CO2-driven ocean acidification repressed the growth of adult sea urchin Strongylocentrotus intermedius by impairing intestine function

Marine Pollution Bulletin 153 (2020) 110944

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CO2-driven ocean acidification repressed the growth of adult sea urchin Strongylocentrotus intermedius by impairing intestine function

T

Yaoyao Zhana, Dongyao Cuia, Dongfei Xinga, Jun Zhangb, Weijie Zhanga, Yingying Lia, Cong Lib, , ⁎ Yaqing Changa, ⁎

a

Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture and Rural Affairs, Dalian Ocean University, Dalian, Liaoning 116023, PR China b College of Basic Medical Science, Dalian Medical University, Dalian, Liaoning 116044, PR China

ARTICLE INFO

ABSTRACT

Keywords: Ocean acidification Strongylocentrotus intermedius Growth Feeding Intestine function

Strongylocentrotus intermedius cultured in the northern Yellow Sea in China was utilized to evaluate the effects of chronic CO2-driven ocean acidification (OA) on adult sea urchins. Based on the projection of the Intergovernmental Panel on Climate Change (IPCC), present natural seawater conditions (pHNBS = 8.10 ± 0.03) and three laboratory-controlled OA conditions (OA1, ΔpHNBS = − 0.3 units; OA2, ΔpHNBS = − 0.4 units; OA3, ΔpHNBS = − 0.5 units) were employed. After 60-day incubation, our results showed that (1) OA significantly repressed the growth of adult S. intermedius; (2) food consumption tended to be decreased with pH decline; (3) intestinal morphology was changed, and activities of intestinal cellulase and lipase were decreased under acidified conditions; (4) expression levels of two immune-related genes (SiTNF14 and SiTGF-β) were altered; (5) rate-limiting enzyme activities of the glycolytic pathway and tricarboxylic acid cycle (TAC) were changed in all OA treatments compared to those of controls.

1. Introduction Ocean acidification (OA) refers to the phenomenon of excessive carbon dioxide (CO2)-driven decreasing of oceanic pH values, thus altering the dynamic equilibrium of ocean carbonate chemistry (Caldeira and Wickett, 2003). According to the forecast of the Intergovernmental Panel on Climate Change (IPCC), the average pH of global ocean surface water will decrease by 0.3–0.5 units by the end of this century (IPCC, 2013). It has been well demonstrated that oceanic pH is one of the most important environmental factors affecting the reproduction (Vehmaa et al., 2013), distribution (Fang et al., 2014), metabolic processes (Torstensson et al., 2015) and behavior of marine organisms (Duarte et al., 2015). Sea urchins are representative invertebrates in many coastal marine ecosystems. As predators and prey, sea urchins play a vital role in maintaining the balance of marine ecosystems (Pearse, 2006). Moreover, some species of sea urchins are of economic importance (Andrew et al., 2003). To date, much research effort on evaluating the effects of ocean acidification has mainly focused on the early developmental stages of sea urchins such as fertilization kinetics (Dupont and Thorndyke, 2014; Hardy and Byrne, 2014), pluteus larval development and survival (Zhan et al., 2016; Zhan et al., 2017), skeleton ⁎

biomineralization (Zhan et al., 2018), gene expression pattern alteration (Hammond and Hofmann, 2012; Martin et al., 2011; Todgham and Hofmann, 2009), and the response of acid-base regulatory systems (Calosi et al., 2013; Stumpp et al., 2012a, 2012b). All observations obtained have indicated a negative effect of OA on early development of the evaluated sea urchin species (Dupont et al., 2010). It is therefore acknowledged that the early life stages of sea urchins will be more vulnerable to OA in the near future OA (Cripps et al., 2014). However, the adult stage of sea urchins has been considered robust to the future effects of OA according to several limited reports (Dupont et al., 2013). The intestine is the main digestive organ in sea urchins, and its morphology and structure can affect the food digestion and absorption efficiency (Chang et al., 2005). It has been reported that OA can induce linear reduction of both gastric pH and digestive efficiency in sea urchin larvae (Stumpp et al., 2013); this result motivated us to explore the impact of OA on intestinal morphology and the digestion related processes of adult sea urchins. The temperate edible sea urchin Strongylocentrotus intermedius, which was introduced from Japan in 1989 by Dalian Ocean University, is widely cultivated along the coastal areas of Liaoning and Shandong Provinces. To date, S. intermedius has been the predominant commercially valuable sea urchin species, with an annual aquaculture output

Corresponding authors. E-mail addresses: [email protected] (C. Li), [email protected] (Y. Chang).

https://doi.org/10.1016/j.marpolbul.2020.110944 Received 1 August 2019; Received in revised form 20 January 2020; Accepted 27 January 2020 Available online 14 February 2020 0025-326X/ © 2020 Elsevier Ltd. All rights reserved.

Marine Pollution Bulletin 153 (2020) 110944

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of > 200 tons (Chang et al., 2004). Our previous study indicated that seawater pH decline can delay early embryonic cleavage, reduce the hatching rate of blastulae, and lower the four-armed larvae survival rate. Furthermore, impaired larval symmetry, shortened larval spicules, and corrosion spicule structure were also observed in larval S. intermedius reared under different OA conditions (Zhan et al., 2016). In the current study, we explored the impact of chronic OA-treatments on the growth, survival, feeding, intestinal morphology, and enzyme activities related to intestinal digestion and energy metabolism in adult S. intermedius, and then analyzed expression alterations of two immune-related genes for preliminarily analysis of the underlying molecular mechanisms. Our observations will provide more information for us to fully evaluate the comprehensive effects of OA on the entire life cycle of sea urchins.

2.3. Growth, survival, and feeding in each experimental group At the beginning of the experiment, 120 active and healthy S. intermedius (average test diameter 4.80 ± 0.12 cm and average wet weight 55.27 ± 3.56 g) were selected at random. We then divided the sea urchins randomly into 12 tanks (length × wide × height = 50 cm × 35 cm × 40 cm) of 10 individuals each (three replicate tanks for each pH value). Sea urchins in each tank were dried with a paper towel and weighed on a digital balance (0.01 g sensitivity; AL204; Mettler Toledo, Shanghai, China) to obtain initial average mass (W0). The specific growth rate (SGR), relative test diameter growth rate (RD), survival rate (SR), average food consumption (FC) of individuals, daily feeding rate (FR), and relative feeding rate (RFR) were calculated using the following formulae:

SGR (%·day 1) = 100 × (ln Wt

2. Materials and methods

RD = (Dt

2.1. Sea urchins and maintenance

ln W0 )/ t;

D0 )/[(Dt + D0 )/2]

SR (%) = 100 × (Nt /N0 );

S. intermedius (average test diameter: 4.80 ± 0.12 cm, average wet weight: 55.27 ± 3.56 g) were transported from Dalian Longwangtang Fisheries Company to the Key Laboratory of Mariculture & Stock Enhancement in the North China Sea, Ministry of Agriculture and Rural Affairs at the Dalian Ocean University in May 2017. Two weeks prior to experimentation, all sea urchins were kept in ~1000-L recirculating sea water tanks and acclimated to default laboratory conditions [20.00 ± 2.00 °C and 31.26 ± 0.09 practical salinity units (PSU)] under natural light. Seawater was sand filtered and continuously aerated. All sea urchins were fed with kelp (Saccharina japonica). The experiments were conducted between June 2017 and August 2017.

FC (mg·individual 1) = (TBt FR (%·day 1) = 100 ×

RBt )/ Nt ;

(TBt /Nt

RBt / Nt )/[(Wt + W0 )/2 × t ];

RFR = FR1/ FR 0 where Wt is the average body weight (g) of live S. intermedius on day t in each tank; t is the duration of the experiment; D0 is the initial average test diameter of live S. intermedius in each tank; Dt is the final average test diameter of live S. intermedius on day t in each tank; N0 is the initial number of live S. intermedius in each tank; Nt is the number of live S. intermedius on day t in each tank; TBt is the bait supplied on day t in each tank; and RBt is the total amount of remaining bait on day t in each tank (Lawrence et al., 2009; Agatsuma, 2000). FR1 is the daily feeding rate of live S. intermedius in the experimental group, and FR0 is the daily feeding rate of live S. intermedius in the control group.

2.2. CO2-driven OA treatments and sea water chemistry We set up one present natural seawater group (Control) and three OAstress groups (OA1, ΔpHNBS = − 0.3 units; OA2, ΔpHNBS = − 0.4 units; OA3, ΔpHNBS = − 0.5 units) in the current study based on the projected ocean pH for 2100 as reported by the IPCC (IPCC, 2013) (Table 1). Sea water of the control group was bubbled with ambient air and filtered but not manipulated as to ambient pH. Sea water of each OA-treatment group was adjusted by injection of synthetic gas (Dalian Special Gases Co., Ltd.) with a CO2-driven OA scenarios system (No. ZL201320267332.7) (Zhan et al., 2013) (Table 1). Conditions of each group were monitored daily by using a pH meter (HI9124, HANNA, Italy) and water quality monitor (6920, YSI, USA) during the entire course of the experiment. The other seawater parameters including temperature (T), and salinity (S) of each experimental group were determined by a water quality monitor (6920, YSI, USA). Total alkalinity (AT) of the sea water in each experimental group was measured by potentiometric titration once a week (Mehrbach et al., 1973). Partial pressure of carbon dioxide (pCO2), and calcite saturation (ΩCa) values for each experimental treatment were calculated based on salinity, temperature, pHNBS and AT data using SWCO2 software (Pierrot et al., 2006). The seawater parameters of each experimental group are reported in Table 1.

2.4. Intestine morphological examination and enzyme activity analyses All living S. intermedius in each replicate per treatment were collected randomly and placed on ice at the end of the 60-day incubation. Intestines of each S. intermedius were dissected carefully, and then divided into three parts: one part for further intestine morphological examination, one part for enzyme activity analyses, and the third part for further molecular expression analysis. For intestine morphological examination, tissue samples were fixed for 24 h in Bouin's Fluid as described by Wu et al. (2007) and then transferred into different concentrations of ethanol for gradient dehydration. Fixed tissues were embedded in paraffin wax, and 5 μm sections were stained with hematoxylin and eosin using routine methods of hematoxylin-eosin staining for further histological analysis. Intestine morphology was detected under an optical microscope (Leica, Germany). Activities of ten enzymes, including three digestive enzymes (amylase, AMS; cellulose, CL; and lipase, LPS), three rate-limiting enzymes of

Table 1 Seawater parameters of each experimental group. Carbonate system T (°C) Control OA1 OA2 OA3

20.00 20.00 20.00 20.00

± ± ± ±

2.00 2.00 2.00 2.00

S (‰) 31.26 31.28 31.30 31.36

± ± ± ±

0.09 0.12 0.11 0.10

pHNBS 8.10 7.82 7.68 7.55

± ± ± ±

−1)

pCO2 (μatm)

10.18 20.04 18.37 19.51

526.22 ± 21.41 1058.92 ± 72.42 1566.05 ± 62.70 2125.14 ± 94.44

AT (μmol kg

0.03 0.03 0.03 0.04

2367.63 2362.58 2366.42 2372.33

± ± ± ±

ΩCa 4.01 2.28 1.66 1.28

± ± ± ±

0.32 0.17 0.14 0.12

T, temperature; S, salinity; AT, total alkalinity; pCO2, partial pressure of carbon dioxide; ΩCa, calcite saturation. Values of pCO2, ΩCa were determined as described in “Materials and methods”. 2

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glycolysis (hexokinase, HK; phosphofructokinase, PFK; and pyruvate kinase, PK), three rate-limiting enzymes of tricarboxylic acid cycle (TAC) (citrate synthetase, CS; succinate dehydrogenase, SDH; and glutamate dehydrogenase, GDH) and lactate dehydrogenase (LDH) were analyzed. Prior to enzyme activity analyses, intestine samples of each experimental group were dried on paper towels and homogenized immediately in an ice bath. Intestine homogenates were then centrifuged at 3500 rpm, 4 °C, for 10 min. Supernatants were stored at −80 °C. Activity analyses of the ten enzymes were carried out by using commercial kits (Nanjing Jiancheng Bioengineering Institute, China; for corresponding commercial kit number, see Table S1). Enzyme activities of intestine homogenates were measured according to the instructions of the corresponding kits. Examined enzyme activities were defined as the unit per gram or unit per milligram of protein (U/gprot or U/ mgprot) as follows:

AMS (U·mgprot 1) = (A 0

CL (U·mgprot 1) = (A3

LPS (U·mgprot 1) = (AT0 HK (U·gprot 1) = (AT1

PK (U·gprot

= (AT0

CS (U·gprot 1) = (AT0 SDH (U·gprot 1) = (AT0

A2)/(A1

=2

[Ct (sample) Ct (reference)] [Ct (control) Ct (reference)]

All data were expressed as the mean ± standard deviation (S. D.). All statistical analyses were performed with SPSS 22.0 (IBM, Shanghai, China). We first confirmed that our data were normally distributed and homogeneous using the Shapiro-Wilk test and the Levene's test. The data of RFR (1–7 d) were not normally distributed, and all the original data on RFR (1–56 d) were converted to a normal distribution by the square root transformation. Statistical differences on SR, SGR, RD, RFR, FC, enzyme activities and gene expression among treatments were determined by one-way ANOVA (factor: pH). The data for RFR (1–7 d, 8–14 d, 22–28 d, 29–35 d, 36–42 d) had unequal variance, and thus Tamhane's T2 test was employed for statistical analysis. We considered p < 0.05 as statistically significant and p < 0.01 as extremely significant.

A 0) × C1 × V1/V0/C0 /T;

AT0)/6.22/T × V1/V0/C0 ; AT1)/C0 × 1000;

AT1)/6.22/T × V1/V0/C0 ;

3. Results

AT1)/13.6 × 10 3/T/25/C0 /10 × 1000;

3.1. Impact of chronic OA on growth, survival, and feeding of S. intermedius

AT1)/0.01/T/V0/C0 × 1000;

A2)/(A1

Ct

2.6. Data analysis

AT1)/A1 × C1 × V1/V0/T/C 0 ;

GDH (U·gprot 1) = 643 × (AT0 LDH (U·gprot 1) = (A3

2

A3)/A 0 × 0.4 × 0.5/10 × 30/7.5/V0/C0 ;

PFK (U·gprot 1) = 450 × (AT0 1)

ROX Reference Dye II, 2 μL cDNA template, and 6 μL ddH2O. The qRTPCR cycling conditions were: 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s; and 60 °C for 30 s. PCR melting curve analysis was employed to confirm unique PCR products. The comparative 2-ΔΔCT method was used to calculate the relative expression level as follows (Livak and Schmittgen, 2001):

AT1)/C0 × 1000;

During the 60-day incubation, pH-dependent decreases in S. intermedius growth, survival, and feeding among all treatments were observed (Fig. 1). Further statistical analysis indicated that the SGR (p < 0.01, F = 358.89; Fig. 1-A) and the RD decreased extremely significantly between control and all OA treatments during the course of incubation (p < 0.01, F = 73.46; Fig. 1-B). High survival rates (> 90%) were also observed across all experimental groups at the end of the 60-day incubation (p > 0.05; Fig. 1-C), whereas significantly reduced average FC of S. intermedius incubated in the OA3-group was observed compared to the control after 56 days of incubation (p < 0.05, F = 4.95; Fig. 1-D). In terms of the relative feeding rate, there were no significant differences among all experimental groups during the 56-day incubation (p > 0.05; Fig. 2).

A 0) × C1/C 0 ;

where A0 is the light absorption value in a blank sample; A1 is the light absorption value of a standard sample; A2 is the light absorption value of a control sample (no reaction); A3 is the light absorption value of an experimental sample (in the reaction); AT0 is the light absorption value before the reaction; AT1 is the light absorption value after the reaction; C0 is the homogenate protein concentration; C1 is the standard sample concentration; V0 is the sample volume; V1 is the total volume of reaction liquid, and T is the catalytic reaction time. Protein concentration of intestine homogenate was determined by the Bradford method with bovine serum albumin as the standard. All determinations were carried out on a multimode reader (SpectraMax i3x, Molecular Devices, USA). 2.5. mRNA expression of SiTNF-14 and SiTGF-β

3.2. Impact of chronic OA on intestinal morphology, digestive enzyme activities, and expression of SiTNF14 and SiTGF-β in S. intermedius

Since TNF14 (LIGHT) and TGF-β play critical roles in inflammation among most eukaryotes (Krause et al., 2014; Yoshimura et al., 2010), we determined and compared the mRNA expression levels of SiTNF14 and SiTGF-β in the intestines of S. intermedius under different seawater pH conditions by using quantitative real-time PCR (qRT-PCR). Total RNA of intestinal tissues of S. intermedius was extracted using a kit for marine animal RNA (TIANGEN, Beijing) following the manufacturer's instructions. The integrity of RNA was evaluated using 1.0% agarose gel electrophoresis. The purity and concentration of total RNA were determined by a nucleic acid purity analyzer (Implen NanoPhotometer, Germany). A PrimeScript RT Enzyme Mix I reverse transcriptase kit (Takara, Japan) was used to synthesize the first chain of cDNA according to the manufacturer's instructions. Primer Premier 5.0 software was used to design primers used for qRT-PCR analyses (Table S2). β-actin was used as a reference gene. The qRT-PCR was performed on an ABI 7500 fluorescent quantitative PCR (ABI, USA) using TB Green TM Premix Ex Taq TM II (Takara, Japan). Total volume of the qRT-PCR reaction system was 20 μL containing 10 μL TB Green Premix Ex Taq II, 0.8 μL (10 μmol/L) of each primer (Table S2), 0.4 μL

Histological images showed that the intestinal plica circulars of S. intermedius cultured in the control condition were arranged neatly and tightly; the inner microstructures of the intestinal wall were compact and tight, while the morphology and histology of intestines were altered in all OA-treated S. intermedius groups. As seawater pH declined, the intestines exhibited the following: (1) the bowel was enlarged with a rounded margin and pale color as seawater pH declined; (2) loosed inner microstructures, swollen cells and deformation (even disappearance) of plica circulars were observed; (3) tissue hollowing was increasingly serious with the decrease in seawater pH; and (4) inflammatory cells were increased in the OA1 and OA2 groups, and more inflammatory cells had infiltrated the inner structure of S. intermedius intestines as seawater pH declined (Fig. 3). The impact of chronic OA on intestinal digestive enzyme activities was determined by calculating activities of amylase, cellulase, and lipase in the intestines of S. intermedius cultured in all experimental groups (Fig. 4). In general, activities of cellulase and lipase in the intestines of S. intermedius exhibited linear declining trends with decreased seawater pH, whereas amylase activity in all experimental 3

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Fig. 1. Impact of chronic CO2-driven ocean acidification on specific growth rate (SGR) (A), relative test diameter growth rate (RD) (B), survival rate (SR) (C) and daily food consumption (FC) (D) in adult Strongylocentrotus intermedius. Control, pHNBS = 8.10 ± 0.03; OA1, ΔpHNBS = − 0.3 units; OA2, ΔpHNBS = − 0.4 units; OA3, ΔpHNBS = − 0.5 units. * Significant differences at p < 0.05 vs. Control; ** extremely significant differences at p < 0.01 vs. Control. Error bars = S. D.; n = 3.

groups did not differ significantly (p > 0.05). Extremely significant reduction of intestinal cellulase activity was observed in all OA-treated groups compared to control (p < 0.01, F = 128.85). Significant reduction of intestinal lipase activity was found between the OA1-treated group and the control (p < 0.05, F = 15.20), and intestinal lipase

activities in both OA2- and OA3-treated groups were extremely significantly reduced (p < 0.01, F = 15.20). Expression alterations of the tumor necrosis factor 14 (here designated as SiTNF14) gene and the transforming growth factor beta (here designated as SiTGF-β) gene in OA-treated groups and the control group

Fig. 2. Impact of chronic CO2-driven ocean acidification on relative feeding rate (RFR) in adult Strongylocentrotus intermedius. Control, pHNBS = 8.10 ± 0.03; OA1, ΔpHNBS = − 0.3 units; OA2, ΔpHNBS = − 0.4 units; OA3, ΔpHNBS = − 0.5 units. Error bars = S. D.; n = 3. 4

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Fig. 3. Impact of chronic CO2-driven ocean acidification on intestinal morphology in adult Strongylocentrotus intermedius. A and A': intestinal morphology images of S. intermedius cultured in the control group (pHNBS = 8.10 ± 0.03); B and B′: The intestinal morphology images of S. intermedius cultured in OA1 group (ΔpHNBS = − 0.3 units); C and C′: The intestinal morphology images of S. intermedius in the OA2 group (ΔpHNBS = − 0.4 units); D and D': The intestinal morphology images of S. intermedius in the OA3 group (ΔpHNBS = − 0.5 units). Arrows indicate plicae circulars; stars indicate the hollowing inner structure. Black squares represent areas for further amplification.

were determined using qRT-PCR (Fig. 5). As shown in the figure, one intestinal SiTNF14 expression peak was observed in the OA1-treated group with an extremely significant upregulation compared to the control (p < 0.01, F = 304.16; Fig. 5-A). In addition, transcript levels of SiTGF-β in the intestines of S. intermedius cultured in all OA-treated groups decreased extremely significantly compared to the control (p < 0.01, F = 43.84; Fig. 5-B).

(Zhan et al., 2016), we also found in this study that the growth of adult S. intermedius was significantly affected by OA stress. All of the above observations suggest that there are negative effects of OA on the growth of sea urchins at each stage of the life cycle. Meanwhile, we also found that most of the adult S. intermedius reared in the OA-treated groups survived during the 60-day incubation, which suggested that chronic OA treatment has no negative effects on the survival of adult S. intermedius. This result is consistent with the reports from Strongylocentrotus droebachiensis and Hemicentrotus pulcherrimus (Kurihara et al., 2013; Stumpp et al., 2012a, 2012b). Contrary to the robustness of adult S. intermedius to OA stress, we demonstrated previously that the survival rates of four-armed larvae of S. intermedius decreased as the seawater pH declined (Zhan et al., 2016). Therefore, all observations obtained previously and in the current study supported the hypothesis that the early development of sea urchins is more sensitive to OA stress, while adult sea urchins (such as S. droebachiensis) were robust to acute OA stress and chronic OA stress (Dupont et al., 2013). Although we did not find the negative effects of chronic OA (60-day incubation) on survival of adult S. intermedius, reduced growth and depressed daily food consumption were observed, especially in the OA3 group’ both measures were significantly lower than those of the natural seawater group (p < .05). In addition, slightly depressed relative feeding rates were found in our study. This result is similar to the change of feeding rate of adult Heliocidaris crassispina and H. pulcherrimus under the conditions of acidification of sea water (Wang et al., 2013; Kurihara et al., 2013).

3.3. Impact of chronic OA on rate-limiting enzyme activities related to intestinal energy metabolism on S. intermedius The responses of the glycolytic pathway (or Embden-Meyerhof pathway, EMP) and TAC to chronic OA stress were determined by comparing rate-limiting enzyme activities in the intestines of S. intermedius among all experimental groups (Fig. 6). For three rate-limiting enzymes of the glycolytic pathway, both HK and PFK exhibited significant activity reduction in the intestines of S. intermedius incubated in all OA-treated groups compared to the control (HK: p < 0.01, F = 23.74; PFK: p < 0.01, F = 158.76), while PK activities in all OAtreated groups were increased in a linear pH-dependent way compared those of the control (p < 0.01, F = 249.80). The activities of the three rate-limiting enzymes of the TAC pathway showed a consistent and extremely significantly decreasing trend as seawater pH declined (CS: p < 0.01, F = 50.02; SDH: p < 0.01, F = 53.52; GDH: p < 0.01, F = 316.88). The activity of LDH was extremely reduced in the OA1-treated group but increased in the OA3treated group (p < 0.01, F = 78.60) compared with the control group, and there was no significant difference in OA2-treated group (p > 0.05).

4.2. Impact of chronic OA on morphology, digestive enzyme activities, and expression of SiTNF14 and SiTGF-β in the intestines of adult S. intermedius

4. Discussion

Altered intestinal histology was observed in S. intermedius cultured in the OA-treated groups, indicating that chronic OA-stress can damage the digestive function of adult S. intermedius by causing inflammation. Combined with the feeding data observed in this study, we therefore assume that the depressed feeding rate and food consumption after 30day incubation might be caused by intestinal function damage. It has

4.1. Impact of chronic OA on growth, survival, and feeding of S. intermedius Our previous study indicated that embryonic cleavage and larval growth of S. intermedius can be delayed and depressed by OA stress

Fig. 4. Impact of chronic CO2-driven ocean acidification on intestinal digestive enzyme activities in adult Strongylocentrotus intermedius. (A) Intestinal amylase activities in S. intermedius incubated in each experimental group; (B) intestinal cellulase activities in S. intermedius incubated in each experimental group; (C) intestinal lipase activities in S. intermedius incubated in each experimental group. Control, pHNBS = 8.10 ± 0.03; OA1, ΔpHNBS = − 0.3 units; OA2, ΔpHNBS = − 0.4 units; OA3, ΔpHNBS = − 0.5 units. * Significant differences at p < 0.05 vs. Control; ** extremely significant differences at p < 0.01 vs. Control. Error bars = S. D.; n = 3.

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(Stumpp et al., 2013). Although we did not investigate the alteration of intestinal pH and digestive efficiency in this study, our data and previous data from pluteus larvae of S. droebachiensis suggest that digestion is a critical process that we need to pay more attention to throughout the life cycle of sea urchins in the context of ocean acidification. Digestive enzyme activities are one of the key factors that may affect digestion in organisms. A linear reduction of cellulase and lipase activities was observed in the intestines of S. intermedius as the seawater pH decreased. The digestion of carbohydrates is the main concern in almost all studies on feeding in sea urchins (Lawrence et al., 2013). It was also reported that sea urchin cellulase activity on native cellulose is minimal; the digestion of native cellulose needs some contribution from gut bacterial enzymes in sea urchins (Lawrence et al., 2013). Thus, further research work should be carried out on the relative contributions to digestion between host enzymes and bacterial enzymes in the intestines of sea urchins under OA conditions. Moreover, it has been documented that the strategy of pluteus larvae of S. droebachiensis is compensatory feeding (Stumpp et al., 2013); interestingly, we did not detect the occurrence of any compensatory feeding in adult S. intermedius reared under OA conditions in this study. We therefore speculate that adult S. intermedius might adopt other strategies in response to OA. All of the above suggest that sea urchins may adopt different strategies in response to OA stress at different stages of the life cycle, further study should be carried out to clarify life stage-specific strategies of sea urchins in response to OA. Inflammation is another factor that is assumed to contribute to the functional damage to the intestines of adult S. intermedius incubated under OA conditions in this study. Several studies have reported that constitutive expression of TNF14 (LIGHT) results in T cell-mediated inflammation that causes tissue destruction (Holmes et al., 2014). Only one significantly increased expression peak was detected in the OA1group compared to control after the 60-day incubation in the current study, while there was no significant difference in SiTNF14 expression in the OA2-group or the OA3-group after the 60-day incubation compared to that of control. We speculate that the stress response of SiTNF14 might vary under different OA conditions. It has been well documented that TGF-β signals play critical roles in inflammation and repair mechanisms, including angiogenesis and in the regulation of host resistance in eukaryotes (Orieux et al., 2013; Vizzini et al., 2016). Our previous study indicated that the expression trend of the TGF-β gene in four-armed larvae of Hemicentrotus pulcherrimus increased significantly with the decrease of seawater pH under different ocean acidification conditions compared to that of natural seawater (Hu, 2017). We also recently reported that the relative expression of intestinal TGF-β mRNA exhibited a trend of first decreasing and then

Fig. 5. Impact of chronic CO2-driven ocean acidification on relative expression level of (A) SiTNF14 and (B) SiTGF-β in intestines of adult Strongylocentrotus intermedius. Control, pHNBS = 8.10 ± 0.03; OA1, ΔpHNBS = − 0.3 units; OA2, ΔpHNBS = − 0.4 units; OA3, ΔpHNBS = − 0.5 units. ** extremely significant differences at p < 0.01 vs. Control. Error bars = S. D.; n = 3.

been reported that linear reduction of both gastric pH and digestive efficiency can be observed when pluteus larvae of Strongylocentrotus droebachiensis are chronically exposed to seawater with a low pH value

Fig. 6. Impact of chronic CO2-driven ocean acidification on intestinal metabolism enzyme activities in adult Strongylocentrotus intermedius. Control, pHNBS = 8.10 ± 0.03; OA1, ΔpHNBS = − 0.3 units; OA2, ΔpHNBS = − 0.4 units; OA3, ΔpHNBS = − 0.5 units. ** extremely significant differences at p < 0.01 vs. Control. Error bars = S. D.; n = 3.

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increasing with the extension of acidification incubation time in the sea urchin Meconocrotus nudus (Li et al., 2019). All these observations suggest that TGF-β and its correlated signal pathways might be one of the most sensitive links throughout the life cycle and may have varied effects in different species of sea urchins in the context of ocean acidification.

Appendix A. Supplementary data

4.3. Impact of chronic OA on rate-limiting enzyme activities related to energy metabolism in the intestines of S. intermedius

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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marpolbul.2020.110944. References

Increasing evidence has accumulated concerning the impact of OA on energy reallocation and limitation in larvae and adults of echinoderm species (Collard et al., 2014; Evans et al., 2017; Gooding et al., 2009). Glycolysis and TAC are two main energy producing pathways regulated by rate-limiting enzymes. Pyruvate kinase (PK) regulates cell energy metabolism via catalyzing the conversion of phosphoenolpyruvate and adenosine diphosphate (ADP) to pyruvate and adenosine triphosphate (ATP) in glycolysis. Subsequently, LDH catalyzes the conversion of pyruvate to lactate, which is crucial for glycolysis to continue production of ATP (Seth et al., 2011). In this study, we found an obvious linear acceleration of PK activity in the intestines of adult S. intermedius with declining seawater pH. This reflects an acceleration of pyruvate production speed to some extent. In addition, increased LDH activities in the intestines of adult S. intermedius incubated under different OA conditions were observed in this study. It has been demonstrated that LDH can accelerate ATP production through anaerobic processes to maintain energy homeostasis under stressful conditions in organisms (Hu et al., 2014). Combining the PK and LDH activity analyses, we thus assume that OA accelerates glycolysis in sea urchins, which is consistent with the reports from Yesso scallop and Atlantic cod (Kreiss et al., 2015; Liao et al., 2018). The TAC is the most productive metabolic pathway in aerobic organisms (Bender, 2003). Significantly reduced TAC rate-limiting enzyme activities indicate the reduction of aerobic processes in the intestines of adult S. intermedius as seawater pH decreased. Altogether, increased PK and LDH activities and reduced TAC rate-limiting enzyme activities observed in this study reflect the enhancement of anaerobic processes and reduction of aerobic processes in the intestines of adult S. intermedius after OA exposure. These results are in line with previous studies suggesting an alteration of energy metabolism in marine organisms under OA-challenging conditions (Cao et al., 2018; Hüning et al., 2012). CRediT authorship contribution statement Yaoyao Zhan: Conceptualization, Methodology, Data curation, Writing - original draft. Dongyao Cui: Investigation, Data curation, Writing - original draft. Dongfei Xing: Investigation, Data curation. Jun Zhang: Investigation, Data curation. Weijie Zhang: Data curation. Yingying Li: Investigation. Cong Li: Conceptualization, Methodology, Data curation. Yaqing Chang: Conceptualization, Methodology, Data curation. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was funded by the Central Public-interest Scientific Institution Basal Research, CAFS & Key Laboratory of Sustainable Development of Marine Fisheries, the Ministry of Agriculture and Rural Affairs, PR China (No. 2019HY-XKQ01), the National Natural Science Foundation of China (No. 31672652) and the Natural Science Foundation of Liaoning Province (No. 20170540104). 8

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