herring and Gawlik 2007; Herring et al. 2011). However, the resistance of HSPs to acute stress triggered by capture and handling has not been systematically compared to those of CORT and H/L ratios in the same study. The tufted titmouse (Baeolophus bicolor) is a good model species to expand research on the relationships between these three stress indicators and their relative sensitivities to handling time. Close relatives of the tufted titmouse, the blue tit and the great tit, have been used in past studies examining both the intracellular and hormonal stress responses (Arriero et al. 2008; Cockrem et al. 2009; del Cerro et al. 2010). The tufted titmouse is a canopy-dwelling, omnivorous permanent resident species of eastern North American deciduous forests (Grubb and Pravosudov 1994). During the winter, they spend time in flocks of about 2 to 5 individuals. Caching food during cold weather, they commonly frequent feeders and carry 1 seed at a time to store within 40 meters of the feeders, allowing them to be easily captured with mist nets (Grubb and Pravosudov 1994). This study compared the sensitivity of HSP60, CORT, and H/L to acute stress induced by the capture and handling of tufted titmice to evaluate their reliability as tools in avian stress research. The relationships between CORT, H/L ̧ and HSP60 and the time elapsed between capture of subjects and blood collection, as well as the relationships among these bioindicators, were analyzed. We predicted that CORT levels, but not H/L ratios or HSP60 levels, would be positively correlated with handling times longer than 2 minutes after capture of subjects. Finally, we examined whether CORT, H/L, and HSP values were correlated within subjects and related to body condition. Virginia Journal of Science, Vol. 65, No. 3, 2014 http://digitalcommons.odu.edu/vjs/vol65/iss3 136 VIRGINIA JOURNAL OF SCIENCE MATERIALS AND METHODS Capture, Handling, Blood Samp