The hypothalamic-pituitary-interrenal (HPI) axis is centrally implicated in stressor mitigation in teleost fishes. Sustained HPI axis activation can be detrimental to the physiological functioning of an organism and can result in fitness-related trade-offs. Predator-induced mortality is known to be higher in stressed fish than in unstressed conspecifics, suggesting a role for the HPI axis in mediating fish behaviour. However, the underlying specific mechanism(s) for this phenomenon is(are) unknown. The purpose of the current study was to address how the HPI axis influences risk-taking, and antipredator behaviours in a wild teleost, the pumpkinseed sunfish (Lepomis gibbosus). Here, individual juvenile pumpkinseed were implanted either with cocoa butter as a sham control or with a biologically-relevant concentration of cortisol. Forty-eight hours post-implantation, fish were assessed for behavioural metrics associated with boldness and risk taking in three sequential behavioural tests: (i) a predation-risk test, (ii) an exploration tendency test, and (iii) a shoaling tendency test, with test order randomized among different trials. Cortisol treatment had no influence on antipredator, exploratory, or shoaling behaviours. However, post-attack swimming duration (in predation-risk test) and exploratory activity (in Z-maze exploration test) were significantly affected by body mass. Collectively, our results indicate that cortisol may not have a role in mediating sociability, boldness, and risk-taking behaviours in pumpkinseed sunfish, at least under the current laboratory conditions. However, cortisol may nonetheless play a role in mediating predator-prey interactions in fishes in more natural environmental settings that were not considered here.
Fish collection and holding conditions
Juvenile pumpkinseed sunfish (mass = 8.4 ± 0.2 g; total length = 81.4 ± 7.5 mm) were haphazardly collected using a hand seine in the shallow nearshore reaches of Lake Opinicon, Ontario, Canada (44.5590° N, 76.3280° W) during June and July 2017 (under Ontario Ministry of Natural Resources permit #1086180). Capture sites were always of the same habitat type, which consisted of a muddy bottom with short vegetation interspersed with woody debris. This was done to avoid potential confounds with specific behavioural phenotypes being associated with habitat type (e.g. Kobler et al., 2011; Wolf and Weissing, 2012). Seine netting was the preferred capture method to avoid any potential biases in the selection of specific personality types (i.e. angling; see Wilson et al., 2011; Gutowsky et al., 2017). Collected fish were transferred to an indoor holding tank (~212 l) at the nearby Queen's University Biological Station (Chaffey's Lock, ON, Canada) and held overnight prior to receiving a cocoa butter implant. Here, fish were maintained on a flow-through circulation (23.82 ± 0.3 °C; O2 > 90% saturation) with independent aeration under a seasonally-appropriate illumination cycle (15 h L: 9 h D). A subset of the captured fish were retained in a large, free-floating net pen (1.3 × 1.3 × 1.1 m) situated in the lake. These fish were used solely as stimulus conspecifics in the shoaling tendency test (see below). All experimental procedures received prior approval of the Carleton University Animal Care Committee (AUP's #104262 & #104281) and therefore are consistent with the guidelines for the care and use of research animals of the Canadian Council on Animal Care and the laws of Canada.
Hunger state commonly influences foraging and risk-taking decisions in teleost fishes (e.g. Godin and Smith, 1988; Gotceitas and Godin, 1991; Godin and Crossman, 1994). Therefore, fish were not fed during the holding period and experimental trials. Test fish were subjected to the implantation of either cocoa butter (5 ml kg−1 body weight [BW]) containing suspended cortisol (hydrocortisone 21-hemisuccinate; 25 mg kg−1 BW) or a sham implant (i.e. no cortisol). Cocoa butter-containing implants are a common and validated means by which cortisol can be elevated in the circulatory system of teleost fishes over semi-chronic durations (Gamperl et al., 1994; Sopinka et al., 2016). We selected the aforementioned dosage based on previous validation work with this species (Lawrence et al., unpubl. data). Here, cortisol levels in experimental fish were on average higher over the first 48 h following implantation (~67 ng ml−1 and 19 ng ml−1 for 24 h and 48 h cortisol-treated fish, respectively) than in sham-control fish (~14 ng ml−1 and 8 ng ml−1, respectively). Preparation of the cortisol-treated cocoa butter followed the methods of Hoogenboom et al. (2011). Fish were selected haphazardly from a pool of available fish and assigned to a treatment group. The order of which fish were implanted with the cortisol or sham treatment was alternated on a daily basis to avoid possible biases in fish selection. Cocoa butter implants were injected intraperitoneally in the fish's abdomen, at a site just posterior to the pelvic fins, using a 1 ml syringe tipped with a 16 G needle. Following implantation, individual fish were immediately transferred to a blacked-out holding chamber (~2.6 l) that was maintained on a flow-through of fresh, aerated lake water (McConnachie et al., 2012). Animals were held in these individual blacked-out (darkened) chambers for 48 h prior to behavioural testing to allow the administered cortisol to reach biologically active concentrations in their blood (McConnachie et al., 2012). Sham and cortisol-treated fish had comparable mean body masses (sham = 8.8 ± 0.3 g, cortisol = 8.1 ± 0.3; t = 1.608, DF = 50, P = 0.114) and total lengths (sham = 82.2 ± 9.9 mm, cortisol = 86.7 ± 11.0 mm; t = 1.029, DF = 50, P = 0.308). Water conditions in the experimental arenas were maintained at >90% O2 saturation and 23.59 ± 0.1 °C.