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Lethal and sublethal responses of native mussels

(Unionidae: Lampsilis siliquoidea and L. higginsii) to elevated carbon dioxide

Journal: Canadian Journal of Fisheries and Aquatic Sciences

Manuscript ID cjfas-2017-0543.R1

Manuscript Type: Article

Date Submitted by the Author: 06-Apr-2018

Complete List of Authors: Waller, Diane; USGS Upper Midwest Environmental Sciences Center,

Bartsch, Michelle; USGS Upper Midwest Environmental Sciences Center Bartsch, Lynn; USGS Upper Midwest Environmental Sciences Center Jackson, Craig; USGS Upper Midwest Environmental Sciences Center

Is the invited manuscript for consideration in a Special

Issue? : N/A

Keyword: freshwater mussel, carbon dioxide, unionid, toxicity

https://mc06.manuscriptcentral.com/cjfas-pubs

Canadian Journal of Fisheries and Aquatic Sciences

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1 For Submission to Canadian Journal of Fisheries and Aquatic Sciences 1

Running title: Responses of juvenile unionid mussels to carbon dioxide exposure. 2

3

Lethal and sublethal responses of native mussels (Unionidae: Lampsilis siliquoidea and L. higginsii) to 4 elevated carbon dioxide 5

*Diane L. Waller, U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 Fanta 6 Reed Road, La Crosse, WI 54603; [email protected]; Telephone: 608-781-6282; Fax: 608-783-6066 7

Michelle R. Bartsch, U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 8 Fanta Reed Road, La Crosse, WI 54603; [email protected] 9

Lynn A. Bartsch, U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 Fanta 10 Reed Road, La Crosse, WI 54603; [email protected] 11

Craig A. Jackson, U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 Fanta 12 Reed Road, La Crosse, WI 54603; [email protected] 13

*Corresponding author 14

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16

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mailto:[email protected]

mailto:[email protected]

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2 Abstract 17

Levels of carbon dioxide (CO2) that have been proposed for aquatic invasive species (AIS) control 18

[24 000 – 96 000 µatm partial pressure CO2 (PCO2); 1 atm = 101.325 kPa] were tested on juvenile 19

mussels, the Fatmucket (Lampsilis siliquoidea) and the U.S. federally endangered Higgins Eye 20

(L. higginsii). A suite of responses (survival, growth, behavior, and gene expression) were measured after 21

28-d exposure and 14-d postexposure to CO2. The 28-d LC20 (lethal concentration to 20%) was lower for 22

L. higginsii (31 800 µatm PCO2, 95% confidence interval (CI) 15 000 – 42 800 µatm) than for 23

L. siliquoidea (58 200 µatm PCO2, 95% CI 45 200 – 68 100 µatm). Treatment-related reductions 24

occurred in all measures of growth and condition. Expression of chitin synthase, key for shell formation, 25

was down-regulated at 28-d exposure. Carbon dioxide caused narcotization and unburial of mussels, 26

behaviors that could increase mortality by predation and displacement. We conclude that survival and 27

growth of juvenile mussels could be reduced by continuous exposure to elevated CO2, but recovery may 28

be possible in shorter duration exposure. 29

30

Key words: freshwater mussel; unionid; carbon dioxide; toxicity; transcriptional and growth response 31

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3 Introduction 34

Elevated atmospheric carbon dioxide (CO2) is most recognized for its role in climate change and 35

ocean acidification, but purposeful infusion into select aquatic systems is being pursued as a tool for 36

control of aquatic invasive species (AIS) such as bighead (Hypophthalmichthys nobilis) and silver carp 37

(H. molitrix) (Cupp et al. 2017) and sea lamprey (Petromyzon marinus). Several studies have 38

demonstrated that CO2 concentrations of 60 to 120 mg·L–1 (24 000 to 75 000 µatm PCO2; 1 atm=101.325 39

kPa) would induce avoidance responses in fish and could be used to corral and harvest fish in a confined 40

area or deter upstream migration through a stream or lock channel (Kates et al. 2012; Dennis et al. 2016; 41

Cupp et al. 2017). Before its widespread deployment in AIS programs, the effects of elevated CO2 on 42

native species is a consideration. 43

Exposure of native freshwater mussels to elevated CO2 is of particular concern given the 44

precarious status of the fauna. Unionid mussels are a high risk faunal group due to a multitude of stressors 45

including habitat loss, overharvest, water quality degradation, and competition with AIS (Williams et al. 46

1993; Lydeard et al. 2004; RéGnier et al. 2009). Native mussel communities play a vital role in nutrient 47

cycling, substrate stabilization, and water filtration in aquatic ecosystems (Vaughn and Hakenkamp 2001; 48

Vaughn et al. 2008; Strayer 2014). Given their relative immobility and reliance on a calcified shell and 49

bicarbonate buffer system, unionid mussels may be especially vulnerable to CO2 exposure and the 50

concomitant production of carbonic acid. 51

Recently, scenarios of CO2 exposure have been tested on several common species of native 52

freshwater mussels (Hannan et al. 2016a, b, c; Jeffrey et al. 2017; Waller et al. 2017; Jeffrey et al. 2018). 53

Adult mussels (Fatmucket Lampsilis siliquoidea and Threeridge Amblema plicata) survived 28-d 54

exposure to 55 000 µatm PCO2, but exhibited physiological signs of respiratory acidosis and alterations 55

in the bicarbonate buffer system (Hannan et al. 2016c). Wabash pigtoe Fusconaia flava also survived 56

exposure to 200 000 and 20 000 µatm PCO2 for 6 h and 32 d, respectively; however, levels of heat shock 57

protein 70 mRNA (gill) and oxygen consumption increased and chitin synthase expression (mantle) 58

decreased in mussels that were exposed for 32 d (Jeffrey et al. 2017). 59

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4

Few studies have compared the effects of elevated CO2 on juvenile and adult freshwater mussels 60

and, to our knowledge, no studies have been conducted with U.S. federally listed species. In separate 61

studies, juvenile (~ 6 mo old; Waller et al. 2017) and adult (L. siliquoidea) mussels (Hannan et al 2016c) 62

were exposed to elevated CO2 for 28 d. Juvenile mussels died at 42 000 µatm PCO2 (Waller et al. 2017), 63

whereas adult mussels survived 55 000 µatm PCO2 (Hannan et al. 2016c). Lampsilis siliquoidea is 64

abundant, geographically widespread (Cummings and Cordeiro 2012), and has become a common species 65

for toxicity testing (e.g., Bringolf et al. 2007a, b; Wang et al. 2007; Wang et al. 2011; and others). Its 66

congener Higgins Eye (L. higginsii Lea, 1857) is a U. S. federally listed species that occurs in the upper 67

Mississippi River drainage. Sites of potential CO2 deployment for AIS control (ACRCC MRW 2014) 68

overlap with essential habitat for L. higginsii 69

(https://www.fws.gov/Midwest/endangered/clams/higginseye/hepmeha.html) and therefore, an evaluation 70

of risk to the species is required before CO2 is approved for use in the field. Toxicity tests with 71

endangered species are infrequent and risk assessments generally rely on responses of surrogate species. 72

In the present study, we simultaneously exposed juvenile L. siliquoidea and L. higginsii to CO2 and 73

determined the suitability of the common species as a surrogate for the latter. 74

We measured a suite of responses in juvenile mussels during long-term 28-d exposure to CO2 75

exposure and after 14-d postexposure (PE) in untreated water. The PCO2 range tested represents that 76

expected from the immediate area of CO2 infusion (~100 000 µatm PCO2) and in a downstream plume 77

(~ 24 000 µatm PCO2). The exposure duration simulates the use of CO2 as a long-term or seasonal barrier 78

to deter fish movement. Mortality, shell growth, and behavioral responses to CO2 were measured on both 79

mussel species and provided comparable data to evaluate interspecies differences (L. higginsii vs L. 80

siliquoidea) and intertrial variation in responses of L. siliquoidea (Waller et al. 2017). Condition indices 81

and transcriptional responses were limited to L. siliquoidea because both analyses required sacrifice of 82

individuals which we avoided with L. higginsii. Additionally, we measured expression levels of several 83

key genes in shell formation, energetics and growth, and calcium regulation at 28 d of CO2 exposure and 84

after 14-d PE. Fold expression was quantified for Chitin synthase (CHS), Calmodulin (CAL), Na+/K+ 85

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https://www.fws.gov/Midwest/endangered/clams/higginseye/hepmeha.html

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5 ATPase (NKA) and a Defensin (DEF). Chitin synthase is a transmembrane glycosyltransferase that plays 86

a role in synthesis of chitin and in formation of the larval bivalve shells (Weiss and Schӧnitzer 2006; 87

Weiss et al. 2006; Fang et al. 2011; Liu et al. 2015). Calmodulin (CAL) is a multifunctional calcium-88

binding messenger protein that mediates many fundamental cellular processes. In bivalves, CAL is 89

integral to Ca2+ uptake from the water by the gills and for shell formation and biomineralization (Ren et 90

al. 2013; Liu et al. 2015; Sun et al. 2015). Na+/K+ ATPase is critical to active transport and as secondary 91

transport of other ions and organic molecules (Giacomin et al. 2013); it may play a role in reducing 92

acidosis by excretion of NH4+ from the mantle (Hüning et al. 2013). Defensins are peptides with 93

antimicrobial activities and have been characterized in several freshwater mussels (Xu and Faisal 2010; 94

Luo et al. 2014). Defensin expression was up-regulated in Uniomerus tetralasmus during emersion and 95

desiccation suggesting it may also be a general stress indicator (Luo et al. 2014). 96

The objectives of the present study were: 1) determine the lethal and sublethal effects of CO2 to 97

juvenile native mussels at levels expected for AIS control, 2) determine the suitability of L. siliquoidea as 98

a surrogate for the endangered species, L. higginsii, and 3) compare organismal and transcriptional 99

responses of juvenile lampsiline mussels to continuous CO2 exposure. 100

101

Materials and Methods 102

Test animals 103

Juvenile L. siliquoidea and L. higginsii (~ 9 months old) were propagated at the U.S. Fish and Wildlife 104

Service, Genoa National Fish Hatchery (Genoa, WI, USA) and transferred to the U.S. Geological Survey, 105

Upper Midwest Environmental Sciences Center (La Crosse, WI, USA). Juveniles of each species came 106

from the same cohort and all animals were cultured under the same conditions. Lampsilis higginsii 107

juveniles ranged from 3.24 to 7.40 mm in shell length (mean = 4.71 mm; standard deviation, SD = 108

0.89 mm, n = 150). Lampsilis siliquoidea juveniles ranged from 4.17 to 9.21 mm in shell length (mean = 109

7.17, SD = 0.92 mm, n = 240). Mussels were held in a raceway with recirculating well water with 10% 110

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6 daily water replacement. Mean (SD) chemical parameters of the well water were: hardness 190.7 (22.0) 111

mg·L–1 as CaCO3, alkalinity 132.8 (4.8) mg·L–1 as CaCO3, and conductivity 412.3 (11.2) µS·cm–1. 112

Mussels were fed a diet of four commercial algal foods (approximate ratio of 2:0.5:1:1, Shellfish diet, 113

Nannochloropsis 3600 Instant Algae, TW1200 and TP1800, Reed Mariculture, Campbell, CA). Food was 114

prepared daily and delivered continuously to the holding raceway and test tanks by a peristaltic pump 115

(Cole Parmer Masterflex Digi-staltic pump, Vernon Hills, IL) at a rate of 0.07 algae dry wt (mg·L–1min-1). 116

Washed sand substrate (Mastercraft® commercial playground sand) was provided for mussels to position 117

themselves upright and bury. Mussels were acclimated from 12 °C to 22 °C at a rate of ≤ 3 °C per day and 118

maintained at 22 °C for 1 week (L. higginsii) and 4 weeks (L. siliquoidea) before initiation of test 119

exposure. 120

Test system and exposure 121

The test system and methods followed those of Waller et al. (2017) with a 28-d exposure period 122

followed by 14-d PE period in untreated water. The test was conducted in three continuous flow-through 123

diluter systems; each diluter served as a replicate. In each system, well water was supplied to a head box 124

followed by a serial dilution box that was partitioned into 10 chambers. The PCO2 was reduced by about 125

20% in each successive chamber of the dilution box. The outflow from the four dilutor chambers with the 126

targeted partial pressure of CO2 drained to a glass tank (25.4 cm × 49.5cm × 30.5 cm, W × L × H) that 127

held test mussels. Outflow from the remaining six dilutor chambers was directed to the effluent drain. The 128

control tank received clean untreated water directly from the head box. Treatments were assigned to each 129

tank within a diluter system using a randomized block design. 130

Tanks were filled to 22 L with 22 °C well water with a mean flow rate of 294 mL·min–1 (about 1 131

tank exchange every 75 min). The algal stock solution was prepared daily, as described in the previous 132

section, and distributed to each of three food reservoirs (one 45-L stainless steel tank per dilutor system). 133

Food was kept in suspension with an electronic mixer and was delivered continuously by peristaltic 134

pumps to each tank at a rate of 110 mL·h–1. Food delivery began 24 h before addition of mussels to the 135

test tanks. Tanks were covered, top and sides, with black polyethylene sheeting (6 mil) to minimize light 136

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7 exposure. Each mussel species was placed into a separate grid on the bottom of a tank to isolate 137

individual mussels and track their movement and position in the substrate. The grid was constructed from 138

plastic light diffusers, cut to produce 3 × 3-cm cells (1.5 cm depth), and 1.5-mm mesh screen to cover the 139

bottom. Each grid cell was filled with 1.2 cm sand substrate (≤ 1.5 mm grain size) to allow movement and 140

burrowing of mussels. 141

Mussels were randomly distributed to the tanks for a total of 10 L. higginsii and 16 L. siliquoidea 142

mussels per test tank. Before placement in the test tank, the shell of each mussel was marked with a 143

unique color dot code using waterproof markers and photographed (side-lying) at 10× under a 144

stereomicroscope. Mussels were placed side-lying into a pre-assigned grid cell in each test tank and 145

allowed to position and acclimate for 2 d before initiation of CO2 infusion. 146

Targeted PCO2 treatments of 18 000, 31 000, 44 000 and 75 000 µatm PCO2 (comparable to 30, 147

50, 70, and 120 mg·L–1 CO2) were based on effective concentrations for deterring silver and bighead carp 148

(i.e., 60 to 100 mg·L–1 CO2) and results of a previous mussel trial (i.e., 28-d LC50 for juvenile 149

L. siliquoidea of 76 mg·L–1 CO2; Waller et al. 2017). Measured PCO2 treatments (28-d mean value) were 150

24 000, 37 000, 61 000 and 96 000 µatm. 151

Food grade CO2 gas was supplied to a pressure differential automatic manifold (Precise 152

Equipment Co., Denton, TX) and then to a CO2 pressure regulator. Outflow from the CO2 regulator was 153

set to 12 L·min–1and was adjusted to each diluter with a 3–way air regulating valve. Carbon dioxide 154

flowed to the dilutor systems through vinyl airline tubing (ID 6.35 mm) and into an airstone (74 mm × 37 155

mm × 37 mm) that was submerged in the first chamber of the dilution box. Carbon dioxide infusion was 156

stopped at 28 d and was at control levels by 8 h PE. Water flow through the diluters and each test tank 157

continued for 14 d PE. 158

Water quality was measured daily in each tank. Dissolved oxygen (mg·L–1) and pH were 159

measured with a Hach LDO IntelliCAL probe and Hach pH probe, respectively, attached to a Hach 160

HQ40d Water Chemistry Multimeter (Hach, Loveland, CO). Temperature (ºC) was measured with a 161

digital thermometer. Total ammonia nitrogen (TAN, µg·L–1) was measured once a week in the control 162

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8 and highest CO2 treatment in each diluter, using a Hach ammonia probe (Model ISENH318101) attached 163

to a Hach HQ40d Water Chemistry Multimeter. Conductivity and hardness were measured on a sample of 164

water from each head box at the beginning and completion of the study. Conductivity (µS·cm–1) was 165

measured with a Fisher Accumet conductivity meter (Fisher Scientific, Waltham, MA) calibrated against 166

a standard solution (APHA 2012). Total hardness (mg·L–1 CaCO3) was determined by titrimetric method 167

with Manver Red indicator (USEPA 1983). Alkalinity was measured on samples from each diluter head 168

box at the beginning and end of the study. Additionally, alkalinity was measured from one randomly 169

selected tank per diluter on the same days that CO2 was measured. Total alkalinity (mg·L–1 CaCO3) was 170

determined by titrimetric method to a pH endpoint of 4.5 (APHA 2012). 171

The partial pressure of CO2 (PCO2, µatm) in each test tank was calculated using the U.S. 172

Geological Survey CO2 calculator (Robbins et al. 2010). Mean total alkalinity of the three sampled tanks 173

and the daily measured pH and temperature value of each test tank were used in the calculation. Carbon 174

dioxide was also measured daily by titration for the first 7 days of exposure and twice a week, thereafter, 175

for the duration of the exposure period. Carbon dioxide (mg·L–1) concentrations were determined by a 176

modified Hach Method 8205 digital titration method using sodium. The titrimetric method consisted of 177

collecting a 100–mL water sample from the test tanks and, while slowly stirring, immediately titrating 178

with 0.363N (0–100 mg·L–1 treatments) or 3.636 N NaOH (≥ 100 mg·L–1treatments) to a pH endpoint of 179

8.3. A modified infrared probe (Vaisala BMP220 and GMT221, St. Louis, MO) was also used to verify 180

PCO2 in each test tank twice during the exposure period. 181

Mussel observations and measurements 182

Daily observations were made of mussel location in the grid, burial status (buried < 10% of shell 183

visible, unburied ≥ 90% of shell visible), gaping, and mortality. Mussels were counted as dead if the 184

valves opened without resistance under light pressure, the valves or foot did not respond to touch, and 185

ciliary movement and filtration activity near the siphons was absent. The daily number of unburied 186

mussels per tank was graphed over days 1–12 of exposure to illustrate initial responses of mussels before 187

onset of mortality which began on day 12. 188

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9

At the end of the 28-d exposure and 14-d PE period, mussels were removed from the tanks for 189

assessment of mortality and growth. A digital photograph of each mussel (side-lying view) was taken 190

under a stereomicroscope at 10×, as described above. Shell growth in length was determined from 191

measurements of live mussels at each sampling period (0-d, 28-d exposure and 14-d PE). Sample size 192

(number of living mussels) varied among treatments because of treatment-specific mortality. 193

Measurements of shell length were made from digital images by 2 independent readers using image 194

analysis software (NIS Elements, Nikon, Melville, NY) after calibration with a photograph of a stage 195

micrometer. Shell length (µm) was defined as the maximum anterior-posterior dimension that is parallel 196

to the hinge line. Measurements taken by the 2 readers were compared and if they varied by > 3% a third 197

reader measured the photograph and the outlying measurement was omitted. The mean shell length 198

determined from the two measurements was used in analyses of shell growth. Percent growth and rate of 199

growth were determined for the 28-d exposure period and the 14-d PE period. The percent growth 200

accounted for differences in the initial length of mussels and was calculated as: percent growth = ((L2 – 201

L1/ L1 × 100), where L1 is shell length (µm) at start of the sampling period (0-d for exposure period, 28-d 202

for PE period), L2 is shell length at the end of the sampling period (28-d for exposure period, 42-d for 203

PE). Daily growth rate (µm·d-1) was calculated as (L2 – L1) / T, where T = number of days in sampling 204

period (T = 28 for exposure, 14 for PE). 205

Tissue and shell condition indices were determined from mussels that were sampled at the end of 206

the PE period. A subsample (n = 4) of L. siliquoidea juveniles from each tank was processed for 207

determination of shell and dry tissue weight. Dead mussels were excluded from analysis of condition 208

which eliminated the highest treatment concentration due to insufficient sample size. Soft tissue was 209

removed from the shell and each component was placed into separate tared aluminum weigh pans. 210

Samples were dried at 80 °C to a constant weight. Dry weights were measured on an analytical balance 211

(Mettler AT200, Columbus, OH). Tissue condition index was defined as: [(1000 × dry tissue weight) / 212

(shell length)]. Shell condition was defined as: [(1000 × dry shell weight) / (shell length)]. 213

Molecular Methods 214

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10

Relative gene expression level was measured in L. siliquoidea mussels at 0-d exposure, 28-d 215

exposure and at 14-d PE. Twelve juveniles were indiscriminately taken on day 0, before distribution of 216

mussels to treatment tanks, for baseline measure of gene expression. Before the onset of CO2 infusion, 217

five mussels were randomly distributed to each control and treatment tank, independent of the mussels 218

that were placed into grids (see section Test system and exposure), and sacrificed at 28-d exposure. At 219

14-d PE, a subsample (n = 4–5) was indiscriminately selected from all live mussels in each tank. Whole 220

individual juvenile mussels were placed into cryovials, then flash frozen in liquid nitrogen and stored 221

at -80 °C. Total RNA was extracted from each mussel sample using the RNeasy Fibrous Tissue Mini Kit 222

(Qiagen, cat# 74704, Valencia, CA) according to the manufacturer’s protocol. Tissues were disrupted and 223

homogenized with a mechanical homogenizer (Geno/Grinder, SPEX Sample Prep, model 2010, 224

Metuchen, NJ) by agitation in 300 µl of lysis buffer containing one 7 mm bead of steel shot for 30 s at 225

1500 rpm. Homogenates in lysis buffer were stored at -80 °C until RNA isolation was completed (one 226

week or less). DNase digestion was performed on-column during RNA isolation, and RNA was 227

subsequently quantified by UV spectrophotometry in nuclease-free water using a MultiSkan Spectrum 228

plate reader (Thermo Fisher, Waltham, MA). RNA samples with A260/A280 ratios between 1.78 and 229

2.23 were considered satisfactory for use in this study. Intact high molecular weight ribosomal RNA 230

bands were observed on a subset of samples analyzed by 2% agarose gel electrophoresis. RNA samples 231

were normalized to a concentration of 20 ng· µL–1 in non-skirted, RNase/DNase/PCR inhibitor-free 96-232

well plates (MidSci, cat# AVRT-N, Valley Park, MO) prior to qRT-PCR. 233

Target genes were selected based on their potential functions in metabolism and energetics, shell 234

formation, ion balance, and stress response. Gene-specific primers were designed based on conserved 235

regions of sequences from several bivalve species using PrimerQuest software 236

(www.idtdna.com/Primerquest) except as noted (Table 1). Ubiquitin (UBQ) is a stably expressed gene 237

that functions in protein degradation and has been widely used as a reference gene for normalization of 238

qPCR data (Bai et al. 2014). The CAL assay was designed from a 726 bp sequence of Hyriopsis cumingii 239

(JQ389856.1) and targets a 101 bp amplicon. The DEF markers were designed from a 302 bp segment of 240

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11 H. cumingii defensin (JN604559.1) and targets a 110 bp amplicon. The NKA assay was designed from a 241

1260 bp sequence of L. cardium (AY303383.1) and targets a 107 bp amplicon. Primer specificity was 242

verified by sequencing the PCR product from each primer set. BLAST search results indicated that all of 243

the primers amplified their intended target genes. 244

Semi-quantitative real-time PCR (qRT-PCR) was used to determine the relative abundances of 245

target RNA molecules in samples of mussels taken before CO2 exposure and from control and treatment 246

tanks at 28-d exposure and 14-d PE. qRT-PCR efficiencies, slopes, and y-intercepts were determined for 247

each primer set from calibration curves of RNA pooled equally from 4 individual mussels. All qPCR 248

efficiencies were calculated to be in the range of 97–102%. 249

qRT-PCR reactions were prepared in duplicate 20 µL volumes using the qScript™ One-Step 250

SYBR® Green qRT-PCR Kit (Quanta Biosciences, cat# 95087, Gaithersburg, MD). Each reaction 251

contained 1X One-Step SYBR Green Master Mix, 100 ng RNA, 250 nM forward and reverse primers, 0.4 252

µL of qScript One-Step Reverse Transcriptase, and nuclease-free water. Thermal cycling was performed 253

using an Eppendorf Mastercycler as follows: cDNA synthesis at 50 °C for 5 min followed by Taq 254

activation at 95°C for 5 min, then 35 PCR cycles of denaturation at 95 °C for 10 s/primer annealing at 255

either 58 °C (NKA, CHS, UBQ) or 60 °C (CAL, DEF) for 20 s/extension at 72 °C for 30 s. Post-PCR 256

melt curve analysis was performed on all samples. A subset of samples (n = 12) was used as template in 257

(-) RT control reactions for each assay and no amplifications were observed. 258

The quantification cycle (Cq) for each sample was determined by averaging the two technical 259

replicate values. All samples with a SD > 0.40 between technical replicates were individually examined 260

using Eppendorf Realplex software, and the discrepant replicate (unstable baseline) was discarded prior to 261

copy number extrapolation. We determined relative expression levels of target genes between treatments 262

using the ΔΔCt method as previously described by Livak and Schmittgen (2001). Briefly, we calculated 263

ΔCt values for each sample by subtracting the mean Cq value for each target gene from the mean Cq 264

value for the reference gene (UBQ). We then calculated ΔΔCt values for each sample by subtracting the 265

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12 ΔCt values from the mean ΔCt value for the controls. Finally, we calculated relative expression using the 266

equation: 2−𝛥𝛥𝛥𝛥𝛥𝛥𝛥𝛥. 267

Statistical analysis 268

For all statistical analyses, differences were considered significant if p < 0.05. The Statistical 269

Analysis Software package (SAS Version 9.4, Cary, NC) was used for analysis of mortality, shell growth, 270

condition indices, and gene expression. The LC20 and LC50 (lethal concentration to 20% and 50% of 271

organisms, respectively) and 95% CI at 28-d exposure and at 14-d PE were determined by probit analysis 272

using the 28-d mean PCO2 (µatm) of each tank. Mortality (28-d and 14-d PE) differences between species 273

were compared in a mixed model with fixed effects of mean PCO2 and species, and dilutor within PCO2 274

treatment as a random effect. We compared the initial length of mussels with analysis of variance and 275

found differences among L. higginsii were significant, but not for L. siliquoidea. Therefore, differences in 276

initial length were accounted for in growth models by: 1) basing growth comparisons on percent growth 277

and 2) including initial shell length as a covariate in models of growth rate. Percent shell growth and 278

growth rate were modeled for each species with a repeated measures nested hierarchical mixed model 279

with fixed effects of mean PCO2, time (T1 = 28-d exposure, T2 = 14-d PE), and the interaction of PCO2 280

and time. Dilutor within mean PCO2 was a random effect with an unstructured covariance. An EC50 281

(median effect concentration) for percent shell growth was determined by fitting a 3-parameter logistic 282

equation to the data as described by Martikainen and Krogh (1999). Parameter estimations and CI were 283

calculated using PROC NLIN. Differences in condition indices among treatments in L. siliquoidea were 284

analyzed with a mixed model with fixed effects of mean PCO2 and dilutor within PCO2 treatment as a 285

random effect. Fold expression (DEF, CHS, NKA, and CAL) in L. siliquoidea at 28-d exposure and 14-d 286

PE was modeled with a nested hierarchical mixed model with fixed effects of mean PCO2, time, and the 287

interaction of PCO2 and time. Dilutor within mean PCO2 was a random effect with an unstructured 288

covariance. Between-with method was used to estimate degrees of freedom at the hierarchical levels of 289

dilutor within PCO2 treatment. The hierarchal nature of the analysis ensured the degrees of freedom 290

represented the correct number of experimental units (i.e., tanks and not the number of mussels). 291

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13 Orthogonal contrasts were constructed to test for difference between controls and individual treatment 292

levels. Mean values and 95% CI were calculated for dissolved oxygen, temperature, alkalinity, hardness 293

and conductivity. Mean pH and 95% CI were calculated as the geometric mean. 294

295

Results and Discussion 296

Water chemistry and exposure conditions 297

Water chemistry parameters were relatively uniform among diluters and did not vary appreciably 298

during the study. Mean (SD) chemical parameters were: hardness 178.7 mg·L–1 (4.7), conductivity 404.5 299

µS·cm–1 (3.8), and alkalinity 135.1 mg·L–1 CaCO3 (4.7). Dissolved oxygen was inversely correlated with 300

CO2 as the increased CO2 concentration caused sparging of oxygen (Table 2); however, dissolved oxygen 301

remained above 7.0 mg·L-1 in the high treatments throughout the study. Total ammonia nitrogen (TAN) 302

remained below acceptable levels throughout the study (mean 39.6 µg·L-1, SD 32.1, range 16.3 – 48.4 303

µg·L-1). The pH was inversely correlated with PCO2 levels, indicating the formation of carbonic acid 304

from the reaction of CO2 with water. Mean tank water flow rates ranged from 164 to 298 mL·min-1. 305

Mortality 306

Survival of mussels in control tanks at the end of the 28-d exposure and 14-d PE period was 307

93.3% and 100% for L. higginsii and L. siliquoidea, respectively, (Fig. 1; Table 3) and exceeded ASTM 308

acceptable criterion of 80% for chronic exposure of juvenile mussels (ASTM 2017). At least two 309

mortalities of L. higginsii occurred in each treatment; however, no tank had 100% mortality of this 310

species (Fig. 1; Table 3). In contrast, 100% mortality of L. siliquoidea occurred in two of three replicates 311

of the highest treatment. Mortality of L. higginsii was greater than that of L. siliqouidea in all treatments, 312

except the highest, at 28-d and 14-d PE (Fig. 1, Table 3). The lethal concentrations (LC20 and LC50) 313

values for L. higginsii were lower than those for L. siliquoidea (Table 4), although confidence intervals 314

for LC50 values overlapped between species. Overall, average mortality was 7 – 8% higher and LC 315

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14 values were 5 000 – 10 000 µatm lower at 14-d PE as mortality of both species continued after exposure 316

(Tables 3 and 4). 317

Previously, we exposed L. siliquoidea juveniles to a wider range and higher levels of PCO2 318

(45 000 – 297 537 µatm) and reported the 28-d LC50 = 87.0 mg·L–1 (91 103 µatm PCO2, 95% CI 81 803 319

– 100 698) and 16-d PE LC50 = 76.0 mg·L–1 (78 039 µatm PCO2, 65 057 – 92 137; Waller et al. 2017). 320

LC50 values and 95% CIs in the present study overlapped with those in Waller et al. (2017), and support 321

our previous estimates of CO2 toxicity to this species. 322

Risk assessment in aquatic toxicology often relies on surrogate species (e.g., fathead minnow, 323

daphnia) to extrapolate to threatened and endangered species (e.g., Sappington et al. 2001) due to limited 324

availability and legal restrictions in testing listed species. The choice of an appropriate surrogate is 325

commonly a congener with similar life history and physiology (Banks et al. 2014). Propagated juvenile 326

L. siliquoidea have become a common toxicity test organism, but this is only the second reported toxicity 327

trial with juvenile L. higginsii (Newton and Bartsch 2007) and the first with ~ 9 mo old animals. In our 328

study, we had the opportunity to simultaneously test juvenile L. higginsii and L. siliquoidea that were 329

propagated at the same facility. However, the U.S. endangered species permit also restricted the number 330

of L. higginsii that could be used in the study, which reduced some statistical power and the response 331

variables that were measured. 332

Mortality of L. higginsii was more variable than that of L. siliquoidea, particularly in the control 333

and lower CO2 treatments. This variability resulted in wide confidence intervals and an unreliable 334

estimate of the 14-d PE LC20 for L. higginsii. Survival of L. higginsii was lower than that of 335

L. siliquoidea in all PCO2 levels, except for the high treatment; but the CI for LC50 values overlapped 336

between species, indicating no significant difference at mid- to high range of PCO2 (Table 4). However, 337

the estimated 28-d LC20 value and CIs of L. higginsii were lower and did not overlap those of 338

L. siliquoidea (Table 4). The model of mortality by species and PCO2 treatment indicated that 28-d 339

mortality of L higginsii was significantly higher than that of L. siliquoidea (F1,14 =6.45, p =0.024), though 340

14-d PE mortality was not different (F1,14 = 3.73, p=0.074). Our results indicate that average survival of 341

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15 the two lampsiline species was similar across treatments and the PE period, but L. higginsii succumbed at 342

lower levels of CO2 than L. siliquoidea. The selection of L. siliquoidea as a surrogate for L. higginsii 343

should account for this difference in sensitivity at various CO2 levels. 344

Shell growth 345

Carbon dioxide treatment (PCO2) significantly reduced percent shell growth of both L. higginsii 346

and L. siliquoidea (and F4,8 = 7.96, p = 0.0068, and F4,7 = 21.49, p = 0.0005, respectively; Table 3). Total 347

percent shell growth of L. higginsii decreased in a dose-dependent pattern from 12.2% (SD 8) in controls 348

to 2.0% (SD 3.0) in 61 000 µatm PCO2 (Table 3). Total percent shell growth of L. higginsii during CO2 349

exposure was significantly less in all PCO2 treatments, except the lowest (24 000 µatm PCO2; Table 3). 350

Total percent shell growth of L. siliquoidea, ranged from 24.3% (SD 4.8) in controls to 1.5% (SD 2.0) in 351

61 000 µatm PCO2 and was significantly less in all PCO2 treatments, relative to the control (Table 3). 352

Models of daily growth indicated significant effects of PCO2 for L. higginsii (F4,8 = 7.06, p = 353

0.0098) and L. siliquoidea (F4,7 = 14.55, p = 0.0017), but time (F1,8 = 14.18, p = 0.0048) and PCO2 × time 354

(F4,8 = 5.49, p = 0.0200) were only significant in L. siliquoidea. Daily growth rate of L. higginsii during 355

CO2 exposure and PE period was significantly less in PCO2 ≥ 61 000 µatm (Fig. 2). Daily growth rate of 356

L. siliquoidea during CO2 exposure was significantly lower in PCO2 ≥ 37 000 µatm (Fig. 2) 357

The 28-d EC50 for shell growth was slightly lower for L. siliquoidea, compared to L. higginsii 358

(Table 4), but the 95% CIs overlapped suggesting that growth was similarly affected in both species. The 359

mixed model did not indicate a significant reduction in growth rate of L. higginsii at 37 000 µatm PCO2, 360

however, the EC50 (32 900 µatm PCO2) suggested otherwise. Growth of L. higginsii juveniles was ~ 361

50% less than L. siliquoidea and was more variable (Table 3, Fig. 2). The statistical power to detect 362

treatment effects on growth of L. higginsii may have been too low with our limited sample size. 363

Previously, we reported that growth of L. siliquoidea juveniles was significantly reduced during exposure 364

to ~ 35 000 µatm PCO2 (43 mg·L-1; Waller et al. 2017). Results of the present study indicate that growth 365

is reduced at pressures as low as 24 000 µatm PCO2 (Tables 3 and 4). 366

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16

During the PE period, percent shell growth of L. higginssi did not differ among treatment groups 367

(Table 3). Daily growth rate in in PCO2 ≥ 61 000 µatm treatments was reduced (Fig. 2), as previously 368

mentioned. Percent growth (PE) of L. siliquoidea was similar in the control and the two lowest CO2 369

treatments (Table 3) and reduced in PCO2 ≥ 61 000 µatm. Compared to the exposure period, PE growth 370

rate of L. siliquoidea was greater in all treatments, except the control and high treatment (Fig. 2). Daily 371

growth rate (PE) of L. siliquoidea was significantly lower than the control in treatments ≥ 37 000 µatm 372

PCO2 (Fig. 2). These results differ from earlier findings of comparable PE growth in L. siliquoidea across 373

treatments (up to 110 mg·L-1 or ~ 85 000 µatm PCO2; Waller et al. 2017). In the same study, PE growth 374

rate of control mussels (L. siliquoidea) was significantly less than the rate during exposure (Waller et al. 375

2017), suggesting that the former rate was influenced by a factor unrelated to CO2 treatment. Based on 376

growth of control mussels, we suggest that results of the present study are more indicative of shell growth 377

following recovery from CO2 exposure. Mussels may recover from low to moderate CO2 exposure and 378

resume growth when the exposure ends, but recovery is less likely at levels that approach lethality. 379

Growth of both species, within and among replicate tanks, was more variable during the PE period than 380

during CO2 exposure (Fig. 2). Variability in growth within a tank may indicate individual differences in 381

recovery and/or response to handling disturbance. Alternatively, the PE growth period was half the 382

duration of the exposure period — variability may be reduced if growth were followed for an equivalent 383

time period (i.e., 28 d). 384

High inherent variability in shell and tissue growth within a cohort of juvenile mussels is 385

common and can reduce ability to distinguish treatment-related effects on growth (Larson et al. 2014). 386

We had the benefit of laboratory-reared juveniles for both species that were of the same cohort, thus 387

reducing some of the inherent variability of mussels held under field conditions. Rapid growth of 388

L. siliquoidea in our study resulted in greater separation of shell sizes in each CO2 treatment and enabled 389

us to detect treatment effects, despite the high degree of variability. Growth of L. higginsii juveniles was 390

~ 50% less than L. siliquoidea and was more variable (Table 3, Fig. 2). These factors may have limited 391

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17 the detection of treatment effects at all CO2 concentrations within our limited sample size (n=10 per test 392

tank). 393

394

Condition 395

Carbon dioxide exposure caused a significant decrease in tissue condition [(dry tissue weight 396

/shell length) (F3,6 = 9.32, p ≤ 0.01; Table 3, Fig. 3a)] and shell condition [(dry shell weight/shell length) 397

(F3,6 = 5.66, p = 0.03; Table 3; Fig. 3b)] of L. siliquoidea with increasing PCO2. Both condition indices 398

were significantly lower than the control at PCO2 ≥ 37,000 µatm. Decreased shell condition is consistent 399

with shell growth response of L. siliquoidea. The decline in tissue condition indicates that CO2 also 400

adversely affected tissue growth. Carbon dioxide exposure may increase the energy needed to maintain 401

homeostasis and reduce energy for shell and tissue growth. Although, body condition index of adult 402

mussels was not reduced after 32-d continuous or intermittent exposure to 20 000 µatm PCO2 (Hannan et 403

al. 2016a, c), physiological and metabolic responses indicated mussels expended energy to maintain acid-404

base and ionic balance (Hannan et al. 2016a, c; Jeffrey et al. 2017). Juvenile mussels likely use the same 405

homeostatic mechanisms as adult mussels, but have less carbonate reserves in the shell to maintain acid-406

base and ionic balance, and therefore experience significant shell and tissue loss. In addition to serving as 407

a bicarbonate source, the shell is the primary means of protection for a mussel. Prolonged CO2 exposure 408

can reduce shell integrity (Waller et al. 2017) and mass, and increase risk of predation and mechanical 409

injury to mussels. 410

Behavior 411

Quantification of behavior during CO2 exposure was limited to the premortality period which 412

included days 0 – 12. Before the start of CO2 infusion, a total of four (2.7%) L. higginsii and zero 413

L. siliquoidea were unburied (Fig. 4). Carbon dioxide infusion induced mussels to unbury, especially in 414

the two highest CO2 treatments (Fig. 4). The mean number of unburied L. higginsii ranged from 3–7/tank 415

(30–70%) in PCO2 ≥ 61 000 µatm after day 3 of exposure, compared to 0−1 mussels/tank in the control 416

and two lowest CO2 treatments. Lampsilis siliquoidea mussels remained buried (> 90%) in all treatments 417

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18 until day 5. The mean number of unburied mussels fluctuated from 1–4/tank (6–25%) on days 5–9 in the 418

highest treatment and then increased to > 5/tank (37%) over the next 3 d. The number of unburied 419

mussels increased (up to an average of 25%) in 61 000 µatm PCO2 on days 10–12; mussels began to die 420

on day 12. Intercell movements were minimal in both species and ranged from 0–3 movements·d-1 in all 421

treatments. Only one intercell movement occurred in the control during the exposure period – one L. 422

higginsii moved into another cell on day 12. All other intercell movements occurred in CO2 treatment 423

tanks; however, no trend was obvious in either species. 424

Abnormal behaviors (agape, extended foot, side lying or positioned on umbo) were absent in 425

control mussels but were observed in the two highest CO2 treatments. About 50% of L. higginsii mussels 426

in PCO2 treatments ≥ 61 000 µatm were narcotized by day 12 of exposure compared to < 20% in 37 000 427

µatm. Lampsilis siliquoidea juveniles were also narcotized in PCO2 treatments of 61 000 (23%) and 428

96 000 µatm (48%) by day 12. 429

Mussels recovered from narcotization within several hours to days after removal of CO2 but the 430

response varied by species and treatment group. At 1-d PE, the total number of unburied L. siliquoidea 431

was one (2.1%) in 37 000 µatm PCO2 and 11 (29.7%) in 61 000 µatm PCO2; all mussels were buried in 432

control and 24 000 µatm PCO2 tanks. At 2-d PE, all but four mussels (11%) were buried in 61 000 µatm 433

PCO2 (not shown). Lampsilis higginsii juveniles took longer to recover and rebury than L. siliquoidea 434

across all treatments (not shown). At 2-d PE, unburied L. higginsii ranged from zero in the control tanks 435

to three (12.5%) in 24 000 µatm PCO2 and 13 (76%) in 61 000 µatm PCO2. Three of the eight (37.5%) 436

mussels that remained alive in the 96 000 µatm PCO2 were unburied. At 4-d PE, L. higginsii remained 437

unburied in 61 000 (n = 5; 29.4%) and 96 000 (n = 2; 25%) µatm PCO2. 438

Behavioral effects of CO2 may impact unionid mussels on several fronts. Carbon dioxide 439

exposure can reduce byssal thread secretion and attachment (Waller and Bartsch, accepted). Combined 440

with the unburying and gaping behavior we observed, this can lead to increased predation and 441

displacement by water current. Carbon dioxide application also may reduce mussel reproduction by 442

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19 causing avoidance behaviors in fish host species (Ross et al. 2001; Clingerman et al 2007; Kates et al. 443

2012) which are required for transformation of the parasitic larval (glochidia) stage. 444

445

Gene Expression 446

Of the four genes that we targeted for qPCR, the most significant treatment-related change was 447

seen in CHS expression (Fig. 5). PCO2 (F3,6 = 12.45, p = 0.0055), time (F1,8 = 35.62, p = 0.0003), and 448

PCO2 × time (F3,8 = 16.79, p = 0.0008) had a significant effect on CHS expression. At 28-d, CHS 449

expression was significantly lower in 61 000 µatm PCO2 (F1,8 = 0.52.78, p < 0.0001) relative to the 450

control and two lowest treatments. There were no significant differences in CHS expression between 451

control and CO2 treatments at 14-d PE, indicating that CHS expression recovered after removal of CO2. 452

A similar response of CHS expression was observed in adult L. siliquoidea. Jeffrey et al. (2018) reported 453

a significant reduction in CHS expression after 7 d exposure to 50 000 µatm PCO2, relative to the control, 454

although differences were not significant at 28 d of exposure. Chitin synthase expression had returned to 455

control levels after 14 d PE. Based on our results, the minimum effect concentration for CHS expression 456

was > 37 000 µatm PCO2. However, Jeffrey et al. (2017) found that 20 000 µatm PCO2 reduced CHS 457

expression in mantle tissue of adult F. flava in a 32-d exposure (Jeffrey et al. 2017). 458

Chitin synthase is integral to the production of chitin, a key component of the shell. Decreased 459

chitin production would adversely affect biomineralization and shell integrity. Down-regulation of CHS 460

in our study coincided with decreased shell growth rate and shell condition in juvenile L. siliquoidea. 461

Reduced shell integrity in juvenile L. siliquoidea after CO2 exposure (Waller et al. 2017) may be another 462

possible consequence of decreased chitin production. The annual shell growth of many adult mussel 463

species is too small (Haag and Rypel 2010) to detect significant effects from a stressor, such as CO2, in 464

30 d; however, CHS expression may be a sensitive indicator of reduced shell growth across a range of life 465

stages and sizes of mussels (Jeffrey et al. 2017; Jeffrey et al. 2018). 466

Transcript responses of CAL, NKA, and DEF in juvenile L. siliquoidea varied by treatment and 467

exposure period. We hypothesized that CO2 exposure would trigger up-regulation of CAL in response to 468

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20 calcium mobilization; but there was no effect of PCO2, time or PCO2 × time on CAL expression (Fig. 469

5b). Likewise, there was no significant effect of PCO2 on NKA expression in L. siliquoidea at 28-d 470

exposure (F3,6 = 0.33, p = 0.8050; Fig. 5c). Mean fold expression was greater in all treatments, except the 471

control, at 14-d PE relative to the 28-d exposure value; however, there was no effect of PCO2 on NKA 472

expression (F1,8 = 2.35, p = 0.1639). Jeffrey et al. (2018) also found no significant change in CAL and 473

NKA levels in gill and mantle of adult L. siliquoidea at 28-d exposure to 50 000 µatm PCO2, but did note 474

decreased expression of both genes at 14 d PE. Lastly, we were unable to detect up or down regulation of 475

DEF gene expression in our study (Fig 5d). 476

Arguably, significant shifts in gene expression may have been evident at the onset of CO2 477

exposure and plateaued over the course of the exposure. For example, CAL levels were significantly 478

reduced at day 1 and 4 in gill tissue of adult L. siliquoidea, but increased to control levels at 28 d (Jeffrey 479

et al. 2018). We may have missed initial changes in the target genes by sampling only at the end of the 480

28-d exposure. However, our objective was to measure the longer-term, adaptive response of mussels to 481

hypercapnia. Additional studies are needed to determine the pattern of genomic responses, and resulting 482

homeostatic mechanisms, of mussels at various CO2 levels and exposure durations. 483

Variability in gene expression was high among individuals within a treatment (Fig. 5), similar to 484

that seen in growth rates and condition indices. N+/K+ ATPase and CAL expression varied by ~ 2 fold 485

among treatments and sampling time. Chitin synthase expression varied > 4-fold among treatments at 28 486

d, but by < 2-fold in 14-d PE samples. Variability was highest for DEF expression, notably in the samples 487

from 61 000 µatm PCO2. In two mussels, DEF expression was 7- and 38-fold lower at 28 d, relative to 488

controls, but fold expression for the other three genes was within the 95% quartile in these same 489

individuals. Extreme fold expression values may indicate survivors versus eventual mortalities. We do not 490

know whether moribund mussels, particularly in the higher CO2 treatments, were the outlier values. 491

Pooling individuals from a treatment would have normalized outliers and reduced the variability in our 492

data; although we found no correlation, we maintained individual samples in order to analyze the 493

relationship between gene expression and growth in individual mussels. 494

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21

Use of whole individuals may have been another source of variability in the expression data. 495

Gene expression can be strongly organ and tissue specific-dependent (Hüning et al. 2013; Ren et al. 2013; 496

Jeffrey et al. 2017). For example, CAL is expressed in a range of tissues, including the mantle, gonad, and 497

adductor mussel, but has highest activity in the gill and foot (Zeng et al. 2012; Ren et al. 2013). 498

Unfortunately, the juveniles were too small to sufficiently sample individual organs. Analysis of 499

transcript levels in individual tissues (e.g., mantle and gill), rather than whole body samples, may be 500

needed to detect significant changes in fold expression related to CO2 treatment. Additionally, a broader 501

transcript profile could be assessed in future studies to identify additional marker genes for hypercapnic 502

stress in juvenile mussels. 503

Application scenarios of CO2 for AIS in natural systems will vary with the management goals 504

and target species in a system. Our test system represents a worst case scenario in which CO2 is applied 505

continuously as a barrier to fish movement. The PCO2 levels that we tested could occur in the immediate 506

zone of infusion (61 000 – 96 000 µatm) and in a downstream plume (24 000 – 37 000 µatm). Shorter 507

infusion periods of CO2 to corral fish into a bay or slough for harvest may be less harmful to native 508

mussels. Mussels can survive acute exposure (e.g., 96 h; Waller and Bartsch, accepted) and recover from 509

longer (e.g., 28 d), low level exposure to CO2 (Waller et al. 2017; Hannan et al. 2016a, c). Another 510

possible application of CO2 is infusion into a navigational lock to deter fish movement at a control point. 511

In this scenario, mussels would be exposed to intermittent pulsed doses of CO2 when the chamber was 512

open. Hannan et al. (2016b) simulated pulsed CO2 exposure of 30 min, 12 times per day, for 28 d and 513

reported no mortality of adult mussels but found physiological responses (e.g., HCO3-, Ca2

+, Na+, Mg2+) 514

remained elevated between doses. 515

Chitinase synthase was a genomic indicator of CO2 exposure in juvenile mussels. A suite of 516

transcriptomes (i.e., RNA seq analysis) is needed to identify a broader genomic response of juvenile 517

mussels to hypercapnia. Additionally, a comparison of mussels that reside in habitats with episodic 518

hypercapnia from natural processes (e.g., reservoirs and lakes) would provide insight on the potential 519

homeostatic strategies used by juvenile mussels and may help determine the relative risk of CO2 exposure 520

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22 to other mussel species. Further work is needed to determine the effects of dose, duration, and frequency 521

of CO2 exposure on the recovery of native mussels. Perhaps the greater concern of sublethal CO2 levels is 522

the behavior of both mussels and native fish. Narcotization of mussels increases their risk of displacement 523

and predation. Mussel reproduction and recruitment would be reduced if fish avoid zones of CO2 524

infusion. Before field deployment of CO2, resource managers will have to weigh its benefits against the 525

risks to native species. Our study provides data on juvenile lampsiline mussels to help inform that 526

decision. 527

Lampsilis siliquoidea was a reasonable surrogate for predicting lethality of CO2 to the endangered 528

L. higginsii mussel across the range of levels tested in our study. Growth and behavioral responses of 529

L. siliquoidea were less predictive of L. higginsii. Lampsilis siliquoidea growth was affected at lower 530

PCO2, but recovery from narcotization was more rapid compared to L. higginsii. We expect that CO2 531

levels that adversely impact L. siliquoidea will similarly affect L. higginsii but also recommend extended 532

monitoring of L. higginsii in any CO2 application. 533

534

ACKNOWLEDGEMENTS 535

The study was funded in part by the Great Lakes Restoration Initiative and the U.S. Geological Survey 536

Ecosystem Mission Area Invasive Species Program. Kerry Weber, Riley Buley, Ann Tronick and 537

Rhiannon Fisher assisted in the laboratory and with data entry. The U.S. Fish and Wildlife Service, Genoa 538

National Fish Hatchery provided mussels. Christopher Merkes, Kim Fredricks and two anonymous 539

reviewers provided insightful comments on earlier versions of the manuscript. 540

Disclaimer 541

Any use of trade, product, or firm names is for descriptive purposes only and does not imply 542

endorsement by the U. S. government. 543

544

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683

684

685

686

687

688

689

690

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Table 1. Gene-specific primer sequences. 691

Gene Primers (5’-3’) Product (bp)

Ubiquitina FWD-TCCAGGACAAAGAAGGGATTCC 166

REV-AGGGCTCTCAAGCTGGGTTCAA

Calmodulin FWD-AGTGGATGCCGATGGTAATG 101

REV-CCTCGCGTAATTCCTCTTCTG

Defensin FWD-GATTTGCCACAAGCAGAAGC 110

REV-TTGCAGTAACCGCCAGTAAA

Na+/K+ ATPase FWD-TGCTGTAGACGAACCTTTCAG 107

REV-GATCCGTGGGAAGGAAGTAATC

Chitin Synthaseb FWD-GAGTCGATTGGCCCAAGACA 104

REV-CCACCTGTTCGTCGAGTTCA

a = sequence taken from Bai et al. (2014) 692

b = sequence taken from Jeffrey et al. (2017) 693

694

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Table 2. Mean (95% confidence interval) water quality parameters (n = 42), CO2 concentration (n = 13) and partial pressure of CO2 ( n= 42)

during the study period.

Relative CO2

treatment

Dissolved oxygen (mg·L-1) pH Temperature (° C)

Titration

CO2 (mg·L-1)

Calculation

PCO2 (×103 µatm)

Exposure

Post exposure

Exposure

Post exposure

Exposure

Post exposure

Control 8.1 (8.1, 8.2)

8.4 (8.3, 8.4)

7.91 (7.89, 7.92)

7.99 (7.97, 8.00)

21.6 (21.6, 21.6)

21.5 (21.4, 21.5)

2.2 (1.9, 2.4)

2.4 (1.9, 2.8)

Low 7.9 (7.8, 8.0)

8.4 (8.3, 8.4)

6.86 (6.84, 6.87)

7.99 (7.98, 8.00)

21.6 (21.6, 21.6)

21.4 (21.4, 21.5)

32.0 (31.0, 32.9)

24.1 (23.3, 24.9)

Medium 7.9 (7.8, 8.0)

8.4 (8.4, 8.5)

6.67 (6.66, 6.68)

7.99 (7.97, 8.00)

21.6 (21.6, 21.7)

21.5 (21.4, 21.5)

46.8 (45.7, 48.0)

37.2 (36.1, 38.3)

Med High 7.8 (7.7, 7.8)

8.4 (8.3, 8.5)

6.46 (6.44, 6.47)

8.00 (7.99, 8.01)

21.7 (21.6, 21.7)

21.5 (21.5, 21.5)

77.0 (74.6, 79.5)

60.6 (59.0, 62.2)

High 7.5 (7.5, 7.6)

8.5 (8.5, 8.6)

6.26 (6.25, 6.27)

7.97 (7.95, 7.98)

21.7 (21.6, 21.7)

21.6 (21.5, 21.6)

119.5 (116.3, 122.7)

95.7 (92.4, 99.0)

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Table 3. Mean and standard deviations (SD) total mortality, percent shell growth (length), tissue and shell condition of juvenile mussels at 28-d

exposure and 14-d postexposure (PE) to CO2.

CO2 treatment

(µatm PCO2)

Total Mortality (%)

Percent growth*

Tissue condition†

Shell condition†

HG FM HG FM Mean Regression Mean Regression

Control

28-d 6.7 (5.8)

0.0 (0.0)

12.2 a (8.02) 24.33a (4.78) 465.3a (132.1)

Y= -8.5 +1.3*L R2 = 0.85

3940.0a (1088.3)

Y= -68 +11*L R2 = 0.89

14-d PE 6.7 (5.8)

0.0 (0.0)

5.421 (5.11) 10.631 (3.07)

Low (24 000)

28-d 23.3 (5.8)

2.1 (3.6)

10.66 a (6.43) 16.58b (4.92) 373.0a (137.0)

Y= -7.6 +1.2*L R2 = 0.93

3122.9ab (1143.3)

Y= -62 +10*L R2 = 0.92

14-d PE 33.3 (11.5)

2.1 (3.6)

6.541 (5.43) 9.801 (5.10)

Medium (37 000)

28-d 13.3 (15.3)

6.3 (6.3)

5.82 b (3.92) 8.00c (4.58) 294.3b (74.0)

Y= -1.4 +0.4*L R2 = 0.60

2345.2b (625.4)

Y= -17 +4*L R2 = 0.73

14-d PE 20.0 (26.5)

8.3 (9.5)

4.451 (4.63) 7.171,2 (4.63)

Med High (61 000)

28-d 40.0 (0.0)

18.8 (6.3)

2.00 b (3.04) 1.51d (2.02) 203.2b (77.0)

Y= -3.5 +0.7*L R2 = 0.63

1983.2b (593.2)

Y= -33 +6*L R2 = 0.84

14-d PE 56.7 (15.3)

29.2 (23.7)

1.091 (1.88) 2.932 (2.10)

High (96 000)

28-d 73.3 (15.3)

75.0 (43.3)

2.71 b (1.21) 4.30d (1.90) NA‡ NA NA NA

14-d PE 73.3 (15.3)

89.6 (18.0)

1.071 (2.78) -0.162 (2.06)

* Initial sample size: Lampsilis higginsii (HG) n = 10; L. siliquoidea (FM) n = 16. See Fig. 2 for 28-d and 14-d PE sample size. Percent growth

values within a column with the same letter (28-d) or number (14-d PE) are not significantly different.

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† Condition indices were determined at 14-d PE. Mean tissue and shell condition of L. siliquoidea at 14-d PE (n = 4). Values within a column with

the same letter are not significantly different.

‡ NA – not determined due to high mortality of treatment group.

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Table 4. Toxicity of CO2 to survival (LC20, lethal concentration to 20%, and LC50, lethal concentration to 50%) and shell growth (EC50, median

effect concentration) of juvenile mussels after 28-d exposure and 14-d postexposure (PE) period*.

Species

28-d LC20 28-d LC50 14-d PE LC20 14-d PE LC50 28-d EC50

Lampsilis higginsii

31 800 (15 600 – 42 800)

71 000 (60 000 – 88 300) Not reported† 61 000

(46 000 – 87 700) 32 900

(26 000 – 39 900)

Lampsilis siliquoidea

58 200 (45 200 – 68 100)

78 200 (68 300 – 92 800)

53 800 (47 200 – 59 100)

69 100 (64 000 – 75 100

28 600 (26 400 – 30 900)

*Mean PCO2 (µatm) with 95% confidence intervals (CI) in parenthesis. †95% CI included 0.

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Figure 1. Cumulative percent mortality of juvenile Lampsilis higginsii and L. siliquoidea in 28-d exposure

to CO2, followed by 14 d postexposure (PE) in untreated water. Partial pressure of carbon dioxide (µatm)

level is the overall mean for each tank.

Figure 2. Mean daily shell growth (length) of juvenile (A) Lampsilis higginsii and (B) L. siliquoidea

mussels during 28-d exposure to CO2 and 14-d PE in untreated water. Mean (horizontal dashed lines

inside boxes), median (horizontal lines inside boxes), interquartile range, 25th to 75th percentiles (box

ends), interquartile range, 5th and 95th percentiles (whiskers), values beyond the 5th and 95th percentile

(dots). Boxplots with the same letter (28-d exposure) or number (14-d PE) are not significantly different

among treatments. Significant differences between 28-d and 14-d PE within a treatment are indicated with

an ‘*’.

Figure 3. (A) Regression of Lampsilis siliquoidea dry tissue weight to shell length for each PCO2

treatment. (B) Regression of L. siliquoidea dry shell weight to shell length by PCO2 treatment. n = 4

mussels/tank; n = 3 tanks/treatment. See Table 4 for regression equations and R2 values.

Figure 4. Mean number of unburied mussels per tank (standard error) during pre-mortality period (12 d)

in CO2. (A) Lampsilis higginsii, n = 10 per tank, (B) L. siliquoidea, n = 16 per tank.

Figure 5. Gene expression fold change in juvenile Lampsilis siliquoidea at 28-d exposure (blue box) to

CO2 and at 14-d PE (red box) in untreated water (A) Chitin synthase (CHS), boxplots with the same letter

(28-d exposure) are not significantly different among treatments, *28-d exposure outlier values= -12 and -

18; (B) Calmodulin (CAL), (C) Na+-K+ ATPase (NKP), (D) Defensin (DEF), *28-d exposure outlier

values = -74 and -388, *14-d PE outlier values = -327. Mean (circle inside box), median (horizontal lines

inside boxes), interquartile range, 25th to 75th percentiles (box ends), interquartile range, 5th and 95th

percentiles (whiskers), values beyond the 5th and 95th percentile (dots). n = 5 mussels/tank; n = 3

tanks/treatment.

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-20

0

20

40

60

80

100

120

-20

0

20

40

60

80

100

120 Lampsilis higginsii

Lampsilis siliquoidea

Gro

wth

in le

ngth

(µm

/day

)

Control 24 37 61

Control 24 37 61

n=24, 15n=29, 27 n=25, 21 n=27, 24

n=44, 33n=48, 48 n=49, 48 n=45, 44

aa

a

b

b

a

11

1

1

1

2

2

3b

a

Gro

wth

in le

ngth

(µm

/day

)

n=15, 5

n=12, 8

96

96

b

2

3*

*

*

b

Partial pressure carbon dioxide (× 103 microatmospheres)

A

B

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A B

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A B

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