Metabolic and functional impacts of hypoxia vary with size in Atlantic salmon

Comparative Biochemistry and Physiology, Part A 231 (2019) 30–38

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This article is a part of the special issue on aquaculture

Metabolic and functional impacts of hypoxia vary with size in Atlantic salmon☆

T

Tina Oldhama,b, , Barbara Nowakb, Malthe Hvasa, Frode Oppedala ⁎

a b

Institute of Marine Research, Matredal, Norway Aquatic Animal Health Group, Institute for Marine and Antarctic Studies, University of Tasmania, Australia

ARTICLE INFO

ABSTRACT

Keywords: Salmo salar Aquaculture Stress Critical swimming speed Oxygen Swim tunnel respirometry Metabolic scaling Cortisol Lactate

The most capricious environmental variable in aquatic habitats, dissolved O2, is fundamental to the fitness and survival of fish. Using swim tunnel respirometry we test how acute exposure to reduced O2 levels, similar to those commonly encountered by fish in crowded streams and on commercial aquaculture farms, affect metabolic rate and swimming performance in Atlantic salmon of three size classes: 0.2, 1.0 and 3.5 kg. Exposure to 45–55% dissolved O2 saturation substantially reduced the aerobic capacity and swimming performance of salmon of all sizes. While hypoxia did not affect standard metabolic rate, it caused a significant decrease in maximum metabolic rate, resulting in reduced absolute and factorial aerobic scope. The most pronounced changes were observed in the smallest fish, where critical swimming speed was reduced from 91 to 70 cm s−1 and absolute aerobic scope dropped by 62% relative to the same measurement in normoxia. In normoxia, absolute critical swimming speed (Ucrit) increased with size, while relative Ucrit, measured in body lengths s−1, was highest in the small fish (3.5) and decreased with larger size (medium = 2.2). Mass specific metabolic rate and cost of transport were inversely related to size, with calculated metabolic scaling exponents of 0.65 for bSMR and 0.78 for bMMR. Metabolic O2 demand increased exponentially with current speed irrespective of fish size (R2 = 0.97–0.99). This work demonstrates that moderate hypoxia reduces the capacity for activity and locomotion in Atlantic salmon, with smaller salmon most vulnerable to hypoxic conditions. As warm and hypoxic conditions become more prevalent in aquatic environments worldwide, understanding local O2 budgets is critical to maximizing the welfare and survival of farmed and wild salmon.

1. Introduction For the ecologically and commercially important Atlantic salmon (Salmo salar, hereafter salmon), both effective conservation and efficient farming require understanding the physiological consequences of exposure to hypoxic conditions. In their natural habitat, the lifecycle of anadromous salmonids necessitates crossing through coastal and estuarine habitats prone to hypoxia, with dissolved O2 levels in spawning streams recorded as low as 16% saturation (Sergeant et al., 2017). Similarly, all environmental examinations of salmon aquaculture cages, spanning studies in Canada, Norway and Australia, have detected hypoxic conditions (Bergheim et al., 2006; Burt et al., 2012; Dempster et al., 2016; Johansson et al., 2007, 2006; Oldham et al., 2018; Oppedal et al., 2017, 2011; Solstorm et al., 2018; Srithongouthai et al., 2006; Stehfest et al., 2017; Wright et al., 2017). A two week study in Norway found that 25% of all dissolved O2 measurements collected throughout

a salmon cage were below levels known to reduce feed intake and growth (Solstorm et al., 2018). In a more extreme example, hypoxic conditions were consistently present throughout an entire two month summer study period in two Australian salmon cages, reaching a minimum of 21% saturation and forcing the fish to remain within a 2 m depth band to survive (Dempster et al., 2016; Stehfest et al., 2017). For salmon, all activities including locomotion, digestion, growth and reproduction, are primarily fuelled by energy generated through aerobic metabolism - a process reliant on O2 availability (Claireaux and Chabot, 2016). As such, the difference between the maximum rate of aerobic metabolism (MMR) and resting metabolic rate, termed aerobic scope for activity (AAS), is a valuable tool for examining the consequences of stress and environmental disturbance (Claireaux and Lefrançois, 2007; McKenzie et al., 2016). O2 demand, however, varies with many factors. In normoxia, the MMR of salmon is largely dictated by temperature (Hvas et al., 2017a;

This article is part of a special issue entitled: Aquaculture, edited by: Dr. Tillmann Benfey, Dr. Inna Sokolova and Dr. Mike Hedrick Corresponding author at: Institute of Marine Research, Matredal, Norway. E-mail address: [email protected] (T. Oldham).

☆ ⁎

https://doi.org/10.1016/j.cbpa.2019.01.012 Received 12 September 2018; Received in revised form 19 January 2019; Accepted 21 January 2019 Available online 25 January 2019 1095-6433/ Crown Copyright © 2019 Published by Elsevier Inc. All rights reserved.

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Wood et al., 2017). However, as dissolved O2 availability declines MMR also declines while SMR remains unchanged until the limiting O2 saturation (LOS) required for basic life support is reached, leading to reduced AAS (Norin and Clark, 2016; Richards et al., 2009). As AAS declines, energetic constraints force fish to minimize all activities nonessential to immediate survival, including feeding and growth (Burt et al., 2013; Eliason and Farrell, 2016; Remen et al., 2016a). Studies of healthy, 300–500 g salmon found that at 19 °C dissolved O2 levels as high as 77% saturation led to reduced feed intake, while LOS was as high as 55% (Remen et al., 2016a, 2013). The O2 requirements of sick or active individuals are even higher (Brett, 1964; Hvas et al., 2017b). In addition to environmental conditions, O2 requirements also vary with size. The metabolic rate of animals increases with body mass according to the power function:

diameter of 36 cm, providing a volume of 252 L. An opening at the rear of the swim section allowed for removal of fatigued fish with minimal disturbance to any fish still swimming. The volume of the entire system was 1905 L. Water currents were generated by a motor-driven propeller with speed, in rotations per minute, controlled through a frequency converter. An acoustic Doppler velocimeter, mounted at the rear of the swim section, was used to find the relationship between propeller speed and current speed in cm s−1. To maintain temperature and dissolved O2 levels, flushing of the tunnel was performed by opening a water inlet valve opposite the swim section which received water from the same reservoir as the holding tanks. A camera and O2 logger (RINKO ARO-FT, JFE Advanced Co., Ltd., Japan) were mounted at the rear of the swim section to allow monitoring with no disturbance to the fish. The respirometer was sterilized prior to beginning trials, and at the experimental mid-point (day 9). No noteworthy background O2 consumption could be detected when the setup was empty, and bacterial respiration was therefore considered negligible.

Y = aM b where Y is the metabolic rate, ɑ is a scaling constant, M is mass and b is a scaling exponent (log-log slope). The ‘metabolic level boundaries hypothesis’ predicts that b varies systematically among species according to environmental and lifestyle factors, and is inversely related to an organisms metabolic activity level (Glazier, 2010; Glazier, 2005). The logic is that in athletic species with higher maintenance costs (due to increased muscle mass, respiratory surface area and heart size) the scaling of metabolic rate with body size is limited by flux of waste and resources across surfaces (b ≈ 2/3), while in less athletic species with lower maintenance costs, surfaces are not limiting and metabolic rate is directly proportional to body mass (b ~ 1) (Killen et al., 2010). Therefore, in athletic species such as migratory salmonids, it is expected that smaller individuals would have a higher mass-specific metabolic demand than larger individuals (Killen et al., 2010), and therefore be more vulnerable to hypoxia (Nilsson and Ostlund-Nilsson, 2008). Despite the physiology of salmon being well-studied, whether or not the metabolic and performance impacts of hypoxia vary throughout the large post-smolt size range remains unknown. To examine the metabolic and locomotor consequences of hypoxia in salmon, we subjected replicate groups of post-smolts of three sizes (0.2 kg - small, 1.0 kg medium and 3.5 kg - large) to a standard Ucrit protocol in moderately hypoxic and normoxic conditions at 16 °C while measuring O2 uptake. We hypothesized that swimming performance of all sizes would be reduced in hypoxia owing to reduced aerobic capacity, but that the small fish would be most severely affected as a result of metabolic scaling.

2.3. Experimental protocols The afternoon prior to measurements, fish were transferred from the holding tank to the swim tunnel by hand net. During an overnight acclimation period of 15 h, water current speed was set to ~ 0.3 BL s−1 with moderate flushing to maintain stable temperature and dissolved O2 levels. After acclimation, the respirometer was sealed and dissolved O2 levels were allowed to drop for 4 h until 45% saturation was reached. The tunnel was then flushed to reach either 95% (normoxic) or 55% (hypoxic) dissolved O2 saturation, at which point the swim trial commenced. During normoxic trials dissolved O2 was maintained at ≥85% saturation, whereas in hypoxic trials dissolved O2 was maintained between 45 and 55% saturation. The critical swimming speed (Ucrit) was determined using a typical swim challenge protocol (Brett, 1964; Plaut, 2001). Water current velocity was increased stepwise, by approximately 0.3 BL s−1 every 20 min, until all fish were fatigued. Flushing was performed during the first 5 min of each speed, after which the system was closed for O2 uptake rate measurements. Ucrit was determined for every individual. Fatigue was defined as when fish were lying against the rear grid of the swim section and were unable to swim despite tactile stimulation. Once fatigued, fish were rapidly removed from the tunnel and euthanized with a blow to the head. Fork length and mass, as well as current speed and time at fatigue, were recorded for every fish (Table 1). In the small and medium groups, blood samples were immediately drawn from the caudal vein of the first and last two fish fatigued in each swim trial with a heparinized syringe and stored on ice until transfer to −80 °C. Due to difficulty fatiguing the large fish, blood samples were only collected from two large fish which reached fatigue in each dissolved O2 treatment. Control blood samples were collected from 9 to 10 fish of each size class following sedation with Finquel MS-222 (10 mg L−1) to minimize handling stress. Dissolved O2 measurements were recorded continuously, every 2 s, throughout all trials. Group numbers for each size class were chosen so that total O2 uptake rates were initially similar in all groups and high enough to provide a reliable trace within the test interval (small = 27–31, medium = 7–10, large = 2). Three replicate tests of each size class were completed in each dissolved O2 treatment, except large, normoxic of which there were only enough fish for two replicates (Table 2).

2. Materials & methods 2.1. Animals Hatchery-reared Atlantic salmon post-smolts were maintained in circular indoor tanks (5.3 m3 volume) at the environmental laboratory of the Institute of Marine Research, Matre, Norway in full-strength filtered and ultra-violet light treated seawater (34 ppt) under a natural photoperiod. A constant inflow of 150 L per minute from a large, thermally regulated reservoir ensured high dissolved O2 levels, removal of waste products and stable temperatures. Salmon of all sizes were maintained at 16 °C for at least one month prior to swim tunnel trials. Fish were fed size-appropriate commercial feed through automated feeding devices. 2.2. Swim tunnel setup

2.4. Calculations

Because salmon frequently utilize a burst-glide gait when swimming anaerobically, we chose to use a large swim tunnel respirometer to evaluate swimming performance of salmon in groups, rather than individually (Remen et al., 2016b). The Brett-type respirometer we used is described in detail by Remen et al. (2016b) and Hvas et al. (2017b). Briefly, the swim section of the tunnel was 248 cm long with an internal

Oxygen uptake rates (MO2) were calculated as the slope of a linear regression of dissolved O2 saturation through time during each closed period, which was then used to calculate the average mass-specific MO2 of the group. To correct for the volume occupied by the fish in the tunnel, a density of 1 kg L−1 was assumed. SMR was estimated by 31

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Table 1 Sample size (N) and mean ± SE of the length, mass and critical swimming speeds (Ucrit) of each size class and dissolved O2 treatment. Because not all large fish reached fatigue, only an approximate Ucrit is provided for those groups. Hypoxic = 45–55% dissolved O2 saturation; Normoxic ≥85% dissolved O2 saturation. Control fish were sedated and sampled directly from holding tanks for determination of baseline plasma parameters. Small

N Length (cm) Mass (g) Ucrit (cm s−1)

Medium

Large

Hypoxic

Normoxic

Control

Hypoxic

Normoxic

Control

Hypoxic

Normoxic

Controlᶧ

91 25.8 ± 0.12 199 ± 3 70 ± 0.7a

88 26.0 ± 0.16 205 ± 4 91 ± 0.7b

10 27.9 ± 0.52 272 ± 11.6 –

23 45.6 ± 0.71 981 ± 51 89 ± 4.9a

28 45.0 ± 0.74 949 ± 53 98 ± 3.4b

10 48.9 ± 1.14 1309 ± 81 –

6 67.3 ± 2.2 3852 ± 322 ≥101

4 64.4 ± 1.2 3371 ± 185 ≥124

9 61.4 ± 1.3 3176 ± 177 –

Statistical differences between treatments within each size class are indicated by superscript letters (Tukey post hoc test, p < .05) ᶧ Sambraus et al. (2018).

exponentially fitting MO2 as a function of steady swimming speeds and extrapolating back to speed zero (Fig. 2; Brett, 1964; Hoar and Randall, 1978). MMR was defined as the highest MO2 measured, which in all cases coincided with the first current speed at which fish began to fatigue. Absolute and factorial aerobic scope (AAS, FAS) were calculated as the difference or factor between MMR and SMR, respectively. The metabolic size scaling exponents were estimated as the slope of least squares linear regressions of the log10 transformed mean SMR and MMR, respectively, and mass of each size class in normoxic conditions (Fig. 1). Ucrit was calculated for each individual according to Brett (1964):

Ucrit = Uf +

3.5 bMMR= 0.78 Log10 (MO2 ,mgO2 h-1)

3

2.5 bSMR= 0.65 2

1.5

t fUi ti

1

where Uf is the last completed current speed, tf is the time spent at the current speed of fatigue, ti is the time interval at each current speed and Ui is the magnitude of each speed increment. The gross cost of transport at each swimming speed, expressed as mgO2 kg−1 km−1, was calculated by dividing MO2 by the corresponding swimming speed. To determine the speed at which cost of transport was lowest (CoTmin), mean gross cost of transport was plotted against all aerobic swimming speeds for each size class and fit with a quadratic function (Fig. 4). The swimming speed with the minimum cost of transport, and thus greatest metabolic efficiency, was determined by finding the vertex of the parabola.

2

2.5

3 Log10 (mass, g)

3.5

4

Fig. 1. – Scaling relationships between log10 transformed MMR (circles), SMR (triangles) and mass (M) of Atlantic salmon in normoxia. Each data point is mean MO2 and mass from group respirometry. Results are MMR (r2 = 0.99): log10MO2 = 0.78(log10M) + 0.40, and SMR (r2 = 0.97): log10MO2 = 0.65(log10M) + 0.13.

For each blood sample, 1 mL blood was centrifuged at 3000 g for 5 min, and the resulting plasma stored at −80 °C until further processing. Plasma lactate concentrations were measured spectrophotometrically with a MaxMat PL, while cortisol concentration was quantified with an ELISA assay kit (IBL International GmbH).

while one-way ANOVA's were used to test for differences in length, weight and plasma parameters between dissolved O2 treatments within each size class. Because the Ucrit data were not normally distributed, a two-way ANOVA was performed on the rank transformed Ucrit values as a function of dissolved O2 treatment and size. When ANOVA detected significant differences, a post hoc Tukey's HSD test was used to determine which groups differed. All data were evaluated for normality and homoscedasticity by the Shapiro-Wilk and Levene's tests, respectively. When appropriate, a Bonferroni correction was applied to account for multiple comparisons, with a significance level of α = 0.05 for all tests. Data presented are mean ± SE unless otherwise stated.

2.6. Statistical analyses

3. Results

Statistical analyses were performed in R version 3.2.0 (www.r-project. org). Metabolic parameters (SMR, MMR, AAS and FAS) were evaluated as a function of dissolved O2 treatment and size using two-way ANOVA's,

Length, weight and density measurements did not differ statistically between the hypoxic and normoxic treatments in any of the size classes (Table 1).

2.5. Stress parameter analyses

Table 2 Sample size (N) and mean ± SE of absolute aerobic scope (AAS) and factorial aerobic scope (FAS) as measured by group respirometry. Hypoxic = 45–55% dissolved O2 saturation; Normoxic ≥85% dissolved O2 saturation. Small

N AAS (mgO2 kg−1 h−1) FAS (AMR/SMR)

Medium

Large

Hypoxic

Normoxic

Hypoxic

Normoxic

Hypoxic

Normoxic

3 230 ± 7a 2.31 ± 0.15a

3 607 ± 23b 4.07 ± 0.17b

3 250 ± 25a 2.40 ± 0.27a

3 397 ± 7b 4.03 ± 0.03b

3 140 ± 27a 2.72 ± 0.48a

2 365 ± 12b 6.31 ± 0.96b

Statistical differences between treatments within each size class are indicated by superscript letters (Tukey post hoc test, p < .05) 32

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3.1. Aerobic capacity

3.3. Stress response

In normoxia, MMR was highest in the small fish and decreased with larger size (799 ± 31, 536 ± 16 and 438 ± 2 mgO2 kg−1 h−1, respectively). MMR was significantly reduced in hypoxia, by > 120 mgO2 kg−1 h−1 in all sizes, while SMR (small = 196 ± 4, medium = 133 ± 4, large = 71 ± 11 mgO2 kg−1 h−1) was unchanged. The calculated scaling exponents based on the relationships between SMR/MMR and size are bSMR = 0.65 ± 0.04 and bMMR = 0.78 ± 0.05 (Fig. 1). The combination of stable SMR with reduced MMR in hypoxia resulted in reduced AAS and FAS in all size classes (Table 2). AAS was reduced the least in the medium size class (41%), and similar amounts in the large (63%) and small (62%) size classes (Table 2). In normoxia, mean FAS ranged from 4.03 ± 0.03 to 6.31 ± 0.96, but was reduced in hypoxia to less than three in all sizes (Table 2).

In all sizes, plasma lactate concentrations were significantly elevated after normoxic swim trials relative to controls, and significantly higher again after hypoxic swim trials compared to normoxic (Table 3). In hypoxia, plasma lactate was lowest in the large fish (8.1 ± 0.19 mmol L−1), but there was no significant difference between the small (22.2 ± 1.4 mmol L−1) and medium fish (21.2 ± 1.7 mmol L−1) (Table 3). Plasma cortisol was elevated after swim trials compared to controls in all sizes, but hypoxic and normoxic treatment had no effect on plasma cortisol in the small and medium fish (Table 3). In the large fish which were exhausted, however, plasma cortisol was lower at the end of the normoxic trials (385 ± 22 ng mL−1) than hypoxic (732 ± 20 ng mL−1). 4. Discussion Acute exposure to moderate hypoxia, well above the estimated critical O2 level of 35% saturation at 16 °C (Remen et al., 2016a), significantly reduced aerobic metabolic capacity in salmon ranging from 200 g to 3.5 kg, causing subsequent declines in swimming performance irrespective of size (Fig. 3, Table 1). The nature and implications of the reduced aerobic capacity resulting from hypoxia exposure, however, varied with size.

3.2. Swimming performance MO2 increased with swimming speed in all sizes (Fig. 2). In normoxia, absolute Ucrit increased with size from 91 ± 0.7 cm s−1 in small fish (N = 88) to 98 ± 3.4 cm s−1 in medium fish (N = 28). For the large fish, two reached fatigue at 92 and 127 cm s−1 in normoxia, while two exceeded the maximum current speed the tunnel could generate of 140 cm s−1. Therefore, while we could not determine a specific Ucrit for the large fish in normoxia, it exceeded 124 cm s−1. Hypoxia reduced Ucrit significantly, by 21 cm s−1 (N = 91) in the small fish and 9 cm s−1 (N = 23) in the medium (Fig. 3, Table 1). Even in hypoxia, one of the large fish exceeded 140 cm s−1, while Ucrit of the other five were 81, 85, 95, 101 and 102 cm s−1, for an approximate average of > 101 cm s−1 (Fig. 3). Relative Ucrit decreased with increasing size (Fig. 3). Cost of transport was highest in the small fish at all measured speeds, and lowest in large fish (Fig. 4). The absolute speed of CoTmin in normoxic conditions was highest in large fish (67 cm s−1) and decreased marginally with size to 60 cm s−1 in the medium fish and 52 cm s−1 in the small fish. Relative speed of CoTmin was highest in the small fish (2.0 BL s−1) and decreased with size (medium = 1.3, large = 1.0 BL s−1). The swimming speed of CoTmin was not significantly changed by hypoxia exposure.

4.1. Performance While exposure to dissolved O2 saturations of 45–55% did not affect the SMR of any group, it caused a significant decrease in the MMR of all groups resulting in reduced AAS and FAS (Table 2). As the primary energetic reservoir for activity, aerobic scope limitation has cascading implications for whole organism performance. By reducing an individual's capacity to produce energy aerobically, hypoxia diminishes the resources available for multi-tasking, and forces prioritization among numerous survival determining activities (Kramer, 1987). Here, FAS was reduced from > 3.5 in all sizes to between 2.3 and 2.7 (Table 2), bordering on the minimum required just for digestion and assimilation in healthy salmon (Millidine et al., 2009; Secor, 2009). Such a reduction of aerobic metabolic capacity, to less than half of the measured capacity in normoxia in all sizes, resulted in concomitant declines in swimming performance as measured by Ucrit (Table 2, Fig. 3). In line with previous studies (Hammer, 1995), small fish had the highest relative swimming capacity in normoxia (max = 4.3 BL s−1) despite having the highest cost of transport, while medium fish only achieved a maximum of 3.0 BL s−1 (Fig. 3). In terms of absolute current speeds relevant to migration and aquaculture, however, the larger fish outperformed smaller individuals, a fact highlighted by our inability to exhaust multiple of the largest individuals at the maximum current speed we could generate of 140 cm s−1, while Ucrit < 100 cm s−1 in both the small and medium fish in normoxia (Fig. 3). Even in hypoxia, one of the six largest fish tested outswam 140 cm s−1. Conversely, previous work has shown that as routine MO2 increases so too does the LOS below which salmon must switch to anaerobic metabolism (Barnes et al., 2011; Remen et al., 2016a, 2013). The high SMR of small fish, more than double that of the large (196 and 71 mg kg−1 h−1, respectively), with a lower FAS, suggests that small salmon are less hypoxia tolerant than larger individuals. While no other studies have examined the impacts of hypoxia across different sized salmonids, Jones (1971) found that similar hypoxic conditions to those used in this study resulted in a 43% decline in maximum swimming speed of 20 g rainbow trout (Oncorhynchus mykiss). Here, we observed that Ucrit was reduced by 23% in the small fish, and only by 9% in the medium fish (Table 1). This would initially appear to be at odds with the finding of Kutty and Saunders (1973) that swimming performance of Atlantic salmon is more dependent on O2 concentration than that of

800 700

MO2 (mgO2 kg-1 h-1)

600

500 400 300

200 100 0 -5

10

25

40 55 Current speed (cm s-1)

70

85

100

Fig. 2. – Exponential regression of the change in MO2 with increasing current speed of the small (black dotted line, r2 = 0.99), medium (dashed grey line, r2 = 0.97) and large (solid grey line, r2 = 0.97) size classes in normoxic conditions. N = 3 for small and medium groups, and N = 2 for the large group. Circles denote mean ± SE of group respirometry measurements, while diamonds represent calculated SMR from back extrapolation to zero swim speed. 33

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135 125

Ucrit (cm s-1)

115 105 95 85 75 65 55 4.5 4.0 3.5

Ucrit (BL s-1)

3.0 2.5 2.0 1.5

1.0 15

25

35

45 Fork Length (cm)

55

65

75

Fig. 3. – Absolute (cm s−1) and relative (BL s−1) critical swimming speed (Ucrit) of each individual in normoxic (solid circle) and hypoxic (open circle) conditions. Triangles denote individuals which outswam the 140 cm s−1 maximum speed of the tunnel.

rainbow trout, however the much smaller size of the fish used in Jones (1971) could well explain the greater observed impact. Plasma lactate and cortisol response also varied with size. In all sizes plasma lactate, a by-product of anaerobic metabolism, was higher after hypoxic swim trials than normoxic, suggesting that in normoxic conditions swimming cessation was not the result of lactate accumulation. In hypoxia, anaerobic metabolism, as evidenced by the lower MO2 in hypoxic compared to normoxic swim trials at the same speeds, began at slowest speeds (40 cm s−1) in the small fish and increased with size to 75 cm s−1 in the medium and 80 cm s−1 in large fish (Fig. 5). Further supporting the hypothesis that hypoxia tolerance increases with size in salmon, plasma lactate concentration in the large fish which were exhausted during hypoxic swim trials was less than half that of either the small or medium fish (Table 3). This agrees with the explanation provided by Nilsson and Ostlund-Nilsson (2008) that the faster metabolic rate of small fish necessitates a faster rate of anaerobic glycolysis in hypoxia to meet energetic requirements, and thus leads to faster lactate accumulation. Cortisol, which serves many functions and is critical to seawater adaptation in fish, is always present in low concentrations (Mommsen et al., 1999). In stressful situations, however, such as hypoxia and intense exercise, catecholamines are released which increase the functional surface area of gills, allowing for higher MO2, but which also

Gross Cost of Transport (mgO2-1 kg-1 km-1)

800

700 600 500 400 300 200 100

0 0.0

0.5

1.0 1.5 Swimming Speed (BL s-1)

2.0

2.5

Fig. 4. – The relationship between gross cost of transport and relative swimming speed in the small (black dotted line, r2 = 0.96), medium (dashed grey line, r2 = 0.88) and large (solid grey line, r2 = 0.97) size classes in normoxic conditions. N = 3 for small and medium groups, and N = 2 for the large group. Data are mean ± SE.

34

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Table 3 Sample size (N) and mean ± SE of plasma lactate, cortisol and glucose of each size class and dissolved O2 treatment. Hypoxic = 45–55% dissolved O2 saturation; Normoxic ≥85% dissolved O2 saturation. Control fish were sedated and sampled directly from holding tanks for determination of baseline plasma parameters. Small

N Lactate (mmol L−1) Cortisol (ng mL−1)

Medium

Large

Hypoxic

Normoxic

Control

Hypoxic

Normoxic

Control

Hypoxic

Normoxic

Controlᶧ

12 22.2 ± 1.4a 445 ± 27a

12 17.4 ± 1.4b 428 ± 27a

10 4.6 ± 1.6c 72.3 ± 30b

12 21.2 ± 1.7a 682 ± 29a

12 18.6 ± 1.7b 690 ± 29a

10 3.7 ± 1.9c 167 ± 32b

2 8.1 ± 0.19a 732 ± 20a

2 5.5 ± 0.23b 385 ± 22b

9 0.9 ± 0.12c 149 ± 34c

Statistical differences between treatments within each size class are indicated by superscript letters (Tukey post hoc test, p < .05) ᶧ Sambraus et al. (2018).

800

finding suggests that although the performance impacts of hypoxia exposure were most pronounced in small fish, the stress on large fish induced by sub-optimal O2 levels during migration could have important implications including reduced reproductive capacity and impaired immune response, and warrant further investigation.

Large

700 600 500 400

4.2. Metabolic scaling

300

For species with an active lifestyle, the metabolic level boundaries hypothesis predicts that scaling of metabolic rate with body size is limited by flux of waste and resources across surfaces, and therefore that bSMR ≈ 2/3 (Glazier, 2010, 2005; Killen et al., 2016, 2010). However, as activity level increases b should shift upward because metabolism is increasingly driven by resource demand, which is limited by muscle mass, rather than by flux across surfaces which can be temporarily alleviated through measures such as O2 storage and tolerance to waste accumulation (Glazier, 2009). Based on swim tunnel respirometry of groups of similarly sized salmon, whose lifestyle necessitates high locomotor capacity, we calculated a bSMR of 0.65 ± 0.04 which increased to 0.78 ± 0.05 for bMMR, findings which align well with the aforementioned predictions (Glazier, 2009, 2005; Killen et al., 2010). As a result of this size-scaling, mass-specific metabolic demand decreases as salmon grow, a pattern typical in fish (Clarke and Johnston, 1999; Killen et al., 2010). Here, AAS of the small fish (607 ± 23 mgO2 kg−1 h−1) was much higher in normoxia than either the medium or large (397 ± 7 and 365 ± 12 mgO2 kg−1 h−1, respectively). Upon initial examination the much higher AAS of the small individuals would seem to suggest they have the most substantial reserve scope for energy production, and thus capacity to handle stressors such as hypoxia or predation. However, given that the energy required to perform aerobic functions is greater per unit mass in smaller individuals (Tucker, 1975; Webb et al., 1984), as evidenced by the nearly six-fold difference in cost of transport between the large and small size classes (Fig. 4), mass-specific AAS is actually a misleading measure of capacity across varied sizes. FAS on the other hand, which provides an estimate of energetic production capacity as a factor of metabolic maintenance requirements (MMR/ SMR), is a more informative measure to compare scope for activity across a wide size range (Farrell, 2016; Killen et al., 2007). Interestingly, FAS was highest in the large size class in both normoxic and hypoxic conditions, despite being potentially underestimated because several individuals did not reach Ucrit (Table 2). Consequently, it then makes sense that the small fish with the lowest FAS in hypoxia experienced the largest decrease in swimming performance (Fig. 3, Table 2).

200 100 0 800

Medium

MO2 (mgO2 kg-1 h-1)

700 600 500 400 300 200 100 0 800

Small

700 600 500 400 300 200 100 0 0

20

40

60

80

100

Current speed (cm s-1) Fig. 5. – The change in O2 uptake rate with current speed of each size group in hypoxic (unfilled circles) and normoxic (solid circles) conditions. N = 3 for all groups other than large normoxic, for which N = 2. Data are mean ± SE.

stimulate excess cortisol production (Sundin and Nilsson, 2002; Wendelaar Bonga, 1997). Elevated cortisol levels, in turn, divert metabolic resources from biosynthetic pathways and suppress reproductive and immune functions (Mommsen et al., 1999; Wendelaar Bonga, 1997). In this study, while plasma cortisol concentrations were similar following hypoxic and normoxic swim trials in the small and medium fish, more than four times higher than that of controls, cortisol levels in the large fish were twice as high after hypoxic swim trials than after normoxic (Table 3). Though caution is required when interpreting the blood parameter data of the large fish due to small sample size, this

4.3. Conservation implications As higher temperatures raise metabolic maintenance costs and dissolved O2 becomes an increasingly limited resource in aquatic systems, salmon of all life stages are ever more exposed to metabolically restricting conditions (Breitburg et al., 2018; Jonsson and Jonsson, 2009; Sergeant et al., 2017). Instances of failed spawning due to poor O2 35

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conditions and/or high flows impeding migration have already been observed on several occasions (Alabaster et al., 1991; Curran and Henderson, 1988; Martins et al., 2012). Our finding that small fish are more affected by hypoxia exposure than large would seem to contradict the findings of Curran and Henderson (1988) that seaward migrating smolt could move through dissolved O2 conditions much lower than larger salmon migrating upstream. However, when considered in conjunction with Kutty and Saunders (1973) finding that maximum swimming speed increases linearly with dissolved O2 concentration, and the knowledge that seaward travelling smolt passively drift with the tide while upstream migration requires actively swimming against currents, it then makes sense that upward migrating salmon would require relatively high dissolved O2 concentrations to succeed (Curran and Henderson, 1988). Indirectly, aerobic scope for activity defines the energetic resources available for survival, and as such is a critical driver of behaviour (i.e. boldness, activity level) and ecology (i.e. foraging, habitat selection, predator-prey interactions) (Killen et al., 2013). While most studies have examined the relationship between metabolism and behaviour in groups of individuals of similar size, we suggest that similar patterns of co-variation may exist as a result of shifting metabolic profiles with size (Biro and Stamps, 2010; Killen et al., 2017; Metcalfe et al., 2016). For example, numerous studies have shown that individuals with high SMR grow more quickly in high resource environments, but lose their advantage and are prone to risk taking in sub-optimal conditions with low food or environmental stressors (Metcalfe et al., 2016; Reid et al., 2012). Perhaps then, for salmon, while the high SMR of smaller individuals confers the capacity for fast growth in times of plenty, the energetic expense of foraging disruptions may make them more likely to increase activity and risk taking behaviour when exposed to hypoxia, thus increasing vulnerability to predation (Killen et al., 2013, 2012; Kramer, 1987). Conversely, larger salmon with low metabolic maintenance costs have greater reserves and can afford to adopt a ‘wait it out’ response to reduced O2 levels, as suggested in Oldham et al. (2017) where large salmon were repeatedly observed to reduce swimming speed during hypoxic conditions. So, while these hypotheses are speculative, what is clear from these data is that the changing metabolic profiles of salmon as they grow will lead to differential impacts of hypoxia throughout their lifecycle, and may have important ecological and evolutionary consequences which require study.

resting metabolic rate, as observed in the small fish, is correlated with the ability to digest a large amount of food quickly in normoxia (Millidine et al., 2009), in hypoxic conditions it becomes a liability. Remen et al. (2016a) demonstrated that the increased metabolic rate resulting from higher temperatures also increases maximum potential feed intake and growth; simultaneously, as temperature and feed intake increase, so too does O2 demand. At 7 °C the maximum potential feed intake was 0.47% biomass and was sustained down to dissolved O2 saturation of 42%; at 19 °C however, maximum potential feed intake was nearly doubled to 0.99% biomass, but was only able to be maintained down to 76% dissolved O2 saturation (Remen et al., 2016a). Similarly, the higher resting metabolic rate of small salmon mean they will reach their reduced feed intake threshold at higher dissolved O2 saturations than larger individuals. Any stressors or disturbances, such as currents, disease, competition or infection with parasites, which frequently occur and incur their own O2 expenses, increase O2 requirements even further. Faced with dual challenges of the energetic demand required to swim against currents and reduced aerobic capacity because of suboptimal dissolved O2, growth will be impeded and survival challenged without mitigation. Barriers could potentially be used to reduce the in cage current speeds, but would reduce O2 replenishment rate and exacerbate hypoxia (Frank et al., 2015; Gansel et al., 2012; Oldham et al., 2017; Stien et al., 2012). More promising are options which adjust fish behavior, such as submerged feeding and lights which can be used to redistribute fish throughout cages, reducing density and attracting them to areas with higher dissolved O2 (Frenzl et al., 2014; Føre et al., 2013; Juell et al., 2003; Stien et al., 2014; Wright et al., 2015). Supplemental oxygenation or aeration to increase mixing could also be used to reduce hypoxic stress, but will be difficult to ensure that the O2 is distributed throughout the cage where it is needed (Bergheim et al., 2006; Endo et al., 2008; Solstorm et al., 2018; Srithongouthai et al., 2006). Ultimately, the most effective strategy to maximize production performance is to adopt a cautious management approach. Autumn transfer, when hypoxic conditions are most likely, should be avoided with novel cage designs and new sites. Further, given the results of this study, novel cage designs and farming sites should be stocked with relatively large fish, which have greater FAS and swimming capacity, at low stocking densities, until local dissolved O2 and current speed dynamics are well understood.

4.4. Aquaculture implications

5. Conclusions

Larger cages designed to withstand the harsh weather of exposed environments and hold as many fish as possible at a single location are currently the trend in salmon aquaculture, but large cages present increased risk of hypoxia relative to current farming practices (Oldham et al., 2018; SalMar, 2016). In what is currently a typical cage (52 m diameter, 20 m deep) with a stocking density of 25 kg m−3, if the fish congregate within a 3 m depth band, as has been previously documented (Dempster et al., 2016), the observed fish density would be 167 kg m−3. In contrast, in the most recently developed offshore cage which is 80 m in diameter and 50 m deep, if stocked at the same density and the fish congregate within a 3 m depth band the observed fish density would be 415 kg m−3. Higher fish densities equal higher O2 demand. Therefore, though hypoxia in salmon cages typically coincides with times of little water movement, as the industry moves towards using larger single cages at offshore sites farmed fish may be simultaneously challenged by metabolically limiting O2 conditions and faster current speeds than what generally occur at protected farm sites (Oldham et al., 2018). Our results show that, in terms of absolute current speeds, larger fish out-perform smaller individuals, a fact highlighted by our inability to exhaust multiple of the largest individuals at the maximum current speed we could generate of 140 cm s−1, while Ucrit < 100 cm s−1 in both the small and medium fish in normoxia (Fig. 3). And while higher

Hypoxia is an ever-present and increasing threat to all salmon, both farmed and wild. These results show that moderate hypoxia of 50% dissolved O2 saturation significantly reduces aerobic capacity and swimming performance in Atlantic salmon. Further, due to their elevated mass-specific metabolic demand and increased cost of transport, smaller post-smolts are more susceptible to reduced dissolved O2 concentrations than larger individuals. Author contribution Study design & conceptualization: T.O, M.H, B·N., F·O.; Data analysis: T.O; Investigation: T.O; Resources: F·O.; Data curation: T.O; Writing - original draft: T.O; Writing - review & editing: T.O, M.H., B·N., F.O.; Supervision: F.O, B.N; Project administration: F.O.; Funding acquisition: F.O. Conflicts of interest None. Ethics All experiments were conducted in accordance with Norwegian 36

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regulations on animal experimentation under permit 9776, and the Australian code of practice for the care and use of animals for scientific purposes under permit A16277.

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