Interrelationship of salinity shift with oxidative stress and lipid metabolism in the monogonont rotifer Brachionus koreanus

Comparative Biochemistry and Physiology, Part A 214 (2017) 79–84

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Interrelationship of salinity shift with oxidative stress and lipid metabolism in the monogonont rotifer Brachionus koreanus

MARK

Min-Chul Leea, Jun Chul Parka, Duck-Hyun Kima, Sujin Kangb, Kyung-Hoon Shinb, Heum Gi Parkc, Jeonghoon Hana,⁎, Jae-Seong Leea,⁎ a b c

Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea Department of Marine Sciences and Convergent Technology, Hanyang University, Ansan 15588, South Korea Department of Marine Resource Development, College of Life Sciences, Gangneung-Wonju National University, Gangneung 25457, South Korea

A R T I C L E I N F O

A B S T R A C T

Keywords: Rotifer Brachionus koreanus Salinity Oxidative stress Lipid metabolism

Salinity is a critical key abiotic factor affecting biological processes such as lipid metabolism, yet the relationship between salinity and lipid metabolism has not been studied in the rotifer. To understand the effects of salinity on the monogonont rotifer B. koreanus, we examined high saline (25 and 35 psu) conditions compared to the control (15 psu). In vivo life cycle parameters (e.g. cumulative offspring and life span) were observed in response to 25 and 35 psu compared to 15 psu. In addition, to investigate whether high salinity induces oxidative stress, the level of reactive oxygen species (ROS) and glutathione S-transferase activity (GST) were measured in a salinity(15, 25, and 35 psu; 24 h) and time-dependent manner (3, 6, 12, 24 h; 35 psu). Furthermore composition of fatty acid (FA) and lipid metabolism-related genes (e.g. elongases and desaturases) were examined in response to different salinity conditions. As a result, retardation in cumulative offspring and significant increase in life span were demonstrated in the 35 psu treatment group compared to the control (15 psu). Furthermore, ROS level and GST activity have both demonstrated a significant increase (P < 0.05) in the 35 psu treatment. In general, the quantity of FA and mRNA expression of the lipid metabolism-related genes was significantly decreased (P < 0.05) in response to high saline condition with exceptions for both GST-S4 and S5 demonstrated a significant increase in their mRNA expression. This study demonstrates that high salinity induces oxidative stress, leading to a negative impact on lipid metabolism in the monogonont rotifer, B. koreanus.

1. Introduction Rotifers (phylum Rotifera), which are microzooplankton, are widely distributed throughout aquatic ecosystems and play a key role as a bridge between producers and higher-level consumers in aquatic food chains (Hutchinson, 1957). Thus, rotifers have been used as a biological indicator for monitoring and evaluating aquatic ecosystem condition. In particular, monogonont rotifers such as Brachionus plicatilis, and B. rotundiformis have been considered as suitable model species and are extensively used in aquaculture, ecology, gerontology, and ecotoxicology research (Dahms et al., 2011; Won et al., 2017). Thus, influences of the abiotic factors (e.g. temperature and salinity) for the growth and reproduction of rotifers (e.g. B. plicatilis and B. rotundiformis), including new species, have been widely studied (Miracle and Serra, 1989; Fielder et al., 2000; Bosque et al., 2001; Chigbu and Suchar, 2006). In general, salinity is a key external factor affecting biological

⁎

processes and can lead to changes in the life cycle parameters (e.g. growth and fecundity) of rotifers (Cervetto et al., 1999; Yin and Zhao, 2008). In the aquatic environment, changes in salinity affect biological processes at organism, population, community, and ecosystem levels (Cervetto et al., 1999; Yin and Zhao, 2008). An understanding of ecophysiological responses to salinity-induced oxidative stress is essential for evaluating the physiological requirements of each species throughout the aquatic food chain (Verity and Smetacek, 1996). In addition, analysis of lipid content and fatty acid (FA) composition has been used to determine physiological state of aquatic animals (e.g. shrimp and fish) including live food organisms (e.g. Artemia and copepod) in the aqua-ecosystems (Chakraborty et al., 2007; Ouraji et al., 2011). Changes in lipid content and FA composition were associated with different salinities. For example, in juvenile American shad Alosa sapidissima, exposed to various salinity condition (7, 12, 21 and 28 psu), increase in the polyunsaturated fatty acids (PUFA) were demonstrated

Corresponding authors. E-mail addresses: [email protected] (J. Han), [email protected] (J.-S. Lee).

http://dx.doi.org/10.1016/j.cbpa.2017.09.014 Received 23 August 2017; Received in revised form 15 September 2017; Accepted 19 September 2017 Available online 23 September 2017 1095-6433/ © 2017 Elsevier Inc. All rights reserved.

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collected to perform the whole experiments. B. koreanus were homogenized in 0.32 mM sucrose containing 20 mM HEPES, 1 mM MgCl2, and 0.5 mM PMSF (pH 7.4). Then, after centrifugation (10,000 × g for 20 min at 4 °C) the supernatant were allowed to react with H2DCFDA and the fluorescence was measured at 485 nm (emission) and 520 nm (excitation) using a multi-channel plate reader (Thermo Scientific Co., Varioscan Flash, Waltham, MA, USA). Intracellular GST activities were measured by following Regoli et al. (1997). Briefly, samples were homogenized in homogenization buffer (2 mM Tris–HCl, containing 20% glycerol, 2 mM mercaptoethanol, and 0.5 mM PMSF [pH 8]), centrifuged at 13,000 × g for 20 min at 4 °C, the supernatants were used for further analysis. For GST activity, the increasing absorbance at 340 nm was measured for the conjugation of GSH with 1-chloro-2,4dinitrobenzene (extinction coefficient of CDNB is 9.6 mM− 1 cm− 1) using a spectrophotometer at 25 °C. The total protein content of the supernatant for each experiment was determined by the dye-binding method (Bradford, 1976) using bovine serum albumin standard (0–200 μg BSA/mL PBS).

with significant enrichment of omega 3 fatty acids (e.g. eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]), while reduction in the monounsaturated fatty acids (MUFA) were shown (Liu et al., 2017). In the razor clam Sinonovacula constricta, a significant increase in the proportion of PUFAs was shown after exposure to high salinity (20–25 psu), despite an optimum salinity of 10–15 psu (Ran et al., 2017). Also, in vivo (e.g. growth and reproduction) analysis of FA composition and the transcriptional regulation of heat shock proteins (hsps) in response to salinity stress was performed to determine physiological condition in the copepod Paracyclopina nana (Lee et al., 2017). Although rotifers (e.g. B. plicatilis and B. rotundiformis) are important food source for higher-level consumers in the aqua-ecosystem, both molecular and biochemical parameters have not been studied easily, as genomic information is not yet available. The monogonont rotifer B. koreanus is a suitable model species for eco-toxicological and eco-physiological studies to examine the effects of marine environmental stressors (e.g. UV-B, gamma radiation, and BDE47) (Kim et al., 2011; Han et al., 2014; Park et al., 2017), due to rapid reproductive rate, high fecundity, and easy maintenance (Dahms et al., 2011). Furthermore, extensive RNA-seq information of B. koreanus provides a better means to investigate effects of environmental stressors at molecular and cellular levels. In this study, we observed life cycle parameters (e.g. cumulative offspring and life span) and measured the level and activity of oxidative stress markers (e.g. reactive oxygen species [ROS] and glutathione Stransferase [GST]) in high salinity. In addition, we investigated the effects of high salinity on fatty acid composition and examined transcriptional regulation of the lipid metabolism-related genes (e.g. elongases and desaturases).

2.4. Effect of salinity on fatty acid composition Variations in FA composition in response to different salinity (15 [control], 25, and 35 psu) were analyzed in B. koreanus as described in Hama and Handa (1987) with minor modifications. Briefly, the lipids were extracted with dichloromethane/methanol 2:1 (v/v). Nonadecanoic acid (C19:0) was added to the extracts as an internal standard. Extraction procedures were repeated thrice with sonication. Lipid fractions were separated from the water-methanol phase and converted into fatty acid methyl esters (FAMEs) by saponification using 0.5 M KOH-methanol, followed by methylation with BF3-methanol. Concentrations and compositions of formed FAMEs were analyzed in a gas chromatograph (GC-2010, Shimadzu, Kyoto, Japan) with a flame ionization detector (FID) using a fused silica capillary column (DB-5, 30 m × 0.25 mm i.d., 0.25-μm film thickness). Helium was used as a carrier gas. Samples were injected in splitless mode at an initial oven temperature of 40 °C, raised to 200 °C at 10 °C/min and, then to 300 °C at 2 °C/min. FAs were identified from the retention times (RT) of standards and from mass spectra from gas chromatograph-mass spectrometer (GCMS-QP2010 Plus; Shimadzu, Kyoto, Japan). All experiments were performed in triplicate.

2. Materials and methods 2.1. Culture and maintenance of Brachionus koreanus The rotifer B. koreanus was collected from a hatchery of East Sea Fisheries Research Institute, Uljin (36°58′43.01″N, 129°24′28.40″E), maintained at the Department of Biological Science, Sungkyunkwan University, Suwon, South Korea, and used for this study. Rotifers were fed with the green marine microalgae Tetraselmis suecica (6 × 104 cells/ mL) every 24 h and maintained in 15-psu filtered artificial seawater (ASW) (TetraMarine Salt Pro, Tetra™, Cincinnati, OH, USA) with a 12:12 h (light:dark) photoperiod at 25 °C. Species identification was confirmed by morphological analysis (Hwang et al., 2013; Mills et al., 2017) and sequencing of the mitochondrial DNA gene CO1 (Hwang et al., 2014).

2.5. Identification of the glutathione S-transferase isoforms and lipid metabolism-related genes To obtain the glutathione S-transferase (GST) and lipid metabolismrelated genes, in silico analysis of B. koreanus RNA-seq information was performed (Lee et al., 2015). Genes were subjected to BLAST analysis in the GenBank non-redundant (NR; including all GenBank, EMBL, DDBJ, and PDB sequence except EST, STS, GSS, and HTGS) amino acid sequence database (http://blast.ncbi.nlm.nih.gov/). The amplicons were sequenced on the ABI PRISM 3700 DNA analyzer and putative transcription factor-binding sites were screened using Geneious (v.10.0.7; Biomatters Ltd., Auckland, New Zealand) (Kearse et al., 2012). To investigate the salinity stress-induced modulation of GST isoforms and lipid metabolism-related genes, we measured mRNA expression levels over 24 h (control, 3, 6, 12 and 24 h) in response to 35 psu. Total RNAs were extracted with TRIZOL® reagent (Invitrogen, Paisley, Scotland, UK) according to the manufacturer's instructions. The quantity and quality were analyzed spectrometrically at 230, 260, and 280 nm (QIAxpert, Qiagen, Hilden, Germany). To synthesize cDNA for a quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), two μg of total RNA and oligo(dT)20 primer were used for reverse transcription (SuperScript™ II RT kit, Invitrogen, Carlsbad, CA, USA). qRT-PCR was conducted under the following conditions: 95 °C/ 4 min; 40 cycles of 95 °C/30 s, 55 °C/30 s, 72 °C/30 s, and 72 °C/10 min using SYBR Green as a probe (Molecular Probes Inc., Eugene, OR, USA)

2.2. Assessment of cumulative offspring and life span in response to different salinity condition To examine life cycle parameters, we recorded the number of offspring and life span. Adult B. koreanus was transferred into a new 24well cell culture plate (SPL Life Science Co. Ltd., Seoul, South Korea) containing 1 mL ASW with T. suecica (6 × 104 cells/mL) at different salinities (15 [control], 25, and 35 psu) every 24 h. The number of newborn rotifers was counted every 12 h to examine fecundity, while the death of mature rotifers was counted to quantify life span. All experiments were performed in triplicate and stereomicroscopy (M205-A, Leica Microsystems, Wetzlar, Germany) was used to observe B. koreanus. 2.3. Measurement of the reactive oxygen species and the enzyme activity of glutathione S-transferase Approximately 5000 healthy individuals were exposed to different salinities in salinity- (15 [control], 25, and 35 psu; 24 h) and time-dependent (3, 6, 12, 24 h; 35 psu only) manner. The samples were 80

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A Cumulative offspring/individual rotifer

in a CFX96™ real-time PCR system (Bio-Rad, Hercules, CA, USA). To confirm the amplification of specific products, melting curve cycles were run at the following conditions: 95 °C/1 min; 55 °C/1 min; and 80 cycles of 55 °C/10 s with a 0.5 °C increase per cycle using qRT-PCR F or R primers (Supplementary Table S1). B. koreanus elongation factor 1alpha (EF1-α) gene, which showed stable expression throughout the experiments, was used as an internal control to normalize expression levels between samples. All experiments were performed in triplicate. The relative fold-change in gene expression compared to the control was calculated by the 2T− ΔΔC comparative method (Livak and Schmittgen, 2001). 2.6. Statistical analysis SPSS ver. 18.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Data are expressed as mean ± S.D. Significant differences between the control and exposed groups were analyzed using the Student's paired t-test and one-way ANOVA followed by Tukey's tests. For life span analysis, Kaplan–Meier survival curves were calculated for significance using R statistical software (version 3.2.1 of the R Foundation for Statistical Computing Platform© 2015). Differences with P < 0.05 were considered significant.

25 20 15 10 15 psu 25 psu 35 psu

5 0 1

2

3

4

5

6

7

8

9

Day

B 100

3. Results

Survival rate (%)

3.1. Effects of salinity on offspring production and life span Cumulative offspring production per individual rotifer was delayed in 25 and 35 psu compared to the control (15 psu) in salinity-dependent manner (Fig. 1A). In terms of life span, only 35 psu demonstrated significant increase (P < 0.05) compared to the control salinity condition (15 psu) (Fig. 1B).

80 60 40

15 25

20

3.2. High salinity-induced reactive oxygen species and glutathione Stransferase enzyme activity

35*

0

To evaluate the cellular level of oxidative stress markers, intracellular level of ROS and GST activity were measured in salinity- (15 [control], 25, and 35 psu; 24 h) and time-dependent manner (3, 6, 12, and 24 h; 35 psu only). The intracellular levels of ROS and GST activity significantly decreased (P < 0.05) in early-exposure time (e.g. 3 and 6 h) but were significantly increased at 24 h and (P < 0.05, Fig. 2).

0

2

4

6

8

10

Day Fig. 1. Effects of salinity on life parameters in Brachionus koreanus exposed to 25 and 35 psu compared to the control (15 psu). (A) Offspring production per individual rotifer, and (B) life span. Asterisk (*) indicates significant differences between exposed and control groups (P < 0.05, Bonferroni's correction).

3.3. Modulation of glutathione S-transferase genes in high saline condition genes were down-regulated after exposure to high salinity, with exceptions of elongase 6 and 8.

To identify the effect of high salinity (35 psu) at the molecular level, the mRNA expression of GST isoforms were measured in time-dependent manner (3, 6, 12, and 24 h). GST-S2 and S3 were decreased significantly (P < 0.05), but GST-S4 and S5 were significantly increased (P < 0.05) and demonstrated the highest expression level at 6 h (Fig. 3).

4. Discussion As the rotifers provide trophic link between phytoplankton and upper trophic organisms, it would be essential to understand how rotifers are affected not only by the environmental pollutants, but the impact they experience from the abiotic factor, salinity. However, studies on rotifers, particularly in a molecular nutritional perspective, are still insufficient. In this study, we have investigated the interrelationship between salinity, oxidative stress, and lipid metabolism. From the results of life cycle parameters, an inverse relationship between offspring production and life span was observed, where in 35 psu, retardation in cumulative offspring was demonstrated in contrast to an increase in their life span compared to 15 psu (control). In general, diverse living organisms can trade-off energy for growth, reproduction, and maintenance of homeostasis in response to environmental stressors (Kooijman and Troost, 2007). In rotifers, survival and reproduction have a negative correlation called the “cost of reproduction” (Snell and King, 1977; Sarma et al., 2002; Stelzer, 2005). This

3.4. Effect of salinity on fatty acid composition The composition of FA was measured in B. koreanus in response to 25 and 35 psu compared to the control (15 psu). All fatty acids (FAs) showed a tendency of reduction (Table 1), but only the total FA, palmitic acid (C16:0), palmitoleic acid (C16:1ω9), and eicosapentaenoic acid (C20:5ω3) showed significant decreases in 35 psu compared to the control (P < 0.05). 3.5. Modulation of lipid metabolism-related genes in high saline condition To examine the effects of salinity on the composition of FA in B. koreanus, the expression of lipid metabolism-related genes were measured in response to 35 psu at 0, 3, 6, 12, and 24 h (Fig. 4). All of the 81

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A

Table 1 The composition of fatty acids exposed to 15, 25, and 35 psu for 24 h was analyzed. The groups are classified by the position of unsaturation. Values are means ± SEM. Different letters indicate significant differences between each group (P < 0.05).

140 15 psu (Control) 25 psu 35 psu

% of Control

120

a b

b

100

SFA

60 40

ω9

0 ROS

ω6

GST

200 Control 3h 6h 12 h 24 h

150

% of Control

15 psu

25 psu

35 psu

C16:0 C18:0 C20:0 C22:0 C24:0 C16:1 C18:1 C20:1 C22:1 C24:1 C18:2 C18:3 C20:3 C20:4 C20:2 C22:2 C20:3 C20:5 C22:6 Total

48.48 ± 6.14a 29.09 ± 10.78 0.51 ± 0.02 0.35 ± 0.07 0.38 ± 0.18 5.02 ± 0.05a 14.17 ± 1.97 9.86 ± 4.04 4.31 ± 2.58 2.49 ± 1.35 26.51 ± 9.61 1.22 ± 0.36 1.47 ± 0.86 2.81 ± 1.31 0.88 ± 0.66 0.46 ± 0.11 1.91 ± 0.93 13.32 ± 0.00a 10.05 ± 6.45 173.26 ± 12.88a

40.66 ± 2.37ab 27.77 ± 20.46 0.43 ± 0.12 0.29 ± 0.05 0.2 ± 0.01 3.59 ± 0.27b 12.56 ± 0.26 8.2 ± 3.02 3.46 ± 1.73 1.96 ± 0.79 21.81 ± 6.78 1.01 ± 0.31 1.23 ± 0.83 2.73 ± 1.61 0.83 ± 0.52 0.46 ± 0.11 1.62 ± 0.98 11.44 ± 0.36ab 8.7 ± 6.57 148.96 ± 12.54ab

30.05 ± 3.56b 12.17 ± 1.9 0.31 ± 0.03 0.2 ± 0.01 0.16 ± 0.02 2.86 ± 0.4b 10.15 ± 1.52 7.56 ± 2.05 3.36 ± 0.83 2.1 ± 0.23 19.15 ± 5.98 0.89 ± 0.3 1.38 ± 0.51 3.2 ± 0.49 1.03 ± 0.44 0.46 ± 0.07 1.72 ± 1.11 10.21 ± 0.7b 7.45 ± 5.45 114.4 ± 10.63b

80

20

B

Type of fatty acid (μg/mg; dry weight)

100

b

c c

b

ω3

a

a

b

b

and Enesco, 2011). In addition, depending on pH, future survival/reproduction had opposing outcomes in the rotifer B. patulus; survival/ reproduction decreased at extreme pH (pH 5 or 9) and increased at a moderate pH (pH 6–8) (Yin and Niu, 2011). In mass cultures of the rotifer B. plicatilis, fasting after hatching (days 1–4) suppressed the reproductive rate and increased life span (Yoshinaga et al., 2003). Salinity is an environmental factor that can modulate physiological processes in rotifers. For example, in the rotifer Colurella dicentra, growth rate was increased at 15 psu compared to 35 psu (Chigbu and Suchar, 2006). Likewise, the marine rotifers Synchaeta cecilia valentina (Oltra and Todoli, 1997) and S. littoralis (Bosque et al., 2001) grew and reproduced optimally at 20–25 psu compared to high salinity (30–37 psu). Taken together, salinity changes can induce adverse effects on life cycle parameters (e.g. growth and reproduction), leading to energy reallocation to the basal metabolic rate for self-maintenance of rotifers. To examine whether high salinity induces oxidative stress, ROS level and activities of GST were measured with salinity (15 [control], 25, and 35; 24 h)- and time (3, 6, 12, and 24 h; 35 psu)-dependent manner. In the 35 psu treatment group, both ROS and GST were significantly decreased at 3 and 6 h, but were significantly increased (P < 0.05) at 24 h. These results indicated that high salinity induces oxidative stress in B. koreanus, resulting in an increasing level of GST enzyme to detoxify oxidative stress. In general, the excessive ROS generation affects biological processes by oxidizing biomolecules, leading to adverse effects on organisms such as oxidative stress-induced signaling pathways, apoptosis, and inflammation (Gniadecki et al., 2000; Valko et al., 2007). As a stress factor, salinity is a well-known abiotic factor which induces stress upon marine organisms For example, in large yellow croaker, Pseudosciaena crocea, hypersalinity (40 psu) increased the content of glutathione, and induced both the enzyme activities and mRNA of superoxide dismutase (SOD), glutathione peroxide (GPx), and glutathione reductase (GR), triggering NF-E2-related nuclear factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signaling pathway compared to the control (26 psu) (Zeng et al., 2017). Furthermore, in the marine intertidal flatworm, Macrostomum lignano, hypersalinity induced O2-formation, with consequential mRNA expression of SOD (Rivera-Ingraham et al., 2016). Taken together, high salinity induces oxidative stress in marine organisms, initiating defense mechanisms against detrimental conditions.

c c 50

a

0 ROS

GST

Fig. 2. Salinity-induced oxidative stress and enzyme activity of glutathione S-transferase in B. koreanus. (A) The level of ROS and GST activity in response to various salinity exposure (15 [control], 25 and 35 psu) at 24 h, and (B) Time-dependent differences in ROS levels and GST activity have been analyzed in 35 psu exposure at (3, 6, 12, and 24 h). The significant differences (P < 0.05) are indicated by different letters in each group after Tukey's post hoc tests.

GST-S1 GST-S2 GST-S3 GST-S4 GST-S5 GST-S6 GST-S7 GST-S8

Control

3h

6h

12 h

0

24 h

1

10

Fig. 3. Transcription profile of 8 glutathione S-transferase isoforms after 35 psu exposure for 3, 6, 12, and 24 h represented in a heat map.

phenomenon can be caused by various environmental factors (e.g. temperature, pH, and fasting). For example, in the rotifer B. calyciflorus, life span was increased at lower temperature (16 °C) compared to higher temperature (29 °C), but led to a reduction in fecundity (Kauler 82

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Con

A

3h

6h

12 h

24 h

patterns of GST genes will be helpful in understanding antioxidant defense mechanisms in response to high salinity in B. koreanus. In contrast to the salinity of 15 psu, being the optimum culture salinity, a high salinity (35 psu) resulted in relatively low fatty acid quality. In 35 psu treated group, total quantity of the FA were decreased significantly (P < 0.05), and among various types of FA, palmitic acid (C16:0), palmitoleic acid (C16:1ω9), and eicosapentaenoic acid (C20:5ω3) were decreased significantly (P < 0.05). As one of the major nutrient, FA plays a vital role in organisms such as in the formation of cell structure, maintenance of homeostasis, and immune function. Also, the FA is known as a sensitive component to stressors and environmental changes (Arts et al., 2009), therefore, modifications in FA content are usually observed when marine organisms were exposed to high salinity. In the marine copepod, Paracyclopina nana, the quantity of FA showed a tendency to decrease when exposed to high salinity condition (25 and 30 psu) compared to the control (15 psu) (Lee et al., 2017). In the razor clam, Sinonovacula constricta, the total fatty acid and monounsaturated fatty acid did not change, while the total content of saturated fatty acids was decreased and polyunsaturated fatty acids were increased, when the salinity increased from 8 to 23 psu (Ran et al., 2017). In association with oxidative stress results, B. koreanus was not likely to synthesize FA in high salinity. Instead, they tried to consume their energy to control oxidative stress. Based on the importance of the FA within the organism, the reduction in total FA was expected to play a negative role in maintaining body homeostasis. In particular, eicosapentaenoic acid (C20:5ω3), one of the n-3 fatty acids, is known to play an important role in the immune system in organisms as well as the precursor of DHA synthesis. Therefore, reduction of eicosapentaenoic acid in B. koreanus can cause a negative effect on the body, resulting in the malfunctioning of the defense mechanisms in high salinity. Almost all of the lipid metabolism-related genes were decreased in their expression at 35 psu exposure. Normally, composition of fatty acid can be modulated by two pathways. One is the internal pathway, which can synthesize fatty acid from glucose (de novo lipogenesis [DNL]), resulting in palmitic acid (C16:0) as the final product. The other one is the external pathway, which absorbs FA from external food sources. In the DNL pathway, a positive correlation between mRNA expression of the DNL pathway (ACLY, ACC, and KAS) and fatty acid composition is present. A decrease in palmitic acid (C16:0) appears to be due to a decrease in the DNL pathway, resulting in a decrease in the total FA as it inhibits the pathway for the production of FA from glucose. After production or absorption of FA, their structure can be modified by elongase and desaturase in the endoplasmic reticulum (Sargent et al., 2002). However, analysis of the expression of elongases and desaturases has demonstrated a reduction in their expression, indicating that the structure of both internal and external FA cannot be changed easily. In addition, in the fatty acid storage part, monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), lipin1 and 2 are responsible for making triacylglycerol (TAG) using three fatty acids and a glycerol backbone (Reue and Zhang, 2008; Yen et al., 2008), their expression had a tendency to decrease. This data indicated that the reduction in the total FA has a negative effect on the storage of fatty acids. As a result, when B. koreanus was exposed to high salinity, the mRNA expression of lipid metabolism-related genes was down-regulated, resulting in the decrease in fatty acid production, alteration, and storage. This study demonstrates detrimental effects of high salinity in life cycle parameters (e.g. cumulative offspring and life span), the elicitation of the oxidative stress marker (e.g. ROS and GST), and lipid metabolism parameters (e.g. FA composition and lipid metabolism-related genes) in the monogonont rotifer B. koreanus. It is suggestive that energy reallocation to the basal metabolism were induced against high salinity to self-sustain. Acute-exposure to high salinity in B. koreanus elicited oxidative stress and low fatty acid quality, resulting in retardation of the cumulative offspring. In return, the retardation in the cumulative offspring led to increase in the life span as a result of energy

ACLY ACC KAS

B

ELO1 ELO2 ELO3 ELO4 ELO5 ELO6 ELO7 ELO8 ELO9 Δ4 DES-1 Δ4 DES-2 Δ5 DES-1 Δ5 DES-2 Δ5 DES-3 Δ5 DES-4 Δ9 DESdf

C

MGAT DGAT Lipin1 Lipin2 0

1

3

Fig. 4. Transcription profile of lipid metabolism-related genes after 35 psu exposure at 3, 6, 12, and 24 h represented in a heat map. (A) de novo lipogenesis genes (ATP-citrate lyase [ACLY], Acetyl-CoA carboxylase [ACC], and beta-keto-acyl-[acyl-carrier-protein] synthase [KAS], (B) fatty acid modification related genes (elongase [ELO] and desaturase [DES]), and (C) triacylglycerol formation-related genes (monoacylglycerol acyltransferase [MGAT], diacylglycerol acyltransferase [DGAT], and lipin1 and 2).

To discover the relationship between enzyme activities and mRNA expression pattern under high salinity, we analyzed mRNA expressions of 8 GST-S isoforms exposed to 35 psu in a time-dependent manner (3, 6, 12, and 24 h). The mRNA expression of GST-S2 and 3 genes were significantly decreased (P < 0.05), but GST-S4 and S5 were significantly increased (P < 0.05). As a phase II detoxification system, GST genes are known as a biomarker for environmental stress (Dourado et al., 2008), thus, GST expressions have been studied in invertebrates. The primary role of GST is detoxification of xenobiotics, therefore, the mRNA expression of GST isoforms showed a tendency to increase, when B. koreanus was exposed to xenobiotics. For example, in response to environmental stressors, such as copper, gamma radiation, and BDE-47, the expression of GST genes were examined in B. koreanus (Han et al., 2013, 2014; Park et al., 2017). Unlike in previous studies treating xenobiotics, the expression pattern of GST isoforms did not show a consistent tendency. However, a significant increase in GST enzymatic activity at 24 h may be due to over-expression of GST-S4 and S5 at earlier times (3 and 6 h). These studies revealed that mRNA expression

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Soc. 82, 113–142. Lee, B.-Y., Kim, H.-S., Hwang, D.-S., Won, E.-J., Choi, B.-S., Choi, I.-Y., Park, H.-G., Rhee, J.-S., Lee, J.-S., 2015. Whole transcriptome analysis of the monogonont rotifer Brachionus koreanus provides molecular resources for developing biomarkers of carbohydrate metabolism. Comp. Biochem. Physiol. D: Genomics Proteomics 14, 33–41. Lee, S.-H., Lee, M.-C., Puthumana, J., Park, J., Kang, S., Hwang, D.-S., Shin, K.-H., Park, H.G., Soussi, S., Om, A.-S., Lee, J.-S., Han, J., 2017. Effects of salinity on growth, fatty acid synthesis, and expression of stress response genes in the cyclopoid copepod Paracyclopina nana. Aquaculture 470, 182–189. Liu, Z.F., Gao, X.Q., Yu, J.X., Qian, X.M., Xue, G.P., Zhang, Q.Y., Liu, B.L., Hong, L., 2017. Effects of different salinities on growth performance, survival, digestive enzyme activity, immune response, and muscle fatty acid composition in juvenile American shad (Alosa sapidissima). Fish Physiol. Biochem. 43, 761–773. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realΔΔC time quantitative PCR and the 2− method. Methods 25, 402–408. T Mills, S., Alcántara-Rodríguez, J.A., Ciros-Pérez, J., Gómez, A., Hagiwara, A., Galindo, K.H., Jersabek, C.D., Malekzadeh-Viayeh, R., Leasi, F., Lee, J.-S., Mark Welch, D.B., Papakostas, S., Riss, S., Segers, H., Serra, M., Shiel, R., Smolak, R., Snell, T.W., Stelzer, C.P., Tang, C.Q., Wallace, R.L., Fontaneto, D., 2017. Fifteen species in one: deciphering the Brachionus plicatilis species complex (Rotifera, Monogononta) through DNA taxonomy. Hydrobiologia 796, 39–58. Miracle, M.R., Serra, M., 1989. Salinity and temperature influence in rotifer life history characteristics. Hydrobiologia 186 (187), 81–102. Oltra, R., Todoli, R., 1997. Effects of temperature, salinity and food level on the life history traits of the marine rotifer Synchaeta cecilia valentina, n. subsp. J. Plankton Res. 19, 693–702. Ouraji, H., Fereidoni, A.E., Shayegan, M., Asil, S.M., 2011. Comparison of fatty acid composition between farmed and wild Indian white shrimps, Fenneropenaeus indicus. Food Nutr. Sci. 2, 824–829. Park, J.C., Han, J., Lee, M.-C., Kang, H.-M., Jeong, C.-B., Hwang, D.-S., Wang, M., Lee, J.S., 2017. Adverse effects of BDE-47 on life cycle parameters, antioxidant system, and activation of MAPK signaling pathway in the rotifer Brachionus koreanus. Aquat. Toxicol. 186, 105–112. Ran, Z., Chen, H., Ran, Y., Yu, S., Li, S., Xu, J., Liao, K., Yu, X., Zhong, Y., Ye, M., Yan, X., 2017. Fatty acid and sterol changes in razor clam Sinonovacula constricta (Lamarck 1818) reared at different salinities. Aquaculture 473, 493–500. Regoli, F., Principato, G.B., Bertoli, E., Nigro, M., Orlando, E., 1997. Biochemical characterization of the antioxidant system in the scallop Adamussium colbecki, a sentinel organism for monitoring the Antarctic environment. Polar Biol. 17, 251–258. Reue, K., Zhang, P., 2008. The lipin protein family: dual roles in lipid biosynthesis and gene expression. FEBS Lett. 582, 90–96. Rivera-Ingraham, G.A., Nommick, A., Blondeau-Bidet, E., Ladurner, P., Lignot, J.H., 2016. Salinity stress from the perspective of the energy-redox axis: lessons from a marine intertidal flatworm. Redox Biol. 10, 53–64. Sargent, J.R., Tocher, D.R., Bell, J.G., 2002. The lipids. In: Halver, J.E., Hardy, R.W. (Eds.), Fish Nutrition, third ed. Academic Press, San Diego, pp. 181–257. Sarma, S.S.S., Nandini, S., Gulati, R.D., 2002. Cost of reproduction in selected species of zooplankton (rotifers and cladocerans). Hydrobiologia 481, 89–99. Snell, T.W., King, C.E., 1977. Lifespan and fecundity patterns in rotifers: the cost of reproduction. Evolution 31, 882–890. Stelzer, C.-P., 2005. Evolution of rotifer life histories. Hydrobiologia 546, 335–346. Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T., Mazur, M., Telser, J., 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39, 44–84. Verity, P.G., Smetacek, V., 1996. Organism life cycles, predation, and the structure of marine pelagic ecosystems. Mar. Ecol. Prog. Ser. 130, 277–293. Won, E.-J., Han, J., Kim, D.-H., Dahms, H.-U., Lee, J.-S., 2017. Rotifers in ecotoxicology. In: Hagiwara, A., Yoshinaga, T. (Eds.), Rotifers: Aquaculture, Ecology, Gerontology, and Ecotoxicology. Springer, Tokyo. Yen, C.-L., Stone, S.J., Koliwad, S., Harris, C., Farese Jr., R.V., 2008. Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis. J. Lipid Res. 49, 2283–2301. Yin, X.W., Niu, C.J., 2011. Testing the cost of reproduction with the rotifer Brachionus patulus at different pH levels. Limnology 12, 145–149. Yin, X.W., Zhao, W., 2008. Studies on life history characteristics of Brachionus plicatilis O.F. Müller (Rotifera) in relation to temperature, salinity and food algae. Aquat. Ecol. 42, 165–176. Yoshinaga, T., Hagiwara, A., Tsukamoto, K., 2003. Life history response and age-specific tolerance to starvation in Brachionus plicatilis O.F. Müller (Rotifera). J. Exp. Mar. Biol. Ecol. 287, 261–271. Zeng, L., Ai, C.X., Wang, Y.H., Zhang, J.S., Wu, C.W., 2017. Abrupt salinity stress induces oxidative stress via the Nrf2-Keap1 signaling pathway in large yellow croaker Pseudosciaena crocea. Fish Physiol. Biochem. 43, 955–964.

trade-off, thus no significant changes between 15 and 35 psu were observed in the total fecundity. This study emphasizes the importance of studying the relationship between salinity and lipid metabolism in the monogonont rotifer B. koreanus. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpa.2017.09.014. Acknowledgement This work was supported by a grant from the National Research Foundation (2017R1A2B4010155) to Jeonghoon Han. References Arts, M.T., Brett, M.T., Kainz, M.J., 2009. Lipids in Aquatic Ecosystems. Springer, New York. Bosque, T., Hernández, R., Pérez, R., Todoli, R., Oltra, R., 2001. Effects of salinity, temperature and food level on the demographic characteristics of the seawater rotifer, Synchaeta littoralis Rousselet. J. Exp. Mar. Biol. Ecol. 258, 55–64. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-gra quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 7, 248–254. Cervetto, G., Gaudy, R., Pagano, M., 1999. Influence of salinity on the distribution of Acartia tonsa (Copepoda, Calanoida). J. Exp. Mar. Biol. Ecol. 239, 33–45. Chakraborty, R.D., Chakraborty, K., Radhakrishnan, E.V., 2007. Variation in fatty acid composition of Artemia salina nauplii enriched with microalgae and baker's yeast for use in larviculture. J. Agric. Food Chem. 55, 4043–4051. Chigbu, P., Suchar, V.A., 2006. Isolation and culture of the marine rotifer, Colurella dicentra (Gosse, 1887), from a Mississippi gulf coast estuary. Aquac. Res. 37, 1400–1405. Dahms, H.-U., Hagiwara, A., Lee, J.-S., 2011. Ecotoxicology, ecophysiology, and mechanistic studies with rotifers. Aquat. Toxicol. 101, 1–12. Dourado, D.F.A.R., Fernandes, P.A., Mannervik, B., Romos, M.J., 2008. Glutathione transferase: new model for glutathione activation. Chem. Eur. J. 14, 9591–9598. Fielder, D.S., Purser, G.J., Battaglene, S.C., 2000. Effect of rapid changes in temperature and salinity on availability of the rotifers Brachionus rotundiformis and Brachionus plicatilis. Aquaculture 189, 85–99. Gniadecki, R., Thorn, T., Vicanova, J., Petersen, A., Wulf, H.C., 2000. Role of mitochondria in ultraviolet-induced oxidative stress. J. Cell. Biochem. 80, 216–222. Hama, T., Handa, N., 1987. Pattern of organic matter production by natural phytoplankton population in a eutrophic lake. I. Intracellular products. Arch. Hydrobiol. 109, 107–120. Han, J., Won, E.-J., Hwang, D.-S., Rhee, J.-S., Kim, I.-C., Lee, J.S., 2013. Effect of copper exposure on GST activity and on the expression of four GSTs under oxidative stress condition in the monogonont rotifer, Brachionus koreanus. Comp. Biochem. Physiol. C 158, 91–100. Han, J., Won, E.-J., Kim, I.-C., Yim, J.H., Lee, S.-J., Lee, J.-S., 2014. Sublethal gamma irradiation affects reproductive impairment and elevates antioxidant enzyme and DNA repair activities in the monogonont rotifer Brachionus koreanus. Aquat. Toxicol. 155, 101–109. Hutchinson, G.E., 1957. A Treatise on Limnology. Wiley, New York. Hwang, D.-S., Dahms, H.-U., Park, H.G., Lee, J.-S., 2013. A new intertidal Brachionus and intrageneric phylogenetic relationships among Brachionus as revealed by allometry and CO1-ITS1 gene analysis. Zool. Stud. 52, 13. Hwang, D.-S., Suga, K., Sakakura, Y., Park, H.G., Hagiwara, A., Rhee, J.-S., Lee, J.-S., 2014. Complete mitochondrial genome of the monogonont rotifer, Brachionus koreanus (Rotifera, Brachionidae). Mitochondrial DNA 25, 29–30. Kauler, P., Enesco, H.E., 2011. The effect of temperature on life history parameters and cost of reproduction in the rotifer Brachionus calyciflorus. J. Freshw. Ecol. 26, 399–408. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Mentjies, P., Drummond, A., 2012. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649. Kim, R.-O., Rhee, J.-S., Won, E.-J., Lee, K.-W., Kang, C.-M., Lee, Y.-M., Lee, J.-S., 2011. Ultraviolet B retards growth, induces oxidative stress, and modulates DNA repairrelated gene and heat shock protein gene expression in the monogonont rotifer, Brachionus sp. Aquat. Toxicol. 101, 529–539. Kooijman, S.A., Troost, T.A., 2007. Quantitative steps in the evolution of metabolic organisation as specified by the dynamic energy budget theory. Biol. Rev. Camb. Philos.

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