Guidelines for reporting methods to estimate metabolic rates by aquatic intermittent-flow respirometry.

Interest in the measurement of metabolic rates is growing rapidly, because of the importance of metabolism in advancing our understanding of organismal physiology, behaviour, evolution and responses to environmental change. The study of metabolism in aquatic animals is undergoing an especially pronounced expansion, with more researchers utilising intermittent-flow respirometry as a research tool than ever before. Aquatic respirometry measures the rate of oxygen uptake as a proxy for metabolic rate, and the intermittent-flow technique has numerous strengths for use with aquatic animals, allowing metabolic rate to be repeatedly estimated on individual animals over several hours or days and during exposure to various conditions or stimuli. There are, however, no published guidelines for the reporting of methodological details when using this method. Here, we provide the first guidelines for reporting intermittent-flow respirometry methods, in the form of a checklist of criteria that we consider to be the minimum required for the interpretation, evaluation and replication of experiments using intermittent-flow respirometry. Furthermore, using a survey of the existing literature, we show that there has been incomplete and inconsistent reporting of methods for intermittent-flow respirometry over the past few decades. Use of the provided checklist of required criteria by researchers when publishing their work should increase consistency of the reporting of methods for studies that use intermittent-flow respirometry. With the steep increase in studies using intermittent-flow respirometry, now is the ideal time to standardise reporting of methods, so that - in the future - data can be properly assessed by other scientists and conservationists.

Shuttle-box systems for studying preferred environmental ranges by aquatic animals.

Animals' selection of environments within a preferred range is key to understanding their habitat selection, tolerance to stressors and responses to environmental change. For aquatic animals, preferred environmental ranges can be studied in so-called shuttle-boxes, where an animal can choose its ambient environment by shuttling between separate choice chambers with differences in an environmental variable. Over time, researchers have refined the shuttle-box technology and applied them in many different research contexts, and we here review the use of shuttle-boxes as a research tool with aquatic animals over the past 50 years. Most studies on the methodology have been published in the latest decade, probably due to an increasing research interest in the effects of environmental change, which underlines the current popularity of the system. The shuttle-box has been applied to a wide range of research topics with regards to preferred ranges of temperature, CO 2 , salinity and O 2  in a vast diversity of species, showing broad applicability for the system. We have synthesized the current state-of-the-art of the methodology and provided best practice guidelines with regards to setup, data analyses, experimental design and study reporting. We have also identified a series of knowledge gaps, which can and should be addressed in future studies. We conclude with highlighting directions for research using shuttle-boxes within evolutionary biology and behavioural and physiological ecology.

Effects of temperature on physiological performance and behavioral thermoregulation in an invasive fish, the round goby.

Invasive species exert negative impacts on biodiversity and ecosystems on a global scale, which may be enhanced in the future by climate change. Knowledge of how invasive species respond physiologically and behaviorally to novel and changing environments can improve our understanding of which traits enable the ecological success of these species, and potentially facilitate mitigation efforts. We examined the effects of acclimation to temperatures ranging from 5 to 28°C on aerobic metabolic rates, upper temperature tolerance (critical thermal maximum, CTmax), as well as temperature preference (Tpref) and avoidance (Tavoid) of the round goby (Neogobius melanostomus), one of the most impactful invasive species in the world. We show that round goby maintained a high aerobic scope from 15 to 28°C; that is, the capacity to increase its aerobic metabolic rate above that of its maintenance metabolism remained high across a broad thermal range. Although CTmax increased relatively little with acclimation temperature compared with other species, Tpref and Tavoid were not affected by acclimation temperature at all, meaning that round goby maintained a large thermal safety margin (CTmax-Tavoid) across acclimation temperatures, indicating a high level of thermal resilience in this species. The unperturbed physiological performance and high thermal resilience were probably facilitated by high levels of phenotypic buffering, which can make species readily adaptable and ecologically competitive in novel and changing environments. We suggest that these physiological and behavioral traits could be common for invasive species, which would only increase their success under continued climate change.

The combined effect of body size and temperature on oxygen consumption rates and the size-dependency of preferred temperature in European perch Perca fluviatilis.

The present study determined the effect of body mass and acclimation temperature (15-28°C) on oxygen consumption rate (MO2 ) and the size dependency of preferred temperature in European perch Perca fluviatilis. Standard metabolic rate (SMR) scaled allometrically with body mass by an exponent of 0.86, and temperature influenced SMR with a Q10 of 1.9 regardless of size. Maximum metabolic rate (MMR) and aerobic scope (MMR-SMR) scaled allometrically with body mass by exponents of 0.75-0.88. The mass scaling exponents of MMR and aerobic scope changed with temperature and were lowest at the highest temperature. Consequently, the optimal temperature for aerobic scope decreased with increasing body mass. Notably, fish <40 g did not show a decrease aerobic scope with increasing temperature. Factorial aerobic scope (MMR × SMR-1 ) generally decreased with increasing temperatures, was unaffected by size at the lower temperatures, and scaled negatively with body mass at the highest temperature. Similar to the optimal temperature for aerobic scope, preferred temperature declined with increasing body mass, unaffectedly by acclimation temperature. The present study indicates a limitation in the capacity for oxygen uptake in larger fish at high temperatures. A constraint in oxygen uptake at high temperature may restrict the growth of larger fish with environmental warming, at least if food availability is not limited. Furthermore, behavioural thermoregulation may be contributing to regional changes in the size distribution of fish in the wild caused by global warming as larger individuals will prefer colder water at higher latitudes and at larger depths than smaller conspecifics with increasing environmental temperatures.

Intra-Specific Difference in the Effect of Salinity on Physiological Performance in European Perch (Perca fluviatilis) and Its Ecological Importance for Fish in Estuaries.

Changes in environmental salinity challenge fish homeostasis and may affect physiological performance, such as swimming capacity and metabolism, which are important for foraging, migration, and escaping predators in the wild. The effects of salinity stress on physiological performance are largely species specific, but may also depend on intra-specific differences in physiological capabilities of sub-populations. We measured critical swimming speed (Ucrit) and metabolic rates during swimming and at rest at salinities of 0 and 10 in European perch (Perca fluviatilis) from a low salinity tolerance population (LSTP) and a high salinity tolerance population (HSTP). Ucrit of LSTP was significantly reduced at a salinity of 10 yet was unaffected by salinity change in HSTP. We did not detect a significant cost of osmoregulation, which should theoretically be apparent from the metabolic rates during swimming and at rest at a salinity of 0 compared to at a salinity of 10 (iso-osmotic). Maximum metabolic rates were also not affected by salinity, indicating a modest tradeoff between respiration and osmoregulation (osmo-respiratory compromise). Intra-specific differences in effects of salinity on physiological performance are important for fish species to maintain ecological compatibility in estuarine environments, yet render these sub-populations vulnerable to fisheries. The findings of the present study are therefore valuable knowledge in conservation and management of estuarine fish populations.

Maximum salinity tolerance and osmoregulatory capabilities of European perch Perca fluviatilis populations originating from different salinity habitats.

Although considered a stenohaline freshwater species, European perch (Perca fluviatilis) inhabit brackish waters. The present study determined the maximum salinity tolerance and osmoregulatory capability on individuals originating from brackish water and from freshwater populations. The fish were acclimated for 3 weeks to salinities of 0, 10, 12.5, 15, 17.5 and 20 after an initial stepwise increase to the target salinity. The maximum salinity tolerance was determined as the test salinity below which the fish could not acclimate and lost equilibrium. Blood plasma osmolality was measured if the fish had not lost equilibrium after the acclimation period. The maximum salinity tolerance was 17.5 for brackish water European perch and 10 for fresh water European perch. The high salinity tolerance of the brackish water European perch was caused by their ability to both hyper- and hypo-osmoregulate, whereas the freshwater originating fish could only hyper-osmoregulate. The results showed that maximum salinity tolerances and osmoregulatory capabilities depends on the origin habitat salinity. Due to genetic differentiation between European perch populations in brackish and fresh water, the possibility of brackish water European perch being a subspecies of European perch is discussed, yet vital knowledge concerning heritability of salinity tolerance traits is still missing. Regardless of species status, within-species plasticity in the ability to cope with varying salinities have substantial ecological and conservation implications and underlines the need for managing brackish water and freshwater European perch stocks separately.

Comparison of the organic matrix found in intestinal CaCO3 precipitates produced by several marine teleost species.

Marine bony fish poses the unique ability to hydrate from imbibed seawater. They accomplish this, in part, by the precipitation of inorganic carbonate mineral in their intestine, which lowers luminal osmotic pressure and allows for water uptake. It has recently been described that in the Gulf toadfish (Opsanus beta) this Ca(Mg)CO3 precipitation occurs under the regulation of an organic matrix. To date no investigations have aimed to determine if this phenomenon applies more generally to marine fish. Here, intestinally derived precipitates were collected from gray snapper (Lutjanus griseus), white grunt (Haemulon plumieri), European flounder (Platichthys flesus), as well as Gulf toadfish, and their matrices were extracted. The ability of these matrices to regulate CaCO3 production was determined using an in vitro calcification assay, which revealed that the matrix derived from each of the tested species increased precipitation at low concentrations, while inhibiting it at higher concentrations in full agreement with the earlier studies on toadfish. Matrix extracted from European flounder precipitates was then analyzed by mass spectrometry, leading to the identification of over 50 unique proteins. When the identities of these proteins were compared to previous investigation of toadfish precipitate matrix, nearly 35% were found to overlap between the flounder and toadfish analyses, suggesting conserved mechanisms of precipitation control. The effects of using different sodium hypochlorite (NaOCl) solutions during precipitate purification on the resulting organic matrix are also discussed.

Evaluating dispersal potential of an invasive fish by the use of aerobic scope and osmoregulation capacity.

Non-indigenous species (NIS) can impact marine biodiversity and ecosystem structure and function. Once introduced into a new region, secondary dispersal is limited by the physiology of the organism in relation to the ambient environment and by complex interactions between a suite of ecological factors such as presence of predators, competitors, and parasites. Early prediction of dispersal potential and future 'area of impact' is challenging, but also a great asset in taking appropriate management actions. Aerobic scope (AS) in fish has been linked to various fitness-related parameters, and may be valuable in determining dispersal potential of aquatic invasive species in novel environments. Round goby, Neogobius melanostomus, one of the most wide-ranging invasive fish species in Europe and North America, currently thrives in brackish and fresh water, but its ability to survive in high salinity waters is unknown to date. We show that AS in round goby is reduced by 30% and blood plasma osmolality increased (indicating reduced capacity for osmoregulation) at salinities approaching oceanic conditions, following slow ramping (5 PSU per week) and subsequent long-term acclimation to salinities ranging between 0 and 30 PSU (8 days at final treatment salinities before blood plasma osmolality measurements, 12-20 additional days before respirometry). Survival was also reduced at the highest salinities yet a significant proportion (61%) of the fish survived at 30 PSU. Reduced physiological performance at the highest salinities may affect growth and competitive ability under oceanic conditions, but to what extent reduced AS and osmoregulatory capacity will slow the current 30 km year-1 rate of advance of the species through the steep salinity gradient from the brackish Baltic Sea and into the oceanic North Sea remains speculative. An unintended natural experiment is in progress to test whether the rate of advance slows down. At the current rate of advance the species will reach the oceanic North Sea by 2018/2019, therefore time for taking preventative action is short.