大西洋鮭魚在高溫下的急性脅迫性死亡與攝氧能力不足有關

主題
對魚類的熱研究可以幫助我們了解它們對氣候變暖的穩健性。大多數實驗都是在較小的個體上進行的，并且由于生理縮放效應，特別是在耐熱性和呼吸能力方面，可能不代表更大的生命階段。在這項研究中，在適應 9 °C 或 19 °C 的海水 3 周后，對大大西洋鮭魚 （Salmo salar）（≈4 kg） 進行了呼吸測定實驗。 此外，還評估了鰓和心臟形態特征。在 9 °C 時，代謝率類似于早期對小魚的研究。然而，在壓力暴露后 19 °C 下，81% 的魚在 ≈6 小時內意外死亡，而幸存的魚則難以恢復基線代謝率。值得注意的是，代謝率在不同溫度下保持相似，而之前發現較小的大西洋鮭魚會提高其最大代謝率，直到接近致命溫度。由于標準代謝率也不可避免地隨著溫度的增加而增加，因此有氧范圍在 19 °C 時會降低。 同時，層狀密度不受影響，表明鰓表面積相似。然而，適應 19 °C 會降低心室圓度和對稱性，而球葉寬度與心室寬度之比增加。這些變化可能反映了對新陳代謝要求更高的環境的適應性反應。然而，在壓力期間，魚似乎無法在 19 °C 時提供足夠的氧氣，我們將其歸因于生理鱗片限制。因此，大西洋鮭魚在高溫下更容易受到壓力引起的死亡，這表明相對于較小的個體，耐熱性降低。這凸顯了將較大的魚納入實驗的必要性，作為體型差異大差異導致熱耐受性變化的基礎。

Thermal studies on fish can help us to understand their robustness to warming climates. Most experiments are performed on smaller individuals and may not represent larger life-stages owing to physiological scaling effects, particularly with regards to thermal tolerance and respiratory capacities. In this study, respirometry experiments were performed on big Atlantic salmon (Salmo salar) (≈4 kg) following 3-weeks acclimation to seawater of 9 °C or 19 °C. Additionally, gill and heart morphology traits were assessed. At 9 °C metabolic rates resembled earlier work on smaller fish. However, at 19 °C following stress exposure, 81 % died unexpectedly within ≈6 h while surviving fish struggled to recover a baseline metabolic rate. Most noteworthy was that maximum metabolic rates remained similar across temperature whereas smaller Atlantic salmon previously were found to increase their maximum metabolic rates until near-lethal temperatures. As standard metabolic rates also inevitably increases with temperature, aerobic scopes become reduced at 19 °C. Meanwhile lamellar density was unaffected, indicating similar gill surface areas. However, acclimation to 19 °C reduced ventricle roundness and symmetry, while bulbus width to ventricle width ratios increased. These changes presumably reflect adaptive responses to more metabolically demanding environments. Yet the fish appeared unable to supply sufficient oxygen at 19 °C during stress, which we attribute to physiological scaling constraints. Big Atlantic salmon were therefore more susceptible to stress-induced mortality at elevated temperatures, indicating reduced thermal tolerance relative to smaller individuals. This highlights the need to include larger fish in experiments as the underlying basis for thermal tolerance changes across large differences in body size.

關鍵字
有氧范圍心臟形態延遲死亡率缺氧耐受性 層密度呼吸測定

Aerobic scope/ Cardiac morphology /Delayed mortality /Hypoxia tolerance /Lamellar density /Respirometry

1. Introduction
Climate change may favor smaller fish sizes owing to faster developmental rates and earlier reproduction ages, together with more limited food resources to support increased energetic demands in warmer environments (Daufresne et al., 2009; Gardner et al., 2011; Sheridan and Bickford, 2011).
A mechanistic framework to explain shrinking body sizes in a warmer future is the gill-oxygen limitation theory which proposes that geometrical scaling constraints of the two-dimensional surface of gill lamellae cannot keep up with the increasing oxygen demand of growing three-dimensional bodies (Pauly and Cheung, 2018; Pauly, 2021). However, this idea has been debated (Lefevre et al., 2017). The main opposing argument is that since gill lamellae are folded surfaces, they are well able to increase appropriately with body size, and moreover, physiological data shows that the respiratory surface areas in fish reflects species-specific adaptations in metabolic requirements and not the other way around (Lefevre et al., 2017; Scheuffele et al., 2021; Skeeles and Clark, 2024).
Another widespread framework to assess thermal tolerance and effects of climate change in fish is aerobic scope models (P?rtner and Farrell, 2008; Clark et al., 2013; Lefevre, 2016). The aerobic scope is the capacity to increase metabolic rates above resting baseline levels, either reported as the absolute or factorial differences between maximum and standard metabolic rates (MMR and SMR, respectively) (Fry and Hart, 1948; Brett, 1971; Claireaux and Lefrancois, 2007; Clark et al., 2013). All physiological functions have an energetic cost, and the aerobic scope then represents the capacity to engage in activities such as foraging, growth, predator avoidance, and reproduction. Environmental conditions that reduce aerobic scopes are therefore less optimal as activities become energetically more restricted. How the aerobic scope is affected by temperature in species of fish has received much scrutiny (Lefevre, 2016). Generally, SMR increases steadily with temperature whereas MMR also tends to increase throughout the thermal niche and sometimes plateaus or even declines at extreme temperatures (Fry and Hart, 1948; Brett, 1964; Claireaux et al., 2006; Norin et al., 2014; Hvas et al., 2017; Leonard and Skov, 2022). The resultant aerobic scope therefore tends to increase with temperature when expressed in absolute units but may decrease when expressed as a factor, leading to some confusion in its interpretation (Halsey et al., 2018). Furthermore, whether ecologically relevant thermal limits of fish can be explained by oxygen supply limitations and thus declining aerobic scopes has been debated (P?rtner et al., 2017; Jutfelt et al., 2018).
Meanwhile, an often overlooked key feature of aerobic scope models is that they change with ontogeny and body size, where the earliest life-stages and the final sexual mature life-stages are expected to have lower aerobic scopes (Killen et al., 2007; P?rtner and Farrell, 2008). Those critical life-stages should therefore be more sensitive to climate change effects. Despite of this, experimental work on thermal fish physiology has mainly been performed on juvenile intermediate life-stages of fish, as it can be technically difficult to experiment on small fish larvae and even more so on larger individuals weighing several kilograms.
A relevant species to consider in terms of temperature effects across body size is the Atlantic salmon (Salmo salar) owing to its cultural importance in recreational fishing, its economic importance in aquaculture, and conservation concerns of wild populations (Krko?ek et al., 2007; Dadswell et al., 2021; Vollset et al., 2021). The thermal niche and limits along with numerous miscellaneous biological temperature effects have been widely studied in this species. Briefly summarized, Atlantic salmon are eurythermal anadromous fish that naturally may encounter temperatures from 0 to 3 °C in their northern distribution (Lacroix, 2013; Reddin, 1985), and occasionally to above 20 °C in summer (Valiente et al., 2011). While they may spend most of their life in nature below 10 °C, the growth optimum under cultured conditions is 10.5 °C–14 °C, depending on size (Handeland et al., 2003, 2008; Fraser et al., 2025). Above 18 °C growth declines owing to lower appetite and feed conversion efficiency (Hevr?y et al., 2015; Kullgren et al., 2013; Wade et al., 2019). Behaviorally Atlantic salmon appear to avoid environments above 16 °C if possible (Johansson et al., 2009; Lacroix, 2013). Long-term survival is generally impossible at chronic temperatures above 22 °C (Hvas et al., 2017; Gamperl et al., 2020), although some genetic variation in upper thermal limits have been documented (Ignatz et al., 2023).
Most experimental data on fish thermal physiology has been obtained on smaller sized individuals and may not represent larger individuals owing to size-scaling constraints in oxygen supply. The purpose of this study was therefore to measure metabolic rate traits and aerobic scopes in larger sized Atlantic salmon (≈4 kg) acclimated to midrange (9 °C) and suboptimal (19 °C) seawater temperatures. Morphological metrics of gills and hearts were also assessed to infer potential links between form and function in respiratory capabilities as both organs are known to display substantial phenotypic plasticity in response to the environment (Crispo and Chapman, 2010; Nilsson et al., 2012; Vindas et al., 2024). Earlier work on smaller sized Atlantic salmon found preserved aerobic scopes at high suboptimal seawater temperatures (Hvas et al., 2017; Hvas, 2022). However, if size-scaling constraints in fact becomes a limiting factor for gill oxygen uptake (e.g., Pauly, 2021), we hypothesized that big Atlantic salmon would display an impaired aerobic scope at the higher acclimation temperature.
2. Materials and methods
2.1. Fish husbandry
Big Atlantic salmon post-smolts of a cultured genotype (Aquagen) hatched and reared on-site at the Matre Research Station, Institute of Marine Research, Norway was used in this study. Two months prior to the experimental trials, approximately 120 fish were transferred and distributed into four indoor circular holding tanks (3 m in diameter, 5.3 m3 in volume). Aerated, filtered, and UV-C treated seawater of 34 ppt was supplied into the holding tanks at a continuous flow-through of 120 l min-1 to provide normoxia and remove waste products. A 12h light/dark photoperiod between 08.00 and 20.00 was used and the fish were fed size appropriate commercial feed pellets (Skretting, Norway) in excess daily via automatic feeding devices. The water temperature was initially maintained at 9 °C in all tanks and was also the test temperature used for the first treatment group. To acclimate fish to the second test temperature of 19 °C in two of the holding tanks, the temperature was first increased from 9 °C to 13 °C, then two days later increased further to 16 °C, and after another two days finally increased to 19 °C. The fish were then maintained at 19 °C for a minimum of three weeks before experimental trials at this temperature. To provide adequate oxygen conditions (>85 %PO2) at 19 °C, oxygen diffusers were added in the holding tanks. The desired water temperatures were maintained precisely via custom made computer software (SDMatre, Normatic AS, Nordfjordeid, Norway) that controlled the automatic mixing of ambient and heated water reservoirs in header tanks above the holding tanks.
This experiment was conducted between February and April 2023 and ethical approval for the use of animals in scientific research was obtained from The Norwegian Food Safety Authorities under permit number 29883.
2.2. Respirometry setup
To measure MO2 in big Atlantic salmon, an automatic static intermittent-flow respirometry system was used (上海瑾瑜 Loligo Systems, Viborg, Denmark). The system consisted of four cylindrical shaped acrylic chambers submerged in their own water tanks so that four individual fish could be tested simultaneously and independently. The respirometry chambers were 125 cm long with a 30 cm internal diameter and were connected to an internal loop with gas-tight PVC tubes that ran through a circulation pump and a flow-through oxygen sensor cell (measuring at 1 Hz). The total closed volume including tubes was 88.88 l. Each chamber was connected to an open loop with a flush pump (Eheim, 20 l min-1) to facilitate intermittent flush periods. Flush pumps, a temperature probe, and oxygen fiber cables were connected to a computer running the AutoResp software (Loligo Systems), and each oxygen sensor had been carefully calibrated according to the manufacturer's instructions before experimental trials started and was checked again prior to the second treatment group. Each water tank containing a respirometer was 150 x 150 cm square shaped and 60 cm high, with an adjusted water height of 45 cm (volume of 1013 l). Furthermore, each tank had its own flow-through water supply of ≈40 l min-1, using the same water supply from the header tank that supplied the holding tanks, ensuring stable temperatures as well as a continuous exchange with clean water. Each respirometry setup was covered with black plastic sheets once a trial with a fish had started. To further mitigate potential disturbances to the fish, the room lights were turned off and other activities in the laboratory hall were not allowed while trials were running.