Effects of reduced water exchange rate and oxygen saturation on growth and stress indicators of juvenile lumpfish (Cyclopterus lumpus L.) in aquaculture

Aquaculture 474 (2017) 26–33

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Effects of reduced water exchange rate and oxygen saturation on growth and stress indicators of juvenile lumpfish (Cyclopterus lumpus L.) in aquaculture Even H. Jørgensen a, Ada Haatuft a, Velmurugu Puvanendran b, Atle Mortensen b,⁎ a b

Faculty of Biosciences, Fisheries and Economy, Department of Arctic and Marine Biology, UiT the Arctic University of Norway, NO 9037, Tormsø, Norway Centre for Marine Aquaculture Research (CMAR), Salarøyvegen 979, N-9100 Kvaløysletta, Nofima AS, Muninbakken 9-13, 9019 Tromsø, Norway

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Article history: Received 9 March 2016 Received in revised form 7 March 2017 Accepted 9 March 2017 Available online 10 March 2017 Keywords: Lumpfish Salmon lice Hypoxia Growth Cortisol Lactate

a b s t r a c t Lumpfish (Cyclopterus lumpus) is currently used as cleaner fish in Norwegian Atlantic salmon (Salmo salar) farming to control parasitic lice on salmon. The need for high numbers of lumpfish necessitates intensive farming of this species but very limited knowledge is available on the environmental requirements of lumpfish in culture. The present study was therefore carried out to investigate the tolerance of lumpfish to varying water exchange rates and oxygen saturations. Four triplicated treatment groups were established at different water exchange rates, which caused oxygen saturations of 55% (0.7 L min−1), 69% (1.2 L min−1), 81% (1.9 L min−1) and 96% (6 L min−1). Decreasing water exchange rate and oxygen saturations negatively affected growth, even in the fish groups held at 81% oxygen saturation. The fish group held at 55% oxygen saturation had almost no growth in body mass and showed sign of fin-infections and were terminated halfway through the experiment. A high workload associated with oxygen extraction and reduced appetite is considered the reason for reduced growth at lower oxygen saturations. While plasma cortisol levels in the groups held at 81 and 96% oxygen saturation corresponded to those considered typical for unstressed fish (b10 ng mL−1), the levels of plasma cortisol of fish in the 69 and 55% oxygen saturation groups were above 20 ng mL−1 in November, indicating a state of chronic stress. A fast, albeit weak, cortisol response to stressors in the lumpfish was confirmed in a separate, 2 h long acute handling and hypoxia experiment. Lack of differences between treatments in plasma lactate levels indicates that the lumpfish reduced their food intake and locomotory activity sufficiently to avoid resorting to anaerobic metabolism when exposed to reduced oxygen saturations. It is concluded that the juvenile lumpfish is sensitive to reduced water oxygen saturations and that oxygen saturations below 80% in aquaculture should be avoided. © 2017 Published by Elsevier B.V.

1. Introduction The salmon lice (Lepeophtheirus salmonis) has been a serious problem for the Atlantic salmon (Salmo salar) farming industry since the 1970s, and has a greater economic impact than any other parasite (Torrissen et al., 2013). Lice infection may result in skin lesions on the host, stress, osmoregulatory problems and an increased vulnerability to infections and diseases (Finstad et al., 2000; Hayward et al., 2011; Tveiten et al., 2010; Torrissen et al., 2013). Traditionally, salmon lice infestations have been treated with chemotherapeutants, however a continuous development of resistance to delousing agents such as pyrethroids (Alphamax®, Betamax® & Excis®), organophospathes ⁎ Corresponding author. E-mail addresses: [email protected] (E.H. Jørgensen), atle.mortensen@nofima.no (A. Mortensen).

http://dx.doi.org/10.1016/j.aquaculture.2017.03.019 0044-8486/© 2017 Published by Elsevier B.V.

(Salmosan®), avermectins (SLICE®) (Burridge et al., 2010) and hydrogen peroxide (Treasurer et al., 2000), has forced the industry to develop and use non-chemical methods such as cleaner fish. In cleaning symbiosis, one species (the cleaner) feed parasites from another species (the host) (Bjordal, 1991). Presently, approximately 64% of Norwegian salmon farmers use cleaner fish as a part of their strategy to keep salmon lice infestations below the national maximum allowable level of 0.5 mature female salmon lice per fish (Heuch et al., 2005). Until recent years, several species of wrasse (Labridus spp) were the only cleaner fish species used in the Norwegian salmon farming industry. However, due to the low tolerance of wrasse species to low water temperatures (Sayer and Reader, 1996), there is a need for cleaner fish species that will maintain grazing of salmon lice in the northernmost areas of the Norwegian coast where temperatures below 10 °C may prevail most of the year. Lumpfish (Cyclopterus lumpus) is distributed on both sides of the north Atlantic, from Spitsbergen in the north to Portugal in the south

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(Blacker, 1983). It is common along the whole Norwegian coastline, however the main part of the population spawns along the northernmost part of the coast (Holst, 1993). After some unreported attempts to use lumpfish as a delousing agent in salmon farms, it was finally reported that lice infestation levels where significantly lower in cages with a lumpfish density of 10–15% of the salmon number (Imsland et al., 2014), indicating that lumpfish is a suitable species for biological delousing of farmed Atlantic salmon. The aquaculture industry has therefore started to use lumpfish as a cleaner fish due to the natural occurrence of this species in the northern part of Norway. The demand for lumpfish is high and cannot be covered by wildcatch. It will therefore be necessary to facilitate commercial farming of lumpfish to meet the increasing demand and there are already several commercial operators who breed lumpfish for the salmon farming industry. Even so, there is still little knowledge of the lumpfish's requirements and tolerance to different conditions in aquaculture such as temperature, oxygen saturation and light. Such knowledge is necessary to obtain, in order to both ensure a well performing fish and an efficient production in captivity. On this background, the focus of the present study was to investigate the physiological responses of lumpfish to reduced water quality in a farming situation. In particular, we have experienced that many farmers are neglectful when it comes to provision of sufficient water exchange rates. To this end, we investigated performance and stress indicators (growth rate, oxygen expenditure and primary and secondary stress responses) in juvenile lumpfish raised in tanks with reduced water exchange rates and, as a consequence, reduced oxygen saturations. 2. Materials and methods 2.1. Fish and research facilities The experiments were carried out at the Centre for Marine Aquaculture Research (CMAR) in Tromsø (69°N) during the period October 14 (day 1) to December 10 (day 58), 2014. Wild, adult lumpfish originating from Northern Norway (69°N) were used as broodstock. Eggs and sperm were stripped and externally fertilized and embryos were incubated at 10 °C until hatch. Once hatched, larvae were transferred to 190 L circular fibre glass tanks and raised using the standard protocol used at the CMAR. The fish were used in two experiments, which both were approved by the Norwegian Committee on Ethics in Animal Experimentation (Id 6872). 2.2. Experimental design 2.2.1. Experiment 1. Long term exposure to reduced water exchange rate and oxygen saturation A total of 444 juvenile lumpfish were randomly distributed among twelve circular, 200 L fibre glass tanks. Four triplicated treatment groups, with each replicate including 37 lumpfish, were established and acclimated for a week before the start of the experiment. Water exchange rates were then adjusted to achieve a range of oxygen saturations, which were kept the same throughout the whole experiment; 0.7 L min− 1 resulting in an oxygen saturation of 55%, 1.2 L min− 1 resulting in an oxygen saturation of 69%, 1.9 L min−1 resulting in an oxygen saturation of 81% and 6 L min−1 resulting in an oxygen saturation of 96%. Measurements of oxygen saturation were conducted daily throughout the experiment (Fig. 1) and the values above represent mean values of flow rates and saturations for the entire experiment. Water temperature was also measured daily and the mean values were 10.28, 10.19, 10.11 and 10.09 °C for the 55, 69, 81 and 96% oxygen saturation groups, respectively. They were held under continuous light and a salinity of 33 ppt. All tanks were fed automatically with a robot every hour (24 times day−1) throughout the entire experiment. Daily feed (2 mm dry pellets,

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Fig. 1. Daily average of water oxygen saturation for the three replicates in the different treatment groups of lumpfish.

Amber Neptune®, Skretting AS, Norway) ration was set to 3% of the tank biomass in all groups (based on tables established at CMAR) however, due to feed accumulation in the tank of the 55 and 69% saturation groups, the rations in these treatment were reduced to 1 and 2% of the tank biomass, respectively. Hand feeding showed that all groups were fed in excess. Initial body mass and -length of the fish were similar among all treatment groups; being 24.3, 24.3, 24.5 and 23.0 g, and 7.2, 7.2, 7.2 and 7.1 cm in the 55, 69, 81 and 96% saturation groups, respectively. Before the start of the experiment, 15 out of the total of 37 fish in each of the 12 groups were anaesthetized (FINQUEL, 0.06 g L−1) and tagged intra-peritoneally with Sokymat® pit-tags (Cyntag, Inc., KY, USA). The pit-tagged individuals were then marked with a fluorescent marker (Visible Implant Elastomer – VIE; Northwest Marine Technology, Inc. WA, USA), injected beneath the transparent skin so that they could be easily identified in the tank. These fish were used for body mass and length measurement, which were performed three times during the experimental period at approximately 4 weeks intervals. On recording dates, tagged fish were removed from the tank and anaesthetized, after which body mass was recorded to the nearest 0.1 g and lengths to the nearest 0.1 cm. Initial biomass in the tanks at the start of the experiment, including both tagged and untagged individuals, varied between 754 and 884 g, representing a biomass of 5.6– 6.6 kg m−3 water. The 3 replicate tanks at 55% saturation were terminated on day 24 due to low appetite and growth, no response to the presence of feed and sign of fin infections. On day 24 (all groups) and 58 (only the 96, 81 and 69% saturation groups), 5 untagged fish were randomly caught from each replicate and immediately anaesthetized and blood was sampled within 4 min of netting, using 2 mL lithium heparin (34 IU) Vacutainer-tubes injected into the caudal vein. After blood-sampling, the fish were killed in a lethal dose of anaesthesia and measured for weight and length. All blood samples were immediately transferred to Eppendorf tubes and centrifuged at 2800 × g for 10 min to separate blood plasma from blood cells. Plasma was pipetted out and distributed among two 0.5 mL Eppendorf tubes and stored at -20 °C until analyses. On days 11, 24, and 58, gill ventilation frequencies of 5 fish from each replicate were measured by counting how many times the gill operculum was opened during one minute. Based on the fish biomass in each

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tank, the water inflow rate and the oxygen saturation measurements on days 24 and 58, the weight-specific oxygen consumption rate of each group was calculated. 2.2.2. Experiment 2. Short-term handling and hypoxia experiment On day 37, a total of 24 lumpfish (mean mass 43.7 g) were subjected to a short-term handling and hypoxia treatment. The experiment started by killing 6 randomly selected fish with a lethal dose of Benzocaine (0.2 g L−1) as an initial pre-stress group (i.e. before handling and hypoxia treatment), after which they were blood sampled and measured for length and body mass. The remaining fish were then placed in a bucket containing 12 L of water (10 °C) and while being in the bucket, they were every 10 min physically agitated by shaking the bucket. After 30, 60 and 120 min, 6 fish were randomly selected, killed as described earlier, blood sampled and measured for body mass and length. All blood samples were taken within 4 min after start of the anaesthesia. The water in the bucket was not exchanged during the course of the experiment. During this period, oxygen saturation was measured regularly and shown to decrease gradually to 30% at the end of the experiment. 2.3. Analyses Blood plasma was analysed for cortisol by a competitive ELISA (Faught et al., 2016). Plasma lactate and glucose concentrations were analysed using the commercial assay kits LAC (LC 2389) and GLUC-HK (GL 1611) from RANDOX Laboratories Limited (UK), respectively, in accordance with the producer's instruction. 2.4. Data treatment and statistical analyses Data are presented as mean ± standard error of mean (SEM). Specific growth rate (SGR) was calculated by the following formula (lnW2 − lnW1) × (t2 − t1)−1, where W2 and W1 are body mass at day 58 and day 1, respectively and t2 − t1 is the number of days between measurements. Specific oxygen consumption rate (mg kg−1 min−1) was calculated by the following formula: (Q × (O2 in-O2 out) × Tb−1), where Q is the rate of water exchange per min, O2 in and O2 out are the oxygen content (mg) in the in- and outflowing water, respectively, and Tb the biomass of the fish in the tank (kg). Statistical calculations were conducted by Systat 13 (Systat Software, Inc., CA, USA) on pooled data from the three replicates. Data on body mass, length, SGR and ventilation frequencies were, with few exceptions, normally distributed and possible differences between treatments and sampling dates in the morphometric data were tested with a two-way analyses of variance (ANOVA), followed by Tukey's Honestly-Significant-Difference Test for pairwise comparisons. Data on plasma

cortisol, glucose and lactate concentrations were generally not normally distributed and they were therefore log-transformed before statistical testing with ANOVA. A probability of p b 0.05 was considered significant. 3. Results 3.1. Experiment 1: Long term exposure to reduced water flow rate and oxygen saturation No mortality was observed during the experiment, however 2 lumpfish from one tank in the 81% saturation groups had fin rot on their tailfin and were therefore euthanized. There were no significant differences in mean body mass (Fig. 2A) among groups at the start of the experiment. On day 24, the fish in the 55% saturation group had a lower body mass than the fish in the 69% saturation group (p b 0.01) and the fish in the 69% saturation group had a lower body mass than the fish in the 96% saturation group (p b 0.05). Body mass of the fish in the 81% saturation group was intermediate, and not significantly different from the fish in the 69 and 96% saturation groups. On day 58 the fish in the 69% saturation group had a lower body mass than the fish in the 81% saturation group (p b 0.01), which had a lower body mass than the fish in the 96% saturation group (p b 0.01). At the start of the experiment, body length (Fig. 2B) did not differ among the four treatment groups. On day 24 there were no significant differences in body length among the fish in the 96, 81 and 69% saturation groups, however, the fish in the 55% saturation group were significantly shorter than the fish in the other groups (p b 0.01). On day 58, there were significant differences (p b 0.05) in length among the 3 remaining saturation groups, which increased with increasing oxygen saturation. In all groups, body length of the fish increased during the course of the study (p b 0.05). The data on SGR (Fig. 3) showed a positive growth throughout the experiment in the different treatment groups (p b 0.05). There was a gradual increase in SGR with increasing water exchange rate and oxygen saturation during the first part of the experiment between days 1 and 24 (p b 0.05). During the last part of the experiment from day 24 and 58, there was a reduced SGR for groups reared at 69% (p b 0.01) and 81% (p b 0.01) oxygen saturation than for the same groups during the first part of the experiment. A small decline in SGR was also seen in the fish from 96% saturation group but this change was not significant. Fish from all three treatment groups differed in SGR from days 24 to 58 (p b 0.05). There were higher plasma cortisol levels (Fig. 5) on day 24 in fish in the groups 69 and 55% saturation than in fish in the 96% saturation group (p b 0.05), while cortisol levels of fish in the 81% saturation

Fig. 2. Mean (±SEM) body mass (A) and body length (B) for individually tagged juvenile lumpfish exposed to different water exchange rates and oxygen saturations on days 1, 24 and 58. Different letters denote significant differences between groups and the +-sign indicate that there had been a significant increase in body mass and -length during the course of the experiment.

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3.2. Experiment 2. Short-term handling and hypoxia experiment At the end of the experiment 2 (water oxygen saturation 30%), the fish had increased ventilation rate due to severe hypoxia. Although some agitation was observed, the fish were mostly calm at the bottom of the bucket. Cortisol levels (Fig. 4) significantly increased (p b 0.01) during the first 30 min from pre-stress level of 0.97 to 33.3 ng mL−1. From that point, plasma cortisol level increased gradually to 47.1 ng mL−1 until the end of the experiment, however, plasma cortisol level at 120 min was not significantly higher than those seen at 30 and 60 min.

4. Discussion 4.1. The effect of reduced water flow rate and oxygen saturation on growth

Fig. 3. Mean (±SEM) specific growth rate of individually tagged juvenile lumpfish exposed to different water exchange rates and oxygen saturations from days 1–24 and days 24–58. Different letters denote significant differences between treatment groups and sampling dates.

group were intermediate and not significantly different from levels in the other groups. On day 58, plasma cortisol levels did not differ among fish in the three remaining saturation groups and these were not different from those on day 24. Plasma lactate concentrations did not differ among saturation groups on day 24 and 58 (Fig. 6A). However fish from the 81% saturation group had higher lactate levels on day 58 than on day 24 (p b 0.01). Plasma glucose concentrations (Fig. 6B) did not differ among groups at any sampling days. Ventilation rates (Table 1) were relatively stable within each treatment group during the course of the experiment. However, there was a gradual, significant increase in ventilation rate with decreasing oxygen saturation at all sampling dates (all p-values b0.001); the only non-significant differences were found between 81% and 96% saturation groups on day 1 and between the 55% and 69% saturation groups on day 24. In lumpfish reared at 55% oxygen saturation, the ventilation rate was 60% higher than the ventilation rate of lumpfish reared at 96% oxygen saturation on both day 1 and 24 and fewer fish in the 55% oxygen saturation group were swimming around than in the other groups. There was a gradual increase in oxygen consumption rate with increasing oxygen saturation on day 24 (Fig. 7) with a tendency to a higher oxygen consumption rate in the 96% than in the 55% saturation group (p = 0.050). On day 58, oxygen consumption rate was lower in fish in the 96% saturation group compared to their oxygen consumption rate on day 24 (p b 0.05).

The results from the present study showed that the growth performance of lumpfish was increasingly compromised by decreasing water exchange rate and oxygen saturation (Fig. 2A and B, Fig. 3). A decrease in water exchange rate may not only reduce oxygen saturation; other factors impairing water quality may also be affected. This was evident in the 55% saturation groups, in which uneaten feed was seen at the bottom of the tank, along with dissolved feed particles in the water. Hence, the reduced growth seen with reduced water exchange rates in the present study was not necessarily related only to reduced oxygen saturation. However, no signs of dissolved feed particles and turbid water were observed in the 81% saturation groups, which grew less than those held at 96% oxygen saturation. The 55% saturation group extracted 4.12 mg O2 L−1 from the water, which would lead to a corresponding increase in CO2 level of maximum 6.67 mg L−1, provided a respiratory quotient (RQ) of 1 (RQ = CO2 eliminated / O2 consumed; RQs for combustion of carbohydrates and saturated fat are 1.0 and 0.7, respectively, while RQ for protein combustion is 0.8–0.9) and a CO2 concentration of the inlet water of 1 mg L−1, corresponding to a partial pressure of CO2 of 0.5 matm (Stiasny et al., 2016). Corresponding values would be 4.90, 3.39 and 3.50 mg CO2 L−1

Table 1 Ventilation frequencies (opercular movements per min; average ± sem) of juvenile lumpfish exposed to different water flows and oxygen saturations at days 1, 24 and 58. Different letters denote groups with significant differences in ventilation frequencies, within sampling days. Treatment

N

Day 1

Day 24

Day 58

55% 69% 81% 96%

15 15 15 15

76.8 ± 1.4a 64. 1 ± 2.3b 50.1 ± 2.4c 47.7 ± 3.1c

76.4 ± 0.9a 71.3 ± 1.2a 64.1 ± 1.7b 44.6 ± 2.5c

– 73.7 ± 1.2a 62.5 ± 1.4b 43.7 ± 2.5c

Fig. 4. Mean (± SEM) plasma cortisol concentrations of juvenile lumpfish exposed to handling disturbance and hypoxia gradually increasing to 30% oxygen saturation at 120 h. Different letters denote significant differences between time points.

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for the 69, 81 and 96% saturation groups, respectively. Few studies have examined the effects of CO2 on growth of marine fish species. In European seabass (Dicentrarchus labrax L.) no negative growth effects were found at a CO2 level of 9 mg L− 1 (Lemarié et al., 2000) or even at 51 mg L− 1 (Petochi et al., 2011). Moreover, Foss et al. (2003) found no effects on growth of juvenile spotted wolfish (Anarhichas minor Olafsen) in the range of 1.1–33.5 mg CO2 L−1. Nothing is known about the hypercapnia tolerance of lumpfish, but judged from the results of the other tested marine species it is unlikely that the CO2 levels experienced by the lumpfish in the present study would have led to a significant reduction in growth rate. We also find it unlikely that accumulation of ammonia had any significant effects of growth rate of the lumpfish. This is partly because theoretical calculations of O2 consumption and ammonium production reveal that negative effects of O2 depletion appear long before the increase in ammonia gives negative effects, and partly because the lumpfish at 55 and 69% oxygen saturation had a reduced feed intake. Hence we consider oxygen saturation to be the main determinant of fish performance in the present study, except for the groups held at the lowest exchange rate (55% saturation), where water quality could be visually characterized as reduced. The growth of the juvenile lumpfish held in normoxic water in the present study were comparable to that previously seen in juvenile lumpfish held at the same water temperature (Nytrø et al., 2014). The reduction in growth with decreasing oxygen saturation levels seen in the present study (Figs. 2 and 3), occurred at a higher oxygen saturation (N 81%) than what Chabot and Dutil (1999) reported for Atlantic cod (Gadus morhua), for which the threshold level for reduced growth was ~ 73% oxygen saturation. The results with lumpfish was, however, in line with what has been found for Atlantic salmon, that had a linear increase in growth rate and feed conversion rate when oxygen saturation increased from 50 to 100%, and even a lower growth rate and feed conversion rate at 85% saturation than at 100% (Crampton et al., 2003). The lumpfish held at 69 and 81% saturation showed a significantly reduced SGR in the second half of the experiment compared to the first period, while only a small and not significant decrease was seen in the 96% saturation groups (Fig. 3). The latter is considered to be due to allometric scaling mechanisms (Kleiber, 1947) with increasing fish size, while the former is considered to reveal an increase in the negative impact of reduced oxygen saturations with time. These findings provide evidence for a very strong sensitivity of juvenile lumpfish to reduced oxygen saturation levels. The minimum oxygen saturation level generally recommended for fish in aquaculture (56–57% oxygen saturation; Thorarensen and Farrell, 2011 and references therein) is obviously much too low for many species, if maximal growth is to be achieved. In lumpfish, it seems that an oxygen saturation above 80% is necessary to achieve maximal growth. As seen among many other fish species (Chabot and Dutil, 1999; Pichavant et al., 2001; Foss et al., 2002; Wang et al., 2009), food intake was reduced for lumpfish during exposure to hypoxia. This was evidenced by an increasing amount of uneaten food with decreasing oxygen saturation in the 69 and 55% saturation groups, when these were fed a ration of 3% of the tank fish biomass in the beginning of the experiment. Further, their feeding response to hand feeding was absent or weak compared to the response of lumpfish held at high oxygen saturations. Hence, it is suggested that reduced food intake (due to reduced appetite) with decreasing oxygen saturation levels was the major determinant of the reduced growth rate seen in lumpfish in the present study. This is supported by Chabot and Dutil (1999), who concluded that food consumption explained 97% of the variation in growth of Atlantic cod exposed to hypoxic conditions. Hyperventilation is a common compensatory response to hypoxia (Perry et al., 2009) and it has previously been shown to be a strong, positive correlation between gill respiration frequency and oxygen consumption rate in pikeperch (Sander lucioperca) exposed to different water temperatures (Frisk et al., 2012). Hence, the gradual increase in ventilation frequency with decreasing oxygen saturation seen in the

present study (Table 1) indicates an increased energy expenditure with decreasing oxygen saturation and hyperventilation. However, the present set-up allowed us to calculate bulk oxygen consumption rate based on the relationship between water exchange rate and oxygen saturation difference between inflowing and outflowing water and these data did not indicate an increased oxygen consumption rate with decreasing oxygen saturation (Fig. 7). We are well aware of the fact that we calculated oxygen consumption rate in tanks without lids and with differing water exchange rates and oxygen saturation levels. Hence, oxygen exchange rate between air and water might have been different between the tanks with different oxygen saturation. Due to a lower oxygen saturation (and hence partial pressure) in the tanks than in the air, it was expected a flux of oxygen into the tank, which would increase with decreasing water exchange rate and oxygen saturation. This would eventually lead to an increasing underestimation of the bulk oxygen consumption rate by the fish in the tanks with decreasing water flow and oxygen saturation. This underestimation is negligible in the 96% saturation group, and the oxygen consumption rate in these fish (1.7 to 2.8 mg kg−1 min− 1) was in the same range as that recorded for another sluggish fish species, the spotted wolfish (1.6 mg kg− 1min−1), held at comparable water temperature (Foss et al., 2002), but lower than the consumption rates recorded for more active species such as salmonids (Christiansen et al., 1991; Thorarensen and Farrell, 2011; Laursen et al., 2013). For the other groups in the present study, the tendency toward lower oxygen consumption rates may partly be related to an underestimation of their actual consumption. In any case, these results were in contrast to the substantial lower oxygen consumption rate in turbot (Scophthalmus maximus) and sea bass (Dicentrarchus labrax) held in hypoxic water than in those held in normoxic water (Pichavant et al., 2001). In the latter study, the difference in oxygen consumption rate was attributed a corresponding difference in food intake (Pichavant et al., 2001) since feeding activity and food processing (digestion, assimilation and processing) are major components of the total energy budget in fish (Jobling, 1994; Jordan and Steffensen, 2007). The small difference in oxygen consumption rates between lumpfish held at 55 and 96% oxygen saturation, despite a substantial difference in food intake and growth (Fig. 3), indicates a high work load associated with oxygen extraction, per se, in the 55% saturation groups. In common carp (Cyprinus carpio) exposed to severe hypoxia (16– 19% oxygen saturation), hyperventilation decreased to normoxic levels within a few days (Moyson et al., 2015), while hyperventilation in the lumpfish in the present study (Table 1) prevailed throughout the whole, two month experimental period. This indicates that the compensatory mechanisms that is seen in response to hypoxia in some fish species (i.e. increase in Hb-O2 affinity and a better oxygen transport capacity; Wood and Johansen, 1972, 1973) is absent or less well developed in lumpfish. This conclusion is supported by their high energy load associated with oxygen extraction suggested above. Taken together, these data indicate that juvenile lumpfish are hypoxia sensitive and may explain why SGR of the lumpfish held at 69 and 81% saturation decreased during the course of the study instead of increasing, which was the case in hypoxia tolerant species such as the spotted wolfish (Anarhichas minor) (Foss et al., 2002). 4.2. The effect of reduced water flow rate and oxygen saturation on stress response Increased ventilation frequency is commonly used as an indicator of a stress response in fish (vanRooij and Videler, 1996). No data have so far been reported on the physiological response to stressors in lumpfish. The short-term handling and hypoxia exposure in the present study showed that plasma cortisol levels were below 10 ng mL−1 before exposure, which is generally considered to be non-stress levels in fish (reviewed by Barton and Iwama, 1991). Upon exposure, plasma cortisol levels increased several fold, and reached what seemed to be maximal levels between 40 and 50 ng mL−1 within 1 h (Fig. 4). Taken into

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Fig. 5. Mean (± SEM) plasma cortisol concentrations of juvenile lumpfish exposed to different water exchange rates and oxygen saturations at day 24 and 58. Different letters denote significant differences between treatment groups and sampling dates.

Fig. 7. Oxygen consumption rate in relation to water oxygen saturation in juvenile lumpfish.

consideration the strong stressor to which they were exposed (repeated handling disturbance and an oxygen saturation decline to 30% toward the end of the experiment), these levels are in the lower part of the scale for cortisol responses to acute stressors in fish in general (Barton and Iwama, 1991). On day 37, fish in the 81 and 96% saturation groups had plasma cortisol levels around, or below, 10 ng mL−1, which indicates that weak hypoxia, per se, did not evoke a primary stress response in lumpfish. However, the cortisol levels in the fish from 55 and 69% oxygen saturation groups were 2- to 3-fold higher (Fig. 5) indicating that low water exchange rate and oxygen saturation not only reduced growth, but also elicited a physiological stress response in juvenile lumpfish. A similar increase in plasma cortisol concentration was seen in Atlantic salmon exposed to 40 and 50% oxygen saturation as compared to the levels in those held in water oxygen saturations from 70 to 90% (Remen et al., 2012). Prolonged secretion of corticosteroids (i.e. chronic

stress) provides a strong indication of a compromised well-being (Huntingford et al., 2006) and maladaptive effects due to tertiary responses (Wendelar Bonga, 1997). For example, it has been shown that cortisol, per se, reduced feed intake and weight gain in channel catfish (Ictalurus punctatus) (Peterson and Small, 2005) and rainbow trout (Oncorhynchus mykiss) (Madison et al., 2015). It is worth noticing that plasma cortisol levels in the 69% saturation groups declined to nonstress levels from the first to the second part of the experiment, and that plasma cortisol levels remained low in the 81% saturation group, despite the fact that growth rate was even more adversely affected by oxygen saturation in the second period than in the first period in these groups (Fig. 5). Plasma lactate concentration (0.2 mmol L 1, Fig. 6a) were an order of magnitude lower in lumpfish than that seen in Atlantic salmon (Remen et al., 2012), but comparable with the levels seen in turbot (Pichavant et al., 2001). Similarly, plasma glucose levels (Fig. 6b) were also an order of

Fig. 6. Mean (±SEM) plasma lactate (A) and glucose (B) concentrations of juvenile lumpfish exposed to different water exchange rates and oxygen saturations at day 24 and 58. Different letters denote significant differences between treatment groups and sampling dates.

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magnitude lower than the levels seen in salmonids (Jørgensen et al., 2002; Remen et al., 2012), but similar to those seen in spotted wolfish (Lays et al., 2009). The low plasma glucose and lactate levels in juvenile lumpfish may reflect their sluggish life-style; in the wild they reside near shore, most of the time attached (with their unique suction disc) to floating seaweed, for shelter and protection but also because seaweed shelters the preferred prey species (Ingólfsson and Kristjánsson, 2002). Increased plasma lactate levels reflect activation of anaerobic metabolism and the lack of any increases with decreasing oxygen saturation seen in the present study (Fig. 6a) suggests that the lumpfish reduced food intake and locomotory activity sufficiently to avoid resorting to anaerobic metabolism. The data indicate that there was a general increase in plasma lactate levels from the first to the second part of the experiment (significant for the 81% saturation groups) but the reason for this increase is not clear. The lack of any response to reduced oxygen saturation on plasma glucose levels (Fig. 6b) was unexpected when taking into account the increase in plasma cortisol levels because the main role of increases in plasma cortisol levels is to mobilize energy by activating gluconeogenesis (Mommsen et al., 1999). However, these results correspond to the lack of changes in plasma lactate and glucose levels during long-term exposure to hypoxia (below 50% oxygen saturation) seen in turbot and sea bass (Pichavant et al., 2001) and in Atlantic cod (Chabot and Dutil, 1999). The lack of plasma glucose changes despite an increase in plasma cortisol levels in the present study may be interpreted as a low capacity for energy mobilisation during stress in lumpfish. However, care should be taken when interpreting differences between species since the measurement of plasma levels of glucose does not reveal the actual turnover rate of glucose. 5. Conclusion The present study showed a reduced growth rate in juvenile lumpfish held at 81% oxygen saturation compared to the growth in fish held at 96% oxygen saturation. In lumpfish held at 55% oxygen saturation appetite and growth were strongly reduced, this treatment group was, therefore terminated halfway through the experiment. The reason for reduced growth rate with decreasing oxygen saturations is considered mainly to be reduced appetite, but also influenced by an increased energy expenditure associated with oxygen extraction at low water oxygen saturations. An increase in ventilation rate measured two months after the start of the experiment indicates that the compensatory mechanisms activated in many fish species when exposed to hypoxia, is lacking or little developed in juvenile lumpfish. The 55 and 69% saturation groups showed elevated plasma cortisol levels, indicating that the rearing environment elicited chronic stress in these fish. Taken together, it is concluded that the juvenile lumpfish is very sensitive to reduced water oxygen saturations and in order to maintain a non-stressed fish with good growth rates, juvenile lumpfish should not be held at oxygen saturations below 80% in farming. Statement of relevance The information in the present manuscript is very relevant for the farming of lumpfish, in a situation with an already huge production of lumpfish and lack of knowledge about the requirements and tolerances of this species in aquaculture. The paper will have high impact. Acknowledgements The authors acknowledge financial support from Skretting Studentfond, which made it possible to go to Canada for the cortisol analyses and the help of Professor Matt Vijayan and PhD student Erin Faught at the University of Calgary, Canada, with the analyses. The staff at CMAR is acknowledged for their proper care of the experimental fish. Thanks also to Chandra Ravuri, who conducted the glucose and lactate analyses.

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