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Integrative Zoology 2017; 12: 361–370 doi: 10.1111/1749-4877.12229

ORIGINAL ARTICLE

Anaerobic metabolism and thermal tolerance: The importance of opine pathways on survival of a gastropod after cardiac dysfunction

Guodong HAN,1,2 Shu ZHANG1 and Yunwei DONG1,2

1State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China and 2Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen University, Xiamen, China

AbstractOrganisms on rocky shores are frequently exposed to high temperatures, which cause impairment of cardiac function and retard cellular oxygen delivery. However, some gastropods can survive at several degrees Celsius higher than their Arrhenius break temperature of cardiac function (ABT), indicating the importance of anaero-bic metabolism for their thermal tolerance. We measured the global molecular responses to heat stress in limpet Cellana toreuma using 454 GS-FLX to investigate the variations of genes involved in anaerobic metabolism at high temperatures. Next, the gene expression levels of 4 anaerobic enzymes and activity of alanopine dehydro-genase (AlDH), which is involved in opine pathway, were measured in response to elevated temperature. A total of 19 heat shock proteins (HSPs) were determined using real-time PCR at different temperatures. At high tem-peratures, the extensive upregulation of HSP genes was an effective but energetically expensive form of pro-tection to prevent thermal damage. The upregulation of hypoxia-inducible factor 1 alpha mRNA indicated the condition of cellular hypoxia and the high gene expression and enzyme activity of AlDH suggested that opine pathway was the main anaerobic pathway. These results implied that anaerobic metabolism was enhanced to provide energy in the face of thermal stress. Our findings highlight the ecological significance of the anaerobic metabolism of gastropods to thermal adaptation. For predicting the ecological impact of global warming on the distribution of gastropods, the role of anaerobic pathways should be evaluated.

Key words: anaerobic metabolism, cardiac function, enzyme activity, thermal stress, transcriptome

Correspondence: Yunwei Dong, State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, 361005, China.Email: dongyw@xmu.edu.cn

INTRODUCTIONGlobal climate change affects all ecological interac-

tions (Solomon 2007; Robinet & Roques 2010; Oswald & Arnold 2012). In fact, intertidal ecosystems have shown noticeable ecological impacts, such as the rap-id biogeographic changes (Barry et al. 1995; Helmuth

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et al. 2006b). Intertidal organisms frequently encounter extreme thermal stress during aerial emersion (Helmuth 2002; Buckeridge 2010; Lima et al. 2010; Zippay & Helmuth 2012), and now they are experiencing tempera-tures at or above their thermal tolerance limits during times when a low tide occurs simultaneously with a heat wave (Helmuth 2002; Stillman 2003; Helmuth et al. 2006a).

Limitations in cardiac function that influence aerobic metabolic capacity have been a central focus in analyz-ing the effects of climate change on marine species (re-viewed by Somero [2012], but see Stenseng et al. [2005], Braby and Somero [2006], Pörtner [2010], Dong and Williams [2011], Marshall et al. [2011] and Han et al. [2013]). Thermal limits of marine ectotherms are con-trolled by the aerobic metabolic capacity, and a mis-match between the demand for oxygen and the capacity of oxygen supply to tissues restricts whole-animal toler-ance to thermal extremes (Pörtner & Knust 2007). In the latest studies, there is also evidence suggesting that the window of thermal tolerance will be widened by the in-creasing contribution of haemocyanin oxygen transport (Giomi & Portner 2013). As the capacity of oxygen sup-ply is closely related to cardiac performance (Frederich & Pörtner 2000), cardiac function in a wide variety of marine taxa may be one of the links in the physiological chain as global change progresses (Somero 2012).

The sensitivity of cardiac function in setting ther-mal limits varies among taxa (Somero 2012). Collapse of cardiac function can cause death in porcelain crabs (genus Pertrolisthes), and once the body temperature exceeds the Arrhenius break temperature (ABT), the temperature at which a sharp discontinuity in slope oc-curs in an Arrhenius plot, heart function cannot recover from heat stress (Somero 2002). In contrast, some gas-tropods can survive at several degrees higher tempera-ture than their ABTs (Stenseng et al. 2005; Dong et al. 2015). As the collapse of cardiac function means a lim-itation of oxygen circulation, these studies suggest that there may be physiological adaptations to anaerobic me-tabolism in gastropods. For these reasons, we suppose that anaerobic metabolism contributes to the survival in high temperature during cardiac dysfunction. Four main anaerobic pathways (Fig. 1), including lactate path-way (end-product lactate), opine pathway (end-product opines), glucose-succinate pathway (end-product suc-cinate) and aspartate-succinate pathway (end-product succinate), are involved in anaerobic metabolism in in-tertidal animals (Hochachka & Somero 1984; Living-stone 1991). Three key enzymes, including cytosolic

malate dehydrogenase (cMDH), lactate dehydrogenase (LDH) and opine dehydrogenase (OpDH), are rate-lim-iting enzymes in these 4 anaerobic pathways, respec-tively. cMDH plays an important role in anaerobic me-tabolism and its kinetics and stability are closely related to species distribution in the face of climate change (Fields et al. 2006; Dong & Somero 2009; Logan et al. 2012). Large and rhythmic variations of cMDH in the transcriptional level were observed in field populations of Mytilus californianus over a couple of tidal cycles (Gracey et al. 2008). Opine pathways are characterized of a low efficiency but high rates of energy production pathway in lower and middle phyla of marine inverte-brates, and use free amino acids as substrates (Living-stone 1991).

The limpet Cellana toreuma (Reeve, 1855) is wide-ly distributed along the Asian coast, and suffers from frequent thermal stress on the rocky shore (Han et al. 2013; Dong et al. 2015; Huang et al. 2015). This lim-pet can survive temperatures >4 °C higher than its ABT (Han et al. 2013; Dong et al. 2014; Zhang et al. 2014). In the present study, a transcriptomic profile was con-ducted using 454 GS-FLX in this species to comprehen-sively understand the transcriptional responses to heat stress, and genes related to heat shock response, cel-lular hypoxia and anaerobic metabolism were also de-termined using real-time PCR. The activity of enzyme AlDH, which represents differential gene expression in response to thermal stress, was also determined. This study aims to test the hypothesis that anaerobic metabo-lism, especially opine pathway, is important for gastro-pods surviving in high temperature during cardiac dys-function and is useful for evaluating and predicting the impacts of climate change on marine gastropods.

MATERIALS AND METHODS454 sequencing, de novo assembly, annotation and differentially expressed genes

A total of 18 individuals of limpet C. toreuma were collected at Xiamen Island (24°25′N, 118°09′E), Fuji-an, China, and were then transported back to the State Key Laboratory of Marine Environmental Science and kept in acclimation conditions. After 1-week acclima-tion at 18 °C, the Arrhenius break temperature (ABT, the temperature at which the heart rate decreases dra-matically) was measured based on the same method de-scribed in (Han et al. 2013). The heartbeat was viewed and recorded using Powerlab AD converter (ADInstru-ments, March-Hugsteeten, Germany). For determining the ABTs, discontinuities in the slopes of heart rate with

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Anaerobic metabolism & thermal tolerance

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temperature were calculated from intersections of fitted 2-phase regressions according to the minimum sum of squares using SigmaPlot 12.5 (SSPS, Point Richmond, CA, USA) (Giomi & Portner 2013).

Then animals were randomly divided into 2 groups with each consisting of 9 limpets. One group (control group) was kept at 18 °C, while the other group (heated group) was heated at a rate of approximately 0.1 °C per min using a water bath. When body temperature exceed-ed the mean ABT, samples were collected and stored in liquid nitrogen immediately. Total RNA was extract-

ed from approximately 100 mg of foot muscle using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) fol-lowing the manufacturer’s recommendations.

The total RNA from each group was sequenced us-ing a 454 GS-FLX sequencer at BGI (Shenzhen, Guang-dong, China). Raw reads generated from the 2 libraries were preprocessed to trim adaptors and remove non-sense reads by BGI (Shenzhen, Guangdong, China). Clean reads from the 2 libraries were assembled using a CLC Genomics Workbench 8 de novo assembly tool (CLC bio, Aarhus, Denmark). Based on the user man-

Figure 1 Changes of main genes involved in anaerobic metabolism pathways in Cellana toreuma. Four main anaerobic pathways, including glucose-succinate pathway, asparate-succinate pathway, lactate pathway and opine pathway are well covered in C. toreu-ma. When temperature reached ABT, the gene expression was determined using transcriptomic profile (the red frame denotes an in-crease, the green frame denotes a decrease and the black frame denotes no differential expression). AlDH, alanopine dehydroge-nase; ALDO, fructose-bisphosphate aldolase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; cMDH, cytosolic malate dehydrogenase; ENO, 2-phosphoglycerate dehydratase; FRD, fumarate reductase; GAPDH, glyceraldehyde-3-phosphate de-hydrogenase; HK, hexokinase; GPI, glucose-6-phosphate isomerase; LDH, Lactate dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; PFK, phosphofructokinase; PGAM, phosphoglycero mutase; PGK, phosphoglycerate kinase; PK, pyruvate kinase; TaDH, taurine dehydrogenase.

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ual, parameters for the assembly were set as follows: word size value was set at 20 and bubble size value was set at 600. Reads were mapped back to the contigs with the default settings. Assembled contigs were annotated using a CLC Genomics Workbench 8 BLAST tool with the default settings. Blastx was chosen as the program and Swiss-prot and Non-redundant protein sequenc-es (Nr) were chosen as the database. The CLC Genom-ics Workbench 8 RNAseq analysis tool was employed to calculate the gene expression value for each group and the contig library was used as the reference tran-scriptome. The gene expression value was normalized to reads per kilobase per million (RPKM) (Mortazavi et al. 2008). Kal’s Z-test (Kal et al. 1999) was chosen to carry out the proportion test. The transcripts were considered as statistically significant different express genes (DEGs) if the false discovery rate (FDR) was smaller than 0.001 and the RPKM values showed at least 2-fold difference between the 2 groups.

Real-time polymerase chain reaction of selected genes

The C. toreuma specimens were collected at Xia-men Island and acclimated at 25 °C for 1 week. Limpets were heated at a rate of approximately 0.1 °C per min in air using a water bath. When the temperature reached the designated temperatures (30, 32, 34, 36, 38, 40, 42 and 44 °C), 4 specimens were randomly collected and recovered in 25 °C seawater for 2 h. Four specimens were kept in 25 °C seawater for 2 h as control. The rel-ative expressions of four genes involving anaerobic me-tabolism (AlDH, cMDH, HIF1A, LDH, TaDH) and 19 heat shock protein genes (Hsp75, HspA12A, Hspbap1, HspA14, Hsp22, Hsc-5, Hsp90, DnajB, Hsp10, Hsc70-4, HspA8, DnajA1, HspA4, Hsp20, Hsp68, CRYAA, Hsp70, CRYAB, Hspbp1) were analyzed using real-time PCR.

Total RNA was isolated from approximately 50 mg of foot muscle using Trizol Reagent (Invitrogen, Grand Island, NY, USA). The cDNA was synthesized using to-tal RNA as a template. Primers were designed using the software Beacon Designer 7 (PREMIER Biosoft, Palo Alto, CA, USA). Real-time PCR was carried out on a CFX96 Touch Real-Time PCR System (Bio-Rad, Berke-ley, CA, USA) in a 20-μL reaction volume containing 10 μL of 2× master mix (DyNAmo Flash SYBR Green qPCR Kit, Thermo Scientific, Waltham, MA, USA), 1 μL of cDNA template, 2 μL of primers and 7 μL of RNase-free water. PCR conditions were as follows: 95 °C 7 min; 40 cycles of 95 °C 20 s, 60 °C 1 min; and a fi-nal melt curve step. The efficiency of each primer pair

was determined by real-time PCR with an appropriate dilution series of cDNA prior to sample analyses (Table S1).

Relative expression of target genes was analyzed us-ing Bio-Rad CFX Manager 3.1 software (Bio-Rad, Berkeley, CA, USA), and 6 genes, including 18S rRNA, β-actin, β-tubulin, Paramyosin, Myosin heavy chain and Triosephosphate isomerase B, were selected as the ref-erence genes and used to normalize the expression of target genes. The statistical analysis of relative gene ex-pression data was conducted using IBM SPSS Statistics 20 (IBM Corporation, Armonk, NY, USA) and Clus-ter 3.0 (de Hoon et al., University of Tokyo, Human Ge-nome Center).

Enzyme activity of alanopine dehydrogenase The C. toreuma specimens were collected at Xia-

men Island and acclimated at 25 °C for 1 week. Limpets were heated at a rate of approximately 0.1 °C per min in air using a water bath. When the temperature reached the designated temperatures (30, 34, 36, 38, 40, 42 and 44 °C), 9 specimens were randomly collected and re-covered in 25 °C seawater for 2 h. Nine specimens were kept in 25 °C seawater for 2 h as control. The ABT for cardiac performance of this batch of C. toreuma speci-mens was determined following the methods described above. For the enzyme assays, 1 g foot muscle from 3 specimens was mixed together as one sample and ho-mogenized in 9 volumes of extraction buffer (50 mM potassium phosphate, KH2PO4/K2PO4, pH = 6.8; 1 mM DTT). Homogenate was centrifuged at 800 g for 1 h at 4 °C and the supernatant was used for the determination of enzyme activity.

The enzyme activity of alanopine dehydrogenase (AlDH) was performed in 200 μM midazole-HCl (pH = 7.0), 0.16 mM NADH, 1 mM pyruvate and 100 mM β-alanine at 25 °C. The change in absorbance during the linear disappearance of NADH at 340 nm was measured in an ultraviolet/visible spectrophotometer (UV-1800, SHIMADZU, Japan). The statistical analysis of enzyme activity was conducted using IBM SPSS Statistics 20 (IBM Corporation, Armonk, NY, USA). The difference in enzyme activity among different heat shock tempera-tures was analyzed using one-way ANOVA.

RESULTS

Sequencing, de novo assembly and differential gene expression

Two libraries were constructed from the control

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group and the heated group (Table 1). A total of 586 019 clean reads were produced from the control group, while 541275 clean reads were produced from the heat-ed group. After de novo assembly, a total of 29 471 con-tigs were generated from both libraries, with an average

size of 814 bp, and the N50 was 993 bp. A total of 2445 contigs showed differential gene expression. Among the 2445 contigs, 1287 genes were upregulated while 1158 genes were repressed in heated group.

Expression of genes related to cellular hypoxia and anaerobic metabolism

Based on the transcriptomic file experiment, 4 key enzymes, including LDH, AlDH, TaDH and cMDH, which are involved in anaerobic metabolism pathways, were identified and only AlDH increased at ABT (Fig. 1). The expression of 5 genes involving glycolysis, in-cluding HK, ALDO, GAPDH, PGAM and ENO, also in-creased at ABT, based on the results of DEGs (Fig. 1, Table S2).

The expression of genes involving cellular hypox-ia and anaerobic metabolism, including AlDH, cMDH, HIF1A, LDH and TaDH, were also analyzed at differ-ent temperatures using real-time PCR (Figs 2 and 3). The expression patterns of these genes were different.

Table 1 Summary of sequencing and de novo assembly of the 454 GS-FLX

SequencingControl Heated

Clean reads 586 019 541 275Average length (bp) 605.11 604.42Q20 percent 69.64% 69.76%

De novo assemblyUnigenes total number 29 471Average length (bp) 814N50 (bp) 993

Figure 2 Changes in relative gene expres-sion of enzymes involving anaerobic me-tabolism exposed to elevated temperatures. Bars with different letters indicate signifi-cantly different means (one-way ANO-VA followed by Tukey’s test, P < 0.05; N = 4 individuals per treatment). Error bars represent ± SE. There are no differenc-es among treatments in cMDH, LDH and TaDH.

AIDH * cMDH

Temperature (°C)

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One-way ANOVA showed that levels of HIF1A and AlDH changed at different temperatures (HIF1A, F8,27 = 3.289, P = 0.009; AlDH, F8,27 = 7.193, P < 0.001). Up-regulation of AlDH occurred at 34 °C and the maximal expression occurred at 38 °C. Upregulation of HIF1A occurred at 32 °C, and there were no significant differ-ences among different temperatures beyond 32 °C. Oth-er genes, including cMDH, TaDH and LDH, did not show significant variations among different tempera-tures (cMDH, F8,27 = 0.994, P = 0.462; TaDH, F8,27 = 1.332, P = 0.270 and LDH, F8,27 = 1.313, P = 0.279).

Alanopine dehydrogenase activity

Alanopine dehydrogenase activity was significantly different at different heat shock temperatures (one-way ANOVA, F7,16 = 8.970, P < 0.001). The enzyme activi-ty significantly increased at 40 °C and the maximal ac-tivity occurred at 44 °C (Fig. 4). The mean ABT for this batch of C. toreuma specimens was 41.62 °C (N = 5).

Heat shock proteins expression

Transcriptomic analysis showed that 86 contigs were heat shock-related proteins. After being blasted to Con-served Domain Database (Marchler-Bauer et al. 2011), these 86 heat shock contigs belonged to 26 heat shock proteins (HSP) in 5 major families of HSPs, including small HSPs, HSP40, HSP60, HSP70 and HSP90 (Ta-ble S3). Among these 86 heat shock-related contigs, 39 showed differential gene expression, and 38 contigs in-

creased after heat stress (Fig. 5, Table S2).The relative expressions of 19 HSP genes were fur-

ther analyzed in a series of temperatures (25, 30, 32, 34, 36, 38, 40, 42 and 44 °C) using real-time PCR. Krus-kal–Wallis’s test (k independent samples, P < 0.05) re-sults showed that levels of 15 HSP genes significantly increased in response to thermal stress (Fig. 6); how-ever, there was no significant upregulation in 4 HSP genes, including hsp75 (P = 0.391), hspbap1 (P = 0.912), hspA12 (P = 0.258) and hspA12A (P = 0.237).

DISCUSSIONThe effective opine pathways can ensure continuous

flux of glycolysis and a constant supply of ATP by pro-viding NAD+ for the intertidal gastropods, protecting against thermal stress during cardiac dysfunction. In the present study, only AlDH mRNA increased to meet the rising NAD+ requirement of glycolysis when the tem-perature reached ABT for cardiac function based on the results of the transcriptomic profile (Fig. 1). Real-time PCR results showed that upregulation of AlDH occurred at 34 °C and the maximal expression occurred at 38 °C (Fig. 2). The increase of AlDH activity was also identi-fied in response to thermal stress when the temperature exceeded ABT (Fig. 4). As one of the OpDHs, AlDH is believed to play an important role in maintaining rates of energy production during hypoxic conditions (Gäde & Grieshaber 1986). The opine pathways are regarded

Figure 3 Changes in relative gene expression of HIF1A ex-posed to elevated temperatures. Bars with different letters indi-cate significantly different means (one-way ANOVA followed by Tukey’s test, P < 0.05; N = 4 individuals per treatment). Er-ror bars represent ± SE.

Figure 4 Changes in activity of AlDH exposed to elevated temperatures. Bars with different letters indicate significantly different means (one-way ANOVA followed by Tukey’s test, P < 0.001; N = 3). Error bars represent ± SE. The dotted line is a Lowess curve for heart rate against temperature for 5 individu-als.

ity (I

Heart rate (beats/m

Temperature (°C) Temperature (°C)

Heart rateAIDH activity

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as analogues to the classical anaerobic glycolytic path-way (lactate pathway) (Harcet et al. 2013), but a sug-gested advantage of the opine pathway is that opine end products will not change the intracellular pH as much as lactate would, and, therefore, the intracellular osmot-ic pressure is constant (Zammit 1978). Intertidal inver-tebrate animals, including limpets, are characterized by containing rich amounts of free amino acid (Livingstone 1991). The upregulation of AlDH indicated the impor-tance of opine pathway and anaerobic metabolism on thermal tolerance of C. toreuma.

The upregulation of AlDH and enhancing anaero-bic metabolism of C. toreuma at high temperatures are adaptive responses to cardiac dysfunction and cellu-lar hypoxia. Hypoxia-inducible factor 1 alpha (HIF1A) is an essential mediator of O2 homeostasis (Semen-za 2000). Real-time PCR results showed that the ex-pression of HIF1A significantly increased at 36 °C (Fig. 3). HIF1A gene has been demonstrated to be affect-ed by the transcriptional regulation response to hypox-ia in marine organisms, and the upregulation of HIF1A

transcript levels is an important component in the adap-tation to hypoxia (Gracey et al. 2011; Liu et al. 2014). The expression patterns of HIF1A provide further evi-dence that high temperatures can induce cellular hypox-ia.

Apart from opine pathway, other anaerobic pathways were not stimulated at the transcriptional level in C. to-reuma during cardiac dysfunction. Neither transcriptom-ic nor real-time PCR results showed differential gene expression of cMDH and LDH in response to thermal stress in C. toreuma. These results suggested that cMDH and LDH may not be regulated at the transcriptional lev-el.

A mismatch between the demand for oxygen and the capacity to supply tissues with oxygen is the major mechanism which limits a species’ tolerance of thermal extremes (Pörtner 2006, 2010; Pörtner & Knust 2007). The thermal window of performance in water breathers matches their window of aerobic scope (Pörtner 2010).

Figure 5 The expression values of 39 heat shock-related con-tigs in control and heated groups. Reads from control group and heated group were mapped to the assembly contigs, re-spectively, and normalized to reads per kilobase per million (RPKM). The contigs were considered to be significantly dif-ferentially expressed genes if the FDR was smaller than 10−3 and the RPKM values showed at least 2-fold differences be-tween the 2 groups.

Figure 6 Heat map of the normalized expression (log-trans-formed data) of 19 heat shock genes exposed to different tem-peratures (25, 30, 32, 34, 36, 38, 40, 42 and 44 °C). An aster-isk (*) means that the expression of Hsp genes significantly increased in response to thermal stress (k independent-samples test, P < 0.05, N = 4 individuals per treatment).

Groups

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However, the survival after cardiac dysfunction suggests that anaerobic metabolism gives gastropods the abili-ty to supply energy to resist thermal stress when the aer-obic metabolism is limited. The upregulation of AlDH in response to thermal stress suggests that limpets can maintain the ATP supply through opine pathway when the cardiac function fails. Our findings highlight the ecological significance of anaerobic metabolism to the thermal adaptation of intertidal organisms.

The extensive upregulation of heat shock relat-ed-proteins also helps to explain why intertidal gas-tropods can survive in high temperatures beyond their ABT. Thirty-eight contigs which were annotated to heat shock related-proteins were upregulated when the temperature rose to ABT in C. toreuma (Fig. 5). Re-al-time PCR results showed that 15 of 19 HSP genes significantly increased in response to thermal stress (Fig. 6). Similar expression patterns of heat shock re-sponse occurred in the other 2 intertidal mollusc spe-cies Crassostrea gigas (Zhang et al. 2012) and Echino-littorina malaccana (Wang et al. 2014). HSP syntheses are activated at the upper temperatures of an organ-ism’s thermal range and are, therefore, thought to be critical for enhancing thermal tolerance limits in ec-tothermic animals (Tomanek 2002; Hofmann 2005). The physiological importance of heat shock proteins is largely due to their cellular role in protein homeosta-sis (Wickner et al. 1999) and protein folding (Hartl & Hayer-Hartl 2002). The massive upregulation of heat shock genes is probably central to intertidal gastro-pods’ adaptation in the highly stressful intertidal zone. In comparison to many oceanic ectotherms that seldom experience acute thermal fluctuations of more than a couple of degrees, eurythermal gastropod species can experience daily temperature fluctuations of more than 20 °C (Marshall et al. 2011; Han et al. 2013). Because the thermal tolerance can be enhanced by the energet-ically costly hsp response (Giomi et al. 2016), limpets will benefit from maintaining the ATP supply through opine pathway in the face of thermal stress.

In conclusion, when the ambient temperature increas-es beyond the ABT, aerobic metabolism is limited and anaerobic metabolism is important for providing energy to meet the extensive energy requirement for heat shock responses. The limpet C. toreuma can maintain the cel-lular metabolism through opine pathway when the car-diac function has failed. The case of limpets suggests that the significance of anaerobic metabolism must be taken into account to predict the ecological impacts of climate change on intertidal organisms.

ACKNOWLEDGMENTSThis work was supported by grants from the Nation-

al Natural Science Foundation of China (41276126, 41476115), the National Basic Research Program of China (2013CB956504), the Program for New Century Excellent Talents of Ministry of Education and Nature Science Funds for Distinguished Young Scholars of Fu-jian Province, China.

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SUPPLEMENTARY MATERIALSAdditional supporting information may be found in

the online version of this article at the publisher’s web-site.

Table S1 The sequences and efficiencies of primer pairs

Table S2 List of differential expression genes in re-sponse to thermal stress

Table S3 Major heat shock proteins identified in Cel-lana toreuma

Han G, Zhang S, Dong Y (2017). Anaerobic metabolism and thermal tolerance: The importance of opine pathways on survival of a gastropod after cardiac dysfunction. Integrative Zoology 12, 361–70.

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