Physiological responses of three species of unionid mussels to intermittent exposure to elevated carbon dioxide.

Freshwater systems are at risk owing to increasing carbon dioxide (CO2) levels, and one of the possible reasons for these elevations is the deployment of non-physical fish barriers to prevent invasive fish movements. Carbon dioxide barriers have the potential to create short, chronic and intermittent exposures of CO2 for surrounding freshwater biota. Although intermittent exposures to a stressor may be more ecologically relevant, the majority of laboratory tests use chronic or short-term time periods to determine how organisms will respond to an environmental stressor. Measurements of the physiological responses of three species of unionid mussel, giant floaters (Pyganodon grandis), threeridge (Amblema plicata) and plain pocketbook (Lampsilis cardium), exposed to control pCO2 (~1000 µatm) or intermittent conditions of pCO2 (ranging from ~1000 to ~55 000 µatm) 12 times per day over a 28 day period were gathered. There was no indication of recovery in the physiological responses of mussels between applications of CO2, suggesting that the recovery time between CO2 pulses (1.5 h) was not sufficient for recovery from the CO2 exposure period (0.5 h). Observations of acid-base and stress responses were consistent with what has been observed in chronic studies of freshwater mussels exposed to elevated pCO2 (i.e. elevations in HCO3-, Ca2+, Na+ and glucose, and decreases in Mg2+ and Cl-). However, species differences were observed across almost all variables measured, which emphasizes the need for multispecies studies.

Introduction

Environmental levels of carbon dioxide (CO2) that are commonly found in freshwater ecosystems have the potential to act as both continuous and intermittent stressors for aquatic organisms. Over the past several decades, levels of CO2 in the atmosphere have been increasing as a result of the anthropogenic burning of fossil fuels, which has led to a concomitant increase in the partial pressure of CO2 gas (pCO2) in marine ecosystems (Shirayama and Thornton, 2005). Unlike marine systems, there is no consensus regarding how pCO2 will change in freshwater as a result of climate change (Hasler ). In freshwater, pCO2 can vary across and within watersheds (Butman and Raymond, 2011), as well as episodically and on seasonal and diel cycles within water bodies (Maberly, 1996). In a review of ~7000 global rivers and streams, the average median value for pCO2 was ~3100 µatm (Raymond ), and in another global review of 47 large rivers the means varied from 679 ± 543 to 35 617 ± 46 757 µatm, with means in the USA ranging from 679 ± 543 to 9475 ± 993 µatm (Cole and Caraco, 2001). In addition to these natural sources of elevated pCO2, recent work has shown that zones of elevated CO2 can act as non-physical fish barriers, thereby providing a management tool to prevent the movement and spread of invasive fish species (Kates ; Noatch and Suski, 2012). Although a specific method for the use of CO2 barriers to deter fish movement has not yet been defined, one potential application is the intermittent addition of CO2 into a navigational lock or approach channel at vulnerable times (i.e. when lock doors are open; United States Army Corps of Engineers, 2014a), resulting in downstream pulses of CO2-rich water. Thus, downstream fluctuations in CO2 might occur, making CO2 a potential intermittent stressor for freshwater organisms.

A taxonomic group of freshwater organisms that may be particularly at risk to CO2 stressors are freshwater mussels (Order Unionoida). Mussels serve many important ecological functions, influence many ecosystem processes (Vaughn and Hakenkamp, 2001) and are often used as indicators of ecosystem health (Williams ). Although North American freshwater ecosystems contain the highest diversity of freshwater mussels in the world (Williams ; Bogan, 2008), more than half (71%) are listed as endangered, threatened or of special concern, largely as a result of anthropogenic stressors, such at habitat alteration and degradation (Williams ; Ricciardi ). Additionally, while mussels are generally considered a homogeneous group of sessile animals, there are four main tribes of mussels in North America (Quadrulini, Lampsilini, Pleurobemini and Amblemini) that all vary in morphology, physiology and reproductive strategies and may thus respond differently to environmental stressors.

At present, there is a paucity of research on the effects of elevated pCO2 on freshwater invertebrates, particularly unionid mussels. Hannan , b) found that mussels experience acid–base regulation in response to short- and long-term exposures to elevated pCO2, and a stress response to long-term exposure to elevated pCO2. Previous studies on marine bivalves indicate that elevated pCO2 causes internal acidosis (Michaelidis ; Bibby ) that is often buffered by increasing HCO3− in the fluids (Pörtner ). Both marine (Michaelidis ) and freshwater mussels (Hannan , b) can increase haemolymph HCO3− by using CaCO3 released from the shell as a result of decreased pH and elevated CO2 (i.e. increases both haemolymph HCO3− and Ca2+) or by reducing the activity of the Cl−–HCO3− exchanger to retain HCO3− at the cost of Cl− uptake (Byrne and Dietz, 1997; Hannan , b). Another strategy to buffer acidosis is to alter the activity of Na+–H+ exchangers to increase removal of H+ ions, thus also increasing Na+ uptake (Byrne and Dietz, 1997; Lannig , Hannan ). Exposure to a chronic elevation in pCO2 also appears to initiate the general stress response in mussels, because a decrease in haemolymph Mg2+ and an increase in haemolymph glucose have been observed in unionid mussels (Hannan , b). More importantly, previous studies (i.e. studies described above) that have quantified CO2 stressors in mussels have used a continuous application of CO2 rather than one that was intermittent as might be expected downstream of a CO2 barrier, and differences may exist between the continuous application of a stressor relative to one applied intermittently (exacerbation, attenuation or no change; Reinert ).

Based on this background, the goal of the present study was to quantify the physiological impacts of intermittent exposures to elevated pCO2 on three species of freshwater mussels each belonging to a different tribe, Pyganodon grandis (tribe Anodontini), Amblema plicata (tribe Amblemini) and Lampsilis cardium (tribe Lampsilini). To accomplish this goal, over a 28 day period the mussels were exposed to either control pCO2 or intermittent increases in pCO2 and then sampled for a suite of physiological parameters related to acid–base status and physiological stress. The results of this study help to clarify further how different exposures to elevated pCO2 affect the acid–base and stress responses of various freshwater mussel species in habitats where pCO2 fluctuates.

Materials and methods

Mussel collection and husbandry

Plain pocketbook (L. cardium) and threeridge mussels (A. plicata) were collected by benthic grab from the Mississippi River, Cordova, IL, USA, in July 2015. Giant floater mussels (P. grandis) were collected by benthic grab from a barrow pit near Champaign, IL, USA, in August 2015. Mussels were taken to the Aquatic Research Facility at the University of Illinois, Champaign-Urbana, IL, USA in coolers (travel time <3 h for L. cardium and A. plicata and <1 h for P. grandis). Upon arrival at the Aquatic Research Facility, all mussels were cleaned of epibionts and tagged for individual identification with a permanent marker (Neves and Moyer, 1988). Once tagged, mussels were placed in three tubs (1136 litres) supplied with water from a 0.04 ha natural, earthen-bottom pond, where they remained for at least 1 week to recover from transport stressors and to acclimate to laboratory conditions (Dietz, 1974; Horohov ; Dietz ). All tubs were equipped with a Teco 500 aquarium chiller (TECO-US, Aquarium Specialty, Columbia, SC, USA) and a low-pressure air blower (Sweetwater, SL24H Pentair, Apopka, FL, USA) to maintain aeration. Fifty per cent water changes using pond water were performed weekly to maintain water quality. Mussels were fed a commercial shellfish diet of the following consituents: Nannochloropsis sp. 1–2 µm and a mixed diet of Isochrysis, Pavlova, Thalassiosira and Tertraselmis spp. 5–12 µm (Instant Algae, Reed Mariculture Inc., Campbell, CA, USA) every other day (American Society of Testing and Materials, 2006; Wang ), although mussels did not receive supplemental food for 24 h prior to sampling. Temperature and dissolved oxygen (DO) were recorded daily across all holding tanks with a portable meter (YSI 550A, Yellow Springs Instruments, Irvine, CA, USA) and averaged 22°C (21.7 ± 0.1°C, mean ± SEM) and 7.50 mg l−1 (7.60 ± 0.06 mg l−1). Water pH was measured using a handheld meter (WTW pH 3310 meter, Germany) that was calibrated regularly, and averaged 8.55 ± 0.01 throughout the acclimation period. Dissolved CO2 and total alkalinity (TA) concentrations were measured using digital titration kits and averaged 4.86 ± 0.04 mg l−1 and 1093.0 ± 27.0 µmol kg−1, respectively (Hach Company, Loveland, CO, USA; Titrator model 16,900 catalogue no. 2272700 and catalogue no. 2271900 for CO2 and TA, respectively).

Fluctuating CO2 exposure

To define the impacts of fluctuating CO2 on mussel physiology, mussels (L. cardium, A. plicata and P. grandis; n = 28) were separated into two recirculating treatment systems (92 litres), each with nine 5 litre tanks (adapted from Hohn and Petrie-Hanson, 2007). Systems were maintained as stated above with the exception that one system received a CO2 treatment. In the CO2 treatment system, pCO2 was turned on every 1.5 h, and increased from ambient (~1000 µatm, 1355 ± 119 µatm; pH = 7.85 ± 0.02) to ~55 000 µatm (56 492 ± 1342 µatm; pH = 6.62 ± 0.03) by bubbling CO2 gas into the water through an air stone (see Supplementary material, Fig. S1). Elevated pCO2 was held constant at ~55 000 µatm for 0.5 h, for a total of 12 fluctuations per day. Thus, animals were held at elevated pCO2 levels for 0.5 h and returned to control levels during the 1.5 h recovery period and then raised back up to elevated conditions for 0.5 h, repeatedly during the course of the experiment. A level of 55 000 µatm was targeted because this level has previously been defined as being a potential target CO2 level that could deter the movement of fishes (Donaldson ) and will possibly be the target level of a CO2 barrier. Twelve fluctuations per day represents the historical lock usage of Brandon Road Lock (41.5054°N, 88.0996°W), a possible site for deployment of a CO2 barrier within the Des Plaines River, IL, USA (United States Army Corps of Engineers, 2014a, 2015). The target pCO2 was maintained with a pH controller (PINPOINT®, American Marine Inc., CT, USA) that automatically bubbled CO2 into the tank system through an air stone should the pH rise above a target level during exposure (Reynaud ; Riebesell ). The level of CO2 was then returned to ~1000 µatm by bubbling in air though an air stone to off-gas excess CO2. An identical recirculating system was used as a control, and mussels in this control system were treated in the same way as animals receiving CO2, except that infused CO2 gas was replaced with compressed air such that mussels were held continuously at ambient ~1000 µatm (876 ± 108 µatm; pH = 8.13 ± 0.02) pCO2. A digital timer (DT620 Heavy Duty Digital Timer, Intermatic Inc Spring Grove, IL, USA) was used to control additions of CO2 and air. A modified infrared probe was used to measure pCO2 (Vaisala GMP220 and GMT221, Vantaa, Finland; Johnson ), along with a CO2 titration kit to determine the concentration of CO2 (Hach Company, catalogue no. 2272700, Loveland, CO, USA). Before and after the 12.00 h exposure, temperature (21.7 ± 0.1°C) and DO (7.60 ± 0.07 mg l−1) were measured as stated above, and the temperature, pH (see above) and TA (2566.3 ± 252.9 µmol kg−1) were entered into CO2calc to verify pCO2 (Robbins ).

Individual mussels were non-lethally and repeatedly sampled for haemolymph on day 1, 4, 7, 14, 21 or 28 of exposure to fluctuating pCO2 or control conditions. Mussels were sampled during the 1.5 h period when CO2 was at ambient levels, not during the 0.5 h when CO2 levels were elevated. Prior to starting this study, it was not known whether sampling mussels immediately prior to the increase in CO2 or immediately after the period of increased CO2 would be optimal to define the impacts of CO2 on physiological parameters. Therefore, mussels were sample during both intervals, and n = 7 animals were sampled immediately prior to the increase in CO2, whereas a second n = 7 animals were sampled immediately following the increase in pCO2, once pCO2 returned to control values. All samples were collected around the 12.00 h CO2 exposure to standardize any potential for diel variation in physiological parameters.

Haemolymph (0.5 ml for L. cardium and A. plicata; and 0.25 ml for P. grandis) was extracted from the anterior adductor muscle with a 1 ml syringe and 26 gauge needle (Gustafson ) and then centrifuged at 12 000 for 2 min. After centrifugation, the supernatant was removed, flash frozen in liquid nitrogen and stored at −80°C until processing. Mussels were sampled for haemolymph only once per sampling day, and were randomly sampled before or after the CO2 exposure on each sampling day over the 28 day period. On day 28 of exposure, mussels were sampled for haemolymph as stated above and then lethally sampled. Lethal mussel sampling included measurements for length, width, depth of the whole mussel using digital callipers (traceable digital carbon fiber calipers, Fisher Scientific, Pittsburg, PA, USA), and weight of the whole mussel (tissue + shell) was collected to the nearest 0.01 g using a balance (HL-300WP, A&D, Ann Arbor, MI, USA). Soft tissue dry weight (in milligrams) was determined by taking mussel soft tissues and drying them at 99°C for 24 h before weighing (Widdows ). If possible, sex was determined for L. siliquoidia and P. grandis using both their external sexual dimorphism and by examination of the gills for glochidia (Trdan, 1981).

The dry weight and length of individuals within each species was not different between control and fluctuating CO2 treatment groups (Student's unpaired t-test, P > 0.05; Table 1). Additionally, mortalities were limited over the exposure period, but occurred for two and five P. grandis from the control and fluctuating pCO2 treatments, respectively, and for two A. plicata and one L. cardium exposed to the fluctuating pCO2 treatment.

Table 1:

Results of Student's unpaired t-test examining the impact of dry weight and length on different pCO2 treatments

Measured variable	Species	d.f.	t	P-value
Dry weight (g)	Threeridge	17.26	−1.247	0.229
Length (cm)		21.65	0.526	0.604
Dry weight (g)	Pocketbook	22.23	0.673	0.508
Length (cm)		20.70	0.218	0.830
Dry weight (g)	Giant floater	16.82	−0.617	0.545
Length (cm)		18.95	0.350	0.730

No significant effects were detected.

Laboratory analyses

Haemolymph Cl−, Mg2+ and Ca2+ concentrations were assayed in duplicate using commercially available kits (QuantiChrom assay kits Cl−, catalogue no. DICL-250; Mg2+, catalogue no. DIMG-250; Ca2+, catalogue no. DICA-500; BioAssay Systems, Hayward, CA, USA). Haemolymph HCO3− and Na+ levels were measured by the diagnostic clinical pathology laboratory at the University of Illinois Urbana-Champaign using a Beckman chemistry analyser (Beckman Coulter AU680, Beckman Coulter, Brea, CA, USA). Quality control testing for this analyser was performed at least every 24 h. Haemolymph glucose concentrations were assayed in duplicate according to the method of Bergmeyer (1974) using a 96-well microplate and a plate spectrophotometer (Molecular Devices, SpectraMax Plus 384, Sunnyvale, CA, USA). For all assays, the inter- and intra-assay coefficients of variability were <10%.

Statistical analyses

The effects of CO2 exposure on haemolymph ion levels and glucose concentrations were quantified using a two-way analysis of variance (ANOVA), with pCO2 (fluctuating or control), sampling day and their interaction (pCO2 × sampling day) entered into each model as fixed effects. Individual mussel identification number (ID), time point (i.e. sampling before or after pCO2), length, dry weight and sex (if applicable) were initially included in the models as cofactors to quantify their potential influence on response variables, but were removed because they had no significant effect on model outputs (Engqvist, 2005; Zuur ). If at least one of the main effects in the ANOVA model was significant, or if the interaction term was significant, a Tukey–Kramer honestly significant difference (HSD) post hoc test was applied to separate means (Rohlf and Sokal, 1995). Finally, a separate Student's unpaired t-test was run on each species to quantify differences in dry weight and length across different pCO2 treatments.

For all statistical analyses, analysis of fitted residuals using a quantile–quantile plot (Anscombe and Tukey, 1963) was used to assess normality, while a Hartley's Fmax test (Hartley, 1950), combined with visual inspection of the distribution of fitted residuals, was used to assess homogeneity of variances. If either normality or homogeneity of variance assumptions were violated (Siegel and Castellan, 1988), data were rank transformed and then re-analysed within the same parametric model described above, and the assumptions of both normality and equal variances were confirmed (Conover and Iman, 1981; Iman ; Potvin and Roff, 1993). All data are presented as means ± SEM where appropriate, all tests were performed using R (version 3.2.2), and differences were considered significant if α was <0.05. For all variables and species, there was no effect of sampling before vs. after CO2 application (i.e. time point) for either treatment (control or fluctuating), so data from mussels sampled before and after CO2 application were combined.

Results

There was a significant interaction between treatment and day for all three species of mussels for haemolymph HCO3− (Tables 2–4). At 14 days of exposure to fluctuating pCO2, P. grandis (treatment × day, F = 13.0, P < 0.001; Fig. 1A) and A. plicata (treatment × day, F = 8.61, P < 0.001; Fig. 1D) had approximately a 2-fold increase in haemolymph HCO3− relative to mussels held at ambient pCO2, and these concentrations remained significantly elevated for the duration of the exposure period. For L. cardium, haemolymph HCO3− was significantly elevated beginning at 4 days of exposure compared with control mussels and throughout the rest of the exposure period (treatment × day, F = 0.52, P < 0.001; Fig. 1G).

Table 2:

Results of two-way ANOVA examining the impact of fluctuating exposure to elevated pCO2 on Pyganodon grandis exposed to one of two different pCO2 treatments [~1000 µatm (ambient); intermittent at ~55 000 µatm] for 28 days

Measured variable	Main effects	Sum of squares	d.f.	F	P-value
HCO3− (mmol l−1)	Treatment	4.84	1	115.59	<0.001
	Day	1.29	4	7.71	<0.001
	Treatment × day	2.18	4	12.99	<0.001
	Residuals	4.61	110
Ca2+ (mg ml−1)	Treatment	40 708	1	61.50	<0.001
	Day	40 630	4	15.34	<0.001
	Treatment × day	22 285	4	8.42	<0.001
	Residuals	79 450	120
Cl− (mg ml−1)	Treatment	16 163	1	21.43	<0.001
	Day	59 308	4	19.66	<0.001
	Treatment × day	17 084	4	5.66	<0.001
	Residuals	90 518	120
Na+ (g l−1)	Treatment	3033	1	3.87	0.0517
	Day	48 876	4	15.58	<0.001
	Treatment × day	9883	4	3.15	0.0170
	Residuals	87 826	112
Mg2+ (mg ml−1)	Treatment	0.001	1	46.21	<0.001
	Day	0.002	4	15.28	<0.001
	Treatment × day	0.002	4	18.33	<0.001
	Residuals	0.003	120
Glucose (µM)	Treatment	2656	1	2.23	0.1385
	Day	12 606	4	2.64	0.0373
	Treatment × day	10 084	4	2.11	0.0838
	Residuals	137 268	115

Bold P-values indicate statistical significance across treatment groups within a measured variable.

Table 4:

Results of two-way ANOVA examining the impact of fluctuating exposure to elevated pCO2 on Lampsilis cardium exposed to one of two different pCO2 treatments [~1000 µatm (ambient); intermittent at ~55 000 µatm] for 28 days

Measured variable	Main effects	Sum of squares	d.f.	F	P-value
HCO3− (mmol l−1)	Treatment	7.28	1	141.70	<0.001
	Day	0.47	4	2.27	0.0653
	Treatment × day	2.10	4	0.52	<0.001
	Residuals	6.48	126
Ca2+ (mg ml−1)	Treatment	0.171	1	377.5	<0.001
	Day	0.024	4	13.36	<0.001
	Treatment × day	0.022	4	12.20	<0.001
	Residuals	0.058	127
Cl− (mg ml−1)	Treatment	479	1	0.35	0.553
	Day	30 987	4	5.73	<0.001
	Treatment × day	4442	4	0.821	0.514
	Residuals	169 113	125
Na+ (g l−1)	Treatment	369.3	1	134.96	<0.001
	Day	10.6	4	0.97	0.4273
	Treatment × day	33.4	4	3.05	0.0194
	Residuals	344.8	126
Mg2+ (mg ml−1)	Treatment	0.001	1	46.24	<0.001
	Day	0.0003	4	2.62	0.038
	Treatment × day	0.0008	4	7.31	<0.001
	Residuals	0.004	127
Glucose (µM)	Treatment	483	1	0.81	0.369
	Day	1938	4	0.81	0.517
	Treatment × day	2369	4	1.00	0.411
	Residuals	69 399	117

Bold P-values indicate statistical significance across treatment groups within a measured variable.

Figure 1:

Concentrations of HCO3−, Ca2+ and Cl− in the haemolymph of Pyganodon grandis (n = 9–14; A–C), Amblema plicata (n = 12–14; D–F) and Lampsilis cardium mussels (n = 13–14; G–I) exposed to two treatments of pCO2, ~1000 µatm (control) or intermittent increase at ~55 000 µatm for 1, 7, 14, 21 or 28 days. Data are presented as means + SEM. *Groups that were significantly different from the control treatment within a time point (two-way ANOVA; see Tables 2–4). For (I), there was no significant interaction between pCO2 treatment and sampling day; a bar above the treatments of a day represents a significant effect of time (two-way ANOVA).

A significant interaction of treatment and day was also found for all three species of mussels with respect to haemolymph Ca2+ concentrations(Tables 2–4). Pyganodon grandis had a significant elevation in haemolymph Ca2+ concentrations beginning at 14 days of exposure to fluctuating levels of CO2 relative to control mussels; however, it should be noted that control mussels also experienced a decrease in haemolymph Ca2+ concentrationsat 28 days compared with 1 day of treatment (treatment × day, F = 8.42, P < 0.001; Fig. 1B). For A. plicata, haemolymph Ca2+ in mussels exposed to fluctuating levels of CO2 was significantly elevated compared with control mussels at 7 days of exposure (treatment × day, F = 4.14, P = 0.003; Fig. 1E). A similar increase in haemolymph Ca2+ in L. cardium occurred in response to fluctuating pCO2, where levels were significantly elevated compared with control mussels for the entire period of exposure (treatment × day, F = 12.2, P < 0.001; Fig. 1H).

For P. grandis and A. plicata (Tables 2 and 3), there was a significant interaction between treatment and day for haemolymph Cl− concentrations, but only a significant effect of day for L. cardium (Table 4). Haemolymph Cl− was lower in P. grandis exposed to fluctuating pCO2 at 7 days of exposure relative to control mussels; however, it is important to note that this might have been attributable to a significant increase in haemolymph Cl− concentrations in control mussels at 7 days compared with control mussels on day 1 of treatment (treatment × day, F = 5.66, P < 0.001; Fig. 1C). In A. plicata, although there was a significant interaction of treatment and day, haemolymph Cl− was not significantly different in mussels exposed to fluctuating levels of CO2 and control mussels at any point throughout the exposure (treatment × day, F = 3.63, P = 0.008; Fig. 1F). In L. cardium, no significant effect of CO2 treatment was detected, and haemolymph Cl− increased overall at 28 days of treatment (day, F = 5.73, P < 0.001; Fig. 1I).

Table 3:

Results of two-way ANOVA examining the impact of fluctuating exposure to elevated pCO2 on Amblema plicata exposed to one of two different pCO2 treatments [~1000 µatm (ambient); intermittent at ~55 000 µatm] for 28 days

Measured variable	Main effects	Sum of squares	d.f.	F	P-value
HCO3− (mmol l−1)	Treatment	8.73	1	118.33	<0.001
	Day	3.12	4	10.57	<0.001
	Treatment × day	2.54	4	8.61	<0.001
	Residuals	9.37	127
Ca2+ (mg ml−1)	Treatment	0.02	1	16.06	<0.001
	Day	0.02	4	4.99	<0.001
	Treatment × day	0.02	4	4.14	0.003
	Residuals	0.13	127
Cl− (mg ml−1)	Treatment	1676	1	1.44	0.232
	Day	47 572	4	10.12	<0.001
	Treatment × day	16 923	4	3.63	0.008
	Residuals	148 094	127
Na+ (g l−1)	Treatment	521	1	211.07	<0.001
	Day	26.5	4	2.68	0.0345
	Treatment × day	126.8	4	12.85	<0.001
	Residuals	313.5	127
Mg2+ (mg ml−1)	Treatment	0.0005	1	16.41	<0.001
	Day	0.0015	4	11.49	<0.001
	Treatment × day	0.0021	4	15.68	<0.001
	Residuals	0.0041	125
Glucose (µM)	Treatment	1.09	1	8.75	0.004
	Day	0.32	4	0.65	0.627
	Treatment × day	0.49	4	0.99	0.416
	Residuals	15.77	127

Bold P-values indicate statistical significance across treatment groups within a measured variable.

With respect to haemolymph Na+, a significant interaction between treatment and day was detected for all three species of mussels (Tables 2–4). Pyganodon grandis exposed to fluctuating pCO2 had a significant elevation in haemolymph Na+ at 28 days of exposure compared with control mussels (treatment × day, F = 3.15, P = 0.017; Fig. 2A). Haemolymph Na+ for both A. plicata (treatment × day, F = 12.85, P < 0.001; Fig. 2B) and L. cardium (treatment × day, F = 3.05, P = 0.0194; Fig. 2C) exposed to the fluctuating pCO2 were significantly elevated compared with control mussels beginning at 4 days and throughout the duration of the exposure period.

Figure 2:

Concentrations of Na+ in the haemolymph of Pyganodon grandis (n = 9–14; A), Amblema plicata (n = 12–14; B) and Lampsilis cardium mussels (n = 13–14; C) exposed to two treatments of pCO2, ~1000 µatm (control) or intermittent increase at ~55 000 µatm for 1, 7, 14, 21 or 28 days. Data are presented as means + SEM. *Groups that were significantly different from the control treatment within a time point (two-way ANOVA; see Tables 2–4).

For haemolymph Mg2+, a significant interaction between treatment and day was also found for all three species of mussels (Tables 2–4). Haemolymph Mg2+ was significantly reduced at 14 and 21 days of exposure to fluctuating pCO2 compared with control mussels for P. grandis (treatment × day, P. grandis, F = 28.33, P < 0.001; Fig. 3A) and A. plicata (treatment × day, F = 15.68, P< 0.001; Fig. 3B), but these concentrations were no longer different from control mussels at 28 days of exposure. Likewise, haemolymph Mg2+ in L. cardium exposed to fluctuating CO2 levels was significantly decreased compared with control mussels on 7 and 14 days but returned to control values after 21 days of exposure (treatment × day, F = 7.31, P < 0.001; Fig. 3C).

Figure 3:

Concentrations of Mg2+ in the haemolymph of Pyganodon grandis (n = 9–14; A), Amblema plicata (n = 12–14; B) and Lampsilis cardium mussels (n = 13–14; C) exposed to two treatments of pCO2, ~1000 µatm (control) or intermittent increase at ~55 000 µatm for 1, 7, 14, 21 or 28 days. Data are presented as means + SEM. *Groups that were significantly different from the control treatment within a time point (two-way ANOVA; see Tables 2–4).

For haemolymph glucose, there was no significant interaction of treatment and day for any species of mussel (Tables 2–4). Haemolymph glucose concentrations of P. grandis and L. cardium were unaffected by pCO2 exposure (treatment, P > 0.05; Fig. 4A and C). Haemolymph glucose of A. plicata was significantly affected by fluctuating pCO2 treatment, but not sampling day, and was elevated throughout the exposure period compared with control mussels (treatment, F = 8.75, P = 0.004; Fig. 4B).

Figure 4:

Concentrations of glucose in the haemolymph of Pyganodon grandis (n = 9–14; A), Amblema plicata (n = 12–14; B) and Lampsilis cardium mussels (n = 13–14; C) exposed to two treatments of pCO2, ~1000 µatm (control) or intermittent increase at ~55 000 µatm for 1, 7, 14, 21 or 28 days. Data are presented as means + SEM. For (B), there was no significant interaction between pCO2 treatment and sampling day; *significant effect of pCO2 treatment between mussels exposed to fluctuating ~55 000 µatm and those exposed to ~1000 µatm (two-way ANOVA; see Table 3).

Discussion

Following exposure to fluctuating elevated pCO2, all three mussel species demonstrated physiological changes indicative of disturbance in acid–base regulation. Exposure to high CO2 often causes the acidification of internal fluids in aquatic animals (Pörtner ), and one strategy for animals to buffer this internal acidosis is to increase HCO3− concentrations (Pörtner ; Pörtner, 2008). Marine mussels are thought to increase haemolymph HCO3− by using CaCO3 released from the shell (Michaelidis ; Bibby ). In the present study, all three mussel species may have used this buffering strategy during intermittent pCO2 exposure, as both haemolymph HCO3− and Ca2+ were elevated. Haemolymph HCO3− can also be increased by reducing activity of Cl−–HCO3− exchangers, thus increasing the retention of HCO3− in the haemolymph but at the cost of Cl− uptake (Byrne and Dietz, 1997). A reduction in haemolymph Cl−, which appears to be a short-term response to elevated pCO2 (Hannan , b), was observed only in P. grandis; however, this difference may have been due to rising Cl− concentrations in the haemolymph of control mussels rather than a decrease in Cl− of CO2-treated mussels. A third strategy that mussels use to buffer acidosis is to mediate activity of Na+–H+ exchangers to increase excretion of H+ ions, thus also increasing Na+ uptake (Byrne and Dietz, 1997; Lannig ; Hannan ). Increases in haemolymph Na+ were observed for all species of mussels exposed to fluctuating pCO2, but the timing of the elevation in haemolymph Na+ was species specific, as haemolymph Na+ concentrations for A. plicata and L. cardium mussels increased after 4 days and for P. grandis after 28 days of exposure. Together, the results of the present study suggest that the three species of mussels used similar mechanisms to deal with acidosis to marine mussels (i.e. manipulating haemolymph HCO3− and H+ concentrations); however, species-specific differences in these responses occurred.

In addition to an acid–base disturbance, the results of our study indicate that the stress response was also activated. An indicator of stress in freshwater mussels is declining Mg2+ of the haemolymph, which has been associated with stressors such as elevated temperature (Fritts ), exposure to heavy metals (Hemelraad ) and chronic exposures to elevated pCO2 (Hannan , b). Haemolymph Mg2+ concentrations decreased by ~2-fold in all mussel species exposed to the fluctuating pCO2 treatment, but returned to control values after 28 days of exposure. In contrast, Hannan did not observe a return of Mg2+ to control values in Fusconaia flava exposed to ~20 000 µatm pCO2 for 32 days. In addition, Lampsilis siliquoidea but not A. plicata exposed to either 20 000 or 55 000 µatm pCO2 showed a decrease in haemolymph Mg2+ during 28 days of exposure, and these values returned to baseline once the CO2 stressor was removed (Hannan ). Although the pCO2 and the species of mussels were not the same in our study and those of Hannan , b), these data suggest that fluctuating exposures to elevated pCO2 have a different effect on the Mg2+ response of unionid mussels compared with a chronic exposure. Haemolymph glucose concentrations, another indicator of stress in freshwater mussels (Patterson ; Fritts ), were elevated only in A. plicata. Increasing glucose in response to stress comes at a cost to non-vital functions, such as growth, reproduction and movement (Patterson ; Fritts ). Although the interaction of pCO2 and sampling time was not significant in the model, the elevation in glucose in A. plicata appeared to return to control levels following 28 days of exposure to fluctuating pCO2, suggesting that A. plicata recovers in terms of this stress marker by the end of the exposure period. A similar increase in haemolymph glucose was also observed for A. plicata exposed to a chronic elevation in pCO2 at 55 000 µatm over a 28 day period (Hannan ), suggesting that fluctuating and long-term exposure to pCO2 may have similar effects on haemolymph glucose in this species. Taken together, changes in haemolymph Mg2+ and glucose concentrations suggest that all three species of mussel experienced physiological stress during exposure to fluctuating pCO2; however, desensitization, acclimation or recovery might have occurred over extended exposure to the intermittent CO2 stressor.

Physiological changes, such as acid–base and stress responses, experienced by animals following a stressor are energetically challenging, and long-term upregulation or maintenance of these responses can lead to less energy availability for non-vital functions, such as growth and reproduction (Wendelaar Bonga, 1997). Following exposure to intermittent or repeated stressors, animals may respond to subsequent exposures in different ways (i.e. exacerbation, attenuation or no change; Reinert ). Our results suggest that the duration and CO2 concentration used in the present study did not permit recovery between pulses of high pCO2, evidenced by the fact that mussels sampled before and after the CO2 exposure were not statistically different from each other. Additionally, the responses of mussels to intermittent pCO2 exposure (i.e. elevations of Ca2+ and Na+ and reduction in Mg2+) were similar to those observed in unionid mussels exposed to a chronically elevated pCO2 (Hannan , b), suggesting that mussels react to the intermittent and chronic CO2 exposures in a similar way. However, differences in the responses of these variables during intermittent (present study) and chronic exposures (Hannan , b) did arise during the later stages of the 28 and 32 days exposure period, respectively. For instance, as mentioned above, the concentration of Mg2+ returned to control values by the end of the intermittent CO2 exposure, whereas in previous studies using either chronic exposure to elevated CO2 (Hannan , b) or elevated temperature (Fritts ), Mg2+ remained reduced throughout the exposure period. In addition, haemolymph Ca2+ (P. grandis and L. cardium) and Na+ (all three mussel species) remained elevated for the intermittent exposure to 55 000 µatm pCO2, whereas these ions returned to control values by 32 days of chronic exposure to ~20 000 µatm pCO2 for F. flava (Hannan ). This sustained increase in haemolymph Ca2+ and Na+, as well as the difference in the dynamics of the haemolymph Mg2+ response in at least two of the mussels species, may suggest that mussels respond differently to intermittent and chronic CO2 exposure. These responses also do not exclude the possibility that the differences might be species specific or driven by the difference in pCO2 used in these two studies. The present study suggests that exposure to intermittent elevations in pCO2 do result in acid–base disturbances and stress responses in unionid mussels that are both attenuated (e.g. Mg2+) and exacerbated (Ca2+ and Na+).

Species-specific responses observed in the present study might have resulted from a combination of differences in the physiology and behaviour of the three mussel species examined. Haemolymph Ca2+ was elevated in both P. grandis and L. cardium for more than half of the treatment period, whereas Ca2+ concentrations were elevated only on day 7 of exposure in A. plicata, suggesting that these species may rely differently on shell CaCO3 stores. In addition, a decrease in haemolymph Cl− was observed only in P. grandis and did not occur in either L. cardium or A. plicata. These differences in the studied unionid mussels suggest that they may use different strategies to retain HCO3− for acid–base regulation. Finally, similar elevations in haemolymph Na+ throughout nearly the entire pCO2 exposure period were observed in L. cardium and A. plicata, whereas haemolymph Na+ in P. grandis was elevated only at 28 days of exposure. This difference in haemolymph Na+ concentrations in response to pCO2 exposure suggests that L. cardium and A. plicata may rely on increased regulation of the Na+–H+ exchanger to buffer acidosis, a mechanism that may be less important for P. grandis until CO2 exposure is extended. In terms of measures of the stress response, a similar transient decrease in Mg2+ was observed across all species; however, haemolymph glucose was elevated only in A. plicata. Taken together, similar responses to intermittent elevation in pCO2 were observed across the three species examined, and the species differences that arose highlight the importance of considering multiple species when testing an organism's reaction to a stressor.

Results obtained in our study increase the understanding of responses of freshwater unionid mussels to fluctuating exposures of elevated pCO2, as modelled after a CO2 barrier to invasive fish movement. There is evidence that, like marine mussels, if freshwater unionid mussels are exposed to elevated pCO2 at either chronically high levels (Hannan , b) or intermittent elevations (pres study), they will experience acid–base disturbances. If unionid mussels were to be exposed to intermittent elevations in pCO2 for an extended period of time, populations might be negatively affected owing to the increased energy demand of acid–base regulation and stress responses that may come at the expense of growth and reproduction. Additionally, resident mussel species may be affected differently, as evidenced by the observed species-specific responses to elevated pCO2, which may arise because of differences in their behaviour and physiology. It is also important to consider that fluctuating elevations in pCO2 may have similar but potentially also differential impacts compared with chronic exposures of elevated pCO2 and that, generally, exposure time and duration between applications of a stressor are important aspects to consider for study design. Taken together, the results of our study suggest that the duration and manner of pCO2 exposure (i.e. chronic vs. intermittent), as well as the species characteristics of resident unionid mussels that may be impacted, are all important factors to consider when designing, implementing and assessing the potential impacts of a CO2 barrier.

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