369 The Journal of Experimental Biology 216, 369-378 © 2013. Published by The Company of Biologists Ltd doi:10.1242/jeb.073023 RESEARCH ARTICLE Low-O2 acclimation shifts the hypoxia avoidance behaviour of snapper (Pagrus auratus) with only subtle changes in aerobic and anaerobic function Denham G. Cook1, Fathima I. Iftikar2, Daniel W. Baker2, Anthony J. R. Hickey2 and Neill A. Herbert1,* 1Leigh Marine Laboratory, The University of Auckland, Leigh, Warkworth 0941, New Zealand and 2School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand *Author for correspondence ([email protected]) SUMMARY It was hypothesised that chronic hypoxia acclimation (preconditioning) would alter the behavioural low-O2 avoidance strategy of fish as a result of both aerobic and anaerobic physiological adaptations. Avoidance and physiological responses of juvenile snapper (Pagrus auratus) were therefore investigated following a 6week period of moderate hypoxia exposure (10.2–12.1kPa PO2, 21±1°C) and compared with those of normoxic controls (PO2=20–21kPa, 21±1°C). The critical oxygen pressure (Pcrit) limit of both groups was unchanged at ~7kPa, as were standard, routine and maximum metabolic rates. However, hypoxia-acclimated fish showed increased tolerances to hypoxia in behavioural choice chambers by avoiding lower PO2 levels (3.3±0.7 vs 5.3±1.1kPa) without displaying greater perturbations of lactate or glucose. This behavioural change was associated with unexpected physiological adjustments. For example, a decrease in blood O2 carrying capacity was observed after hypoxia acclimation. Also unexpected was an increase in whole-blood P50 following acclimation to low O2, perhaps facilitating Hb–O2 off-loading to tissues. In addition, cardiac mitochondria measured in situ using permeabilised fibres showed improved O2 uptake efficiencies. The proportion of the anaerobic enzyme lactate dehydrogenase, at least relative to the aerobic marker enzyme citrate synthase, also increased in heart and skeletal red muscle, indicating enhanced anaerobic potential, or in situ lactate metabolism, in these tissues. Overall, these data suggest that a prioritization of O2 delivery and O2 utilisation over O2 uptake during long-term hypoxia may convey a significant survival benefit to snapper in terms of behavioural low-O2 tolerance. Key words: preconditioning, Sparidae, oxygen affinity, metabolism, swimming, activity, haemoglobin, mitochondria, respiration. Received 26 March 2012; Accepted 20 September 2012 INTRODUCTION undoubtedly help to ensure the survival of fish facing hypoxic The prevalence of low oxygen (environmental hypoxia) has conditions (Herbert et al., 2011; Poulsen et al., 2011). Some fish increased in coastal regions (Diaz and Rosenberg, 2008), and thus engage these strategies before encountering their respective Pcrit it is now more important than ever to understand how different fish (Herbert and Steffensen, 2005; Poulsen et al., 2011), but the New species will respond to environmental change. Because hypoxia Zealand snapper (Pagrus auratus, Sparidae) does not show these presents a significant metabolic challenge for most aquatic animals, responses (Cook et al., 2011). Snapper are late to leave hypoxic physiological adaptations to chronic hypoxia generally involve water because low O2 avoidance commences below their Pcrit (Cook enhancing the capacity for O2 uptake and delivery (Richards, 2009). et al., 2011). Moreover, snapper do not show any significant change For example, many fish species studied to date increase red blood in swimming speed during hypoxic exposure (Cook et al., 2011). cell numbers and haemoglobin (Hb) concentrations to boost O2 The snapper used in this study are presumed to have never carrying capacity (Wells, 2009; Wells et al., 1989), restructure gill experienced low O2, raising the possibility that behavioural morphologies to enhance gas exchange (Sollid et al., 2003), increase responses and resulting physiological impacts could differ with Hb–O2 binding affinities to increase O2 uptake (Wood and Johansen, previous exposure as a result of a loss of naivety and/or acclimatory 1972; Wood et al., 1975) or even modify cardiac function to improve responses (i.e. physiological adaptation). low O2 performance (Petersen and Gamperl, 2010). When combined, To date, the effects of hypoxic acclimation on behaviour of fish cellular and tissue modifications should enhance the whole animal’s in low O2 conditions remain largely untested. Moreover, little direct capacity to tolerate hypoxia, and be evident as a lowering of the evidence identifies how adaptations associated with hypoxia critical O2 tension (Pcrit), which defines the partial pressure of O2 preconditioning influence fish behaviour. Therefore, the present (PO2) above which basal metabolic demand (maintenance) is study aimed to resolve whether the behavioural avoidance and satisfied (Timmerman and Chapman, 2004). swimming speed response of snapper would differ after acclimation Fish engage an array of physiological mechanisms to combat low to long-term hypoxia. The physiology of snapper was also oxygen, but may also use behavioural strategies to counter investigated in detail using both novel and commonly applied deleterious effects (Richards, 2009). This is particularly true for measures of aerobic and anaerobic physiology to gauge how species that have a limited capacity to adapt physiologically physiological changes (cellular, organ and whole animal) integrate (Pichavant et al., 2003). Reductions in locomotory activity and early with behavioural responses. We hypothesised that snapper would avoidance of low O2 represent two notable behaviours that would show one of two responses. Firstly, long-term hypoxia could THE JOURNAL OF EXPERIMENTAL BIOLOGY 370 The Journal of Experimental Biology 216 (3) convey a degree of low-O2 experience to fish whereby they are actively move between these two flows via a square port (10×10cm) simply less naive and employ a more cautious avoidance strategy positioned centrally on the divider. Diffusers and baffles were used −1 well above Pcrit limits. In this scenario, snapper would adjust their to create rectilinear flow in the BA (4800lh combined flow rate) behavioural strategy and simply avoid low O2 earlier without any and each side of the BA received water from a large (400l) gassing major changes in physiology. Alternatively, long-term hypoxia could tower. PO2 on either side of the BA was therefore manipulated by provide greater low-O2 tolerance across a number of physiological purging nitrogen gas (BOC Gas Supplies), or compressed air, levels (e.g. improved Hb–O2 transport potential). In this scenario, through the towers. Oxygen set-points were controlled by two fish might remain in hypoxia and avoid even lower levels of O2, Oxyreg units (Loligo Systems, Tjele, Denmark) coupled to a DAQ feasibly without enlarged levels of low O2 stress. The present study device (miniLAB 1008, Measurement Computing, Norton, MA, therefore set out to resolve how physiological and behavioural USA) under the control of Labview software (v. 8.6, National changes might interact following chronic hypoxia. Instruments, Austin, TX, USA). The behavioural activity of fish in the choice box was quantified using a digital camera (Fire-I, MATERIALS AND METHODS Unibrain, San Ramon, CA, USA) that streamed video to a PC Fish handling and acclimation procedures running ‘Swistrack’ software (Correll et al., 2006). The movements Juvenile Pagrus auratus (Forster 1801) (Sparidae; common name of fish were sampled at a rate of 15Hz and were used to resolve snapper or red bream; 150–300g) were captured by line and avoidance behaviour and activity variables (including swimming barbless hooks from coastal waters around Leigh (36°19′S, speed and spontaneous turning rates). Water temperatures within 174°48′E, Northland, New Zealand). Following capture, fish were the choice chamber were actively maintained at 21.0±0.3°C (mean housed in one of two 500l tanks (maximum of 50 individuals per ± 95% CI). tank) at the Leigh Marine Laboratory. Fish were provided with a Fish were transferred to the BA at least 18h prior to continuous flow of high-quality, aerated seawater for at least experimentation. Following this period, behavioural variables were 6weeks before experimentation. After this period, one tank was determined over a 1h control period, during which any preference designated for hypoxia acclimation and the other as a normoxic for side and normoxic swimming activity was determined. During control. A reduced PO2 (10.2–12.1kPa) was maintained in the this 1h control period, all fish formed a strong preference for one hypoxia tank using an oxygen controller (Model PR514, PR particular water flow (i.e. >80% of time spent on one particular side Electronics, Rønde, Denmark) and an Oxyguard oxygen electrode of the divider) and excursions into the alternate channel were (Mini probe, Technolab, Mornington, Tasmania, Australia) infrequent and never in excess of 15s. This behavioural routine positioned in the centre of the holding tank. The PO2 level of formation presented investigators with the opportunity to ‘drive’ 10.2–12.1kPa was deemed a significant level of hypoxia for this fish from their preferred side and identify clear avoidance thresholds species because it reduces their aerobic scope by >50% at lower (Cook et al., 2011). Following the 1h control period, the preferred experimental temperatures (Cook et al., 2011). An on/off relay channel was deoxygenated progressively