Introduction

As temperature is a key environmental factor that can induce ecophysiological responses in many organisms [1], global warming is a great concern in marine organisms over the last few decades [2]. Even a moderate temperature shift can induce vital physiological and biochemical adjustments in organisms, leading to many variations in physiological and life cycle parameters (growth and reproduction) and fatty acid (FA) compositions [3,4,5]. Copepods, as the climate change indicator species [6], are the dominant component of marine mesozooplankton and the major link of trophic interactions [7, 8], which demonstrates rapid adaptation response caused by global warming [9]. To date, the response of temperature on physiological and life parameters of copepods have been demonstrated. For example, in the copepod Pseudocalanus newmani, egg production was suppressed at high temperatures (20 °C) compared to low temperatures [4]. On the other hand, high egg production and hatching rate were shown in the temperature of 25 °C compared to low temperatures (18 and 20 °C) in the copepod Pseudocalanus pelagicus [5]. Also, in the copepod Centropages chierchias, egg production rate was highter at higher temperatures (19 and 24 °C) than 13 °C [8]. However, further investigations are required to gain a better understanding of the mechanistic aspects of temperature-induced physiological and biochemical variations in copepods.

FA compositions in the cell membrane are critical for maintaining membrane fluidity in response to temperature shift [10]. Among FAs, polyunsaturated fatty acids (PUFAs) conferred temperature tolerance in the copepod Calanoides acutus [10]. Fatty acid synthase (FAS), a cytosolic multifunctional enzymatic complex, is responsible for de novo lipogenesis (DNL) of saturated fatty acids (SFAs) from acetyl-CoA (up to C16:0) and further converts to mono- and polyunsaturated fatty acids (MUFA and PUFA) by desaturases and elongases located in the endoplasmic reticulum (Fig. 1) [11]. The biosynthesis of PUFAs could either occur by desaturation of the aliphatic chain of a FA and/or elongation, which adds two carbon units in a controlled manner [12,13,14]. The changes in FA compositions in response to lower temperature have been reported in the calanoid copepod Calanoides acutus [10], the krill Euphausia superba [15,16,17], the scallop Placopecten magellanicus [18], and the water flea Daphnia pulex [19]. In general, the lipid content in copepods is higher for those inhabiting in colder oceans compared to those in warmer environment [20, 21].

Fig. 1
figure 1

Illustration of lipogenesis pathway in the copepod Paracyclopina nana. EcR ecdysone receptor, SREBP sterol regulatory element binding protein, ChREBP carbohydrate regulatory element binding protein, ACLY ATP-citrate lyase, ACC acetyl-CoA carboxylase, KAS β-ketoacyl-acyl-carrier-protein synthase, PA palmitic acid, SA stearic acid, OA oleic acid, LA linoleic acid, GLA γ-linoleic acid, DGLA dihomo-γ-linoleic acid, ARA arachidonic acid, ALA α-linoleic acid, SDA stearidonic acid, ETE eicosatrienoic acid, ETA eicosatetraenoic acid, EPA eicosapentaenoic acid, DPA docosapentaenoic acid, DHA docosahexaenoic acid

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As a key species in the trophic level of marine systems [22], climate change-driven phenological changes in copepods are well known [23]. Moreover, change of ambient temperature resulting in reduced performance and altered life-cycle events in copepods have also been investigated particularly on egg production, hatching rate, growth, fecundity, metabolism, and respiration [24,25,26,27,28]. Despite suitable indicators of the functional structure of marine ecosystems, the temperature effects on copepods also reflect impacts on higher trophic levels. Moreover, molecular studies on the effects of temperature shifts can reveal the optimum culture conditions for the copepod. Although in vivo (e.g. growth and reproduction) analysis of fatty acid composition and the transcriptional regulation of heat shock proteins (hsps) in response to salinity stress was performed to determine the modulation of fatty acids in the copepod Paracyclopina nana [29], the molecular and biochemical mechanism for fatty acid and optimum culture condition of copepods are still lacking.

The cyclopoid copepod P. nana as a poikilothermic model animal has several ideal traits for climate change studies [6] including planktonic nature, small size (<0.6 mm), high fecundity, short generation time (~14 days), and ease of maintenance. Also, P. nana is sensitive to a broad range of environmental conditions (e.g., temperature, salinity, pollutants) and stressors (e.g., heavy metal, radiation), and is used for environmental risk assessment of marine organisms [30, 31]. The available whole genome sequence (85 Mb) and RNA-seq data [30] could also be useful for understanding the genetic expression of P. nana in response to temperature changes.

In this paper, the gene expression patterns involved in lipogenesis pathway in P. nana were investigated. To discover the correlation between gene expression and physiological findings, the area of lipid droplets, which store various lipids (e.g., triacylglycerol and sterols) in phospholipid monolayer, were measured using Nile red and the composition of FA was analyzed in response to different temperature treatment. Furthermore, life parameters (growth, and fecundity) were measured to observe physiological changes due to changes in temperature. This study will provide a better understanding of how temperature affects lipogenesis in the cyclopoid copepod P. nana and their ecophysiological responses to a temperature shift.

Materials and methods

Culture and maintenance

The copepod P. nana was collected from Lake Songji (Gangneung, South Korea, 38°20′9.89″N, 128°30′55.17″E), maintained in the Department of Biological Science, Sungkyunkwan University, Suwon, South Korea, and was used for this study. The animals were fed green marine microalgae Tetraselmis suecica (~6×104 cells/mL) every 24 h and maintained in filtered artificial seawater (ASW) (TetraMarine Salt Pro, Tetra™, Cincinnati, OH, USA) under confined laboratory conditions of 15 practical salinity unit (psu) salinity, and a 12:12 h (light:dark) photoperiod at 25 °C. The species identity of P. nana was confirmed by morphometric analysis followed by molecular characterization of cytochrome oxidase 1 (CO1) DNA of mitochondrial genome [32].

Effect of temperature on lipogenesis pathway genes

To obtain the lipogenesis pathway genes, in silico analysis of P. nana RNA-seq information was performed [30]. Lipogenesis genes were subjected to BLAST analysis in the GenBank non-redundant (NR; including all GenBank, EMBL, DDBJ, and PDB sequence except EST, STS, GSS, or HTGS) amino acid sequence database (http://blast.ncbi.nlm.nih.gov/) to confirm the identity (Table 1).

Table 1 List of lipogenesis genes from Paracyclopina nana and the corresponding primer sequences
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To investigate the expression profile of lipogenesis pathway genes, we measured mRNA expression levels of lipogenesis genes (EcR, SREBP, ChREBP, ACC, ACLY, KAS, elongases [Elongases 1, 2, and 3], and desaturases [Δ4-, Δ5-, and Δ9-desaturase]) over 120 min (0, 20, 40, 60, and 120 min) in response to three temperatures (15, 20, and 30 °C) with a control (25 °C).

Briefly, total RNAs were extracted with TRIZOL® reagent (Invitrogen, Paisley, Scotland, UK) according to the manufacturer’s instructions. Total RNA quantity and quality were analyzed spectrometrically at 230, 260, and 280 nm (Ultrospec 2100pro, Amersham Bioscience, Freiburg, Germany). To synthesize cDNA for real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR), 2 µg of total RNA and oligo (dT)20 primer were used for reverse transcription (SuperScript™ II RT kit, Invitrogen, Carlsbad, CA, USA). Real-time RT-PCR was conducted under the following conditions: 95 °C/4 min; 40 cycles of 95 °C/30 s, 56 °C/30 s, 72 °C/30 s; 72 °C/10 min using SYBR Green as a probe (Molecular Probes Inc., Eugene, OR, USA) 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; 80 cycles of 55 °C/10 s with a 0.5 °C increase per cycle using RT-PCR forward (F) or reverse (R) primers (Table 1). P. nana 18S rRNA gene was used as an internal control to normalize the expression levels among the samples. All experiments were performed in triplicates and used approximately 300 adult copepods in each temperature. The relative fold changes of gene expression compared to control was calculated by the \( 2^{{ - \Delta \Delta {\text{C}}_{\text{T}} }} \) method [33].

Formation of lipid droplet in response to temperature shift

To examine the effects of temperature shift on lipid accumulation in vivo, Nile red staining method for the detection of lipid droplets (LDs) was performed. Briefly, four groups of P. nana were exposed with 15, 20, 25, and 30 °C for 96 h. The group exposed at 25 °C was considered as the control group. The Nile red (1 mg/mL) in acetone, a stock solution of lipophilic stain, was prepared just before use. Copepods were placed in the mixture of formaldehyde (4%) and Nile red (final conc. 2.5 μg/mL) for 5 min. Fixed and stained copepods were viewed under a confocal laser scanning microscope (LSM 510 META; Zeiss, Oberkochen, Germany) at 543 and 560–615 nm excitation and emission wavelengths respectively. Accumulated LDs were assesed as red fluorescent bodies, and the temperature-dependent accumulated LDs were compared to that of the control (25 °C). The area of lipid droplets was analyzed with a LAS image analysis tool (ver. 4.3; Leica, Wetzlar, Germany). Measured area is further represented by relative values compared to the control (25 °C). For each test group, 30 copepods were used and incubated in 50 ml of ASW with T. suecica (~6×104 cells/mL).

Effect of temperature on fatty acid composition

The composition of saturated and unsaturated (n-3, -6, and -9) fatty acids were measured in P. nana in response to different temperature (15, 20, and 30 °C) and compared to that of the control (25 °C). Variations in FA composition were analyzed according to the protocol of Hama and Handa [34] with minor modifications. Briefly, the lipids from various temperature groups (15, 20, and 30 °C) and control (25 °C) 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 boron trifluoride methanol solution. Concentrations and compositions of FAMEs formed were analyzed by a gas chromatography (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, and raised to 200 °C at 10 °C/min and, finally, to 300 °C at 2 °C/min. FAs were identified from the retention times (RT) of standards and mass spectra from gas chromatograph-mass spectrometer (GCMS-QP2010 Plus, Shimadzu, Kyoto, Japan). Experiments were performed in triplicates and used approximately 300 adult copepods in each temperature fed T. suecica (~6×104 cells/mL).

Effects of temperature on development and reproduction

The developmental stages from nauplius (N) to copepodid (C) and to adult (A) of P. nana in response to different in temperatures (15, 20, and 30 °C) were investigated and compared to that of the control (25 °C). For each experiment, ten nauplii/4 mL ASW (<12 h after hatching) were transferred to 12-well cell culture plate (30012, SPL Life Science Co. Ltd., South Korea) and maintained at different temperatures (25 [Control], 15, 20, and 30 °C). Developmental stages were observed once every 24 h for 25 days.

To examine effects of rapid temperature shift on fecundity, adult ovigerous female was exposed to three temperatures (15, 20, and 30 °C) with a control (25 °C) in a 12-well culture plate containing 4 ml ASW, repeated in 10 wells. The number of newly developed nauplii was counted every 24 h for 10 days.

During development and fecundity experiments, 50% of the ASW was replaced with T. suecica (~6×104 cells/mL) once per 24 h throughout the experiments. All experiments were performed in triplicate and stereomicroscope (M205-A, Leica Microsystems, Wetzlar, Germany) was used to observe P. nana.

Statistical analysis

All data were expressed as the mean value with standard error. The Levene’s test verified the normal distribution and homogeneity of variances of data. Significant differences among observations of control and the test groups were analyzed using one-way ANOVA followed by Tukey’s new multiple range test (P < 0.05). All the statistical analyses were performed using SPSS® software (SPSS Inc., Chicago, IL, USA).

Results

Effect of temperature on lipogenesis pathway genes

The genes involved in de novo lipogenesis (EcR, SREBP, ChREBP, ACLY, ACC, and KAS) demonstrated insignificant differences in response to different temperature groups (Fig. 2a–c). Temperature below ambient (15 °C), the expression of three isoforms of elongase genes (Elongase1, 2, and 3) were significantly up-regulated (P < 0.05) at 120 min of exposure with markedly elevated expression of Elongase3 gene (Fig. 2d). However, at temperatures above the ambient (30 °C), all of the elongase and desaturase genes were significantly down-regulated (P < 0.05) at 120 min compared to the control (Fig. 2d, e).

Fig. 2
figure 2

Transcription profiles of lipogenesis genes in response to change in temperature in comparison with 25 °C (ambient temperature as control) represented as heat map diagram. a mRNA transcripts level of ECR, SREBP, ChREBP1, ChREBP2, ACLY, ACC, and KAS genes in Paracyclopina nana exposed to 15 °C, b 20 °C, and c 30 °C for 2 h. d Transcription profile of three isoforms of Elongase genes (Elongase1, 2, and 3) identified from P. nana in response to 15, 20, and 30 °C for 120 min. E) mRNA transcripts of gene encoding desaturase enzyme (∆4-, ∆5-, and ∆9-desaturase) in P. nana in response to different temperature (15, 20, and 30 °C) for 120 min

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Formation of lipid droplet in response to change in temperature

The relative areas of LDs were significantly increased (P < 0.05) in P. nana exposed to 15 and 20 °C, while about 50% reduction was observed at 30 °C (Fig. 3).

Fig. 3
figure 3

Lipid accumulation in Paracyclopina nana exposed to different temperatures. a Confocal photomicrograph of Nile red-stained P. nana exposed to 15, 20, 25, and 30 °C. The fluorescent red color indicates the accumulated lipid droplets in vivo, represented by a bright field, b fluorescence, and c merged. b Accumulation of lipid droplets in P. nana exposed to different temperature represented as a percentage (%) of the relative area against that of control (25 °C) using LAS image analysis tool (n = 30). Significant differences from control value are indicated by different letters on the data bar (P < 0.05) analyzed by Tukey’s post hoc analysis

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Effect of temperature on fatty acid composition

Saturated fatty acids (SFAs, Fig. 4a) and two types of unsaturated fatty acids (USFAs), which are n-3 and -6 (Fig. 4c, d), were significantly higher (P < 0.05) in the control group and below ambient temperature group compared to high temperature (30 °C). Among USFAs, n-3 FAs were significantly higher (P < 0.05) in animals exposed to low temperature (Fig. 4d).

Fig. 4
figure 4

The quantity of fatty acids in Paracyclopina nana exposed to different temperatures (15, 20, 25, and 30 °C) represented as a percentage of control. a Saturated fatty acids. b n-9 fatty acids. c n-6 fatty acids. d n-3 fatty acids. Significant differences from control values are indicated by different letters on the data bar (P < 0.05) analyzed by ANOVA and Tukey’s post hoc analysis. Data are the mean ± SE of duplicates

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Effects of temperature on development and reproduction

Developmental time from N-C and N-A of P. nana exposed to 15 °C showed ~9.5 and 21.7 days, respectively, whereas those at 30 °C were ~5.5 and 11.7 days, respectively (Fig. 5a). Each developmental stage (N-C/N-A) was significantly delayed (P < 0.05) in response to 15 and 20 °C compared to 25 and 30 °C. Fecundity was significantly decreased (P < 0.05) (~20%) at 15 and 20 °C compared with 25 and 30 °C (Fig. 5b).

Fig. 5
figure 5

Effects of temperature on the life parameters of Paracyclopina nana. a Developmental time from nauplius to copepodite (N-C) and nauplius to adult (N-A) at various temperatures (15, 20, 25, and 30 °C). b Fecundity of ovigerous female in terms of a number of hatched nauplii per individual per day over 10 days at different temperatures (15, 20, 25, and 30 °C). Significant differences were analyzed by ANOVA (Tukey’s post hoc test; P < 0.05) and are indicated with different letters. Data are mean ± SE of replicates (n = 10)

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Discussion

Although adaptation potential of the copepods in response to environmental changes have been used as a good indicators for ecotoxicity [6], studies on the composition of fatty acids, as well as fatty acid synthesis are still insufficient as of yet. In this study, we focused on the genes involved in lipogenesis and compositional changes of the fatty acid using the cyclopoid copepod P. nana. By setting the ambient temperature as a standard set point, we have observed correlation between n-3 fatty acid composition and the area of lipid droplet in different temperature (15, 20, and 30 °C). In addition, using Nile red staining technique, we have analyzed actual composition of the fatty acids and its mutual relationship between the area of lipid droplet. Furthermore, life parameters (growth and fecundity) changes were investigated. Thus, this study will provide a better understanding of how temperature affects in vivo life cycle parameters with the underlying molecular responses of lipogenesis in the copepod P. nana.

The result of mRNA expression has demonstrated that genes involved in DNL pathway (EcR, SREBP, ChREBP, ACLY, ACC, and KAS) had minimal changes in response to temperature changes (Fig. 2a–c). In general, DNL pathway functions by breaking down glucose to synthesize lipid strictly under positive nutrition status. In DNL pathway, LXR (liver X receptor), SREBP, and ChREBP were known to act as transcription factor in vertebrates [35], but in recent studies, EcR has demonstrated its potential as a substitute transcription factor for LXR in the copepod Tigriopus japonicus [36, 37]. Thus, we might predict that de novo lipogenesis via acetyl-CoA would not have occurred, as only minimal changes were evident in DNL pathway. However, while both elongases and desaturases were reduced in response to high temperature (30 °C), only elongases demonstrated high expression at low temperature (15 °C) (Fig. 2d, e). The elongation is a simpler enzymatic step than desaturation, as desaturases are frequently rate limiting in PUFA synthesis [38]. Among desaturases, Δ9-desaturase is responsible for the production of monounsaturated fatty acids (MUFAs) (palmitoleic acid and oleic acid) from SFAs [39] by introducing double bond between C-9 and C-10 [15]. Also, the desaturases and elongases involved in the biosynthesis of PUFA demonstrated their role in the harpacticoid copepod Tisbe holothuriae [40, 41]. We could further anticipate that lipogenesis has actually proceeded by different elongases and desaturases mRNA expression, in response to temperature changes. Overall, the DNL pathway, responsible for making fatty acid from glucose, was not affected, indicating that the fatty acids were not direct products of dietary food consumption in our experimental condition. However, the high expression of both elongase and desaturase genes have been demonstrated, and they play a role in the formation of fatty acids from external dietary sources. Taken together, our data supports that temperature-dependent modulations of both elongase and desaturase genes indeed modify the external dietary source.

Nile red staining technique is widely used to identify triacylglycerol and we have used this staining technique to analyze the total area of lipid droplet through confocal microscopy. In comparison to ambient temperature, the total area in low temperature group (15, and 20 °C) was significantly increased, while 50% reduction was observed in high temperature group (30 °C) (Fig. 3). Lipid droplets are mainly composed of triacylglycerol with its surface covered with phospholipid monolayer [42]. The close correlation between the total quantity of fatty acids and lipid droplets exists due to the nature of triacylglycerol structure where three fatty acids are bound to the glycerol backbone. In general, the lipid contents in copepods living in low temperature are higher than those inhabiting in warmer [20, 21], and are stored in the form of LDs [43]. Thus, Nile red staining was used to identify the LDs and also observed modulation along with the developmental stages and xenobiotic exposure in copepod [37, 44, 45]. Consequently, all these thermal adaptation leads to specific changes in the area of LDs accumulation. Thus, we suggest that moderate change on temperature have a profound impact on lipid formation in P. nana.

Through the analysis of fatty acid changes upon temperature shifts, the quantity of fatty acids have significantly increased compared to the temperature at 30 °C with the exception of n-9 fatty acids (Fig. 4 and Table S1). As the biosynthetic pathway of PUFAs is determined by the set of desaturases and elongases present in the cell, the expression pattern of these genes is critical [14]. Moreover, activation of the desaturases is critical for the modifications of FAs composition during cold acclimation [46]. The desaturation of fatty acids in membrane lipids is considered as acclimatory or adaptational changes of organisms in response to low temperature and increased the proportion of PUFAs that is vital for survival [47]. Consequently in P. nana, the reduction in the mRNA expression of elongases and desaturases at high temperature could have negatively impacted on the synthesis of FAs. However, the induction of elongases at lower temperature (15 °C) could have influenced the synthesis of n-3 fatty acids. In normal conditions, n-3 content is comparatively higher than n-6 and -9 fatty acids (Control, Table S1), which could be an advantage for enhancing the tolerance elicited upon stressors (i.e. temperature changes) by synthesizing more n-3 fatty acids in order to form LDs at which the elongases may have played its role. Also, n-3 fatty acids (e.g., eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) are critical in determining food quality in the aquaculture industry [48,49,50,51]. Taken together, exposure to temperature conditions of 15 °C would be an asset to acquire the lipid-rich cultured P. nana (i.e. high n-3 content).

Adverse effects such as decreased developmental time and reduced fecundity in response to below ambient temperatures (15 and 20 °C) was observed compared to higher temperatures (25 and 30 °C) (Fig. 5). In natural conditions, a trade-off in the energy budget between growth and reproduction for maintaining homeostasis in response to environmental factors is common phenomena (e.g., starvation and temperature) [52]. For example, in the rotifer Brachionus plicatilis, lifespan was extended, while fecundity was reduced during starvation [53, 54]. Also, in the copepod Centropages chierchiae, low temperature determines the lower limit of optimal conditions for the distribution due to low productivity [8]. Also, deleterious effects of non-optimal temperature on metabolism [25], respiration [55], fecundity [24], and growth [27] have been reported in copepods. Copepods are adapted to survive within a “thermal window” and change of ambient temperature, resulting in reduced performance that influences the life-cycle events [28]. Thus, temperature changes lead to the adverse effects on life cycle parameters, leading to the energy trade-off for maintaining homeostasis in the copepod P. nana.

Lipids are essential to copepods [43] and their FA compositions in the cell membrane are critical to maintaining membrane order in response to change in physical environment [10]. Temperature shift in P. nana showed both the positive and negative consequences, but it seems to be within the adaptable limits for this species. Taken together, lipogenesis, growth, and reproduction were temperature dependent, leading to a modulated effect at individual and population level in P. nana. Although mRNA expression, FA composition, and the area of LDs demonstrated their close association to one another, it is not clear whether the observed changes in FA composition could contribute to growth and fecundity. In this study, we have provided physiological changes and influence on the lipogenesis in response to temperature changes. This study will warrant further study to a better understanding of the responses of a copepod and the population to temperature shift.