Anthropogenic CO2-mediated freshwater acidification limits survival, calcification, metabolism, and behaviour in stress-tolerant freshwater crustaceans

Dissolution of anthropogenic CO2 is chronically acidifying aquatic ecosystems. Studies indicate that ocean acidification will cause marine life, especially calcifying species, to suffer at the organismal and ecosystem levels. In comparison, freshwater acidification has received less attention rendering its consequences unclear. Here, juvenile Chinese 15 mitten crabs, Eriocheir sinensis, were used as a calcifying model to investigate the impacts of CO2-mediated freshwater acidification. Our integrative approach investigating changes in the animal’s acid-base homeostasis, metabolism, calcification, locomotory behaviour, and survival rate indicate that the crab will face energetic consequences from future freshwater acidification. These energetic trade-offs allow the animal to maintain its acid-base homeostasis at the cost of reduced metabolic activity, exoskeletal calcification, and locomotion reducing the animal’s overall fitness and increasing its mortality. Results 20 suggest that present-day calcifying invertebrates could be heavily affected by freshwater acidification similar to their marine organisms and emphasizes the importance in understanding the long-term implications of freshwater acidification on species fitness.

acidification conditions. Therefore, we hypothesized that E. sinensis would be well-adapted to counteract challenges associated with fluctuating pCO2 resulting from anthropogenic activity and not experience detrimental physiological or behavioural impairment.

Animal Maintenance 70
Wild-caught male and female juvenile Chinese mitten crab (Eriocheir sinensis 10-20 g) were purchased from the Chinese mitten crab Breeding Association of Taiwan. Crabs were maintained at the Academia Sinica Institute of Cellular and Organismal Biology aquatics facility (Taipei, Taiwan) in three 120-L aquariums with flow through dechlorinated Taipei tap water (in µmol l -1 Na + 237, K + 16, Ca 2+ 216, Mg 2+ 213, Cl -201; Y.C. Tseng pers. comm. See ringer measurement methods below) on a 14:10 h light-dark cycle with temperature ranging from 23 to 25 ºC. Water parameters for these holding tanks 75 were the same as that of the control water used in the experimental acclimations. Juvenile crabs in non-experimental holding tanks were maintained at a density of roughly 100 individuals per tank with PVC pipes for shelter and a constant flow of freshwater to prevent the build-up of metabolic wastes. Crabs were fed ad libitum with oatmeal and mollusc meat three times per week and monitored for activity level and the presence of disease as general health indicators. Diet was selected to maintain an omnivorous diet as seen in the wild (Czerniejewski et al., 2010) and based on what is fed by our crab supplier (Y.C. Tseng 80 pers. comm.). Crabs were fasted for a minimum of 48 hours before sampling to minimize the effects of dietary intake on measured parameters.

Freshwater acidification
For experimental acclimation, crabs were sampled upon removal from the holding tanks (0-day time point) and transferred to flow through 10-L experimental tanks (6-7 crabs per tank, 4 tanks per treatment) containing either control or acidified 85 freshwater (Table 1) with PVC pipes added for shelter. Acidified freshwater was achieved by injection of CO2 directly into the experimental tanks by air stone to maintain environmental pH (pH controller, Aqua-MACRO). The pH controller system used in this study required that each tank had a pH probe, pH controller, CO2 tank, gas regulating solenoid and air stone, thus meaning each tank in this study was independently pH/CO2 regulated. CO2 bubbling rate and freshwater flow rate were adjusted to minimize overshooting the target pCO2 level. Following injection of CO2 to regulate water pCO2 we recorded a 90 brief pCO2 overshoot to a maximum level of 5625 µatm resulting from direct CO2 injection into the experimental tanks by the pH controller. Water pH, total alkalinity, and temperature were regularly measured in the experimental tanks throughout the study. Water pH (NBS scale) and temperature were measured with a pH electrode (Accumet AP55 pH/ATC electrode, Ohio, USA) connected to a portable pH meter (Accumet AP71, Ohio, USA) calibrated with pH buffers (pH 4.00, 7.00, and 10.01) traceable to NIST standard reference material (Thermofisher Orion). Water alkalinity was measured by spectrophotometric 95 assay on a Nanodrop 2000c (Thermo scientific, Wilminton, DE, USA) according to previously established protocols (Sarazin et al., 1999). Water pCO2 was calculated with the CO2SYS excel add-in (Lewis and Wallace, 1998) using measured water temperature, pH and total alkalinity. Constants used for pCO2 calculations include freshwater carbonate dissociation constants (K1 and K2) from Millero (1979), and KHSO4 constants from Dickson (1990).

Hemolymph Acid-Base Status 105
Hemolymph acid-base experiments were conducted over seven days to determine if crabs could actively regulate acid-base status in the presence of future freshwater acidification conditions. Hemolymph samples (100 µL per crab) were taken at the base of a walking leg with a sterile syringe according to previous protocols for E. sinensis (Truchot, 1992). Samples from 2 to 3 crabs were pooled together (200-300 µL pooled hemolymph per n value) to get a sufficient volume for downstream analyses of ammonia, pH, and total carbon. Pooled hemolymph samples were gently mixed by slowly pipetting to avoid off gassing of 110 CO2 and disrupting hemolymph acid-base parameters. Measurements of pH and total carbon were performed immediately after hemolymph collection and the remaining hemolymph was frozen at -20 °C for later analysis of ammonia. Hemolymph pH (200-300 µL samples) was measured in NBS scale using an InLab micro pH electrode calibrated with pH buffers traceable to NIST standard reference material (Thermofisher Orion). Hemolymph total carbon was measured in duplicate (50 µL per measurement) using the Corning 965 carbon dioxide analyzer (± 0.2 mmol l -1 precision) calibrated with NaHCO3 standards 115 ranging from 0 to 20 mmol l -1 to produce a standard curve with a minimum R 2 of 0.99. Hemolymph pCO2 and HCO3were calculated using a rearrangement of the Henderson-Hasselbalch equation with pK1 and αCO 2 values derived for E. sinensis hemolymph at 23 ºC (pK1= 6.079773, αCO 2= 0.00031263 mmol l -1 Pa -1 (Truchot, 1976(Truchot, , 1992. Hemolymph ammonium was measured in triplicate (25 µL hemolymph per measurement) with a microplate reader (Molecular Devices, SpectraMax, M5) using an orthophthaldialdehyde fluorometric assay which is insensitive to amino acids and proteins (Holmes et al., 1999). 120 Ammonia standards were made from NH4Cl in E. sinensis ringer (pH 8.1) containing (in mmol l -1 ): 185 NaCl, 16 CaCl 2 , 6 MgCl 2 , 7 KCl, and 13 NaHCO 3 . The ion concentrations for the ringer were based on ion composition measurement done on 4 juvenile Chinese mitten crabs in this study (in mmol l -1 Na + 191, K + 7.2, Ca 2+ 16.3, Mg 2+ 5.9, Cl -252). Concentrations of Na + , K + , Ca 2+ , Mg 2+ were measured by flame absorption spectrophotometry (Polarized Zeeman Atomic Absorption Spectrophotometer Z-5000, Hitachi High-Technologies, Tokyo, Japan), Clwas measured spectrophotometrically using the 125 mercury (II) thiocyanate method (Florence and Farrar, 1971). HCO3and pH values for the ringer were based on measurements taken from control crabs in this study and measured as described above.

Ammonia excretion and oxygen consumption
Ammonia excretion and oxygen consumption were measured over a seven-day acclimation to control and acidified freshwater.
These two parameters were measured on individual crabs haphazardly selected from the four control and four acidified 130 freshwater aquaria. Ammonia excretion and oxygen consumption measurements were performed in parallel to hemolymph sampling however, crabs were first randomly selected and placed into respirometry chambers before selecting crabs for hemolymph sampling to avoid using crabs recently sampled for hemolymph. Ammonia excretion experiments were performed in plastic Tupperware filled with 200 mL of filtered control or acidified freshwater. Crabs were given 30 minutes to acclimate to the experimental chambers before initiation of water sampling, as ammonia excretion is elevated for a short time directly 135 after handling (Hans et al., 2014). Water samples (1 mL) for ammonia analysis were collected directly after 30 and 90 minutes of being placed in the experimental chambers. Ammonia concentrations of the water at the 30 and 90-minute time points were determined using the aforementioned orthophthaldialdehyde fluorometric assay (Holmes et al., 1999). Ammonia excretion rates were calculated according to the Eq. (1): where Amm90 is the water ammonia concentration at 90 minutes, Amm30 is the water ammonia concentration at 30 minutes, V is the chamber volume during the flux period in litres, t is the flux time in hours, and m is the fresh weight of the crab in grams.
The oxygen consumption rate was measured by closed-system respirometry in custom-made 3 L glass respiration chambers containing filtered (0.2 µm) freshwater. To achieve the correct experimental CO2 tension, respirometry chambers were 145 submerged in large 18 L buckets of filtered freshwater and a pH controller (Aqua-MACRO) was used to regulate the injection of CO2 as described above for experimental tanks. Crabs were transferred to the submerged respirometry chambers and given 15 minutes to adjust to fully oxygenated respiration chambers before being sealed. Chambers were placed horizontally, allowing for lateral crab movement in the chamber, and oxygen saturation was measured continuously every 15 seconds for 30 minutes at 23 °C. The oxygen sensor (PreSens oxygen micro optode, type PSt1, PreSens Precision Sensing GmbH, 150 Regensburg, Germany) was attached to the top of the chamber and connected to an OXY-4 mini multichannel fibre optic oxygen transmitter (PreSens Precision Sensing GmbH, Regensburg, Germany). Oxygen saturation was always maintained above 80%. Respiration chambers without a crab were used to determine any potential background bacterial respiration for each trial. Preliminary trials demonstrated that crab movement and ventilation rate in the chamber was sufficient to mix the water within the chamber and prevent oxygen stratification, as indicated by a linear decline in oxygen availability. While this 155 approach allows for the measurement of oxygen consumption, some limitations must be considered. Logistical constraints prevented the use of an intermittent flow respirometry approach where the animal could have been given a long amount of time to acclimate to the respirometry chamber. This technical limitation means that the reported measurements cannot be considered a resting metabolic rate as the handling stress, brief air exposure and transfer to a novel environment may have influenced the animal's metabolic rate. However, we would like to point out that in previous trials from our lab using an 160 intermittent flow respirometry setup on green crabs Carcinus maenas, crayfish Procambarus clarkii, and lobsters Homarus americanus, that crustaceans placed in respirometry chambers will stabilize oxygen consumption to a resting rate in under 30 minutes (Gwangseok R. Yoon pers. comm).

Carapace calcification
To assess carapace calcification, changes in the calcium content relative to carapace mass was measured at one, two, three and 165 six weeks of high CO2 exposure according to previously established protocols (Spicer and Eriksson, 2003). In brief, a piece of carapace (ca. 2.5 cm 2 , 15.2 ± 0.4 mg) was removed from the dorsal carapace. The weighed piece of carapace was digested in HNO3 (13.1 N) at 60°C for 16 hours. Digested samples were then diluted to a final HNO3 concentration of 2 % v/v. The carapace Ca 2+ content was measured by atomic absorption spectrophotometer (Z-8000; Hitachi). Standard solutions from Merck (Darmstadt, Germany) were used to make the Ca 2+ standard curve. 170

Locomotory Behaviour Assay
A 24 x 24 cm square, novel, opaque tank was used in the open field test to assess changes in movement of juvenile crabs after a seven-day exposure to control and freshwater acidified conditions. Acclimated crabs were transferred to the novel tank containing control or acidified freshwater and given 5 minutes to acclimate, as done in previous crustacean behavioural studies (Robertson et al., 2018). After acclimation, crab activity was recorded with a digital camera (UI-3240CP Rev.2, Ids, Germany) 175 for 5 minutes (300 seconds) and videos of the movement were processed with the image analysis Ethovision XT motion tracking software (v. 7.0, Noldus, Netherlands). In this study, four factors were measured; distance covered (cm), velocity (cm/s), movement (time in movement, seconds) and mobility (time in mobile state, seconds). We defined movement as the duration for which the central body point (whole body) was changing location. Mobile state was defined as the duration in which crabs exhibited any movement, even if the center point of the animals remained in the same location, for example, 180 appendage movement.

Statistical analysis
Statistical analyses were conducted using JMP Pro 16 (Cary, NC, USA) and GraphPad Prism 8.4.2 (San Diego, CA, USA).
Data were analyzed for outliers by the ROUT test with a Q value of 1 %. For all data, heterogeneity of variance was tested by Levene's test and normal distribution of residuals by Shapiro-Wilk test. Two transformations were done so that data could meet the assumptions of normal distribution and homogeneity of variance. A Johnson SB transformation was applied to hemolymph pCO2 data and a square root transformation was applied to ammonia excretion rate data. In this study, hemolymph parameters, ammonia excretion, and oxygen consumption data were analyzed by a two-way ANOVA post hoc Dunnett's test.
For Dunnett's test, comparisons were made to the zero-day control with time and pCO2 values as fixed factors. Carapace calcification data were analyzed by two-way ANOVA post hoc Tukey HSD with time and pCO2 values as the fixed factors. 190 Behavioural data displayed a high degree of co-linearity between dependent variables, violating the assumptions of the MANOVA test. Therefore, we analyzed behavioural data using a student's t-test except for appendage movement time.
Appendage movement time data violated assumptions of student's t-test, so were analyzed by the Wilcoxon test. Survival curves were analyzed for significant differences by the Mantel-Cox test. The survival curve hazard ratio was determined by the Mantel-Haenszel test. For all data sets, p values ≤ 0.05 were considered significant. Data are presented as mean ± standard 195 error (SEM). Statistical output results are written in text or summarized in Table 2.

Probability of Survival
The effect of freshwater acidification on survival was determined by generating survival curves for crabs in control and acidified freshwater (Fig. 1). There was a significant difference in the probability of survival between the control and acidified 200 freshwater environments (Mantel-Cox log rank test, X 2 1=9.41, p=0.0022, Fig. 1). Crabs in the acidified freshwater tanks had a 50% mortality compared to 15 % mortality in control freshwater tanks. Calculation of the Mantel-Haenszel hazard ratio indicates that crabs in acidified freshwater have a 3.68 times greater probability of mortality than the crabs held under control conditions.

Acid-base status
Chinese mitten crab maintained in control freshwater showed no changes in hemolymph pH, bicarbonate, pCO2, or ammonia throughout the experimental time course ( Fig. 2; Table 2). In contrast, acidified freshwater had a significant effect on hemolymph pH, bicarbonate, pCO2, or ammonia ( Fig. 2; Table 2). Exposure to acidified freshwater induced a respiratory acidosis indicated by a decline in hemolymph pH (pH 8.11 ± 0.015 to 8.03 ± 0.0019) and an increase in hemolymph pCO2 215

Metabolism
Metabolic changes were quantified through ammonia excretion rate and oxygen consumption rate. Ammonia excretion rate was used as an indicator of potential shifts in protein catabolism. Oxygen consumption rate was used as an indicator of changes 230 in aerobic metabolism. Control crabs exhibited steady oxygen consumption rates and ammonia excretion rates throughout the measured time course (Fig. 3; Table 2). Crabs exposed to freshwater acidification experienced a significant reduction in oxygen consumption rate within six hours that was maintained throughout the rest of the time course ( Fig. 3a; Table 2). Ammonia excretion rates were also significantly affected by freshwater acidification (Fig. 3b; Table 2.). Initially, ammonia excretion rates were unchanged until the second day of exposure (Fig. 3b). On the second day of exposure, ammonia excretion rates 235 doubled and remained elevated for the duration of the seven-day time course (Fig. 3b).

Carapace calcification
Changes in calcification were quantified as the change in the crab's exoskeletal calcium content following exposure to 245 freshwater acidification conditions. Calcification was measured several times over a six-week acclimation, as several studies on marine crustaceans report changes in calcification after 20+ days of acclimation (Long et al., 2013;Ries et al., 2009;Taylor et al., 2015). Overall, there was a significant time, pCO2 and interactive time and pCO2 effect on calcification (Table 2). Post hoc analysis suggests there were no significant changes in carapace calcification in the first two weeks of exposure to freshwater acidification (Fig. 4). However, after three and six weeks of exposure, a significant decline in carapace calcium 250 content to 84.1 ± 2.9 % and 85.2 ± 3.3 % of control crab levels was observed (Fig. 4).  (pH 7.4, 1389 µatm pCO2) or CO2-acidified (pH 6.8, 4634 µatm pCO2) freshwater. Data are presented as mean +/-SEM. (N=6-12). Statistical significance was assessed by two-way ANOVA followed by a post-hoc Tukey HSD test with * indicating significant 255 difference between control and acidified FW crabs for each respective week.

Locomotory Behaviour Assay 265
An open field test was used to quantify locomotory behavioural changes over a five-minute recording period in a novel arena (Table 3). Crabs exposed to acidified freshwater covered less distance than crabs in control freshwater (student's t-test, t35=-2.5, p=0.017, Table 3). Crabs in acidified freshwater also had a lower velocity than crabs in control freshwater after the sevenday exposure (student's t-test, t35=-2.37, p=0.024, Table 3). Movement and mobility were also quantified. Movement was defined as the crab changing its relative location in the arena. Mobility was defined as the movement of body appendages even 270 if the crab's location did not change. There was a significant decrease in movement (student's t-test, t35=-2.55, p=0.015, Table   3) and mobility (Wilcoxon test, Z=2.08, p=0.037, Table 3) following the seven-day exposure to acidified freshwater.

Discussion
Anthropogenically driven aquatic acidification has the potential to negatively impact both freshwater and marine life. Metaanalyses of biological responses to ocean acidification suggest that marine crustaceans generally experience minimal consequences to pCO2 tensions (~1000 µatm) predicted to occur by the year 2100 (Kroeker et al., 2013;Melzner et al., 2009;280 Wittmann and Pörtner, 2013). Acidification to levels expected for the year 2300 (~2000 µatm) negatively impacts about half of the studied marine crustaceans (Wittmann and Pörtner, 2013). In contrast, the biological responses of any freshwater invertebrate to realistic future CO2 mediated freshwater acidification remain unknown. In the present study, we aimed to demonstrate the physiological and behavioural consequences of a future CO2 mediated freshwater acidification scenario on a juvenile freshwater crustacean, the Chinese mitten crab, Eriocheir sinensis. Our results suggest that freshwater juvenile 285 Chinese mitten crab experience significant impairment of metabolism, calcification, locomotory behaviour and survival when exposed to freshwater acidification (4633-5109 µatm pCO2). The high energetic demands to sustain essential physiological processes such as acid-base regulation may cause energetic reallocation that impairs several physiological processes and alters animal fitness.

Plausibility of Freshwater Acidification Conditions 290
Modelling of future CO2 mediated freshwater acidification for the year 2100 is nearly non-existent, making the plausibility of the pCO2 levels used in this study difficult to assess. The control pCO2 levels used in this study reflect the average pCO2 measured in 13 stations along the mainstem of the Yangtze River system (excluding Nanjing station which is at the mouth of the river and influenced by coastal upwelling) (Ran et al., 2017). The future freshwater acidification conditions used in this study represents a 3500+ µatm increase in pCO2 from control levels. This increase is roughly 1000+ µatm higher than the 295 highest average level recorded by the 13 stations along the mainstem of the Yangtze river (Ran et al., 2017). While future CO2 mediated acidification models are not available for the Yangtze River, the relationship between changes in freshwater pCO2 in other freshwater systems as a response to changes in atmospheric pCO2 may provide indications of plausible future increases in pCO2. Weiss et al. (2018) tracked changes in pCO2 of four freshwater bodies in Germany between 1981-2015 and reported that freshwater pCO2 increased by an average of 561 µatm over this time period while atmospheric pCO2 increased by ~60 300 µatm from 340 to 399 µatm (National Oceanic and Atmospheric Administration; www.esrl.noaa.gov/gmd/dv/iadv). This relationship suggests that for every 1 µatm increase in atmospheric pCO2, these freshwater bodies increased by 9.35 µatm.
Since atmospheric pCO2 is projected to rise to approximately 985 µatm by the year 2100 (IPCC, 2013) this would mean that freshwater pCO2 in these systems could rise by as much as 5469 µatm. Assuming this relationship is accurate, the pCO2 levels used in this study would be within a range that could feasibly occur in the Chinese mitten crab's native environment by the 305 year 2100. Further, it should be noted that while freshwater systems average pCO2 levels of 3100 µatm (streams and rivers) and 1410 µatm (lakes), the pCO2 levels used for acidified freshwater in this study are within ranges that can already be seen in freshwater systems globally (Raymond et al., 2013). For example, the Mackenzie, Mississippi, Ohio and Elbe rivers suggesting that acidification scenario used in this study is conceivable for freshwater (Cole and Caraco, 2001;Raymond et al., 2013). 310

Probability of Survival
Sensitivity to aquatic acidification is quite variable in marine crustaceans. In mid to high intertidal and burrowing species including porcelain crabs (Petrolisthes cinctipes, Petrolisthes manimaculus, and Porcellana platycheles), burrowing shrimp (Upogebia deltaura), and barnacles (Semibalanus balanoides and Elminius modestus), minimal changes in survival probability are reported at pCO2 tensions ranging from 1395 to 2707 µatm (Donohue et al., 2012;Findlay et al., 2010;Page et al., 2017). 315 Presumably, the variability in CO2 levels experienced in burrows and intertidal zones has driven the evolution of adaptation for greater CO2 tolerance in these groups of crustaceans. We predicted that juvenile Chinese mitten crab would also have an elevated CO2 tolerance and face minimal changes in survival probability because the Yangtze river normally fluctuates by as much as 3000 µatm (Ran et al., 2017). Despite being a freshwater organism with strong ionoregulatory capabilities, our results show a sharp decrease in survival rate of Chinese mitten crabs over 14 days of exposure to 4633 µatm pCO2 (Fig 1). Such rapid decreases in survival have also been observed in non-burrowing crustaceans or crustaceans that do not inhabit high intertidal regions including brine shrimp (Artemia sinica), red king crab (Paralithodes camtschaticus), and low intertidal longclawed porcelain crab, (Pisidia longicornis), exposed to 1500, 1637, and 5821 µatm pCO2, respectively (Long et al., 2013;Page et al., 2017;Zheng et al., 2015). It might be tempting to conclude that low survival in Chinese mitten crabs compared to tolerant mid to high intertidal and burrowing marine crustaceans is simply due to the greater pCO2 tensions used in the present 325 study (4633 µatm). However, in the intertidal broad-clawed porcelain crab (Porcellana platycheles) pCO2 levels of 5821 µatm have been shown to not affect the probability of survival after 24 days of exposure (Page et al., 2017). It should also be mentioned that for all mortalities in this experiment there were no obvious signs of disease and intact bodies of deceased crabs were collected, suggesting that the elevated CO2 treatment and not disease or cannibalism was the reason for increased mortality. Therefore, the low survival rates in the present study suggest a high susceptibility to acidification and contradict our 330 hypothesis that inhabiting a highly fluctuating CO2 environment would confer tolerance to future freshwater acidification.

Physiological Responses
Juvenile Chinese mitten crab effectively recovered extracellular pH following respiratory acidosis resulting from freshwater acidification by the accumulation of extracellular HCO3as a buffer (Fig. 2). Compensation of acid-base homeostasis under freshwater acidification was not surprising given that strong acid-base regulatory capabilities are typically seen in highly active 335 organisms such as fish, cephalopods and crustaceans (Melzner et al., 2009). Similar recovery of extracellular pH to elevated environmental CO2 has also been observed in Dungeness crab (Metacarcinus magister) and velvet crab (Necora puber) exposed to even higher pCO2 tensions (10000+ µatm; Pane and Barry, 2007;Spicer et al., 2007). In contrast, green crab (Carcinus maenas) and blue crab (Callinectes sapidus) have been shown to not fully compensate extracellular pH at 10000+ µatm CO2 levels (Cameron, 1978;Fehsenfeld and Weihrauch, 2016). However, measurements in these species were only done over 48 340 hours and more time may have been required for the animals to recover, as seen in our study, where recovery was only observed after seven days. The compensatory responses to acidosis in crustaceans generally includes respiratory CO2 excretion, H + excretion typically through Na + /H + or NH4 + exchange and accumulation of extracellular HCO3as a buffer, where HCO3is derived through either branchial Cl -/HCO3exchange and, to a lesser degree, from calcified structures (e.g. exoskeleton) (Wheatly and Henry, 1992). In freshwater crustaceans, acid-base regulation occurs mainly within the gills (Henry et al., 2012), 345 where the Na + /K + -ATPase and H + -ATPase generate the electrochemical gradients that drive ion exchange (Leone et al., 2017).
The Na + /K + -ATPase alone may already account for over 20 % of an animal's energetic budget (Milligan and McBride, 1985).
The increase in ion transport that must occur to re-establish and maintain acid-base homeostasis in the face of freshwater acidification could pose an increased energetic demand. In fact, in sea urchin larvae pCO2 tensions of 800 µatm have been shown to double ion transport ATP demands (Pan et al., 2015). It is therefore conceivable that the energetic cost for long-term 350 maintenance of acid-base homeostasis under freshwater acidification may come at substantial energetic cost, which could have negative implications on other physiological parameters and thereby animal fitness.
Heightened energetic demands to maintain crucial physiological processes during exposure to environmental CO2 acidification can occur through reallocation of energy budgets or through modification of metabolism to increase energy supplies. In fact, in the marine brittle star, Amphiura filiformis, exposure to CO2 tensions ranging from 1000 to 8000 µatm for 40 days caused 355 an increase in metabolic rate (increased energy budget) (Wood et al., 2008). This metabolic change was postulated to fuel increased calcification observed in this species (Wood et al., 2008). In contrast, the metabolic rate of juvenile European lobster (Homarus gammarus) remained unchanged when exposed to 1100 and 8000 µatm CO2 (Small et al., 2020). However, in H. gammarus, branchial Na + /K + ATPase activity was increased, demonstrating a reallocation of energy supplies despite maintaining an unchanged energy budget (Small et al., 2020). Unlike juvenile European lobster and brittle star, juvenile 360 Chinese mitten crabs experienced a decrease in oxygen consumption (potentially decreased energy budget). Despite reductions in oxygen consumption, crabs could still re-establish extracellular pH through HCO3accumulation, suggesting a potential reallocation of energy supplies to essential ionoregulatory processes.
Typically, a reduction in oxygen consumption, as seen in the present study, is observed when an organism cannot compensate for a reduction in extracellular pH (Pörtner et al., 2004). While in juvenile Chinese mitten crabs, this could be the case at the 365 initial two days of the time course, by day seven extracellular pH was fully compensated yet, oxygen consumption rates were reduced. It is known that high environmental pCO2 levels can trigger an accumulation of compounds such as adenosine that can lead to reduced oxygen consumption as observed in the peanut worm, Sipunculus nudus (Reipschläger et al., 1997). A similar mechanism could conceivably be in place that led to reduced oxygen consumption in the Chinese mitten crab as a strategy to conserve energy supplies to promote survival upon exposure to short-term stressors like high environmental pCO2 370 levels. Such an adaptation may be present in Chinese mitten crab as these crabs would regularly experience short-term fluctuations in environmental CO2 of their natural habitat. In fact, in the Mediterranean mussel (Mytilus galloprovincialis) chronically reduced oxygen consumption rates lasting up to 90 days have been observed to allow survival following exposure to ocean acidification (5026 µatm pCO2, Michaelidis et al., 2005). Reducing oxygen consumption is a viable strategy used by many organisms to survive short-term periods of environmental stress (Guppy and Withers, 1999). However, it is a less viable 375 long-term strategy as reduction in metabolic rate reduces energy availability for costly physiological processes such as calcification and protein synthesis which would ultimately affect growth and reproductive success as reported in freshwater pink salmon (Oncorhynchus gorbuscha) and marine amphipod (Gammarus locusta) (Borges et al., 2018;Ou et al., 2015).
Besides reduced oxygen consumption, freshwater acidification led to an increase in extracellular concentrations and excretion of ammonia, a metabolic product of protein catabolism. Elevated excretion of ammonia may function as an excretable acid 380 equivalent to assist the maintenance of pH homeostasis, a mechanism suggested for the brackish water green crab (Carcinus maenas) and hydrothermal vent crab (Xenograpsus testudinatus) (Allen et al., 2020;Fehsenfeld and Weihrauch, 2013). µatm pCO2, 28 days), where catabolism of amino acid such as glutamine may provide metabolic bicarbonate to further help sustain pH homeostasis (Hu et al., 2014;Michaelidis et al., 2005). While potentially beneficial for sustaining acid-base status, elevated protein catabolism requires a consistent source of protein through either a high protein diet or increased food consumption which if not met could cause muscle wastage an effect seen in brittle star during heightened energetic demands 390 of ocean acidification (Wood et al., 2008). Interestingly, feeding rate has been shown in juvenile European lobster (H. gammarus) and green crab (C. maenas) to decline as a result of elevated environmental CO2 making a greater reliance on protein catabolism during energetically constricted times a potentially precarious situation for juvenile Chinese mitten crab (Appelhans et al., 2012;Small et al., 2020).
Carapace calcification is an energetically costly process related to growth and predation defence in crustaceans that freshwater 395 acidification and the associated metabolic changes could impair. Decapod crustaceans are believed to be the least susceptible of calcifying organisms to aquatic acidification as their exoskeletal CaCO3 exists in the more stable calcite form providing greater resilience to dissolution in contrast to bivalves and corals (Ries et al., 2009). Indeed, the marine crustacean carapace is well protected from aquatic acidification mediated dissolution with reports of either no change or an increase in calcification being typically observed (Kroeker et al., 2013;Ries et al., 2009;Whiteley, 2011). However, in the present study, juvenile 400 Chinese mitten crab had reduced levels of carapace calcification as reflected by a lower carapace calcium content after three and six weeks of exposure (Fig. 4). While not as common, examples of reductions in carapace calcification have been observed in marine crustaceans, including several porcelain crabs and the tanner crab, Chionoecetes bairdi (Long et al., 2013;Page et al., 2017). In crustaceans, carapace dissolution may occur to support extracellular pH buffering that normally occurs through branchial HCO3uptake by providing an alternative source of HCO3 - (Cameron, 1985;Defur et al., 1980). In the present study, 405 extracellular pH was recovered long before carapace dissolution was apparent, therefore it is less likely that the carapace is mobilized as a source of HCO3 -. Instead, reductions in carapace calcium content most likely reflect an alteration in the rate of calcification or acid mediated dissolution of the carapace. As carapace formation and maintenance is an energetically expensive process requiring careful ion regulation by numerous organs, the aforementioned changes in whole animal energetics due to freshwater acidification could have negative implications on animal fitness either by weakening the exoskeleton or impairing 410 post-moult calcification which can hamper growth and leave animals vulnerable to predation.

Behavioural Responses
Elevated freshwater pCO2 altered locomotory behaviour in juvenile Chinese mitten crabs. Crabs in acidified freshwater covered less total distance during movement and did so at a lower velocity. No studies have previously examined changes in crustacean distance covered in the presence of elevated environmental CO2. However, reduced speed of movement has also 415 been reported in Shiba shrimp (Metapenaeus joyneri) exposed to CO2 levels of 9079 µatm; however, unlike in Chinese mitten crab, this shrimp did not experience a reduction in oxygen consumption rate correlated with locomotory impairment (Dissanayake and Ishimatsu, 2011). While not measured in our study, in Shiba shrimp there was a reduction in aerobic scope, which would likely lead to reduced aerobic performance and reduced movement (Dissanayake and Ishimatsu, 2011). Similar alterations in aerobic scope could partially be behind the reductions in velocity seen in juvenile Chinese mitten crab, however, addition to moving slower, Chinese mitten crab spent less time moving their entire body throughout the novel arena and less time moving only their appendages while staying at a fixed location. Reduced movement time and appendage movement were also seen in the hermit crab (Pagurus bernhardus) exposed to 12000 µatm CO2 (de la Haye et al., 2011). In contrast, the isopod (Paradella dianae) experienced no change in swim time or crawling time when exposed to 2085 µatm CO2 despite a measured 425 metabolic depression (Alenius and Munguia, 2012). Differences in the effect of CO2 on movement time may result from the CO2 levels employed, but further studies on a greater variety of species are required to determine potential patterns for crustaceans. It is plausible that overall locomotory behaviour is reduced in this study due to alterations in neurological function resulting from ionic imbalances or other CO2-mediated effects that may occur from elevated environmental CO2 (For review of neural effects of aquatic acidification see Tresguerres and Hamilton, 2017). With a potential reduction in overall energy 430 availability, crabs may reduce energy expenditure through locomotion to conserve energy stores for physiological processes more crucial to surviving the physiological distress caused by freshwater acidification. The overall reductions in locomotion observed in juvenile Chinese mitten crab could have negative consequences for their survival, as reduced movement would make these crabs more vulnerable to predation, reduce migratory capabilities and reduce foraging ability.

Conclusion 435
In conclusion, we found impairment of survival, metabolism, calcification, and locomotion with exposure to a potential future CO2 mediated freshwater acidification scenario. Energy availability was reduced despite heightened ionoregulatory energetic demands. Changes in the animals' energy budgets likely result in a greater dependency on protein catabolism as an energy source to allow for extracellular pH recovery at the cost of reducing their exoskeletal calcification and locomotion. We found that despite successful acid-base compensation, survivals rates declined with 3.8 times greater probability of mortality under 440 acidified freshwater conditions. While our study suggests negative impacts of freshwater acidification, these results should be assessed with caution as the assumed acidification levels are based on a relationship between changes in atmospheric CO2 and freshwater CO2 which remains to be more effectively modelled. Nevertheless, this study shows that despite inhabiting an environment that experiences regular fluctuations in pCO2 the Chinese mitten crab may be at risk to future freshwater acidification. This emphasizes the importance of modelling acidification in freshwater systems to accurately assess biological 445 consequences of global change. Based on our findings that a physiologically robust species displays sensitivity to future freshwater acidification, further research investigating the effect of freshwater acidification on a wide range of freshwater species from all phyla are required to better identify the effects of anthropogenic CO2 accumulation on freshwater ecosystems.