Canadian Journal of Fisheries and Aquatic Sciences
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;Draft USGS Upper Midwest Environmental Sciences Center Jackson, Craig; USGS Upper Midwest Environmental Sciences Center
Is the invited manuscript for consideration in a Special N/A Issue? :
Keyword: freshwater mussel, carbon dioxide, unionid, toxicity
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1 For Submission to Canadian Journal of Fisheries and Aquatic Sciences 2 Running title: Responses of juvenile unionid mussels to carbon dioxide exposure. 3 4 Lethal and sublethal responses of native mussels (Unionidae: Lampsilis siliquoidea and L. higginsii) to 5 elevated carbon dioxide 6 *Diane L. Waller, U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 Fanta 7 Reed Road, La Crosse, WI 54603; [email protected]; Telephone: 608-781-6282; Fax: 608-783-6066 8 Michelle R. Bartsch, U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 9 Fanta Reed Road, La Crosse, WI 54603; [email protected] 10 Lynn A. Bartsch, U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 Fanta 11 Reed Road, La Crosse, WI 54603; [email protected] 12 Craig A. Jackson, U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 Fanta 13 Reed Road, La Crosse, WI 54603; [email protected] 14 *Corresponding author 15 16 Draft
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17 Abstract
18 Levels of carbon dioxide (CO2) that have been proposed for aquatic invasive species (AIS) control
19 [24 000 – 96 000 µatm partial pressure CO2 (PCO2); 1 atm = 101.325 kPa] were tested on juvenile
20 mussels, the Fatmucket (Lampsilis siliquoidea) and the U.S. federally endangered Higgins Eye
21 (L. higginsii). A suite of responses (survival, growth, behavior, and gene expression) were measured after
22 28-d exposure and 14-d postexposure to CO2. The 28-d LC20 (lethal concentration to 20%) was lower for
23 L. higginsii (31 800 µatm PCO2, 95% confidence interval (CI) 15 000 – 42 800 µatm) than for
24 L. siliquoidea (58 200 µatm PCO2, 95% CI 45 200 – 68 100 µatm). Treatment-related reductions
25 occurred in all measures of growth and condition. Expression of chitin synthase, key for shell formation,
26 was down-regulated at 28-d exposure. Carbon dioxide caused narcotization and unburial of mussels,
27 behaviors that could increase mortality by predation and displacement. We conclude that survival and 28 growth of juvenile mussels could be reducedDraft by continuous exposure to elevated CO2, but recovery may 29 be possible in shorter duration exposure.
30 31 Key words: freshwater mussel; unionid; carbon dioxide; toxicity; transcriptional and growth response 32
33
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34 Introduction
35 Elevated atmospheric carbon dioxide (CO2) is most recognized for its role in climate change and
36 ocean acidification, but purposeful infusion into select aquatic systems is being pursued as a tool for
37 control of aquatic invasive species (AIS) such as bighead (Hypophthalmichthys nobilis) and silver carp
38 (H. molitrix) (Cupp et al. 2017) and sea lamprey (Petromyzon marinus). Several studies have
–1 39 demonstrated that CO2 concentrations of 60 to 120 mg·L (24 000 to 75 000 µatm PCO2; 1 atm=101.325
40 kPa) would induce avoidance responses in fish and could be used to corral and harvest fish in a confined
41 area or deter upstream migration through a stream or lock channel (Kates et al. 2012; Dennis et al. 2016;
42 Cupp et al. 2017). Before its widespread deployment in AIS programs, the effects of elevated CO2 on
43 native species is a consideration.
44 Exposure of native freshwater mussels to elevated CO2 is of particular concern given the
45 precarious status of the fauna. Unionid musselsDraft are a high risk faunal group due to a multitude of stressors
46 including habitat loss, overharvest, water quality degradation, and competition with AIS (Williams et al.
47 1993; Lydeard et al. 2004; RéGnier et al. 2009). Native mussel communities play a vital role in nutrient
48 cycling, substrate stabilization, and water filtration in aquatic ecosystems (Vaughn and Hakenkamp 2001;
49 Vaughn et al. 2008; Strayer 2014). Given their relative immobility and reliance on a calcified shell and
50 bicarbonate buffer system, unionid mussels may be especially vulnerable to CO2 exposure and the
51 concomitant production of carbonic acid.
52 Recently, scenarios of CO2 exposure have been tested on several common species of native
53 freshwater mussels (Hannan et al. 2016a, b, c; Jeffrey et al. 2017; Waller et al. 2017; Jeffrey et al. 2018).
54 Adult mussels (Fatmucket Lampsilis siliquoidea and Threeridge Amblema plicata) survived 28-d
55 exposure to 55 000 µatm PCO2, but exhibited physiological signs of respiratory acidosis and alterations
56 in the bicarbonate buffer system (Hannan et al. 2016c). Wabash pigtoe Fusconaia flava also survived
57 exposure to 200 000 and 20 000 µatm PCO2 for 6 h and 32 d, respectively; however, levels of heat shock
58 protein 70 mRNA (gill) and oxygen consumption increased and chitin synthase expression (mantle)
59 decreased in mussels that were exposed for 32 d (Jeffrey et al. 2017).
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60 Few studies have compared the effects of elevated CO2 on juvenile and adult freshwater mussels
61 and, to our knowledge, no studies have been conducted with U.S. federally listed species. In separate
62 studies, juvenile (~ 6 mo old; Waller et al. 2017) and adult (L. siliquoidea) mussels (Hannan et al 2016c)
63 were exposed to elevated CO2 for 28 d. Juvenile mussels died at 42 000 µatm PCO2 (Waller et al. 2017),
64 whereas adult mussels survived 55 000 µatm PCO2 (Hannan et al. 2016c). Lampsilis siliquoidea is
65 abundant, geographically widespread (Cummings and Cordeiro 2012), and has become a common species
66 for toxicity testing (e.g., Bringolf et al. 2007a, b; Wang et al. 2007; Wang et al. 2011; and others). Its
67 congener Higgins Eye (L. higginsii Lea, 1857) is a U. S. federally listed species that occurs in the upper
68 Mississippi River drainage. Sites of potential CO2 deployment for AIS control (ACRCC MRW 2014)
69 overlap with essential habitat for L. higginsii
70 (https://www.fws.gov/Midwest/endangered/clams/higginseye/hepmeha.html) and therefore, an evaluation
71 of risk to the species is required before CO2Draft is approved for use in the field. Toxicity tests with 72 endangered species are infrequent and risk assessments generally rely on responses of surrogate species.
73 In the present study, we simultaneously exposed juvenile L. siliquoidea and L. higginsii to CO2 and
74 determined the suitability of the common species as a surrogate for the latter.
75 We measured a suite of responses in juvenile mussels during long-term 28-d exposure to CO2
76 exposure and after 14-d postexposure (PE) in untreated water. The PCO2 range tested represents that
77 expected from the immediate area of CO2 infusion (~100 000 µatm PCO2) and in a downstream plume
78 (~ 24 000 µatm PCO2). The exposure duration simulates the use of CO2 as a long-term or seasonal barrier
79 to deter fish movement. Mortality, shell growth, and behavioral responses to CO2 were measured on both
80 mussel species and provided comparable data to evaluate interspecies differences (L. higginsii vs L.
81 siliquoidea) and intertrial variation in responses of L. siliquoidea (Waller et al. 2017). Condition indices
82 and transcriptional responses were limited to L. siliquoidea because both analyses required sacrifice of
83 individuals which we avoided with L. higginsii. Additionally, we measured expression levels of several
84 key genes in shell formation, energetics and growth, and calcium regulation at 28 d of CO2 exposure and
85 after 14-d PE. Fold expression was quantified for Chitin synthase (CHS), Calmodulin (CAL), Na+/K+
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86 ATPase (NKA) and a Defensin (DEF). Chitin synthase is a transmembrane glycosyltransferase that plays
87 a role in synthesis of chitin and in formation of the larval bivalve shells (Weiss and Schӧnitzer 2006;
88 Weiss et al. 2006; Fang et al. 2011; Liu et al. 2015). Calmodulin (CAL) is a multifunctional calcium-
89 binding messenger protein that mediates many fundamental cellular processes. In bivalves, CAL is
90 integral to Ca2+ uptake from the water by the gills and for shell formation and biomineralization (Ren et
91 al. 2013; Liu et al. 2015; Sun et al. 2015). Na+/K+ ATPase is critical to active transport and as secondary
92 transport of other ions and organic molecules (Giacomin et al. 2013); it may play a role in reducing
+ 93 acidosis by excretion of NH4 from the mantle (Hüning et al. 2013). Defensins are peptides with
94 antimicrobial activities and have been characterized in several freshwater mussels (Xu and Faisal 2010;
95 Luo et al. 2014). Defensin expression was up-regulated in Uniomerus tetralasmus during emersion and
96 desiccation suggesting it may also be a general stress indicator (Luo et al. 2014).
97 The objectives of the present study Draftwere: 1) determine the lethal and sublethal effects of CO2 to
98 juvenile native mussels at levels expected for AIS control, 2) determine the suitability of L. siliquoidea as
99 a surrogate for the endangered species, L. higginsii, and 3) compare organismal and transcriptional
100 responses of juvenile lampsiline mussels to continuous CO2 exposure.
101
102 Materials and Methods
103 Test animals
104 Juvenile L. siliquoidea and L. higginsii (~ 9 months old) were propagated at the U.S. Fish and Wildlife
105 Service, Genoa National Fish Hatchery (Genoa, WI, USA) and transferred to the U.S. Geological Survey,
106 Upper Midwest Environmental Sciences Center (La Crosse, WI, USA). Juveniles of each species came
107 from the same cohort and all animals were cultured under the same conditions. Lampsilis higginsii
108 juveniles ranged from 3.24 to 7.40 mm in shell length (mean = 4.71 mm; standard deviation, SD =
109 0.89 mm, n = 150). Lampsilis siliquoidea juveniles ranged from 4.17 to 9.21 mm in shell length (mean =
110 7.17, SD = 0.92 mm, n = 240). Mussels were held in a raceway with recirculating well water with 10%
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111 daily water replacement. Mean (SD) chemical parameters of the well water were: hardness 190.7 (22.0)
–1 –1 –1 112 mg·L as CaCO3, alkalinity 132.8 (4.8) mg·L as CaCO3, and conductivity 412.3 (11.2) µS·cm .
113 Mussels were fed a diet of four commercial algal foods (approximate ratio of 2:0.5:1:1, Shellfish diet,
114 Nannochloropsis 3600 Instant Algae, TW1200 and TP1800, Reed Mariculture, Campbell, CA). Food was
115 prepared daily and delivered continuously to the holding raceway and test tanks by a peristaltic pump
116 (Cole Parmer Masterflex Digi-staltic pump, Vernon Hills, IL) at a rate of 0.07 algae dry wt (mg·L–1min-1).
117 Washed sand substrate (Mastercraft® commercial playground sand) was provided for mussels to position
118 themselves upright and bury. Mussels were acclimated from 12 °C to 22 °C at a rate of ≤ 3 °C per day and
119 maintained at 22 °C for 1 week (L. higginsii) and 4 weeks (L. siliquoidea) before initiation of test
120 exposure.
121 Test system and exposure Draft 122 The test system and methods followed those of Waller et al. (2017) with a 28-d exposure period
123 followed by 14-d PE period in untreated water. The test was conducted in three continuous flow-through
124 diluter systems; each diluter served as a replicate. In each system, well water was supplied to a head box
125 followed by a serial dilution box that was partitioned into 10 chambers. The PCO2 was reduced by about
126 20% in each successive chamber of the dilution box. The outflow from the four dilutor chambers with the
127 targeted partial pressure of CO2 drained to a glass tank (25.4 cm × 49.5cm × 30.5 cm, W × L × H) that
128 held test mussels. Outflow from the remaining six dilutor chambers was directed to the effluent drain. The
129 control tank received clean untreated water directly from the head box. Treatments were assigned to each
130 tank within a diluter system using a randomized block design.
131 Tanks were filled to 22 L with 22 °C well water with a mean flow rate of 294 mL·min–1 (about 1
132 tank exchange every 75 min). The algal stock solution was prepared daily, as described in the previous
133 section, and distributed to each of three food reservoirs (one 45-L stainless steel tank per dilutor system).
134 Food was kept in suspension with an electronic mixer and was delivered continuously by peristaltic
135 pumps to each tank at a rate of 110 mL·h–1. Food delivery began 24 h before addition of mussels to the
136 test tanks. Tanks were covered, top and sides, with black polyethylene sheeting (6 mil) to minimize light
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137 exposure. Each mussel species was placed into a separate grid on the bottom of a tank to isolate
138 individual mussels and track their movement and position in the substrate. The grid was constructed from
139 plastic light diffusers, cut to produce 3 × 3-cm cells (1.5 cm depth), and 1.5-mm mesh screen to cover the
140 bottom. Each grid cell was filled with 1.2 cm sand substrate (≤ 1.5 mm grain size) to allow movement and
141 burrowing of mussels.
142 Mussels were randomly distributed to the tanks for a total of 10 L. higginsii and 16 L. siliquoidea
143 mussels per test tank. Before placement in the test tank, the shell of each mussel was marked with a
144 unique color dot code using waterproof markers and photographed (side-lying) at 10× under a
145 stereomicroscope. Mussels were placed side-lying into a pre-assigned grid cell in each test tank and
146 allowed to position and acclimate for 2 d before initiation of CO2 infusion.
147 Targeted PCO2 treatments of 18 000, 31 000, 44 000 and 75 000 µatm PCO2 (comparable to 30, –1 Draft 148 50, 70, and 120 mg·L CO2) were based on effective concentrations for deterring silver and bighead carp
–1 149 (i.e., 60 to 100 mg·L CO2) and results of a previous mussel trial (i.e., 28-d LC50 for juvenile
–1 150 L. siliquoidea of 76 mg·L CO2; Waller et al. 2017). Measured PCO2 treatments (28-d mean value) were
151 24 000, 37 000, 61 000 and 96 000 µatm.
152 Food grade CO2 gas was supplied to a pressure differential automatic manifold (Precise
153 Equipment Co., Denton, TX) and then to a CO2 pressure regulator. Outflow from the CO2 regulator was
154 set to 12 L·min–1and was adjusted to each diluter with a 3–way air regulating valve. Carbon dioxide
155 flowed to the dilutor systems through vinyl airline tubing (ID 6.35 mm) and into an airstone (74 mm × 37
156 mm × 37 mm) that was submerged in the first chamber of the dilution box. Carbon dioxide infusion was
157 stopped at 28 d and was at control levels by 8 h PE. Water flow through the diluters and each test tank
158 continued for 14 d PE.
159 Water quality was measured daily in each tank. Dissolved oxygen (mg·L–1) and pH were
160 measured with a Hach LDO IntelliCAL probe and Hach pH probe, respectively, attached to a Hach
161 HQ40d Water Chemistry Multimeter (Hach, Loveland, CO). Temperature (ºC) was measured with a
162 digital thermometer. Total ammonia nitrogen (TAN, µg·L–1) was measured once a week in the control
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163 and highest CO2 treatment in each diluter, using a Hach ammonia probe (Model ISENH318101) attached
164 to a Hach HQ40d Water Chemistry Multimeter. Conductivity and hardness were measured on a sample of
165 water from each head box at the beginning and completion of the study. Conductivity (µS·cm–1) was
166 measured with a Fisher Accumet conductivity meter (Fisher Scientific, Waltham, MA) calibrated against
–1 167 a standard solution (APHA 2012). Total hardness (mg·L CaCO3) was determined by titrimetric method
168 with Manver Red indicator (USEPA 1983). Alkalinity was measured on samples from each diluter head
169 box at the beginning and end of the study. Additionally, alkalinity was measured from one randomly
–1 170 selected tank per diluter on the same days that CO2 was measured. Total alkalinity (mg·L CaCO3) was
171 determined by titrimetric method to a pH endpoint of 4.5 (APHA 2012).
172 The partial pressure of CO2 (PCO2, µatm) in each test tank was calculated using the U.S.
173 Geological Survey CO2 calculator (Robbins et al. 2010). Mean total alkalinity of the three sampled tanks
174 and the daily measured pH and temperatureDraft value of each test tank were used in the calculation. Carbon
175 dioxide was also measured daily by titration for the first 7 days of exposure and twice a week, thereafter,
176 for the duration of the exposure period. Carbon dioxide (mg·L–1) concentrations were determined by a
177 modified Hach Method 8205 digital titration method using sodium. The titrimetric method consisted of
178 collecting a 100–mL water sample from the test tanks and, while slowly stirring, immediately titrating
179 with 0.363N (0–100 mg·L–1 treatments) or 3.636 N NaOH (≥ 100 mg·L–1treatments) to a pH endpoint of
180 8.3. A modified infrared probe (Vaisala BMP220 and GMT221, St. Louis, MO) was also used to verify
181 PCO2 in each test tank twice during the exposure period.
182 Mussel observations and measurements
183 Daily observations were made of mussel location in the grid, burial status (buried < 10% of shell
184 visible, unburied ≥ 90% of shell visible), gaping, and mortality. Mussels were counted as dead if the
185 valves opened without resistance under light pressure, the valves or foot did not respond to touch, and
186 ciliary movement and filtration activity near the siphons was absent. The daily number of unburied
187 mussels per tank was graphed over days 1–12 of exposure to illustrate initial responses of mussels before
188 onset of mortality which began on day 12.
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189 At the end of the 28-d exposure and 14-d PE period, mussels were removed from the tanks for
190 assessment of mortality and growth. A digital photograph of each mussel (side-lying view) was taken
191 under a stereomicroscope at 10×, as described above. Shell growth in length was determined from
192 measurements of live mussels at each sampling period (0-d, 28-d exposure and 14-d PE). Sample size
193 (number of living mussels) varied among treatments because of treatment-specific mortality.
194 Measurements of shell length were made from digital images by 2 independent readers using image
195 analysis software (NIS Elements, Nikon, Melville, NY) after calibration with a photograph of a stage
196 micrometer. Shell length (µm) was defined as the maximum anterior-posterior dimension that is parallel
197 to the hinge line. Measurements taken by the 2 readers were compared and if they varied by > 3% a third
198 reader measured the photograph and the outlying measurement was omitted. The mean shell length
199 determined from the two measurements was used in analyses of shell growth. Percent growth and rate of 200 growth were determined for the 28-d exposureDraft period and the 14-d PE period. The percent growth
201 accounted for differences in the initial length of mussels and was calculated as: percent growth = ((L2 –
202 L1/ L1 × 100), where L1 is shell length (µm) at start of the sampling period (0-d for exposure period, 28-d
203 for PE period), L2 is shell length at the end of the sampling period (28-d for exposure period, 42-d for
-1 204 PE). Daily growth rate (µm·d ) was calculated as (L2 – L1) / T, where T = number of days in sampling
205 period (T = 28 for exposure, 14 for PE).
206 Tissue and shell condition indices were determined from mussels that were sampled at the end of
207 the PE period. A subsample (n = 4) of L. siliquoidea juveniles from each tank was processed for
208 determination of shell and dry tissue weight. Dead mussels were excluded from analysis of condition
209 which eliminated the highest treatment concentration due to insufficient sample size. Soft tissue was
210 removed from the shell and each component was placed into separate tared aluminum weigh pans.
211 Samples were dried at 80 °C to a constant weight. Dry weights were measured on an analytical balance
212 (Mettler AT200, Columbus, OH). Tissue condition index was defined as: [(1000 × dry tissue weight) /
213 (shell length)]. Shell condition was defined as: [(1000 × dry shell weight) / (shell length)].
214 Molecular Methods
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215 Relative gene expression level was measured in L. siliquoidea mussels at 0-d exposure, 28-d
216 exposure and at 14-d PE. Twelve juveniles were indiscriminately taken on day 0, before distribution of
217 mussels to treatment tanks, for baseline measure of gene expression. Before the onset of CO2 infusion,
218 five mussels were randomly distributed to each control and treatment tank, independent of the mussels
219 that were placed into grids (see section Test system and exposure), and sacrificed at 28-d exposure. At
220 14-d PE, a subsample (n = 4–5) was indiscriminately selected from all live mussels in each tank. Whole
221 individual juvenile mussels were placed into cryovials, then flash frozen in liquid nitrogen and stored
222 at -80 °C. Total RNA was extracted from each mussel sample using the RNeasy Fibrous Tissue Mini Kit
223 (Qiagen, cat# 74704, Valencia, CA) according to the manufacturer’s protocol. Tissues were disrupted and
224 homogenized with a mechanical homogenizer (Geno/Grinder, SPEX Sample Prep, model 2010,
225 Metuchen, NJ) by agitation in 300 µl of lysis buffer containing one 7 mm bead of steel shot for 30 s at 226 1500 rpm. Homogenates in lysis buffer wereDraft stored at -80 °C until RNA isolation was completed (one 227 week or less). DNase digestion was performed on-column during RNA isolation, and RNA was
228 subsequently quantified by UV spectrophotometry in nuclease-free water using a MultiSkan Spectrum
229 plate reader (Thermo Fisher, Waltham, MA). RNA samples with A260/A280 ratios between 1.78 and
230 2.23 were considered satisfactory for use in this study. Intact high molecular weight ribosomal RNA
231 bands were observed on a subset of samples analyzed by 2% agarose gel electrophoresis. RNA samples
232 were normalized to a concentration of 20 ng· µL–1 in non-skirted, RNase/DNase/PCR inhibitor-free 96-
233 well plates (MidSci, cat# AVRT-N, Valley Park, MO) prior to qRT-PCR.
234 Target genes were selected based on their potential functions in metabolism and energetics, shell
235 formation, ion balance, and stress response. Gene-specific primers were designed based on conserved
236 regions of sequences from several bivalve species using PrimerQuest software
237 (www.idtdna.com/Primerquest) except as noted (Table 1). Ubiquitin (UBQ) is a stably expressed gene
238 that functions in protein degradation and has been widely used as a reference gene for normalization of
239 qPCR data (Bai et al. 2014). The CAL assay was designed from a 726 bp sequence of Hyriopsis cumingii
240 (JQ389856.1) and targets a 101 bp amplicon. The DEF markers were designed from a 302 bp segment of
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241 H. cumingii defensin (JN604559.1) and targets a 110 bp amplicon. The NKA assay was designed from a
242 1260 bp sequence of L. cardium (AY303383.1) and targets a 107 bp amplicon. Primer specificity was
243 verified by sequencing the PCR product from each primer set. BLAST search results indicated that all of
244 the primers amplified their intended target genes.
245 Semi-quantitative real-time PCR (qRT-PCR) was used to determine the relative abundances of
246 target RNA molecules in samples of mussels taken before CO2 exposure and from control and treatment
247 tanks at 28-d exposure and 14-d PE. qRT-PCR efficiencies, slopes, and y-intercepts were determined for
248 each primer set from calibration curves of RNA pooled equally from 4 individual mussels. All qPCR
249 efficiencies were calculated to be in the range of 97–102%.
250 qRT-PCR reactions were prepared in duplicate 20 µL volumes using the qScript™ One-Step 251 SYBR® Green qRT-PCR Kit (Quanta Biosciences,Draft cat# 95087, Gaithersburg, MD). Each reaction 252 contained 1X One-Step SYBR Green Master Mix, 100 ng RNA, 250 nM forward and reverse primers, 0.4
253 µL of qScript One-Step Reverse Transcriptase, and nuclease-free water. Thermal cycling was performed
254 using an Eppendorf Mastercycler as follows: cDNA synthesis at 50 °C for 5 min followed by Taq
255 activation at 95°C for 5 min, then 35 PCR cycles of denaturation at 95 °C for 10 s/primer annealing at
256 either 58 °C (NKA, CHS, UBQ) or 60 °C (CAL, DEF) for 20 s/extension at 72 °C for 30 s. Post-PCR
257 melt curve analysis was performed on all samples. A subset of samples (n = 12) was used as template in
258 (-) RT control reactions for each assay and no amplifications were observed.
259 The quantification cycle (Cq) for each sample was determined by averaging the two technical
260 replicate values. All samples with a SD > 0.40 between technical replicates were individually examined
261 using Eppendorf Realplex software, and the discrepant replicate (unstable baseline) was discarded prior to
262 copy number extrapolation. We determined relative expression levels of target genes between treatments
263 using the ΔΔCt method as previously described by Livak and Schmittgen (2001). Briefly, we calculated
264 ΔCt values for each sample by subtracting the mean Cq value for each target gene from the mean Cq
265 value for the reference gene (UBQ). We then calculated ΔΔCt values for each sample by subtracting the
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266 ΔCt values from the mean ΔCt value for the controls. Finally, we calculated relative expression using the
267 equation: 2 . −𝛥𝛥𝛥𝛥𝛥𝛥𝛥𝛥
268 Statistical analysis
269 For all statistical analyses, differences were considered significant if p < 0.05. The Statistical
270 Analysis Software package (SAS Version 9.4, Cary, NC) was used for analysis of mortality, shell growth,
271 condition indices, and gene expression. The LC20 and LC50 (lethal concentration to 20% and 50% of
272 organisms, respectively) and 95% CI at 28-d exposure and at 14-d PE were determined by probit analysis
273 using the 28-d mean PCO2 (µatm) of each tank. Mortality (28-d and 14-d PE) differences between species
274 were compared in a mixed model with fixed effects of mean PCO2 and species, and dilutor within PCO2
275 treatment as a random effect. We compared the initial length of mussels with analysis of variance and 276 found differences among L. higginsii were significant,Draft but not for L. siliquoidea. Therefore, differences in 277 initial length were accounted for in growth models by: 1) basing growth comparisons on percent growth
278 and 2) including initial shell length as a covariate in models of growth rate. Percent shell growth and
279 growth rate were modeled for each species with a repeated measures nested hierarchical mixed model
280 with fixed effects of mean PCO2, time (T1 = 28-d exposure, T2 = 14-d PE), and the interaction of PCO2
281 and time. Dilutor within mean PCO2 was a random effect with an unstructured covariance. An EC50
282 (median effect concentration) for percent shell growth was determined by fitting a 3-parameter logistic
283 equation to the data as described by Martikainen and Krogh (1999). Parameter estimations and CI were
284 calculated using PROC NLIN. Differences in condition indices among treatments in L. siliquoidea were
285 analyzed with a mixed model with fixed effects of mean PCO2 and dilutor within PCO2 treatment as a
286 random effect. Fold expression (DEF, CHS, NKA, and CAL) in L. siliquoidea at 28-d exposure and 14-d
287 PE was modeled with a nested hierarchical mixed model with fixed effects of mean PCO2, time, and the
288 interaction of PCO2 and time. Dilutor within mean PCO2 was a random effect with an unstructured
289 covariance. Between-with method was used to estimate degrees of freedom at the hierarchical levels of
290 dilutor within PCO2 treatment. The hierarchal nature of the analysis ensured the degrees of freedom
291 represented the correct number of experimental units (i.e., tanks and not the number of mussels).
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292 Orthogonal contrasts were constructed to test for difference between controls and individual treatment
293 levels. Mean values and 95% CI were calculated for dissolved oxygen, temperature, alkalinity, hardness
294 and conductivity. Mean pH and 95% CI were calculated as the geometric mean.
295
296 Results and Discussion
297 Water chemistry and exposure conditions
298 Water chemistry parameters were relatively uniform among diluters and did not vary appreciably
299 during the study. Mean (SD) chemical parameters were: hardness 178.7 mg·L–1 (4.7), conductivity 404.5
–1 –1 300 µS·cm (3.8), and alkalinity 135.1 mg·L CaCO3 (4.7). Dissolved oxygen was inversely correlated with
301 CO2 as the increased CO2 concentration caused sparging of oxygen (Table 2); however, dissolved oxygen
302 remained above 7.0 mg·L-1 in the high treatmentsDraft throughout the study. Total ammonia nitrogen (TAN)
303 remained below acceptable levels throughout the study (mean 39.6 µg·L-1, SD 32.1, range 16.3 – 48.4
-1 304 µg·L ). The pH was inversely correlated with PCO2 levels, indicating the formation of carbonic acid
-1 305 from the reaction of CO2 with water. Mean tank water flow rates ranged from 164 to 298 mL·min .
306 Mortality
307 Survival of mussels in control tanks at the end of the 28-d exposure and 14-d PE period was
308 93.3% and 100% for L. higginsii and L. siliquoidea, respectively, (Fig. 1; Table 3) and exceeded ASTM
309 acceptable criterion of 80% for chronic exposure of juvenile mussels (ASTM 2017). At least two
310 mortalities of L. higginsii occurred in each treatment; however, no tank had 100% mortality of this
311 species (Fig. 1; Table 3). In contrast, 100% mortality of L. siliquoidea occurred in two of three replicates
312 of the highest treatment. Mortality of L. higginsii was greater than that of L. siliqouidea in all treatments,
313 except the highest, at 28-d and 14-d PE (Fig. 1, Table 3). The lethal concentrations (LC20 and LC50)
314 values for L. higginsii were lower than those for L. siliquoidea (Table 4), although confidence intervals
315 for LC50 values overlapped between species. Overall, average mortality was 7 – 8% higher and LC
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316 values were 5 000 – 10 000 µatm lower at 14-d PE as mortality of both species continued after exposure
317 (Tables 3 and 4).
318 Previously, we exposed L. siliquoidea juveniles to a wider range and higher levels of PCO2
–1 319 (45 000 – 297 537 µatm) and reported the 28-d LC50 = 87.0 mg·L (91 103 µatm PCO2, 95% CI 81 803
–1 320 – 100 698) and 16-d PE LC50 = 76.0 mg·L (78 039 µatm PCO2, 65 057 – 92 137; Waller et al. 2017).
321 LC50 values and 95% CIs in the present study overlapped with those in Waller et al. (2017), and support
322 our previous estimates of CO2 toxicity to this species.
323 Risk assessment in aquatic toxicology often relies on surrogate species (e.g., fathead minnow,
324 daphnia) to extrapolate to threatened and endangered species (e.g., Sappington et al. 2001) due to limited
325 availability and legal restrictions in testing listed species. The choice of an appropriate surrogate is 326 commonly a congener with similar life historyDraft and physiology (Banks et al. 2014). Propagated juvenile 327 L. siliquoidea have become a common toxicity test organism, but this is only the second reported toxicity
328 trial with juvenile L. higginsii (Newton and Bartsch 2007) and the first with ~ 9 mo old animals. In our
329 study, we had the opportunity to simultaneously test juvenile L. higginsii and L. siliquoidea that were
330 propagated at the same facility. However, the U.S. endangered species permit also restricted the number
331 of L. higginsii that could be used in the study, which reduced some statistical power and the response
332 variables that were measured.
333 Mortality of L. higginsii was more variable than that of L. siliquoidea, particularly in the control
334 and lower CO2 treatments. This variability resulted in wide confidence intervals and an unreliable
335 estimate of the 14-d PE LC20 for L. higginsii. Survival of L. higginsii was lower than that of
336 L. siliquoidea in all PCO2 levels, except for the high treatment; but the CI for LC50 values overlapped
337 between species, indicating no significant difference at mid- to high range of PCO2 (Table 4). However,
338 the estimated 28-d LC20 value and CIs of L. higginsii were lower and did not overlap those of
339 L. siliquoidea (Table 4). The model of mortality by species and PCO2 treatment indicated that 28-d
340 mortality of L higginsii was significantly higher than that of L. siliquoidea (F1,14 =6.45, p =0.024), though
341 14-d PE mortality was not different (F1,14 = 3.73, p=0.074). Our results indicate that average survival of
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342 the two lampsiline species was similar across treatments and the PE period, but L. higginsii succumbed at
343 lower levels of CO2 than L. siliquoidea. The selection of L. siliquoidea as a surrogate for L. higginsii
344 should account for this difference in sensitivity at various CO2 levels.
345 Shell growth
346 Carbon dioxide treatment (PCO2) significantly reduced percent shell growth of both L. higginsii
347 and L. siliquoidea (and F4,8 = 7.96, p = 0.0068, and F4,7 = 21.49, p = 0.0005, respectively; Table 3). Total
348 percent shell growth of L. higginsii decreased in a dose-dependent pattern from 12.2% (SD 8) in controls
349 to 2.0% (SD 3.0) in 61 000 µatm PCO2 (Table 3). Total percent shell growth of L. higginsii during CO2
350 exposure was significantly less in all PCO2 treatments, except the lowest (24 000 µatm PCO2; Table 3).
351 Total percent shell growth of L. siliquoidea, ranged from 24.3% (SD 4.8) in controls to 1.5% (SD 2.0) in 352 61 000 µatm PCO2 and was significantly lessDraft in all PCO2 treatments, relative to the control (Table 3).
353 Models of daily growth indicated significant effects of PCO2 for L. higginsii (F4,8 = 7.06, p =
354 0.0098) and L. siliquoidea (F4,7 = 14.55, p = 0.0017), but time (F1,8 = 14.18, p = 0.0048) and PCO2 × time
355 (F4,8 = 5.49, p = 0.0200) were only significant in L. siliquoidea. Daily growth rate of L. higginsii during
356 CO2 exposure and PE period was significantly less in PCO2 ≥ 61 000 µatm (Fig. 2). Daily growth rate of
357 L. siliquoidea during CO2 exposure was significantly lower in PCO2 ≥ 37 000 µatm (Fig. 2)
358 The 28-d EC50 for shell growth was slightly lower for L. siliquoidea, compared to L. higginsii
359 (Table 4), but the 95% CIs overlapped suggesting that growth was similarly affected in both species. The
360 mixed model did not indicate a significant reduction in growth rate of L. higginsii at 37 000 µatm PCO2,
361 however, the EC50 (32 900 µatm PCO2) suggested otherwise. Growth of L. higginsii juveniles was ~
362 50% less than L. siliquoidea and was more variable (Table 3, Fig. 2). The statistical power to detect
363 treatment effects on growth of L. higginsii may have been too low with our limited sample size.
364 Previously, we reported that growth of L. siliquoidea juveniles was significantly reduced during exposure
-1 365 to ~ 35 000 µatm PCO2 (43 mg·L ; Waller et al. 2017). Results of the present study indicate that growth
366 is reduced at pressures as low as 24 000 µatm PCO2 (Tables 3 and 4).
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367 During the PE period, percent shell growth of L. higginssi did not differ among treatment groups
368 (Table 3). Daily growth rate in in PCO2 ≥ 61 000 µatm treatments was reduced (Fig. 2), as previously
369 mentioned. Percent growth (PE) of L. siliquoidea was similar in the control and the two lowest CO2
370 treatments (Table 3) and reduced in PCO2 ≥ 61 000 µatm. Compared to the exposure period, PE growth
371 rate of L. siliquoidea was greater in all treatments, except the control and high treatment (Fig. 2). Daily
372 growth rate (PE) of L. siliquoidea was significantly lower than the control in treatments ≥ 37 000 µatm
373 PCO2 (Fig. 2). These results differ from earlier findings of comparable PE growth in L. siliquoidea across
-1 374 treatments (up to 110 mg·L or ~ 85 000 µatm PCO2; Waller et al. 2017). In the same study, PE growth
375 rate of control mussels (L. siliquoidea) was significantly less than the rate during exposure (Waller et al.
376 2017), suggesting that the former rate was influenced by a factor unrelated to CO2 treatment. Based on
377 growth of control mussels, we suggest that results of the present study are more indicative of shell growth
378 following recovery from CO2 exposure. MusselsDraft may recover from low to moderate CO2 exposure and 379 resume growth when the exposure ends, but recovery is less likely at levels that approach lethality.
380 Growth of both species, within and among replicate tanks, was more variable during the PE period than
381 during CO2 exposure (Fig. 2). Variability in growth within a tank may indicate individual differences in
382 recovery and/or response to handling disturbance. Alternatively, the PE growth period was half the
383 duration of the exposure period — variability may be reduced if growth were followed for an equivalent
384 time period (i.e., 28 d).
385 High inherent variability in shell and tissue growth within a cohort of juvenile mussels is
386 common and can reduce ability to distinguish treatment-related effects on growth (Larson et al. 2014).
387 We had the benefit of laboratory-reared juveniles for both species that were of the same cohort, thus
388 reducing some of the inherent variability of mussels held under field conditions. Rapid growth of
389 L. siliquoidea in our study resulted in greater separation of shell sizes in each CO2 treatment and enabled
390 us to detect treatment effects, despite the high degree of variability. Growth of L. higginsii juveniles was
391 ~ 50% less than L. siliquoidea and was more variable (Table 3, Fig. 2). These factors may have limited
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392 the detection of treatment effects at all CO2 concentrations within our limited sample size (n=10 per test
393 tank).
394
395 Condition
396 Carbon dioxide exposure caused a significant decrease in tissue condition [(dry tissue weight
397 /shell length) (F3,6 = 9.32, p ≤ 0.01; Table 3, Fig. 3a)] and shell condition [(dry shell weight/shell length)
398 (F3,6 = 5.66, p = 0.03; Table 3; Fig. 3b)] of L. siliquoidea with increasing PCO2. Both condition indices
399 were significantly lower than the control at PCO2 ≥ 37,000 µatm. Decreased shell condition is consistent
400 with shell growth response of L. siliquoidea. The decline in tissue condition indicates that CO2 also
401 adversely affected tissue growth. Carbon dioxide exposure may increase the energy needed to maintain 402 homeostasis and reduce energy for shell andDraft tissue growth. Although, body condition index of adult
403 mussels was not reduced after 32-d continuous or intermittent exposure to 20 000 µatm PCO2 (Hannan et
404 al. 2016a, c), physiological and metabolic responses indicated mussels expended energy to maintain acid-
405 base and ionic balance (Hannan et al. 2016a, c; Jeffrey et al. 2017). Juvenile mussels likely use the same
406 homeostatic mechanisms as adult mussels, but have less carbonate reserves in the shell to maintain acid-
407 base and ionic balance, and therefore experience significant shell and tissue loss. In addition to serving as
408 a bicarbonate source, the shell is the primary means of protection for a mussel. Prolonged CO2 exposure
409 can reduce shell integrity (Waller et al. 2017) and mass, and increase risk of predation and mechanical
410 injury to mussels.
411 Behavior
412 Quantification of behavior during CO2 exposure was limited to the premortality period which
413 included days 0 – 12. Before the start of CO2 infusion, a total of four (2.7%) L. higginsii and zero
414 L. siliquoidea were unburied (Fig. 4). Carbon dioxide infusion induced mussels to unbury, especially in
415 the two highest CO2 treatments (Fig. 4). The mean number of unburied L. higginsii ranged from 3–7/tank
416 (30–70%) in PCO2 ≥ 61 000 µatm after day 3 of exposure, compared to 0−1 mussels/tank in the control
417 and two lowest CO2 treatments. Lampsilis siliquoidea mussels remained buried (> 90%) in all treatments
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418 until day 5. The mean number of unburied mussels fluctuated from 1–4/tank (6–25%) on days 5–9 in the
419 highest treatment and then increased to > 5/tank (37%) over the next 3 d. The number of unburied
420 mussels increased (up to an average of 25%) in 61 000 µatm PCO2 on days 10–12; mussels began to die
421 on day 12. Intercell movements were minimal in both species and ranged from 0–3 movements·d-1 in all
422 treatments. Only one intercell movement occurred in the control during the exposure period – one L.
423 higginsii moved into another cell on day 12. All other intercell movements occurred in CO2 treatment
424 tanks; however, no trend was obvious in either species.
425 Abnormal behaviors (agape, extended foot, side lying or positioned on umbo) were absent in
426 control mussels but were observed in the two highest CO2 treatments. About 50% of L. higginsii mussels
427 in PCO2 treatments ≥ 61 000 µatm were narcotized by day 12 of exposure compared to < 20% in 37 000
428 µatm. Lampsilis siliquoidea juveniles were also narcotized in PCO2 treatments of 61 000 (23%) and
429 96 000 µatm (48%) by day 12. Draft
430 Mussels recovered from narcotization within several hours to days after removal of CO2 but the
431 response varied by species and treatment group. At 1-d PE, the total number of unburied L. siliquoidea
432 was one (2.1%) in 37 000 µatm PCO2 and 11 (29.7%) in 61 000 µatm PCO2; all mussels were buried in
433 control and 24 000 µatm PCO2 tanks. At 2-d PE, all but four mussels (11%) were buried in 61 000 µatm
434 PCO2 (not shown). Lampsilis higginsii juveniles took longer to recover and rebury than L. siliquoidea
435 across all treatments (not shown). At 2-d PE, unburied L. higginsii ranged from zero in the control tanks
436 to three (12.5%) in 24 000 µatm PCO2 and 13 (76%) in 61 000 µatm PCO2. Three of the eight (37.5%)
437 mussels that remained alive in the 96 000 µatm PCO2 were unburied. At 4-d PE, L. higginsii remained
438 unburied in 61 000 (n = 5; 29.4%) and 96 000 (n = 2; 25%) µatm PCO2.
439 Behavioral effects of CO2 may impact unionid mussels on several fronts. Carbon dioxide
440 exposure can reduce byssal thread secretion and attachment (Waller and Bartsch, accepted). Combined
441 with the unburying and gaping behavior we observed, this can lead to increased predation and
442 displacement by water current. Carbon dioxide application also may reduce mussel reproduction by
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443 causing avoidance behaviors in fish host species (Ross et al. 2001; Clingerman et al 2007; Kates et al.
444 2012) which are required for transformation of the parasitic larval (glochidia) stage.
445
446 Gene Expression
447 Of the four genes that we targeted for qPCR, the most significant treatment-related change was
448 seen in CHS expression (Fig. 5). PCO2 (F3,6 = 12.45, p = 0.0055), time (F1,8 = 35.62, p = 0.0003), and
449 PCO2 × time (F3,8 = 16.79, p = 0.0008) had a significant effect on CHS expression. At 28-d, CHS
450 expression was significantly lower in 61 000 µatm PCO2 (F1,8 = 0.52.78, p < 0.0001) relative to the
451 control and two lowest treatments. There were no significant differences in CHS expression between
452 control and CO2 treatments at 14-d PE, indicating that CHS expression recovered after removal of CO2. 453 A similar response of CHS expression was Draftobserved in adult L. siliquoidea. Jeffrey et al. (2018) reported
454 a significant reduction in CHS expression after 7 d exposure to 50 000 µatm PCO2, relative to the control,
455 although differences were not significant at 28 d of exposure. Chitin synthase expression had returned to
456 control levels after 14 d PE. Based on our results, the minimum effect concentration for CHS expression
457 was > 37 000 µatm PCO2. However, Jeffrey et al. (2017) found that 20 000 µatm PCO2 reduced CHS
458 expression in mantle tissue of adult F. flava in a 32-d exposure (Jeffrey et al. 2017).
459 Chitin synthase is integral to the production of chitin, a key component of the shell. Decreased
460 chitin production would adversely affect biomineralization and shell integrity. Down-regulation of CHS
461 in our study coincided with decreased shell growth rate and shell condition in juvenile L. siliquoidea.
462 Reduced shell integrity in juvenile L. siliquoidea after CO2 exposure (Waller et al. 2017) may be another
463 possible consequence of decreased chitin production. The annual shell growth of many adult mussel
464 species is too small (Haag and Rypel 2010) to detect significant effects from a stressor, such as CO2, in
465 30 d; however, CHS expression may be a sensitive indicator of reduced shell growth across a range of life
466 stages and sizes of mussels (Jeffrey et al. 2017; Jeffrey et al. 2018).
467 Transcript responses of CAL, NKA, and DEF in juvenile L. siliquoidea varied by treatment and
468 exposure period. We hypothesized that CO2 exposure would trigger up-regulation of CAL in response to
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469 calcium mobilization; but there was no effect of PCO2, time or PCO2 × time on CAL expression (Fig.
470 5b). Likewise, there was no significant effect of PCO2 on NKA expression in L. siliquoidea at 28-d
471 exposure (F3,6 = 0.33, p = 0.8050; Fig. 5c). Mean fold expression was greater in all treatments, except the
472 control, at 14-d PE relative to the 28-d exposure value; however, there was no effect of PCO2 on NKA
473 expression (F1,8 = 2.35, p = 0.1639). Jeffrey et al. (2018) also found no significant change in CAL and
474 NKA levels in gill and mantle of adult L. siliquoidea at 28-d exposure to 50 000 µatm PCO2, but did note
475 decreased expression of both genes at 14 d PE. Lastly, we were unable to detect up or down regulation of
476 DEF gene expression in our study (Fig 5d).
477 Arguably, significant shifts in gene expression may have been evident at the onset of CO2
478 exposure and plateaued over the course of the exposure. For example, CAL levels were significantly
479 reduced at day 1 and 4 in gill tissue of adult L. siliquoidea, but increased to control levels at 28 d (Jeffrey 480 et al. 2018). We may have missed initial changesDraft in the target genes by sampling only at the end of the 481 28-d exposure. However, our objective was to measure the longer-term, adaptive response of mussels to
482 hypercapnia. Additional studies are needed to determine the pattern of genomic responses, and resulting
483 homeostatic mechanisms, of mussels at various CO2 levels and exposure durations.
484 Variability in gene expression was high among individuals within a treatment (Fig. 5), similar to
485 that seen in growth rates and condition indices. N+/K+ ATPase and CAL expression varied by ~ 2 fold
486 among treatments and sampling time. Chitin synthase expression varied > 4-fold among treatments at 28
487 d, but by < 2-fold in 14-d PE samples. Variability was highest for DEF expression, notably in the samples
488 from 61 000 µatm PCO2. In two mussels, DEF expression was 7- and 38-fold lower at 28 d, relative to
489 controls, but fold expression for the other three genes was within the 95% quartile in these same
490 individuals. Extreme fold expression values may indicate survivors versus eventual mortalities. We do not
491 know whether moribund mussels, particularly in the higher CO2 treatments, were the outlier values.
492 Pooling individuals from a treatment would have normalized outliers and reduced the variability in our
493 data; although we found no correlation, we maintained individual samples in order to analyze the
494 relationship between gene expression and growth in individual mussels.
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495 Use of whole individuals may have been another source of variability in the expression data.
496 Gene expression can be strongly organ and tissue specific-dependent (Hüning et al. 2013; Ren et al. 2013;
497 Jeffrey et al. 2017). For example, CAL is expressed in a range of tissues, including the mantle, gonad, and
498 adductor mussel, but has highest activity in the gill and foot (Zeng et al. 2012; Ren et al. 2013).
499 Unfortunately, the juveniles were too small to sufficiently sample individual organs. Analysis of
500 transcript levels in individual tissues (e.g., mantle and gill), rather than whole body samples, may be
501 needed to detect significant changes in fold expression related to CO2 treatment. Additionally, a broader
502 transcript profile could be assessed in future studies to identify additional marker genes for hypercapnic
503 stress in juvenile mussels.
504 Application scenarios of CO2 for AIS in natural systems will vary with the management goals
505 and target species in a system. Our test system represents a worst case scenario in which CO2 is applied
506 continuously as a barrier to fish movement.Draft The PCO2 levels that we tested could occur in the immediate
507 zone of infusion (61 000 – 96 000 µatm) and in a downstream plume (24 000 – 37 000 µatm). Shorter
508 infusion periods of CO2 to corral fish into a bay or slough for harvest may be less harmful to native
509 mussels. Mussels can survive acute exposure (e.g., 96 h; Waller and Bartsch, accepted) and recover from
510 longer (e.g., 28 d), low level exposure to CO2 (Waller et al. 2017; Hannan et al. 2016a, c). Another
511 possible application of CO2 is infusion into a navigational lock to deter fish movement at a control point.
512 In this scenario, mussels would be exposed to intermittent pulsed doses of CO2 when the chamber was
513 open. Hannan et al. (2016b) simulated pulsed CO2 exposure of 30 min, 12 times per day, for 28 d and
- + + 2+ 514 reported no mortality of adult mussels but found physiological responses (e.g., HCO3 , Ca2 , Na , Mg )
515 remained elevated between doses.
516 Chitinase synthase was a genomic indicator of CO2 exposure in juvenile mussels. A suite of
517 transcriptomes (i.e., RNA seq analysis) is needed to identify a broader genomic response of juvenile
518 mussels to hypercapnia. Additionally, a comparison of mussels that reside in habitats with episodic
519 hypercapnia from natural processes (e.g., reservoirs and lakes) would provide insight on the potential
520 homeostatic strategies used by juvenile mussels and may help determine the relative risk of CO2 exposure
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521 to other mussel species. Further work is needed to determine the effects of dose, duration, and frequency
522 of CO2 exposure on the recovery of native mussels. Perhaps the greater concern of sublethal CO2 levels is
523 the behavior of both mussels and native fish. Narcotization of mussels increases their risk of displacement
524 and predation. Mussel reproduction and recruitment would be reduced if fish avoid zones of CO2
525 infusion. Before field deployment of CO2, resource managers will have to weigh its benefits against the
526 risks to native species. Our study provides data on juvenile lampsiline mussels to help inform that
527 decision.
528 Lampsilis siliquoidea was a reasonable surrogate for predicting lethality of CO2 to the endangered
529 L. higginsii mussel across the range of levels tested in our study. Growth and behavioral responses of
530 L. siliquoidea were less predictive of L. higginsii. Lampsilis siliquoidea growth was affected at lower
531 PCO2, but recovery from narcotization was more rapid compared to L. higginsii. We expect that CO2
532 levels that adversely impact L. siliquoidea willDraft similarly affect L. higginsii but also recommend extended
533 monitoring of L. higginsii in any CO2 application.
534
535 ACKNOWLEDGEMENTS
536 The study was funded in part by the Great Lakes Restoration Initiative and the U.S. Geological Survey
537 Ecosystem Mission Area Invasive Species Program. Kerry Weber, Riley Buley, Ann Tronick and
538 Rhiannon Fisher assisted in the laboratory and with data entry. The U.S. Fish and Wildlife Service, Genoa
539 National Fish Hatchery provided mussels. Christopher Merkes, Kim Fredricks and two anonymous
540 reviewers provided insightful comments on earlier versions of the manuscript.
541 Disclaimer
542 Any use of trade, product, or firm names is for descriptive purposes only and does not imply
543 endorsement by the U. S. government.
544
545 References
https://mc06.manuscriptcentral.com/cjfas-pubs Page 23 of 39 Canadian Journal of Fisheries and Aquatic Sciences
23
546 ACRCC MRW. 2014. Monitoring and response plan for Asian carp in the upper Illinoi River and
547 Chicago Area Waterway System. Asian Carp Regional Coordinating Committee Monitoring and
548 Response Workgroup. Available from http://www.asiancarp.us/documents/MRP2014.pdf
549 [accessed 3 January 2016].
550 American Public Health Association (APHA). 2012. Standard methods for the examination of water and
551 wastewater. In 22nd edition. American Public Health Association, Washington, D.C.
552 ASTM International. 2017. Standard guide for conducting laboratory toxicity tests with freshwater
553 mussels (E2455-06 (2013). In Annual Book of ASTM Standards, Volume 11.06. ASTM
554 International, West Conshohocken, PA
555 Bai, Z., Lin, J., Ma, K., Wang, G., Niu, D., and Li, J. 2014. Identification of housekeeping genes suitable
556 for gene expression analysis in the pearl mussel, Hyriopsis cumingii, during biomineralization. 557 Mol. Genet. Genom. 289(4): 717–725.Draft doi:10.1007/s00438-014-0837-1. 558 Banks, J., Stark, J. Vargas, R. and Ackleh, A. 2014. Deconstructing the surrogate species concept: a life
559 history approach to the protection of ecosystem services. Ecol. Appl. 24(4):770-778.
560 Bringolf, R.B., Cope, W.G., Eads, C.B., Lazaro, P.R., Barnhart, M.C., and Shea, D. 2007a. Acute and
561 chronic toxicity of technical-grade pesticides to glochidia and juveniles of freshwater mussels
562 (unionidae). Environm. Toxicol. Chem. 26(10): 2086–2093.
563 Bringolf, R.B., Cope, W.G., Mosher, S., Barnhart, M.C., and Shea, D. 2007b. Acute and chronic toxicity
564 of glyphosate compounds to glochidia and juveniles of Lampsilis siliquoidea (Unionidae).
565 Environ. Toxicol. Chem. 26(10): 2094–2100.
566 Clingerman, J., Bebak, J., Mazik, P.M., and Summerfelt, S.T. 2007. Use of avoidance response by
567 rainbow trout to carbon dioxide for fish self-transfer between tanks. Aquacul. Eng. 37(3): 234–
568 251. doi:10.1016/j.aquaeng.2007.07.001.
569 Cummings Kevin, and Cordeiro, J. 2012. Lampsilis siliquoidea. The IUCN Red List of Threatened
570 Species. Available from http://dx.doi.org/10.2305/IUCN.UK.2012-
571 1.RLTS.T189448A1926029.en [accessed 17 August 2017].
https://mc06.manuscriptcentral.com/cjfas-pubs Canadian Journal of Fisheries and Aquatic Sciences Page 24 of 39
24
572 Cupp, A.R., Erickson, R.A., Fredricks, K.T., Swyers, N.M., Hatton, T.W., and Amberg, J.J. 2017.
573 Responses of invasive silver and bighead carp to a carbon dioxide barrier in outdoor ponds. Can.
574 J. Fish. Aquat. Sci.:74(3):297-305. doi:10.1139/cjfas-2015-0472.
575 Dennis, C.E., Wright, A.W., and Suski, C.D. 2016. Potential for carbon dioxide to act as a non-physical
576 barrier for invasive sea lamprey movement. J. Great Lakes Res. 42(1): 150–155.
577 doi:10.1016/j.jglr.2015.10.013.
578 Fang, D., Xu, G.R., Hu, Y.L., Pan, C., Xie, L.P., and Zhang, R.Q. 2011. Identification of genes directly
579 involved in shell formation and their functions in pearl oyster, Pinctada fucata. PloS ONE 6.
580 doi:10.1371/journal.pone.0021860.
581 Giacomin, M., Gillis, P.L., Bianchini, A., and Wood, C.M. 2013. Interactive effects of copper and
582 dissolved organic matter on sodium uptake, copper bioaccumulation, and oxidative stress in 583 juvenile freshwater mussels (LampsilisDraft siliquoidea). Aquat. Toxicol. 144: 105–115. 584 doi:10.1016/j.aquatox.2013.09.028.
585 Haag, W., and Rypel, A. 2010. Growth and longevity in freshwater mussels: evolutionary and
586 conservation implications. Biol. Rev. 86(1):225-247. doi:10.1111/j.1469-185X.2010.00146.x.
587 Hannan, K.D., Jeffrey, J.D., Hasler, C.T., and Suski, C.D. 2016a. Physiological effects of short-and long-
588 term exposure to elevated carbon dioxide on a freshwater mussel, Fusconaia flava. Can. J. Fish.
589 Aquat. Sci. 73(10): 1538–1546.
590 Hannan, K.D., Jeffrey, J.D., Hasler, C.T., and Suski, C.D. 2016b. Physiological responses of three species
591 of unionid mussels to intermittent exposure to elevated carbon dioxide. Conserv. Physiol. 4(1):
592 cow066. doi:10.1093/conphys/cow066.
593 Hannan, K.D., Jeffrey, J.D., Hasler, C.T., and Suski, C.D. 2016c. The response of two species of unionid
594 mussels to extended exposure to elevated carbon dioxide. Comp. Biochem. Physiol. Part A Mol.
595 Integr. Physiol. 201: 173–181.
596 Hüning, A.K., Melzner, F., Thomsen, J., Gutowska, M.A., Krämer, L., Frickenhaus, S., Rosenstiel, P.,
597 Pörtner, H.-O., Philipp, E.E.R., and Lucassen, M. 2013. Impacts of seawater acidification on
https://mc06.manuscriptcentral.com/cjfas-pubs Page 25 of 39 Canadian Journal of Fisheries and Aquatic Sciences
25
598 mantle gene expression patterns of the Baltic Sea blue mussel: implications for shell formation
599 and energy metabolism. Mar. Biol. 160(8): 1845–1861. doi:10.1007/s00227-012-1930-9.
600 Jeffrey, J.D., Hannan, K.D., Hasler, C.T., and Suski, C.D. 2017. Responses to elevated CO2 exposure in a
601 freshwater mussel, Fusconaia flava. J. Comp. Physiol. B 187(1): 87–101. doi:10.1007/s00360-
602 016-1023-z.
603 Jeffrey, J.D., Hannan, K.D., Hasler, C.T., and Suski, C.D. 2018. Chronic exposure of a freshwater mussel
604 to elevated pCO2: Effects on the control of biomineralization and ion-regulatory responses:
605 Responses of Lampsilis siliquoidea to chronically elevated pCO2. Environ. Toxicol. Chem. 37(2):
606 538–550. doi:10.1002/etc.3991.
607 Kates, D., Dennis, C., Noatch, M.R., and Suski, C.D. 2012. Responses of native and invasive fishes to
608 carbon dioxide: potential for a nonphysical barrier to fish dispersal. Can. J. Fish. Aquat. Sci. 609 69(11): 1748–1759. Draft 610 Larson, J., Eckert, J, and Bartsch, M. 2014. Intrinsic variability in shell and soft tissue growth of the
611 freshwater mussel Lampsilis siliquoidea. PLoS One 9(11):e112252.
612 doi:10.1371/journal.pone.0112252
613 Liu, J., Yang, D., Liu, S., Li, S., Xu, G., Zheng, G., Xie, L., and Zhang, R. 2015. Microarray: a global
614 analysis of biomineralization-related gene expression profiles during larval development in the
615 pearl oyster, Pinctada fucata. BMC Genomics 16(1): 325. doi:10.1186/s12864-015-1524-2.
616 Livak, K.J., and Schmittgen, T.D. 2001. Analysis of Relative Gene Expression Data Using Real-Time
617 Quantitative PCR and the 2−ΔΔCT Method. Methods 25(4): 402–408.
618 doi:10.1006/meth.2001.1262.
619 Luo, Y., Li, C., Landis, A.G., Wang, G., Stoeckel, J., and Peatman, E. 2014. Transcriptomic Profiling of
620 Differential Responses to Drought in Two Freshwater Mussel Species, the Giant Floater
621 Pyganodon grandis and the Pondhorn Uniomerus tetralasmus. PLoS ONE 9(2): e89481.
622 doi:10.1371/journal.pone.0089481.
623 Lydeard, C., Cowie, R.H., Ponder, W.F., Bogan, A.E., Bouchet, P., Clark, S.A., Cummings, K.S., Frest,
624 T.J., Gargominy, O., Herbert, D.G., Hershler, R., Perez, K.E., Roth, B., Seddon, M., Strong, E.E.,
https://mc06.manuscriptcentral.com/cjfas-pubs Canadian Journal of Fisheries and Aquatic Sciences Page 26 of 39
26
625 and Thompson, F.G. 2004. The Global Decline of Nonmarine Mollusks. BioScience 54(4): 321–
626 330.
627 Martikainen, E.A.T., and Krogh, P.H. 1999. Effects of soil organic matter content and temperature on
628 toxicity of dimethoate to Folsomia fimetaria (Collembola: Isotomiidae). Environ. Toxicol. Chem.
629 18(5): 865–872.
630 Newton, T.J., and Bartsch, M.R. 2007. Lethal and sublethal effects of ammonia to juvenile Lampsilis
631 mussels (unionidae) in sediment and water-only exposures. Environ. Toxicol. Chem. 26(10):
632 2057–2065.
633 RéGnier, C., Fontaine, B., and Bouchet, P. 2009. Not Knowing, Not Recording, Not Listing: Numerous
634 Unnoticed Mollusk Extinctions. Conserv. Biol. 23(5): 1214–1221. doi:10.1111/j.1523-
635 1739.2009.01245.x. 636 Ren, G., Hu, X., Tang, J., and Wang, Y. 2013.Draft Characterization of cDNAs for calmodulin and 637 calmodulin-like protein in the freshwater mussel Hyriopsis cumingii: Differential expression in
638 response to environmental Ca2+ and calcium binding of recombinant proteins. Comp. Biochem.
639 Physiol. Part B Biochem. Mol. Biol. 165(3): 165–171. doi:10.1016/j.cbpb.2013.04.003.
640 Robbins, L.L., Hansen, M.E., Kleypas, J.A., and Meylan, S.C. 2010. CO2calc: A user-friendly seawater
641 carbon calculator for Windows, Mac OS X, and iOS (iPhone). U.S. Geological Survey Open-File
642 Report, U.S. Geological Survey.
643 Ross, R.M., Krise, W.F., Redell, L.A., and Bennett, R.M. 2001. Effects of dissolved carbon dioxide on
644 the physiology and behavior of fish in artificial streams. Environ. Toxicol. 16(1): 84–95.
645 doi:10.1002/1522-7278(2001)16:1<84::AID-TOX100>3.0.CO;2-1.
646 Sappington, L., Mayer, F., Dwyer, F., Buckler, D., Jones, J., and Ellersieck, M. 2001. Contaminant
647 sensitivity of threated and endangered fishes compared to standard surrogate species. Environ.
648 Toxicol. Chem. 20(12):2869-2876.
649 Strayer, D.L. 2014. Understanding how nutrient cycles and freshwater mussels (Unionoida) affect one
650 another. Hydrobiologia 735(1): 277–292. doi:10.1007/s10750-013-1461-5.
https://mc06.manuscriptcentral.com/cjfas-pubs Page 27 of 39 Canadian Journal of Fisheries and Aquatic Sciences
27
651 Sun, X., Yang, A., Wu, B., Zhou, L., and Liu, Z. 2015. Characterization of the Mantle Transcriptome of
652 Yesso Scallop (Patinopecten yessoensis): Identification of Genes Potentially Involved in
653 Biomineralization and Pigmentation. PloS ONE 10(4): e0122967.
654 doi:10.1371/journal.pone.0122967.
655 US Environmental Protection Agency (USEPA). 1983. Methods for chemical analysis of water and
656 wastes. Total hardness, method 130.2 (Titrimetric EDTA). EPA/600/4-79/020.
657 Vaughn, C.C., and Hakenkamp, C.C. 2001. The functional role of burrowing bivalves in freshwater
658 ecosystems. Freshw. Biol. 46(11): 1431–1446.
659 Vaughn, C.C., Nichols, S.J., and Spooner, D.E. 2008. Community and foodweb ecology of freshwater
660 mussels. J. N. Am. Benthol. Soc. 27(2): 409–423. doi:10.1899/07-058.1.
661 Waller, D.L., Bartsch, M.R., Fredricks, K.T., Bartsch, L.A., Schleis, S.M., and Lee, S.H. 2017. Effects of 662 carbon dioxide on juveniles of the freshwaterDraft mussel (Lampsilis siliquoidea [Unionidae]). 663 Environ. Toxicol. Chem. 36(3):671-681. doi:10.1002/etc.3567.
664 Wang, N., Consbrock, R.A., Ingersoll, C.G., and Barnhart, M.C. 2011. Evaluation of influence of
665 sediment on the sensitivity of a unionid mussel (Lampsilis siliquoidea) to ammonia in 28-day
666 water exposures. Environ. Toxicol. Chem. 30(10): 2270–2276. doi:10.1002/etc.616.
667 Wang, N., Ingersoll, C.G., Greer, I.E., Hardesty, D.K., Ivey, C.D., Kunz, J.L., Brumbaugh, W.G., Dwyer,
668 F.J., Roberts, A.D., Augspurger, T., and others. 2007. Chronic toxicity of copper and ammonia to
669 juvenile freshwater mussels (Unionidae). Environ. Toxicol. Chem. 26(10): 2048–2056.
670 Weiss, I.M., and Schönitzer, V. 2006. The distribution of chitin in larval shells of the bivalve mollusk
671 Mytilus galloprovincialis. J. Struct. Biol. 153(3): 264–277. doi:10.1016/j.jsb.2005.11.006.
672 Weiss, I.M., Schonitzer, V., Eichner, N., and Sumper, M. 2006. The chitin synthase involved in marine
673 bivalve mollusk shell formation contains a myosin domain. Febs Letters 580.
674 doi:10.1016/j.febslet.2006.02.044.
675 Williams, J.D., Warren Jr, M.L., Cummings, K.S., Harris, J.L., and Neves, R.J. 1993. Conservation status
676 of freshwater mussels of the United States and Canada. Fisheries 18(9): 6–22.
https://mc06.manuscriptcentral.com/cjfas-pubs Canadian Journal of Fisheries and Aquatic Sciences Page 28 of 39
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677 Xu, W., and Faisal, M. 2010. Defensin of the zebra mussel (Dreissena polymorpha): Molecular structure,
678 in vitro expression, antimicrobial activity, and potential functions. Mol. Immunol. 47(11–12):
679 2138–2147. doi:10.1016/j.molimm.2010.01.025.
680 Zeng, L.., Wang, J., Li, Y., Sheng, J., Gu, Q., and Hong, Y. 2012. Molecular characteristics and
681 expression of calmodulin cDNA from the freshwater pearl mussel, Hyriopsis schlegelii. Genet.
682 Mol. Res. 11(1): 42–52. doi:10.4238/2012.January.9.5.
683
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687 688 Draft 689
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691 Table 1. Gene-specific primer sequences.
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-GATCCGTGGGAAGGAAGTAATCDraft
Chitin Synthaseb FWD-GAGTCGATTGGCCCAAGACA 104 REV-CCACCTGTTCGTCGAGTTCA
692 a = sequence taken from Bai et al. (2014)
693 b = sequence taken from Jeffrey et al. (2017)
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.
Dissolved oxygen (mg·L-1) pH Temperature (° C) Relative Titration Calculation
CO2 -1 Post Post Post CO2 (mg·L ) PCO2 treatment 3 Exposure exposure Exposure exposure Exposure exposure (×10 µatm)
Control 8.1 8.4 7.91 7.99 21.6 21.5 2.2 2.4 (8.1, 8.2) (8.3, 8.4) (7.89, 7.92) (7.97, 8.00) (21.6, 21.6) (21.4, 21.5) (1.9, 2.4) (1.9, 2.8)
Low 7.9 8.4 6.86 Draft7.99 21.6 21.4 32.0 24.1 (7.8, 8.0) (8.3, 8.4) (6.84, 6.87) (7.98, 8.00) (21.6, 21.6) (21.4, 21.5) (31.0, 32.9) (23.3, 24.9)
Medium 7.9 8.4 6.67 7.99 21.6 21.5 46.8 37.2 (7.8, 8.0) (8.4, 8.5) (6.66, 6.68) (7.97, 8.00) (21.6, 21.7) (21.4, 21.5) (45.7, 48.0) (36.1, 38.3)
Med High 7.8 8.4 6.46 8.00 21.7 21.5 77.0 60.6 (7.7, 7.8) (8.3, 8.5) (6.44, 6.47) (7.99, 8.01) (21.6, 21.7) (21.5, 21.5) (74.6, 79.5) (59.0, 62.2)
High 7.5 8.5 6.26 7.97 21.7 21.6 119.5 95.7 (7.5, 7.6) (8.5, 8.6) (6.25, 6.27) (7.95, 7.98) (21.6, 21.7) (21.5, 21.6) (116.3, 122.7) (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 Total Mortality (%) Percent growth* Tissue condition† Shell condition† (µatm
PCO2) HG FM HG FM Mean Regression Mean Regression
6.7 0.0 a a a a Control 28-d 12.2 (8.02) 24.33 (4.78) 465.3 Y= -8.5 +1.3*L 3940.0 Y= -68 +11*L (5.8) (0.0) (132.1) R2 = 0.85 (1088.3) R2 = 0.89 1 1 6.7 0.0 5.42 (5.11) 10.63 (3.07) 14-d PE (5.8) (0.0) 23.3 2.1 a Draftb a ab Low 28-d 10.66 (6.43) 16.58 (4.92) 373.0 Y= -7.6 +1.2*L 3122.9 Y= -62 +10*L (24 000) (5.8) (3.6) (137.0) R2 = 0.93 (1143.3) R2 = 0.92 33.3 2.1 6.541 (5.43) 9.801 (5.10) 14-d PE (11.5) (3.6) 13.3 6.3 b c b b Medium 28-d 5.82 (3.92) 8.00 (4.58) 294.3 Y= -1.4 +0.4*L 2345.2 Y= -17 +4*L (15.3) (6.3) (37 000) (74.0) R2 = 0.60 (625.4) R2 = 0.73 20.0 8.3 1 1,2 14-d PE 4.45 (4.63) 7.17 (4.63) (26.5) (9.5) 40.0 18.8 b d b b Med High 28-d 2.00 (3.04) 1.51 (2.02) 203.2 Y= -3.5 +0.7*L 1983.2 Y= -33 +6*L (61 000) (0.0) (6.3) (77.0) R2 = 0.63 (593.2) R2 = 0.84 56.7 29.2 1.091 (1.88) 2.932 (2.10) 14-d PE (15.3) (23.7) d High 73.3 75.0 b 4.30 (1.90) NA‡ NA NA NA 28-d 2.71 (1.21) (96 000) (15.3) (43.3) 73.3 89.6 1.071 (2.78) -0.162 (2.06) 14-d PE (15.3) (18.0) * 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
31 800 71 000 61 000 32 900 Lampsilis higginsii Not reported† (15 600 – 42 800) (60 000 – 88 300) (46 000 – 87 700) (26 000 – 39 900)
58 200 78 200 53 800 69 100 28 600 Lampsilis siliquoidea (45 200 – 68 100) (68 300 – 92 800) (47 200 – 59 100) (64 000 – 75 100 (26 400 – 30 900)
Draft *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 siliquoideaDraft 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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A
120 Lampsilis higginsii
100
80 1 1
1 60 a a 40 a 2
20 b 2 b Growth in length (µm/day) length in Growth 0
-20 n=29, 27 n=25, 21 n=27, 24 n=24, 15 n=12, 8 Control 24Draft 37 61 96
B
1 120 1 Lampsilis siliquoidea 100 a * 2 80 a
60 * b 3 40 b
20 b * 3 Growth in length (µm/day) length in Growth
0
-20 n=48, 48 n=49, 48 n=45, 44 n=44, 33 n=15, 5 Control 24 37 61 96
Partial pressure carbon dioxide (× 103 microatmospheres)
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A B
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A B Draft
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