In the face of a changing world, there has been increasing interest in the behavioural and physiological responses of wild animals to stressors. Many factors can influence stress responsiveness, but two that have not been extensively studied during the stress-induced phase are environmental complexity and the presence of conspecifics. Using wild pumpkinseed sunfish (Lepomis gibbosus (L., 1758)) collected from limnetic and littoral sites, we tested whether glucose and cortisol were affected by environmental complexity and the density of conspecifics during the period of maximum response following a standardized air stressor. Overall, environmental complexity and conspecific density did not have a significant effect on maximum stress. However, in the environmental complexity experiment, fish collected from the littoral site had significantly higher concentrations of maximum glucose and cortisol, and tended to have higher glucose and cortisol responsiveness, than limnetic fish. This indicates that although the collection site did not affect a fish’s baseline values, intraspecific variation in site use is associated with divergent sensitivity of the hypothalamic–pituitary–interrenal axis to stressors. The importance of capture location on maximal response from stressors represents a potential sampling bias and source of variation, and may be even more pronounced in species that are habitat specialists.
Environmental complexity experiment
This study was performed from 26 June through 2 July 2014 at the Queen's University Biological Station located approximately 50 km north of Kingston, Ontario, Canada. Working from a 6 m research vessel, pumpkinseeds were collected by angling from Lake Opinicon, Ontario, Canada (44[degrees]56'N, 76[degrees]33'W), from both limnetic and littoral locations. Locations were alternated to ensure fish were sampled in each location during both morning and afternoon to remove diurnal confounds on Cortisol concentrations. Surface water temperature throughout the 9-day period remained fairly stable (25-28 [degrees]C) and mean temperatures were similar between habitats (littoral mean of 26.4 [degrees]C, limnetic mean of 25.8 [degrees]C). All fish were captured using small pieces of dew worm on barb less hooks. Only fish that were between 150 and 238 mm in length, hooked in the jaw, and showed no evidence of bleeding or other significant angling-related injury were included. All fish were fought for 10 s to standardize the capture stressor. Upon landing, the fish underwent a 3 min standardized air stressor. During the air-exposure period, a baseline blood sample was obtained using a 1 mL heparinized syringe and a 25 gauge, 11/2 inch (38.1 mm) needle. Approximately 0.2-0.3 mL of blood was taken from the caudal vasculature, directly posterior to the anal fin. Any fish that was not bled within the 3 min air-exposure period was excluded from the experiment, because, in general, increases in Cortisol concentrations are only detectable in teleost fish after that time (Pankhurst 2011); specifically, in pumpkinseeds, samples collected under 3 min represent baseline values (S.J. Cooke, unpublished data). Once the 3 min had elapsed, fish were introduced into one of four treatment tanks. All treatments took place in 68 L Rubbermaid bins (64.77 cm x 44.45 cm x 39.05 cm) filled to 30 cm with water collected from the lake that was replaced after every trial. The four treatments were (1) barren (a bin with just water), (2) a bin with cobble substrate (approximately 2.5 cm deep covering the bottom of the bin), (3) a bin with cobble substrate and approximately 50% vegetative cover collected from the lake (a combination of water milfoil (genus Myriophyllum L.) and pond-weed (Potamogeton L.)), and (4) a bin with cobble substrate and approximately 90% vegetative cover. A lid with holes was placed on top of the bin and the assigned fish was left for 42 min (45 min total from start of air exposure). During that period, water temperatures remained stable and disturbance (e.g., boat motor noise, talking) was minimized. Working with the congeneric bluegill, Cook et al. (2012) reported that Cortisol concentrations peaked at that time period. Following the 42 min, the fish were removed and a second blood sample was taken. If this second sample was not completed within 2 min, then the fish was removed from the study. This second blood sample represented maximum Cortisol and glucose concentrations, whereas the first blood sample represented baseline concentrations.
Prior to being returned to the lake, total length (to the nearest millimetre) was recorded and the caudal fin was clipped in case of recapture. All blood samples were kept in the capped syringes and placed in an ice-water slurry for no longer than 2 h. Whole-blood glucose levels were read using a handheld glucose reader (Accu-Chek Compact Plus blood glucose meter; Roche Diagnostics) that had been previously validated for use on fish (Stoot et al. 2014). Blood samples were then centrifuged at 2000g for 5 min (Fisher Scientific Mini Centrifuge) and the resulting plasma was pipetted into two separate 1.5 mL screw-cap vials. Vials were kept in liquid nitrogen until they could be transferred to a -80 [degrees]C freezer where they remained until analysis.
Conspecific density experiment
This study was also performed at the Queen's University Biological Station from 3 July through 6 July 2014. Pumpkinseeds were collected using angling from Lake Opinicon from both limnetic and littoral locations as noted above. Surface water temperature throughout the 4-day period remained fairly constant (24-25 [degrees]C) and mean temperature was similar between habitats (littoral mean of 25.1 [degrees]C, limnetic mean of 24.5 [degrees]C). Blood sampling and sample processing was identical to the environmental complexity experiment, but here environment was standardized and only fish density was manipulated. Bins were left empty of substrate and vegetation (barren) and were filled to 30 cm with lake water. The three treatments were (1) no conspecifics, (2) one conspecific, and (3) five conspecifics that varied in length between 125 and 218 mm. Baseline and maximum glucose and Cortisol samples were collected as above.