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Interactive Effects of Seawater Acidification and Elevated Temperature on the Transcriptome and Biomineralization in the Pearl Oyster Pinctada fucata Shiguo Li, Jingliang Huang, Chuang Liu, Yangjia Liu, Guilan Zheng, Liping Xie, and Rongqing Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05107 • Publication Date (Web): 04 Jan 2016 Downloaded from http://pubs.acs.org on January 9, 2016
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Environmental Science & Technology
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Interactive Effects of Seawater Acidification and Elevated Temperature on the
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Transcriptome and Biomineralization in the Pearl Oyster Pinctada fucata
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Shiguo Li , Jingliang Huang , Chuang Liu , Yangjia Liu , Guilan Zheng , Liping Xie ∗, Rongqing Zhang ∗
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†
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Sciences, Tsinghua University, Beijing 100084, China.
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§
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100084, China.
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Institute of Marine Biotechnology, Collaborative Innovation Center of Deep Sea Biology, School of Life
Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing
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ABSTRACT
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Interactive effects of ocean acidification and ocean warming on marine calcifiers vary among species, but
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little is known about the underlying mechanisms. The present study investigated the combined effects of
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seawater acidification and elevated temperature (ambient condition: pH 8.1 × 23 °C, stress conditions: pH
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7.8 × 23 °C, pH 8.1 × 28 °C and pH 7.8 × 28 °C, exposure time: two months) on the transcriptome and
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biomineralization of the pearl oyster Pinctada fucata, which is an important marine calcifier.
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Transcriptome analyses indicated that P. fucata implemented a compensatory acid-base mechanism,
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metabolic depression and positive physiological responses to mitigate the effects of seawater acidification
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alone. These responses were energy-expensive processes, leading to decreases in the net calcification rate,
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shell surface calcium and carbon content, and changes in the shell ultrastructure. Elevated temperature
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(28 °C) within the thermal window of P. fucata did not induce significant enrichment of the sequenced
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genes and conversely facilitated calcification, which was detected to alleviate the negative effects of
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seawater acidification on biomineralization and the shell ultrastructure. Overall, this study will help
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elucidate the mechanisms by which pearl oysters respond to changing seawater conditions and predict the
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effects of global climate change on pearl aquaculture.
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INTRODUCTION
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The concentration of anthropogenically derived carbon dioxide (CO2) in the atmosphere is acutely
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increasing. CO2 absorption by the world’s oceans has increased the partial pressure of CO2 (pCO2), altered 1
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the dissolved inorganic carbon (DIC, the sum of inorganic carbon species in seawater), and decreased the
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pH value of surface seawater, leading to ocean acidification (OA) 1. Simultaneously, another consequence
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of increasing atmospheric CO2 is ocean warming 2. The pH of surface seawater has declined by an average
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of 0.1 units since 1750 relative to the preindustrial value of 8.18, representing a 30% increase in pCO2. The
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oceans are predicted to experience further pH decreases of 0.3–0.5 units concomitant with seawater
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warming of 2–6 °C by the end of this century
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chemistry and carbonate saturation, which have the potential to affect the physiology and behavior of a
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range of organisms in marine ecosystems
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calcifiers are more sensitive to these environmental stressors 9.
3, 4
5-10
. OA and ocean warming have led to a shift in seawater
. As the calcifying taxa in marine ecosystems, marine
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Increasing evidence has indicated that OA does not act in isolation; rather, it usually interacts with
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marine pollutants 11, salinity 12, light 13, 14, and, particularly, ocean warming 12, 15, 16. Importantly, the effects
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of concurrent OA and ocean warming on marine calcifiers differ among species (adverse and/or beneficial).
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For example, a meta-analysis suggested that ocean warming enhanced the sensitivity of most marine
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calcifiers (e.g., corals, sea urchins, oysters and mussels) to OA
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mussel Mytilus edulis when temperature stress was combined with pCO2 stress 17. Conversely, increasing
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the temperature from 24 °C to 30 °C reduced the adverse effects of OA (pH 7.4) on attachment and
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metamorphosis in the oyster Crassostrea gigas 12. In the sea urchin Tripneustes gratilla, the force needed to
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crush live urchins increased when the temperature rose from 22 °C to 28 °C under OA conditions 15. Given
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the interdependence among climate change stressors, their combined effects on marine calcifiers are
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complex and remain poorly understood. The thermal window of marine organisms is proposed to be the
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main factor in determining the extent of the interactive effects of OA and ocean warming. Increased
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temperatures may exacerbate the negative effects of OA when they exceed the thermal window and,
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conversely, may alleviate the threats 16, 18. Elucidating the mechanisms underlying changes in physiological
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performance is critical to better understand these complex responses. Recently, transcriptome analysis has
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become an excellent approach for investigating the responses of marine organisms to environmental
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changes 19, which can be used in the studies mentioned above.
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. No shell growth was measured in the
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The pearl oyster Pinctada fucata, an economically and ecologically important marine calcifier that is
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distributed along the coast of the Indo-Pacific region and South China Sea (SCS), is threatened by the 2
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changing seawater environment. Since it emerged as the best-studied species for the mechanisms of pearl
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and shell formation, the majority of investigations on P. fucata have focused on biomineralization (or
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calcification), which involves the biologically controlled deposition of calcium carbonate (CaCO3) 20. The
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success of pearl aquaculture greatly relies on the biomineralization process of adult pearl oysters. Early
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studies have reported detrimental effects of seawater acidification on the biomineralization
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mechanical properties
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interactive effects on pearl oysters, especially on biomineralization, is unknown. This is particularly
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important in the SCS, which is the center for pearl production in China and worldwide. Moreover, mantle is
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the most important tissue involved in biomineralization in pearl oysters and the mantle epithelia play
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important role in ionic regulation. The mantle transcriptomes have been successfully sequenced 24, which
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lay the foundation for revealing the response mechanisms of these species to climate stressors 25.
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and metabolism
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, shell
of adult P. fucata. However, whether climate stressors have
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The aims of this study are to reveal the responses of P. fucata to the interaction of seawater acidification
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and elevated temperature and to determine the mechanisms underlying these physiological changes. To
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achieve these aims, adult P. fucata were reared at different pH vales (pH 8.1 and pH 7.8) and temperatures
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(23 °C and 28 °C) for two months. The conditions mimicked predicted climate change scenarios in the year
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2100. Seawater chemistry, x-ray photoelectron spectroscopy, scanning electron microscopy, and
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transcriptome sequencing were employed to measure changes in the net calcification rate, shell surface
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CaCO3 content, shell ultrastructure, and transcriptome responses. In the SCS, the pH and temperature of the
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surface seawater fluctuate from pH 8.1 and 19 °C in winter to pH 7.6 and 30 °C in summer
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large fluctuations require pearl oysters to possess the ability to adapt to changing seawater conditions.
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Therefore, we propose that elevated temperatures are beneficial for the ability of P. fucata to resist the
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threat of OA due to the adaptability of this species to temperature changes.
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MATERIALS AND METHODS
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Study Organism
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Adult pearl oysters (Pinctada fucata, 6-7 cm shell length, 2 years old) were collected during January 2015
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from the pearl oyster aquaculture farm in Leizhou Peninsula, China (20°24′ N, 110°0′E). The average
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seawater conditions of the collection area were 23.25 ± 0.48 °C, 33.58 ± 0.34 psu and pH 8.07 ± 0.03. The
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pearl oysters were immediately transported to the laboratory and acclimatized at a temperature of 23.00 °C, 3
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a salinity of 33.00 psu and pH 8.10 for 2 weeks in a 500-L aquarium filled with artificial seawater (Formula
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Grade A Reef Sea Salt, Formula, Japan). The pearl oysters were fed twice daily with mixed microalgae (50%
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Platymonas subcordiformis and 50% Isochrysis zhanjiangensis) at a concentration of 2.0 × 104 cells/mL.
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Experimental Design
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The experiments were conducted in 50-L seawater circulation tanks with the seawater temperature and pH
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levels automatically maintained by heating the seawater and continuously pumping a CO2-gas mixture into
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the tanks
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designed to mimic the predicted levels for the ocean scenarios in the year 2100 (IPCC, 2007). These values
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also fall within the natural seawater pH and temperature fluctuations of the sampling area. Pearl oysters
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exposed to these conditions were divided into four groups: 28.0 °C, pH 7.8 and salinity 33.0 psu (28.0 °C ×
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pH 7.8); 28.0 °C, pH 8.1 and salinity 33.0 psu (28.0 °C × pH 8.1); 23.0 °C, pH 7.8 and salinity 33.0 psu
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(23.0 °C × pH 7.8); and 23.0 °C, pH 8.1 and salinity 33.0 psu (23.0 °C × pH 8.1). The ambient condition
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(23.0 °C × pH 8.1) served as the control. These treatments represented the individual and interactive effects
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of temperature and pH on pearl oysters. Prior to the experiment, the pearl oysters were acclimatized to the
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experimental conditions by gradually increasing the seawater temperature from 23 °C to 28 °C (1 °C • day-1)
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and gradually decreasing the pH from 8.1 to 7.8 (0.05 units • day-1). All treatments (including the control
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group) were performed for 60 days. Each treatment comprised three biological replicates in three
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independent tanks, and each tank contained 30 individuals. Details of the experimental treatments are listed
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in the supporting information (SI) Table S1. The pearl oysters were fed the microalgae mentioned above.
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. The two pH levels (pH 8.1 and pH 7.8) and two temperatures (23.0 °C and 28.0 °C) were
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The temperature and pH value (NBS scale) were monitored continuously in each tank and recorded twice
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daily. Total alkalinity (TA) was measured by Gran titration on 25 mL sample with a Kloehn digital syringe
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pump (Kloehn, Las Vegas, Nevada, with a precision of 0.1%) after filtering the seawater samples through a
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0.45-µm Millipore filter. Total dissolved inorganic carbon (DIC) was determined using a DIC analyzer
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(AS-C3, Apollo SciTech Inc., GA, USA) after filtering the seawater samples. The partial pressure of
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carbon dioxide (pCO2) and other carbonate system parameters were calculated by the software CO2SYS
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using pH, temperature, salinity, TA, and DIC with the dissociation constants K1, K2 and KSO4. The seawater
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carbonate chemistry conditions were stable throughout the treatments (Table S2).
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Transcriptome Sequencing 4
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Ten pearl oysters were selected randomly from each tank. Equal mantles (including edge, pallial and center)
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were cut from each individual, and the ten mantle samples were pooled together to obtain one mixture. The
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mantle mixtures from the four treatments were washed with RNase-free water and immediately immersed
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in liquid nitrogen. Total RNA was extracted using the TRIZOL Reagent (Life Technologies, Carlsbad, CA,
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USA) following the manufacturer’s instructions. The concentration and integrity of each RNA sample were
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determined using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and
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1.0% formaldehyde-denatured agarose gel electrophoresis, respectively. cDNA library construction,
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transcriptome sequencing and sequence assembly were performed by BGI-Shenzhen (Shenzhen, China)
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using Illumina HiSeq™ 2000 (Illumina, Inc. USA) using protocols that have been successfully employed
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for this species
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National Center for Biotechnology Information (NCBI) and are accessible through accession number
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SRP064812. The sequences were aligned to the non-redundant (nr), nucleotide (nt) and Swiss-Prot
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databases (e < 1.00e-5) in NCBI and annotated by retrieving known nucleotide and protein sequences with
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the highest similarity to mollusks and mammals using Blastn and Blastx.
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. The transcriptome data were deposited in the Sequence Read Archive (SRA) at the
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To identify differentially expressed unigenes (DEGs) between each treatment and the control, MARS
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(MA plot-based methods using a random sampling model) in the DEGseq R package was used to analyze
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RPKM (reads per kilobase of transcript per million uniquely mapped reads) values 31. The false discovery
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rate (FDR)-adjusted p value was used to estimate the representative DEGs (Benjiamini-Hochberg
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correction). Genes with p < 0.001 and a fold change > 2 were considered differentially expressed. The
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DEGs were assigned to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to analyze
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metabolic pathway enrichment (e < 1.00e-5). The Bi-directional Best-Hit (BBH) method was used to query
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the set of organisms representative for eukaryotes with default settings. Fisher's exact test was implemented
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to determine DEG enrichment. An FDR-adjusted p value < 0.001 was used to identify the most
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representative pathways. To analyze the enrichment of functional categories, Gene Ontology (GO)
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annotations were conducted using the Blast2GO program with default parameters (e < 1.00e-5)
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addition to KEGG and GO, specific DEG categories (including ion and acid-base regulation,
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biomineralization and thermal stress responses) were also classified artificially to reveal the responses of
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pearl oysters to seawater environment changes based on the literature 33-36. 5
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To validate the gene expression differences obtained from transcriptome sequencing, ten genes (5 up-
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regulated and 5 down-regulated) in each treatment were analyzed by real-time quantitative PCR (RT-
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qPCR). The degree of agreement between the transcriptome sequencing and RT-qPCR was evaluated by
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least-squares linear regression
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listed in SI, Table S3 and Table S4.
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Net Calcification Rate (NCR)
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To analyze the changes in calcification in pearl oysters exposed to the pH and temperature stressors, the
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alkalinity anomaly technique
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in TA values during the 60-day treatment and compared between each treatment and the control at the
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corresponding time points. The NCR was expressed as µmol CaCO3 •g-1 d-1.
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X-Ray Photoelectron Spectroscopy (XPS)
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To assess shell-surface CaCO3 deposition in pearl oysters exposed to the different treatments, shells from
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three P. fucata from each tank were randomly collected and pooled to obtain one biological replicate. The
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shells were washed with deionized water and immersed in 5% sodium hydroxide for 12 h to remove
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organic components attached to the shell surface. The shells were then washed thoroughly with deionized
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water, air-dried and stored in a desiccator. The calcium and carbon contents of the nacreous and prismatic
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layers in the inner surface of the shells near the nacre-prism transition region (Figure 3A) were measured
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by XPS (ESCALAB 250Xi, Thermo Scientific, USA) with monochromatic Al Ka radiation (1486.7 eV)
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and an incident angle of 45°. The binding energies were referenced to the adventitious C 1s peak at 285 eV;
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the accuracy of the binding energy measurements was 0.2 eV. High-resolution C 1s and Ca 2p spectra were
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obtained at a pass energy of 50 eV. Three areas were determined on each shell, and three biological
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replicates were used in each treatment. The element content was represented as the peak area.
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Scanning Electron Microscopy (SEM)
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To observe changes in the ultrastructure of newly formed shells in P. fucata exposed to pH and temperature
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stresses, shells were collected and pre-treated as described above for XPS. The inner surface near the nacre-
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prism transition region (Figure 3A) was processed for SEM (FEI Quanta 200, Netherlands) 22. Three areas
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were observed for the nacreous and prismatic layers on each shell sample, and three biological replicates
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were used in each treatment. The characterizations of the shell ultrastructures were recorded.
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. Full details regarding gene selection, primer design and RT-qPCR are
was used to estimate the NCR. The NCR was calculated using the changes
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Statistical Analyses
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Statistical analyses were conducted using SPSS version 18.0 for Windows (SPSS Inc., Chicago, IL, USA).
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The interactive effects of seawater acidification and elevated temperature on the NCR and element content
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were assessed by two-way analysis of variance (two-way ANOVA). The assumptions of normality and
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homogeneity of variance were assessed by residual analysis. The NCR, element content and transcriptome
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analysis were visualized in SigmaPlot version 12.5 (Systat Software, San Jose, CA, USA). SEM images
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were processed using Adobe Photoshop CS4.0 (Adobe Systems, San Jose, CA, USA). All results were
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presented as the mean and standard deviation (mean ± SD).
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RESULTS AND DISCUSSION
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The sequencing and assembly qualities for the mantle transcriptomes of P. fucata were excellent, which
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were evident in the clean reads, the clean nucleotides, Q20, mean length and N50 (Table S5 and Table S6).
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Significant correlations were obtained between the transcriptome and RT-qPCR results, confirming the
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reliability of the sequencing results (Figure S1). A total of 16924, 6243 and 9244 up-regulated DEGs were
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found at 23 °C × pH 7.8, 28 °C × pH 8.1 and 28 °C × pH 7.8, respectively, whereas a total of 10242, 10815
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and 7960 DEGS were down-regulated. These results indicated that more genes responded to seawater
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acidification (Figure S2), implying greater effects of this stressor on the pearl oysters.
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Effects of Seawater Acidification on Pearl Oysters
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Studies tend to focus on the DEGs involved in “ion and acid-base regulation” (Table S7) because ion- and
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acid-base homeostasis determine the sensitivity and adaptability of marine organisms to OA
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expression was significantly up-regulated at 23 °C × pH 7.8, including the sodium-driven chloride
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bicarbonate exchanger (NCBE), sodium/hydrogen exchanger (NHE), anion exchanger (AE), chloride
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channel (CLC), sodium channel (Na+ channel), potassium channel (K+ channel), sarco-endoplasmic
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reticulum calcium transport ATPase (SERCA), vacuolar type H+-ATPase (V-ATPase), sodium/calcium
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exchanger (NCX), sodium/potassium/calcium exchanger (NKCK), calmodulin (CAM), calmodulin-like
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protein (CAML), calreticulin (CRT) and carbonic anhydrase (CA) genes.
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. Gene
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Although marine bivalves are regarded as weak acid-base regulators, the transcriptome data may indicate
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a potential mechanism underlying the response of P. fucata to seawater acidification (Figure 1). Based on 7
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functional studies in other taxa, particularly in marine calcifiers, we propose that the mantle epithelia of
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pearl oysters are equipped with ion-regulatory machinery that is useful in controlling acid-base
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disturbances. CA appears to be sensitive to OA in marine calcifiers and could catalyze the interconversion
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between CO2 and carbonate to produce more H+ and HCO3- 33. The redundant H+ may then be excreted into
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the extracellular fluid by the apical V-ATPase and NHE 40, while the redundant HCO3- is enriched in the
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interstitial fluid or hemolymph through basolateral CLC
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HCO3- in these areas can neutralize the hypercapnia induced by elevated CO2 42. The increased Na+ channel,
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K+ channel, NCX and NKCX levels provide transmembrane Na+ and K+ gradients for all exchangers. Cl-
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recycling may occur transcellularly via basolateral CLC 35, AE 35 and NCBE 41. The specific compensation
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diminishes the acid-base imbalance caused by seawater acidification in the mantle epithelia. This
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compensation is also observed in sea urchin S. purpuratus larvae
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bivalves. Therefore, our study provides the first evidence that P. fucata may respond to seawater
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acidification through tight control of ion and acid-base regulation. Further investigations are needed to
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confirm these changes at physiological level.
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, AE
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and NCBE
33, 34
41
. The accumulation of
but has not been found in marine
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Significant changes in the genes involved in “vacuole”, “lysosome”, “autophagic cell death”, “histolysis”
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and “tissue death” in the ontology of “cellular component” and “biological process” were observed in the
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GO enrichment analyses in the 23 °C × pH 7.8 group (Table 1, p < 0.001), suggesting that seawater
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acidification induced mantle cell injuries. These responses were consistent with those in the sea urchin S.
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purpuratus 34. Notably, genes associated with the “cell adhesion molecules”, “fatty acid biosynthesis” and
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“ubiquitin mediated proteolysis” pathways were significantly enriched by this treatment (Table 2, p
