J Comp Physiol B (2017) 187:87–101 DOI 10.1007/s00360-016-1023-z ORIGINAL PAPER Responses to elevated CO2 exposure in a freshwater mussel, Fusconaia flava Jennifer D. Jeffrey1 · Kelly D. Hannan1 · Caleb T. Hasler1 · Cory D. Suski1 Received: 7 April 2016 / Revised: 29 June 2016 / Accepted: 19 July 2016 / Published online: 29 July 2016 © Springer-Verlag Berlin Heidelberg 2016 Abstract Freshwater mussels are some of the most reduce their investment in non-essential processes such as imperiled species in North America and are particularly shell growth. susceptible to environmental change. One environmen- tal disturbance that mussels may encounter that remains Keywords Chitin synthase · Heat shock protein 70 · understudied is an increase in the partial pressure of CO2 Metabolic rate · Bivalve (pCO2). The present study quantified the impacts of acute (6 h) and chronic (up to 32 days) exposures to elevated pCO2 on genes associated with shell formation (chitin syn- Introduction thase; cs) and the stress response (heat shock protein 70; hsp70) in Fusconaia flava. Oxygen consumption (MO2) Freshwater mussels have their highest abundance and was also assessed over the chronic CO2 exposure period. diversity in North America, and provide many important Although mussels exhibited an increase in cs following ecological functions (Williams et al. 1993; Bogan 2008). an acute exposure to elevated pCO2, long-term exposure For example, freshwater mussels filter large volumes of resulted in a decrease in cs mRNA abundance, suggest- water daily, remove bacteria and particles from the water ing that mussels may invest less in shell formation during column, and generate nutrient-rich areas (Vaughn and chronic exposure to elevated pCO2. In response to an acute Hakenkamp 2001; Hauer and Lamberti 2007). In addi- elevation in pCO2, mussels increased hsp70 mRNA abun- tion, freshwater mussels provide an important resource as dance in mantle and adductor muscle and a similar increase food for other aquatic and terrestrial animals (Vaughn and was observed in the gill and adductor muscle in response to Hakenkamp 2001; Hauer and Lamberti 2007). Notably, a chronic elevation in pCO2. A chronic elevation in pCO2 freshwater mussel populations are on the decline, in both also increased mussel MO2. This overall increase in hsp70 species richness and biomass (Williams et al. 1993; Lyd- mRNA levels and MO2 in F. flava indicates that exposure eard et al. 2004; Regnier et al. 2009). Alterations in flow to elevated pCO2 initiates activation of the general stress regimes, land-use changes, invasive species such as zebra response and an increased energy demand. Together, the mussels, and climate change are all thought to have contrib- results of the present study suggest that freshwater mussels uted to these declines (Strayer et al. 2004; Vaughn 2010). respond to elevated pCO2 by increasing processes neces- With only a small percentage of stable freshwater mussel sary to ‘deal with’ the stressor and, over the long-term, may populations remaining (Williams et al. 1993) and continued degradation of freshwater ecosystems, there is an increased need to understand the vulnerabilities of these animals to Communicated by H.V. Carey. environmental stressors, and the mechanisms underlying * Jennifer D. Jeffrey their physiological responses to these stressors (e.g., Jeffrey [email protected] et al. 2015). One environmental stressor that is currently understudied 1 Department of Natural Resources and Environmental Science, University of Illinois at Urbana-Champaign, 1102 in the freshwater environment is the impact of elevations in South Goodwin Avenue, Urbana, IL 61801, USA the partial pressure of carbon dioxide (pCO2). In the context 1 3 88 J Comp Physiol B (2017) 187:87–101 of climate change and rising atmospheric CO2, the impact 1983). The shell is comprised of a mineral phase (95–99 % of ocean acidification (i.e., elevated pCO2) on marine cal- predominantly calcium carbonate; CaCO3) and an organic cifying organisms has been investigated to a large extent matrix (1–5 %). Chitin is an insoluble polysaccharide that (reviewed by Fabry et al. 2008; Gazeau et al. 2013); how- forms the highly structured organic framework of mol- ever, virtually northing is known about the responses of lusc shells within which CaCO3 minerals are deposited freshwater bivalves to increased pCO2 (Hasler et al. 2016). (Weiner et al. 1984; Levi-Kalisman et al. 2001; Weiss and Upon entering freshwater, CO2 results in a decrease in water Schonitzer 2006). The enzymes involved in chitin synthesis pH due to the production of carbonic acid (H2CO3), lead- are important not only for mechanical strength and tough- ing to the release of H+ and, thus, the weak acidification of ness of the shell, but also for coordination of mineraliza- water. Levels of freshwater pCO2 can vary for a variety of tion processes and shell formation (Schonitzer and Weiss reasons including, terrestrial productivity, precipitation, and 2007). Chitin synthase (CS) is a key enzyme involved in local geology (Cole et al. 1994; Maberly 1996; Butman and the synthesis of chitin (Fang et al. 2011), and inhibition of Raymond 2011), resulting in CO2 levels that can fluctuate CS during early development has been shown to negatively both seasonally and daily, and that can exceed atmospheric affect rates of shell development, solubility of the shell, and levels (i.e., water bodies can be supersaturated with CO2). survival in Mytilus galloprovincialis larvae (Schonitzer and River environments can thus experience a wide range of Weiss 2007). Furthermore, changes in the mRNA abun- pCO2 over the course of a year (from less than 100 to over dance of cs occurred in mantle of adult Laternula elliptica, 15,000 µatm), with higher values being observed in warmer, a marine bivalve, in response to changes in environmen- dryer periods (Cole and Caraco 2001). Although less well tal pCO2 (and thus pH) (Cummings et al. 2011). Due to understood than for the marine environment, freshwater its importance in the biological control of shell formation pCO2 may increase as a result of increased atmospheric and evidence of its regulation in response to situations of CO2, greater terrestrial primary productivity, increased pre- ocean acidification, cs provides a useful target to assess the cipitation, and longer periods of dry conditions—although impact of environmental stressors, such as elevated pCO2, the magnitude of change is not known (Phillips et al. 2015; on shell formation in adult freshwater mussels. Hasler et al. 2016; Perga et al. 2016). Levels of freshwa- The impacts of elevated pCO2 on other cellular func- ter CO2 can also be intentionally elevated in the context tions, such as mediators of cellular stress, have also been of generating non-physical barriers to deter the movement investigated to some extent in marine bivalves (e.g., Cum- of invasive fishes (Noatch and Suski 2012). The form that mings et al. 2011). Heat shock proteins (HSPs) are among such a non-physical barrier may take has not yet been well the most evolutionarily conserved proteins, and are induced defined, but CO2 levels would likely dissipate as the dis- by a number of factors beyond heat-stress that affect cell tance from the CO2 infusion site increases, thus mussels protein structure and functioning (Feder and Hofmann may be exposed to a gradient of CO2 depending on their 1999; Sørensen et al. 2003). A key role of HSPs is to pro- proximity to the barrier. Together, freshwater mussels may tect and repair cellular proteins damaged by exposure to experience periods of elevated pCO2 due to both natural and stressors, and to minimize protein aggregation (Feder and anthropogenic sources, and with pCO2 expected to rise in Hofmann 1999). Heat shock protein 70 (HSP70) is the the future, this necessitates a need for a better understanding most abundant family of HSPs, and consists of the consti- of the consequences for freshwater mussels. tutively expressed HSC70 and inducible HSP70 that are In the marine environment, a major consequence of ubiquitously distributed in eukaryotic cells (Feder and Hof- exposure to elevated pCO2 for bivalves is a reduction in mann 1999). The key role of HSPs in mechanisms of cellu- both shell growth and biomineralization (reviewed by lar protection renders them good markers of the stress sta- Gazeau et al. 2013). The mollusc shell provides an impor- tus of an organism. In this way, HSPs provide information tant external structure to support living tissues, protect about the general condition and health of an organism as against predators, and exclude mud and sand from the well as the sub-lethal effects (i.e., early warning signs) of a mantle cavity of burrowing species (Gazeau et al. 2013). stressor, before more complex functions are compromised Changes in the integrity of the shell have occurred due to (reviewed by Fabbri et al. 2008). The inducible HSP70 is exposure to conditions of ocean acidification, and dissolu- widely up-regulated in response to a variety of stressors in tion of the shell as a result can have consequences for the bivalves (e.g., Franzellitti and Fabbri 2005; Toyohara et al. health and survival of bivalves (reviewed by Gazeau et al. 2005; Cellura et al. 2006; Cummings et al. 2011; Chen 2013). The mantle, a thin secretory epithelial tissue lining et al. 2014; Luo et al. 2014) and may represent a useful bio- the inner surface of the shell, is responsible for mollusc marker in examining elevated CO2 as a potential stressor in shell formation, and shell calcification occurs in a small freshwater mussels. compartment (i.e., extrapallial cavity) located between the In addition to the impacts of elevated pCO2 on cel- calcifying outer mantle and the shell (Wilbur and Saleuddin lular function, elevations in pCO2 also have the potential 1 3 J Comp Physiol B (2017) 187:87–101 89 to affect whole-animal energetics and metabolism.