The critical oxygen threshold of Yellowtail Kingfish (Seriola lalandi)

Journal Pre-proof The critical oxygen threshold of Yellowtail Kingfish (Seriola lalandi) Caroline L. Candebat, Mark Booth, Jane E. Williamson, Igor Pirozzi PII:

S0044-8486(19)31442-5

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734519

Reference:

AQUA 734519

To appear in:

Aquaculture

Received Date: 7 June 2019 Revised Date:

13 September 2019

Accepted Date: 15 September 2019

Please cite this article as: Candebat, C.L., Booth, M., Williamson, J.E., Pirozzi, I., The critical oxygen threshold of Yellowtail Kingfish (Seriola lalandi), Aquaculture (2019), doi: https://doi.org/10.1016/ j.aquaculture.2019.734519. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

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The critical oxygen threshold of Yellowtail Kingfish (Seriola lalandi) Caroline L. Candebata,b*, Mark Boothc, Jane E. Williamsona, Igor Pirozzic,d a

Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109 Australia b Institute of Marine Ecosystem and Fishery Science, University of Hamburg, Olbersweg 24, 22767 Hamburg, Germany c NSW Department of Primary Industries & Aquafin CRC, Port Stephens Fisheries Institute, Taylors Beach, 2316 NSW, Australia. d Centre for Sustainable Tropical Fisheries and Aquaculture, James Cook University, 1 James Cook Drive, Townsville, 4811, Queensland, Australia ∗Corresponding author: [email protected] Centre for Sustainable Tropical Fisheries and Aquaculture, James Cook University, 1 James Cook Drive, Townsville, 4811, Queensland, Australia.

Declarations of interest: none Abstract Low concentrations of dissolved oxygen are one of the most limiting abiotic factors in land-based

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and marine aquaculture, impacting the welfare of target-species. Yellowtail Kingfish (Seriola

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lalandi) (YTK) is a high energy demanding species and its commercial aquaculture is rapidly

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expanding globally yet no information on its hypoxia tolerance is available. YTK is commonly

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cultured in sea pens, in which abiotic factors such as temperature and ambient oxygen can

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fluctuate substantially. The move away from marine fish oils to more sustainable terrestrial oil

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sources in aquafeeds implies a change in fatty acid intake. This shift in fatty acid concentrations

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and temperature fluctuations can impart physiological effects, impacting the animals stress

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tolerance. The critical oxygen threshold is a common method to quantify the lower, tolerated

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threshold of oxygen concentrations for an organism. This study assessed the critical oxygen

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threshold in fasted, juvenile YTK with respect to acclimation temperature (15°C & 20°C) and

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dietary lipid source (fish oil & poultry oil). Additionally, observations on the visual and behavioral

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hypoxia responses in YTK were made. This study demonstrated that YTK could regulate their

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oxygen consumption down to 2.92-1.84 mg dissolved oxygen L-1, but this strongly depends on the

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acclimation temperature, and to a lesser extent dietary oil source. At dissolved oxygen

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concentrations below this level, YTK became oxyconformers, unable to maintain an optimum rate

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of oxygen uptake. Warmer acclimation temperatures led to significantly less hypoxia tolerance

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compared to YTK held in cooler temperatures. Dietary oil source had no significant effect on the

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critical oxygen threshold; however, YTK fed a poultry-oil based diet displayed less hypoxia

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tolerance and greater deviation around the mean, attributing the non-significant difference to YTK

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fed a fish oil-based diet. Additionally, hypoxia triggered behavioral responses were initiated earlier

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in YTK fed the poultry oil diet. First behavioral responses, after passing the critical oxygen

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threshold, were attempted aquatic surface respiration, increased opercular frequency, and

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gulping, followed by darkening of skin coloration. We recommend rapid oxygenation of the rearing

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system if dissolved oxygen levels approach 2.92 mg L-1 at 20°C or at first sign of these changes.

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Further onset, such as rush or rest behavior, may rapidly lead to the final stages of hypoxia. These

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results expand knowledge on YTK physiology and behavioral responses to low dissolved oxygen

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environments and provide information for farm managers to ensure adequate levels of dissolved

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oxygen throughout rearing, handling, bathing or transportation procedures.

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Key words: Hypoxia tolerance; temperature; fatty acids; oxyregulator; behavior

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Highlights

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1) YTK regulate their oxygen consumption down to a dissolved oxygen concentration of ~1.9 – 2.6

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mg O2 L-1 (equiv. ~22 – 39% saturation) at 20°C and 15°C, respectively, after which they

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become oxyconformers and transition to a hypoxic state.

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2) The critical oxygen threshold is strongly dependent on the acclimation water temperature. Warmer acclimation temperatures decrease the hypoxia tolerance of YTK. 3) The dietary oil-source (fish oil versus poultry oil) had no significant effect on the critical oxygen

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threshold of YTK in this study. However, YTK fed a poultry-oil diet showed a relatively large

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deviation in routine metabolic rate and critical oxygen threshold.

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4) YTK show a consistent series of behavioral responses to the onset of hypoxia which is initiated

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with attempted aquatic surface respiration, increased opercular frequency, and gulping. Rapid

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oxygenation of the rearing system at first indications of these behavioral changes is strongly

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recommended.

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1. Background The critical oxygen threshold ([O2]crit) is the point at which an oxyregulating organism

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transitions to being an oxyconformer. This phase shift can be relatively abrupt or gradual

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depending on the species and environmental condition (Lagos et al., 2017; Wood, 2018).

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Throughout the oxyregulating phase, organisms maintain an optimal oxygen consumption rate

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independently of the ambient oxygen concentration. Throughout the oxyconforming phase,

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organisms are directly relying upon the ambient oxygen concentration and are typically unable to

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maintain an optimal oxygen consumption rate. Most aquatic organisms maintain a relatively

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constant oxygen consumption rate with decreasing ambient oxygen concentration until the [O2]crit

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is reached (Perry et al., 2009; Rogers et al., 2016). There are exceptions such as Inanga (Galaxias

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maculatus) where oxygen consumption decreases linearly from a normoxic through to a hypoxic

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environment, indicating that it is a true oxyconformer (Pörtner and Grieshaber, 1993; Urbina and

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Glover, 2013). Decreasing environmental oxygen past the [O2]crit provokes behavioral (Kramer,

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1987), physiological (Barton and Zwama, 1991; Claireaux and Chabot, 2016; Pörtner and Peck,

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2010), molecular (Richards, 2009; Soitamo et al., 2001) and genetic (Nikinmaa and Rees, 2005;

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Wenger, 2000) responses which can help to mitigate oxygen delivery by conserving energy to

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maintain aerobic ATP production.

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The sensitivity to environmental hypoxia is species and individual specific (Rogers et al.,

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2016), correlating with abiotic factors such as temperature (Beamish, 1964) and salinity (Ern et al.,

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2014) and biotic factors such as routine metabolic rate, life-stage, biomass, and body shape

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(Burton et al., 1980; Ferguson et al., 2013; Lagos et al., 2017). Abiotic and biotic factors alter the

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oxygen supply, demand, and energy partitioning for and in the animal. Exposure to low DO will

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cause a decrease in amino acid utilization efficiencies in YTK (Pirozzi et al., 2019). Elevated

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temperatures cause most fish to be less hypoxia tolerant by inducing an increase of the standard

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metabolic rate (SMR) and reduced oxygen supply through decreased oxygen solubility in the

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water, causing an imbalance of energy portioning (Rogers et al., 2016).

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Feeding induces postprandial metabolic responses which are highly influenced by

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temperature in fish (Pirozzi and Booth, 2009a). Further, diet composition can have a significant

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effect on the short-term, rapid post-prandial increase in metabolic rate (Fu et al., 2005).

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Nevertheless, the effect of diet on [O2]cri has been little studied. In European eel (Anguilla anguilla)

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a supplementation of commercial diet with 15% menhaden oil revealed a significantly lower

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routine metabolic rate than eel fed 15% coconut oil; however, there was no statistical effect on

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[O2]crit (9.6±1.1 kPa & 7.6±1.1 kPa respectively) (McKenzie et al., 2000). Essential fatty acids alter

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the routine metabolic rate (RMR) and [O2]crit in larval and juvenile Dover sole (Solea solea)

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(McKenzie et al., 2008). Dietary lipids for fast-growing, carnivorous marine fish are mainly

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provided by fish oil, which is extracted from capture fisheries for the aquafeed industry and is rich

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in long-chain polyunsaturated fatty acids (LC-PUFA), such as eicosapentaenoic acid (20:5n-3, EPA),

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docosahexaenoic acid (22:6n-3, DHA), and arachidonic acid (20:4 n-6, AA). Research on the lipid

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requirements of carnivorous marine fish has shown the inability of some species to endogenously

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convert lipids to LC-PUFA, or at least at a rate to meet requirement, and consequently LC-PUFA

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have to be provided via dietary supplementation to maintain growth, health, and physiological

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functioning (Tocher, 2010). Despite fish oil’s essentiality, it has become an unsustainable and

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costly source of LC-PUFA (FAO, 2015). Currently, the aquaculture industry utilizes alternative

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dietary oils, including blends of fish and terrestrial animal and/or plant oils. One of these sources is

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poultry oil (PO) which is rich in monounsaturated fatty acids (MUFA) and total n-6 PUFA, but

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devoid of EPA and DHA (Higgs et al., 2006). Considering the growing importance of sustainable

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alternative feed ingredients for carnivorous marine species in aquaculture it is important to know

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if dietary oils have a significant impact on physiological stress thresholds. Hulbert and Lewis Else

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(1999) hypothesized that an organism’s metabolism is linked to membrane bilayers, emphasizing

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that DHA is an important building block of the bilayer.

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YTK is an important emerging species to Australian large-scale mariculture and is grown in

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New South Wales, South Australia and Western Australia. However, to date, there is no

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information on the critical oxygen threshold in YTK. The impact of temperature on the [O2]crit is of

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particular interest for the aquaculture industry in terms of acute, seasonal, and climate change

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related fluctuations. Further, poultry oil is now routinely being used in commercial diets to

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partially replace fish oil for this species.

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The objective of this study was to quantify the [O2]crit and hypoxia tolerance of YTK at

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seasonally relevant temperatures (15 and 20°C) fed one of two diets, containing fish or poultry oil,

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to further elucidate the effect of dietary oil source on oxygen metabolism. The results will provide

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a better understanding of the physiological thresholds of YTK.

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2. Material and Methods

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The experiment was performed under the NSW DPI Fisheries Animal Care & Ethics (ACEC)

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Research Authority known as ‘Aquaculture Nutrition ACEC 93/5–Port Stephens’. Care, husbandry

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and termination of fish were carried out according to methods outlined in ‘A Guide to Acceptable

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Procedures and Practices for Aquaculture and Fisheries Research’ ACEC (2017).

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2.1 Experimental design 5

A 2 x 2 factorial design was applied to test the effect of two acclimation temperatures (15°C

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or 20°C) and two dietary oils (fish oil or poultry oil) on the hypoxia tolerance of YTK. The hypoxia

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tolerances of the four treatments were determined over six days. Each treatment was tested in

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three replicated respirometers in which four individual YTK (483.3 ± 55.1 g) were stocked. Each

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hypoxia tolerance measurement of each treatment was run for approximately 2.5 to 4 h and

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repeated twice on different individuals and days. Daily measurements of the six respirometers were

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each started in 40-50 min delays to prevent simultaneous ending of experiment and conducted

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between 0900 and 1600h.

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2.2 Experiment diets Two isonitrogenous (55% crude protein) and isoenergetic (25 MJ kg-1) diets were formulated

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using practical ingredients (Table 1). The dietary nutrient profile was chosen as a commercially

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relevant specification and to meet the nutritional requirement for this species (Booth et al., 2010).

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Except for the dietary oil all ingredient inclusions were consistent between the diets. Ingredients were first milled to <650 µm particles, ensuring homogenous blending. Dry

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ingredients were then mixed in a batch mixer for 45 min (Ernest Fleming Machinery & Equipment

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Pty Ltd). Dietary oil and water were then added and thoroughly mixed (Hobart Food Equipment Co.,

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LTD. Mixer). The soft dough was then mechanically extruded with an electric mincing machine to

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obtain pellets of the desired diameter (6 mm) and strands were then manually broken to <5mm

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pieces. Moist pellets were then dried on perforated trays in convection drier at 38°C for 8.5 h

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(moisture content < 10%). Dried pellets were stored in re-sealable containers and kept frozen at -

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18°C until used. Feeds were sampled and analyzed for proximate (i.e. moisture, total nitrogen, crude

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lipid, and ash content), energy and fatty acid composition. Diet compositions are presented in Table

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1.

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2.3 Temperature and diet acclimation

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Prior to determining [O2]crit, YTK were acclimated to an allocated diet and temperature

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regime over a period of 51 days. The acclimation phase was as follows. Eight groups of forty YTK

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(initial mean body weight; 250.9 ± 34.5 g) were each stocked into 1m3 net-cages (i.e. ~10 kg fish m-

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aquaculture systems. YTK were initially stocked at an ambient temperature of 18°C. The

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temperature of each RAS was adjusted up or down at ~1°C per day until the target temperatures

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of 15°C or 20°C were achieved. Each experimental diet was randomly allocated to two net-cages

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per temperature treatment. Throughout the acclimation period YTK were fed ad libitum once daily

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at 1200h. Water quality parameters were monitored daily for ammonia (1.1± 1.1 ppm), dissolved

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oxygen (DO) saturation (104± 18.5 %), pH (8.0± 0.25), and salinity (32± 0.85 g L-1). The light regime

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was based on the natural light regime of the season (11L: 13D). Due to a technical error, YTK, held

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in the warm water system, were briefly exposed to low DO resulting in eight mortalities.

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Remaining YTK recuperated quickly, showed no adverse behavior, and fed normally for three

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weeks and 3 days until utilization for the [O2]crit trials.

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2.4 Respirometer

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). Four net-cages were each situated in one of two 10m3 holding tanks on separate recirculating

The experimental inventories consisted of one circular-shaped 10m-3 temperature bath

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holding six floating, 200 L flat-bottomed respirometers (Figure 1). Respirometers were utilized to

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measure the hypoxia tolerance of YTK using a closed static system. The warming/cooling bath was

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temperature regulated by a reverse cycle heat pump (HWP20-3P, Rheem Pool Heating, Liverpool,

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NSW, Australia), stabilizing the experiment temperature. Each respirometer was airtight sealed and

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fitted with a clear Perspex lid (5mm thickness). DO was infused through a ceramic stone, placed

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beneath the pump in the water bath, supplying the respirometers with fresh oxygenated seawater

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for the experimental acclimation phase and the post-experiment recuperation phase of the YTK.

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The air-water interface in an open-top respirometer has a sufficient boundary layer to

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reliably measure oxygen consumption in YTK (Pirozzi and Booth, 2009b). Nevertheless, additional

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tests on the hypoxia tolerance of YTK require adjustments of respirometers, as larger YTK tend to

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break the air water interface during hypoxic conditions. To prevent mixing events respirometers

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were therefore sealed with Perspex lids.

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DO (mg L-1) in the respirometers was measured with two FireStingO2 devices (Pyro Science

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GmbH, Aachen, Germany). DO concentrations were later converted to oxygen saturation to

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compare the research outcome of other fish species (U.S. Geological Survey, 2011). Three optical

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fibre cables (SPFIB, Pyro Science GmbH, Aachen, Germany) were connected to each FireStingO2

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device transferring the admitted red light source to lens spot adapters (SPADLNS, Pyro Science

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GmbH, Aachen, Germany). Lens spot adapters were carefully fixed in place with adhesive glue (Sika

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Australia Pty. Ltd.) to the outer surface of the Perspex lid. Lens spot adapters have an integrated

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collimating lens, which allows working with Perspex thicknesses of 2 mm to 6 mm. Contactless

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oxygen sensor spots (COSS, OXSP5, Pyro Science GmbH, Aachen, Germany) were attached with clear

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silicone glue to the inner side of the Perspex lid. COSS allow measurements within closed containers

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through a transparent barrier and provide exact measurements. COSS have no intrinsic O2

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consumption and therefore no correction of data was required. Before each experimental run, each

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sensor was individually 2-point calibrated (0% & 100% calibration) in seawater. Black corflute sheets

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were installed in-between the respirometers, shielding YTK from external disturbances.

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After each experiment the background biological oxygen demand (BOD) in the respirometer

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was measured. None or low levels of background BOD were found, but where necessary oxygen

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consumption rates were corrected for background respiration.

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2.5 Critical oxygen threshold determination

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The [O2]crit level of YTK acclimated to different temperatures and dietary oils was assessed

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using a total of 88 YTK (483.3 ± 55.1 g) of which four YTK were stocked in each respirometer. There

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were n=3 replicate respirometers per treatment.

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YTK were fasted for 36 h (20°C treatment) or 48 h (15°C treatment) prior to respirometry to ensure

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fish were not in an elevated postprandial metabolic state. Fasting times were based on previous

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work in our laboratory on gut transit time in YTK which indicated that a single feed of commercial

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pellets would be digested and excreted over these time periods at each respective temperature

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(Candebat, unpub. data). On the same day of hypoxia tolerance determination, fiber-optic sensors (OXSP5, Pyro

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Science GmbH, Germany) were calibrated [two-point in water at the beginning of the experiment

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(O2 at 100% & 0%)]. Additionally, salinity, temperature, and pressure were measured by a Hach

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HQ40D portable meter. Variables were then manually entered in the Pyro Oxygen Logger to refine

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measurements (FireStingO2, Pyro Science GmbH, Germany). After the calibration procedure, oxygen saturation was maintained at 100% by controlling

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the inflow rate of oxygenated water into the respirometers. Respirometers were then systematically

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stocked with four YTK each over 25 min increments to facilitate a time shifted start for

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measurements and the respirometers were carefully sealed with Perspex lids and spring clamps. YTK

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were habituated in the normoxic water (O2 ~100%) until the oxygen consumption stabilized (~45

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min). After the habituation phase, respirometers were isolated by stopping the inflow of oxygenated

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water and outflow of water. Remnant air was removed through an air-outlet on the Perspex lid,

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which was afterwards sealed. Automatic, computational live measurements recorded oxygen

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concentration in the water (Pyro Oxygen Logger Software version >3.3). The level of oxygen in the

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static water was decreased by the oxygen consumption of the YTK until the [O2]crit threshold was

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passed.

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2.6 Hypoxia related behavioral observations Behavioral responses of grouped YTK in respirometers were recorded throughout the determination

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of [O2]crit. Behavioral and visual responses included a series of changes in ventilation, skin

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coloration, rush/rest behavior, and the loss of equilibrium. Classifications were adapted from Cook

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and Herbert (2012) and Urbina et al. (2011). Change in ventilation ([O2]vent) was identified as the oxygen level at which YTK exhibited

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attempted aquatic surface respiration (ASR), increased opercular frequency, and gulping (Urbina et

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al., 2011). Change in skin coloration ([O2]col) was identified as the oxygen level at which individual

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YTK exhibited dark patches or turned noticeably darker. Rush or rest behavior ([O2]R/R) was the

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oxygen level at which YTK displayed repetitive disoriented burst swimming and resting behavior

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(Cook and Herbert, 2012). The loss of equilibrium [O2]LOE was defined as the point at which YTK

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started to sink to the bottom of the tank, with no attempt to swim to the water surface. Immediately after the display of [O2]LOE in an individual YTK, the respirometer was opened,

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and water was quickly sampled for subsequent determination of CO2 levels (OxyGuard CO2

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Analyser). CO2 concentration never exceeded 6 ppm. Then, fresh O2 saturated water (100%) was

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quickly pumped into the respirometers. After 15 min, YTK were removed from the respirometer to

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record body weight and length. All YTK fully recovered from the hypoxia exposure.

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2.7 Data analyses The [O2]crit and routine metabolic rate (RMR) were determined by averaging outcomes of

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each experimental run and were then analyzed via GraphPad Prism ver. 6 (La Jolla, CA, USA). The following formula was used to calculate the oxygen consumption (mg kg–1·h–1) of each

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run: =

–

∗

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248

where; [O2]t refers to the oxygen concentration (mg L–1) at time point t, [O2]t+1 is the oxygen

∗

Eq. 1

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concentration at the next time point (the oxygen measurements were calculated according to the

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O2 solubility coefficient in water under corresponding temperature, pressure, and salinity), v is the 10

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volume in liters of the respirometer, k is the time interval between time points t and t+1, and m is

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the body mass of the YTK in kg. The collected oxygen consumption values were subject to non-linear

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regression analysis (NLS): =

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+,/) .

Eq. 2

and segmental linear regression ´broken stick` analysis (BSR):

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− ! ∗ ∗ ln (1 + exp *

1

=2

3 3

+ 4 7+ ( 4 1

1

5 3 ≤ 7− 4 8) + 4 8 5 3 >

Eq. 3

Models were then cross-validated for quality and fit via Akaike information criterion (AIC). Statistical analyzes were performed using R software environment for statistical computing

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(2.13.). The effects and interactions of thermal acclimation and dietary oil on RMR, [O2]crit, body

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mass, [O2]LOE, [O2]vent, [O2]col, [O2]R/R were statistically analyzed via two-way analysis of variance.

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Assumptions of homogeneity and normality were tested via Levene’s and Shapiro-Wilk test. In case

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assumptions were not met, data were either log or square-root transformed and then statistically

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analyzed.

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3. Results

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3.1 Critical oxygen threshold ([O2]crit)

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[O2]crit values are presented in Table 3. [O2]crit was determined by NLS or BSR and then

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statistically analyzed for differences in regression models. Results indicated a significant impact on

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[O2]crit by the analysis method (p<0.05). All [O2]crit determined by BSR analysis were significantly

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higher than [O2]crit values analyzed via NLS (p<0.05). Therefore, [O2]crit analyzed through BSR

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indicate YTK which are less hypoxia tolerant than [O2]crit analyzed through NLS. Models were

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compared through the AIC criterion and results indicate NLS as the better fit as a measure of [O2]crit.

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Hereafter, stated [O2]crit values are the result of NLS model analysis (Figure 2).

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No interaction of dietary oil source and temperature on [O2]crit was detected. YTK acclimated to

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different temperatures showed significant differences in [O2]crit (p<0.05). YTK were less hypoxia

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tolerant in warm water than YTK held in colder water [O2]crit; fish oil diet 2.33 ± 0.11 mg L-1 and

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poultry oil diet: 2.80 ± 0.79 mg L-1), than YTK acclimated to 15°C ([O2]crit; fish oil diet 1.37 ± 0.14 mg

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L-1 and poultry oil diet: 1.66 ± 0.55 mg L-1). While different dietary oils did not significantly affect [O2]crit, trends were notable (p<0.07).

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YTK fed diets containing more fish oil tolerate hypoxia better than YTK fed diets containing poultry

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oil. Additionally, YTK fed the poultry oil diets demonstrated greater standard deviations than the YTK

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fed the fish oil diets (Figure 2). The elapsed time until [O2]crit thresholds were reached differed significantly between the

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different temperature treatments (p<0.01; Table 3). The oxygen consumption in YTK acclimated to

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warmer temperatures was faster than in YTK acclimated to colder temperatures. Additionally,

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[O2]crit was reached faster in YTK acclimated to warmer temperatures and YTK reached the loss of

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equilibrium at higher DO contents in the ambient water.

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3.2 Routine metabolic rate

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RMR data are presented in Table 3 and Figure 2. No interactions between temperature and

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dietary oil on the routine metabolic rate of YTK were detected. Nevertheless, the routine metabolic

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rates of YTK differed significantly between temperatures (p<0.05) and were almost double for YTK

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acclimated to 20°C (poultry oil: 254.06 ± 4.73 mg DO L-1 kg-1 h-1 and fish oil: 263.75 ± 35.26 mg DO L-1

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kg-1 h-1; mean±SD) compared to YTK acclimated to 15°C (poultry oil: 167.11 ± 8.94 mg DO L-1 kg-1 h-1

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and fish oil: 179.29 ± 11.51 mg DO L-1 kg-1 h-1; mean±SD). No significant difference in routine

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metabolic rate was detected in YTK acclimated to different dietary oils at either temperature (Table

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3).

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3.3 Behavioral responses to hypoxia A consistent sequence of behavioral and visual responses across all treatments to hypoxia

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was observed. As DO steadily decreased into a hypoxic condition, YTK exhibited ASR, increased

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opercular frequency, gulping, rush and rest behavior, dorsal cranial and spinal patches of dark skin

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and loss of equilibrium (Table 4).

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No interaction between dietary oil and temperature considering behavioral and visual

302

responses were shown. Acclimation temperature and lipid-source had no significant effect on the

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ventilation behavior of YTK. Ventilatory changes commenced at the [O2]crit stage (Table 4, Fig. 2).

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Skin discoloration then occurred which included dark patches appearing around the dorsal, cranial

305

and spinal areas. Change of coloration was not significantly impacted by dietary lipid but showed

306

trends in terms of the respective acclimation temperature (p=0.05; Table 4). YTK acclimated to

307

warmer temperatures exhibited patches at earlier stages of hypoxia than YTK acclimated to the

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colder temperature. YTK fed fish oil-based diets at warmer temperatures changed coloration at

309

lower DO concentrations (1.68±0.11 mg L-1) than fish fed poultry oil-based diets (1.92±0.36 mg L-1).

310

Following the change of coloration, YTK exhibited repetitive rush or resting behavior. Dietary

311

lipid did not significantly affect the commencement of rush or resting behavior. Acclimation

312

temperature significantly affected the DO concentration at which YTK started to rush and rest.

313

Colder acclimation temperatures led to lower DO concentrations at which YTK started to rush and

314

rest, while YTK which were acclimated to warmer temperatures showed earlier burst swimming

315

behavior at higher DO concentrations (Table 4).

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Shortly after rush and rest, YTK were observed to lose equilibrium. The acclimation

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temperature, but not dietary lipid, had a significant effect at what DO concentration lost equilibrium

318

occurred (p<0.05). YTK acclimated to colder temperatures were experiencing loss of equilibrium at

319

much lower DO concentrations than YTK acclimated to warmer temperatures. YTK displayed loss of

320

equilibrium at DO concentration of 1.22 mg·L-1 to 1.48 mg·L-1 at 15°C and 20°C respectively (Table 4).

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4. Discussion

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4.1 Critical oxygen threshold ([O2]crit)

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This study provides information on the hypoxia tolerance of YTK under different temperature and

324

dietary regimes, showing that YTK are able to regulate their oxygen consumption down to a DO

325

threshold of 2.92 – 1.84 mg L-1 (equiv. 38.94% – 22.24% saturation at 19.2°C - 14.8°C°C respectively).

326

These results indicate that the hypoxia tolerance in YTK is strongly linked to ambient water

327

temperature. Similar results were found in the Atlantic salmon (Salmo salar) which can regulate the

328

oxygen consumption down to ~50–35% saturation between 22 and 14°C respectively; below these

329

levels salmon are oxyconformists (Barnes et al., 2011).

330

The impact of the ambient water temperature on the hypoxia tolerance is well known and

331

incorporated in most studies to measure the species-specific interaction of temperature and

332

hypoxia tolerance. Warmer temperatures usually cause a fish to be less hypoxia tolerant (Barnes et

333

al., 2011; Collins et al., 2013; Rogers et al., 2016) however the magnitude of impact varies from

334

species to species. For example, a 3°C increase dramatically reduced the hypoxia tolerance in

335

cardinalfish (Ostorhinchus doederleini), while an increase of temperature by 20°C only subtly

336

reduced the hypoxia tolerance in carp only (Nilsson and Renshaw, 2004; Ott et al., 1980). Similar to

337

the common dentex (Dentex dentex), YTK show a relatively strong reduction of hypoxia tolerance to

338

an increase of 5°C (Cerezo et al., 2006).

339

Dietary oil did not significantly affect hypoxia tolerance of YTK; however, there was a

340

nominal increase of hypoxia tolerance when YTK were fed fish oil at both water temperatures. The

341

non-significant effects of oil sources on [O2]crit in YTK may be partially explained by the inclusion of

342

fish meal in both diets, approximating current commercial YTK diets. Fish meal contains fish oil

343

(~10% lipid dry matter basis) which contributed to the overall amount of essential fatty acids in the

344

test diets (Table 2). In larval and juvenile Dover sole essential fatty acid enriched diets significantly

345

improved the hypoxia tolerance when acclimated to approximately 18°C (McKenzie et al., 2008),

14

346

while deficiencies in polyunsaturated fatty acids reduced hypoxia tolerance and led to significantly

347

higher mortalities when exposed to 10% oxygen saturation (Logue et al., 2000). Even though no

348

significant effect of dietary oil on hypoxia tolerance of YTK was demonstrated in this study, the high

349

variability in hypoxia tolerance of YTK fed the poultry oil-based diet in comparison to the YTK fed the

350

fish oil-based diet was noticeable (Figure 2). It is not clear why this occurred and is an area that

351

requires further investigation.

352

This study measured [O2]crit in closed, static respirometers in which a group of four fish collectively

353

depleted oxygen through respiration. YTK are a pelagic species which exhibit strong schooling

354

behavior (Kailola et al., 1993) and are farmed in cohorts in aquaculture settings. Fish densities in

355

respirometers can influence routine metabolism through species-specific behavior (Ruer and Cech,

356

1987; Umezawa et al., 1983). Schooling fish exhibited relatively lower metabolic rates when

357

measured in groups as opposed to individual measurements (Parker, 2004), whereas the metabolic

358

rate of solitary and territorial species increased when measured in groups (Umezawa et al., 1983).

359

The [O2]crit of individual Atlantic salmon is 35% - 50% saturation held at 6°C - 18°C (Barnes et al.,

360

2011), which is similar to measurements of groups of Atlantic salmon (34 ind. tank-1) of 30% - 55%

361

saturation held at 14°C - 22°C (Remen et al. (2013). It is not known if solitary housing of YTK will

362

influence the average [O2]crit value however the collective measurements of YTK would likely

363

represent more closely the ecological and aquacultural relevant inflection points.

364

The rate of decline in ambient oxygen to an hypoxic environment can significantly affect [O2]crit at

365

which longer acclimation time could potentially induce some relative tolerance to hypoxia (Rogers et

366

al., 2016). In Atlantic salmon (Remen et al., 2013) and snapper (Cook et al., 2013) a 33 and 42 day

367

hypoxia acclimation had no significant effect on [O2]crit. The time to reach [O2]LOE for YTK acclimated

368

to 15°C and 20°C was approximately 5.3 h and 2.3 h respectively. While YTK at 15C° were

369

effectively held for twice the time as those at 20C°, it is unlikely that this would have 15

370

provided sufficient acclimation time to significantly influence the results, particularly when

371

considering the work of Remen et al., (2013) and Cook et al., (2013) on Atlantic salmon and

372

snapper.

373

[O2]crit can be estimated by using segmented linear regression, also called piecewise regression or

374

broken-stick regression (BSR) (Cerezo et al., 2006; Nilsson et al., 2010).This mathematical approach

375

assumes an abrupt change to occur in the animal as it moves from being an oxyregulator to an

376

oxyconformer. In a biological sense, this approach does not seem particularly appropriate for a

377

biochemical and biophysical process. The NLS describes [O2]crit as the point at which a slope of a

378

function starts to flatten out and at which the slope approaches zero (i.e. the asymptote). Marshall

379

et al. (2013) compared [O2]crit assessed by BSR and several NLSs’, confirming that the respective

380

regression analysis can cause significant differences in the assessment of [O2]crit. It is recommended

381

to choose a regression analysis which results in the best fit by comparing models visually as well as

382

statistically using appropriate criterion such as AIC. Clearly, biphasic slopes, describing the

383

relationship between oxygen consumption and decreasing oxygen availability, show more similar

384

results in [O2]crit between the two techniques. The smoother a slope becomes, the more it appears

385

that [O2]crit assessed by NLS and BSR differ (Figure 2). NLS incorporates the smoothness of a function

386

with decreasing oxygen availability (Marshall et al., 2013). Results from this study confirm that the

387

selection of regression analysis for the determination of [O2]crit must be carefully considered. The

388

BSR analysis tended to shift the [O2]crit further towards the right on the x-axis (Figure 2), indicating a

389

lower degree of hypoxia tolerance while the [O2]crit assessed by NLS indicated that YTK had a

390

relatively greater degree of hypoxia tolerance. Due to a better statistical fit, results on the [O2]crit

391

assessed via NLS were considered more appropriate.

392 393

The results from this study base on YTK which were fasted for 36- 48 h prior to the [O2]crit investigation. Feeding of YTK might accelerate the depletion of oxygen through increased energetic 16

394

costs and might decrease the hypoxia tolerance of YTK (Pirozzi and Booth, 2009b). Therefore,

395

caution is advised to apply these results to YTK that have been fed. YTK exposed to a saturation level

396

of 60% showed an impairment of amino acid utilization and nutrient digestibility (Pirozzi et al.,

397

2019). Bowyer et al. (2014) also observed depressed growth of YTK held at <70%. Therefore, it is

398

recommended to maintain ambient saturations of 100% which can provide an equivalent normal

399

blood saturation and, depending on the situation, may provide some time to respond in the event of

400

an aquaculture system failure.

401 402

4.2 Routine metabolic rate (RMR) The effect of dietary lipid source on the routine metabolic rate was statistically insignificant

403

(Table 3), indicating that the dietary lipid-source had no effect on the RMR. Nevertheless, the

404

statistical non-significance might be the result of the high standard deviation of YTK fed the poultry

405

oil-based diet at both acclimation temperatures. Interestingly, the average RMR in YTK acclimated

406

to colder temperatures was higher when fed a poultry oil-based diet than in YTK fed a fish oil-

407

based diet as opposed to YTK acclimated to warmer temperatures. YTK acclimated to warmer

408

temperatures and fed the poultry oil-based diet, had a lower average RMR than YTK fed a fish oil-

409

based diet.

410

The effect of acclimation temperature on the RMR of YTK was statistically significant. YTK

411

acclimated to warmer temperatures had higher RMR than YTK acclimated to colder temperatures.

412

Past research on YTK showed that the RMR of YTK is linearly dependent on the ambient water

413

temperature, steadily increasing with increasing temperature (Pirozzi and Booth, 2009b).

414

Furthermore, RMR results from (Pirozzi and Booth, 2009b) and RMR results from this study strongly

415

correlate indicating a nearly twofold increase in RMR when acclimation temperatures were

416

increased from 15°C to 20°C.

417

YTK is a pelagic fish species which RMR is expected to be quite high in comparison to other

17

418

aquaculture target species. However, RMR results on southern bluefin tuna (Thunnus maccoyii),

419

another highly pelagic fish species, and Atlantic salmon indicate that the RMR of YTK is more

420

comparable to the RMR of 150.7 g Atlantic salmon acclimated to 18°C (203.9 mg kg-1 h−1) (Barnes et

421

al., 2011) than to the RMR of 19.6 kg southern bluefin tuna, where RMR is two to three times higher

422

(460 mg kg−1 h−1) (Fitzgibbon et al., 2008).

423 424

4.3. Behavioral responses to hypoxia YTK exposed to a gradual decrease of DO into progressive hypoxia exhibited a consistent

425

sequence of behavioral and visual responses across all treatments. Classifications of responses were

426

previously observed in other studies on Dover sole (McKenzie et al., 2008), Inanga (Urbina et al.,

427

2011), and YTK (Cook and Herbert, 2012) exposed to hypoxia. We also considered the change of

428

coloration and gulping. As DO deceased YTK began to first exhibit ASR, increased opercular

429

frequency and gulping. These responses can increase the oxygen uptake by switching to oxygen rich

430

surface-water and by increasing the volume of water flowing across the gills, respectively (Soares et

431

al., 2006). Oral water gulping might also increase the volume of water flowing across the gills. YTK

432

exhibited patches of dark spots or complete skin darkening during advanced hypoxia. The

433

physiological mechanisms explaining hypoxia related discoloration in fish are not well understood.

434

The hypoxia caused discoloration of tissue might be a form of cyanosis, caused by low oxygen

435

concentrations near the skin surface (Lundsgaard et al., 1923). In guppy (Poecilia reticulata), stress

436

induced the darkening of the meninges (Gibson et al., 2009). In Arctic Charr (Salvelinus alpinus)

437

social stress led to skin darkening (Höglund et al., 2000), potentially mediated by cortisol release

438

dispersing chromatophores in skin tissue (Fujii and Oshima, 1986). YTK exhibited repetitive rush and

439

rest behavior, perhaps in attempt to avoid the hypoxic zone (McKenzie et al., 2008) and/or to ram

440

ventilate, increasing the flow of water over the gills. However, tradeoff of rush or rest is that it

441

increases the energy demand, subsequently increasing the aerobic metabolism in a hypoxic

18

442

environment. In contrast to YTK, juvenile Dover sole show strong burst swimming at 2.18 mg O2 L-1

443

post [O2]crit, while YTK’s showed rush or rest at 1.50-1 mg O2 L-1. The early sign of change in

444

ventilation in Dover sole might present enough time to rectify a hypoxia condition in aquaculture,

445

while the late rush or rest behavior might be too late for DO interventions as it is shortly followed by

446

the loss of equilibrium in YTK. In the absence of real-time DO monitoring systems or regular DO

447

measurements, awareness of these early visual hypoxia responses may provide an opportunity for

448

early intervention preventing mortalities to hypoxia in aquaculture systems.

449

5. Conclusion

450

This study quantified the critical DO threshold of fasted YTK. Concomitant with this threshold are a

451

consistent sequence of behavioral responses to hypoxia which are initiated with increased opercular

452

frequency, gulping, ASR, and end with the loss of equilibrium. Standard management practices

453

should ensure aquaculture systems remain saturated (100% DO) at all times; however, if the

454

initiation of these behavioral responses is observed in culture situations, rapid re-oxygenation must

455

be implemented to avoid the onset of lethal hypoxic conditions. Fasted YTK are hypoxia sensitive,

456

especially when held at warmer water temperatures; at a temperature of 20°C a DO concentration

457

of below 2.6 mg O2 L-1 (~38% saturation) will induce hypoxia. YTK have an elevated RMR in warm

458

water compared to YTK held in cool water. The time taken to deplete normoxic saturated (100%)

459

water to [O2]crit levels at 15°C is more than double that of YTK at 20°C; this has significant

460

implications on the reaction time to implement re-oxygenation of a rearing system should a system

461

failure occur. The influence of dietary oil source did not have a significant effect on respiration rates

462

of YTK; however, the consistent trend observed when comparing [O2]crit of YTK fed poultry oil diets

463

to YTK fed fish oil diets warrants further investigation.

464 465

6. Acknowledgements This project is supported by funding from the Australian Government Department of

19

466

Agriculture and Water Resources as part of its Rural R&D for Profit program, the Fisheries Research

467

and Development Corporation (FRDC), South Australian Research and Development Institute

468

(SARDI), Clean Seas Seafood, New South Wales Department of Primary Industries (NSW DPI) and

469

Huon Aquaculture. Ridley and Skretting Australia have also contributed actively to the project

470

through the input of technical information. The financial support of Macquarie University and the

471

University of Hamburg is gratefully acknowledged.

472

We would like to thank Dr. Michael Salini from Ridley Aquafeed for provision of several of

473

the raw materials used in the experimental diets and Dr. Jodie Rummer from James Cook University

474

for the provision of a FireStingO2 device. We also thank Dr. Basseer Codabaccus, Brendan Findlay

475

and Ian Russel who provided valuable technical support and assistance in running experiments and

476

constructing respirometers (NSW DPI). We would also like to thank the team at CSIRO Agriculture

477

and Food (especially Barney Hines) for the chemical analysis of diets.

478 479 480 481 482 483 484 485 486 487 488 489

7. Reference ACEC, 2017. A guide to acceptable procedures and practices for aquaculture and fisheries research, 4th Edition, NSW DPI Animal Care and Ethics Committee. Barnes, R., King, H., Carter, C.G., 2011. Hypoxia tolerance and oxygen regulation in Atlantic salmon, Salmo salar from a Tasmanian population. Aquaculture 318, 397–401. Barton, B.A., Zwama, G.K., 1991. Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of Corticosteids. Annu. Rev. Fish Dis. 3–26. Beamish, F.W.H., 1964. Respiration of fishes with special emphasis on standard oxygen consumption. Can. J. Zool. 42, 177–188. Booth, M.A., Allan, G.L., Pirozzi, I., 2010. Estimation of digestible protein and energy requirements of yellowtail kingfish Seriola lalandi using a factorial approach. Aquaculture 307, 247–259.

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Claireaux, G., Chabot, D., 2016. Responses by fishes to environmental hypoxia: Integration through Fry’s concept of aerobic metabolic scope. J. Fish Biol. 88, 232–251. Collins, G.M., Clark, T.D., Rummer, J.L., Carton, A.G., 2013. Hypoxia tolerance is conserved across

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Table 1. Experiment diet formulations (dry matter basis). Ingredient (%) Fishmeal Wheat flour Dehulled lupin Taurine Vit/min premix Monosodium phosphate Choline chloride 70%

Fish oil diet 55.0 15.0 10.0 1.0 0.5 0.15 0.05

Poultry oil diet 55.0 15.0 10.0 1.0 0.5 0.15 0.05

Rovimix Stay-C 35%

0.05

0.05

Fish oil Poultry oil

18.25 -

18.25

610 611

26

612

Table 2. Measured proximate and fatty acid content of experimental diets (dry matter basis). Proximates (%) Fish oil-based diet Poultry oil-based diet Moisture 6.05 6.23 Crude lipid 22.60 23.89 Crude protein 56.23 53.26 Carbohydrate 13.22 14.85 Ash 7.95 7.99 Total nitrogen 9.00 8.52 -1 Gross energy (MJ kg ) 25.08 24.66 Fatty acid (% of total fatty acids) C12:0 0.0 0.1 C14:0 4.0 1.6 C14:1n-5 0.0 0.2 C15:0 0.7 0.3 C16:0 19.0 21.3 C16:1n-7 4.9 5.0 C17:0 0.5 0.4 C17:1 0.6 0.3 C18:0 4.2 6.0 C18:1n-9T 0.2 0.2 C18:1n-9C 22.9 38.1 C18:1n-7 3.5 2.6 C18:2n-6T 0.2 0.1 C18:2n-6C 3.2 12.0 C19:0 0.2 0.2 C18:3n-6 0.0 0.0 C18:3n-3 1.3 2.1 C18:4n-3 1.0 0.4 C20:0 0.3 0.2 C20:1n-9 7.1 0.8 C20:1n-7 0.5 0.1 C20:2n-6 0.4 0.2 C20:4n-6 1.1 0.6 C20:3n-3 0.3 0.1 C20:5n-3 6.8 1.8 C22:0 0.3 0.2 C22:1n-9 1.1 0.1 C23:0 0.1 0.0 C22:4n-6 0.2 0.1 C24:0 1.8 0.4 C22:5n-3 0.1 0.1 C22:6n-3 12.6 4.4 C22:1n-9 1.0 0.1 Total saturated 31.7 30.9 Total monoenes 41.7 47.4 Total PUFA 27.1 21.9 Total n-3 PUFA 22.0 8.8 Total n-6 PUFA 5.2 13.1 n-3/n-6 4.2 0.7

613

27

Table 3. Mean values (±SD) for routine metabolic rate (RMR) under normoxic conditions, duration of [O2]crit trial in h, critical oxygen level using non-linear (NLS) and segmental ‘broken-stick’ linear (BSR)regression model in YTK fed either poultry or fish oil and acclimated to either 20 or 15°C. Different superscript letters within columns indicate a significant difference. Diet Temp.(°C) Harvest RMR Time BSR - [O2]crit NLS - [O2]crit -1 weight (mg O2 kg h (h) 1 -1 -1 (g) ) (mg·L ) (mg·L ) (%saturation) a a a a Fish oil 20 538±77 264.0±44.90 2.30 2.50±0.07 2.24±0.13a 29.09±1.94a Poultry a a a a a a 20 515±77 255.68±33.71 2.25 3.04±0.60 2.92±0.73 38.94±9.61 oil b b b b b Fish oil 15 445±81 166.95±8.93 5.10 2.33±0.67b 1.84±0.19 22.24±2.60 Poultry b b b b b b 15 421±56 179.05±11.47 5.33 2.25±0.46 2.00±0.33 23.77±4.15 oil Temp. <0.01 <0.01 <0.01 <0.05 <0.05 <0.05 p-value Diet NS NS NS NS NS NS p-value Diet x NS NS NS NS NS NS Temp.

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Table 4. Mean values (± SD) for hypoxia induced visual and behavioral changes and the loss of equilibrium in YTK fed different dietary oils and acclimated to different temperatures. Different superscript letters within columns indicate a significant difference. Behavior 1 2 3 4 Diet Temp. (°C) [O2]vent [O2]col [O2]R/R [O2]LOE a a Fish oil 20 2.14±0.30 1.68±0.11 1.30±0.09 1.57±0.15a a Poultry oil 20 2.48±0.44 1.92±0.36 1.48±0.21a 1.90±0.22a a b Fish oil 15 2.15±0.39 1.54±0.33 1.22±0.08 1.52±0.03b a b Poultry oil 15 2.39±0.15 1.59±0.19 1.29±0.08 1.50±0.06b Temp. p-value NS =0.05 <0.05 <0.05 Diet p-value NS NS NS NS 5 Diet x Temp. NS NS NS NS 1 [O2]vent, oxygen level at which individual YTK exhibited ASR, increased opercular frequency and oral water gulping 2 [O2]col, oxygen level at which individual YTK showed a change in color 3 [O2]R/R, oxygen level at which individual YTK exhibited repetitive rush or rest behavior 4 [O2]LOE, loss of equilibrium 5 NS, non-significant.

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Figure 1. Closed static set-up for hypoxia tolerance measurements in YTK. One large tank (light grey) housed six respirometer units (dark grey). Respirometer units held four fish and were sealed with clear perspex lids. Within each respirometer a small submersible pump circulated water. On the inside of each perspex lid a fibre-optic oxygen sensor-spot was attached and connected to the FireSting unit. Water temperature was maintained via heating/chilling unit. Additionally, water was treated with industrial oxygen ensuring 100% DO in seawater prior to respirometer.

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Figure 2. Respective [O2]crit plots of YTK acclimated to (a) 20°C and a fish oil based diet, (b) 20°C and a poultry oil based diet, (c) 15°C and a fish oil based diet, (d)15°C and a poultry oil based diet. Data points indicate the mean mass-specific oxygen consumption rate over a 6 min period. The [O2]crit was assessed via multiphasic linear modelling indicated by the vertical solid line. The grey zone indicates the mean ±95% confidence interval. Routine metabolic rate (RMR) is indicated by the horizontal regression line. The mean of four different behavioral responses to low DO are indicated as; (α) [O2]LOE, (β) [O2]R/R, (χ) [O2]col, and (δ) [O2]vent.

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Author statement Author Caroline L . Candebat Mark Booth Jane E. Williamson Igor Pirozzi

Credit Conceptualization, Methodology, Formal Analysis, Investigation, Writing – Original Draft, Visualization Conceptualization, Methodology, Resources, Writing – Review & Editing, Supervision, Funding Conceptualization, Resources, Writing – Review & Editing, Supervision Conceptualization, Methodology, Formal Analysis, Investigation, Resources, Writing – Review & Editing, Supervision

Declaration of interests ☒The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.