Identification and molecular characterization of dorsal ...

MARGEN-00369; No of Pages 9 Marine Genomics xxx (2015) xxx–xxx

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Identification and molecular characterization of dorsal and dorsal-like genes in the cyclopoid copepod Paracyclopina nana Chang-Bum Jeong a,b,1, Min Chul Lee a,1, Kyun-Woo Lee b, Jung Soo Seo c, Heum Gi Park d,⁎, Jae-Sung Rhee e,⁎, Jae-Seong Lee a,⁎ a

Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746, South Korea Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea Pathology Team, National Fisheries Research & Development Institute, Busan 619-902, South Korea d Department of Marine Resource Development, College of Life Sciences, Gangneung-Wonju National University, Gangneung 210-702, South Korea e Department of Marine Science, College of Natural Sciences, Incheon National University, Incheon 406-772, South Korea b c

a r t i c l e

i n f o

Article history: Received 3 March 2015 Received in revised form 8 July 2015 Accepted 7 August 2015 Available online xxxx Keywords: Copepod Paracyclopina nana Dorsal Lipopolysaccharide Culture conditions

a b s t r a c t To date, knowledge of the immune system in aquatic invertebrates has been reported in only a few model organisms, even though all metazoans have an innate immune system. In particular, information on the copepod's immunity and the potential role of key genes in the innate immune systems is still unclear. In this study, we identified dorsal and dorsal-like genes in the cyclopoid copepod Paracyclopina nana. In silico analyses for identifying conserved domains and phylogenetic relationships supported their gene annotations. The transcriptional levels of both genes were slightly increased from the nauplius to copepodid stages, suggesting that these genes are putatively involved in copepodid development of P. nana. To examine the involvement of both genes in the innate immune response and under stressful conditions, the copepods were exposed to lipopolysaccharide (LPS), different culture densities, salinities, and temperatures. LPS significantly upregulated mRNA expressions of dorsal and dorsal-like genes, suggesting that both genes are transcriptionally sensitive in response to immune modulators. Exposure to unfavorable culture conditions also increased mRNA levels of dorsal and dorsal-like genes. These findings suggest that transcriptional regulation of the dorsal and dorsal-like genes would be associated with environmental changes in P. nana. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Copepods have a number of promising characteristics for invertebrate immunity studies (Huq et al., 1983; Kurtz, 2007; Raisuddin et al., 2007). However, in copepods, the innate immune system and the role of immune-relevant genes have as yet rarely been studied. Aquatic invertebrates have a primitive immune response system and also a potential role as intermediate hosts of parasites to upper predators according to their trophic position in the marine food web (Cáceres et al., 2014). Previously, the memorizing capability of the copepod defense system was observed in response to consecutive exposures to antigenically similar parasites (Kurtz and Franz, 2003) and a recent study revealed that cathepsin superfamily is conserved and responsive upon LPS exposure (Jeong et al., 2015), implicating that the host defense mechanism of copepods is a more complex system than expected (Huq et al., 1983; Kurtz, 2007). ⁎ Corresponding authors. E-mail addresses: [email protected] (H.G. Park), [email protected] (J.-S. Rhee), [email protected] (J.-S. Lee). 1 These authors equally contributed to this work.

To date, immunity studies (e.g., recognition of pathogens, cell-free or cellular responses, biomarker development against pathogens, and the integration of immune mechanisms) in aquatic invertebrates has focused on large mollusks or crustaceans including crabs, lobsters, and shrimps, as they are important economically (Mydlarz et al., 2006), while the specific role of each immune-relevant gene is not yet clearly compared to that of the mammalian immune system. In small crustaceans including copepods, several key immune components have been reported with characterization of gene/protein expression of immunity-relevant genes (Decaestecker et al., 2011; Kim et al., 2014; McTaggart et al., 2009), although the basic molecular mechanisms of innate immunity of small crustaceans are still missing and only little attention is given to these tiny species. Thus, the understanding of copepod innate immunity and its sensitivity in response to immune modulators would be comparable with those of large mollusks or crustaceans. The NF-κB and Rel subfamily constitute the Rel/NF-κB superfamily. The Rel/NF-κB members are transcription factors involved in numerous cellular responses to diverse stimuli and function in the host defense system of invertebrates to control the expression of genes encoding immune-relevant proteins (Hetru and Hoffmann, 2009). The Rel

http://dx.doi.org/10.1016/j.margen.2015.08.002 1874-7787/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Jeong, C.-B., et al., Identification and molecular characterization of dorsal and dorsal-like genes in the cyclopoid copepod Paracyclopina nana, Mar. Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.08.002

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C.-B. Jeong et al. / Marine Genomics xxx (2015) xxx–xxx

homology domain (RHD) is conserved for DNA binding, dimerization, and cellular localization (Siebenlist et al., 1994). In insects and crustaceans, dorsal and dorsal-related immunity factor (Dif) proteins have a typical Rel homology domain, translocating to the nucleus to stimulate subsequent gene expression with stimulus-induced degradation of the inhibitor protein Cactus, a homolog to the vertebrate inhibitor of the NF-κB (IκB) gene (Belvin and Anderson, 1996). In copepods, the Rel/ NF-κB gene (annotated as dorsal in this study) was recently identified in the intertidal copepod Tigriopus japonicus and its transcriptional response was analyzed after exposure to lipopolysaccharide (LPS) and two Vibrio sp. (Kim et al., 2014). However, to date, there is no additional dorsal gene information in copepods. The Rel/NF-κB gene family functions as a central regulator of stress responses including environmental or physiological stress (Pahl, 1999). Furthermore, the Rel/NF-κB gene family is involved in the control of diverse signaling pathways beyond the immune response (Oeckinghaus and Ghosh, 2009). Activation of Rel/NF-κB gene family occurs in response to hypoxia, physical stress (i.e. UV-B, gamma radiation) or to oxidative stress (Koong et al., 1994; de Martin et al., 1999; Li and Karin, 1999; Morgan and Liu, 2011). Although the cellular role of Rel/ NF-κB gene family upon environmental stress in vertebrates and fruitfly are well established, the gene information of Rel/NF-κB gene family and molecular response in response to environmental condition still remains yet unclear in aquatic invertebrates. Paracyclopina nana Smirnov 1935 (Cyclopinidae) is a planktonic brackish water cyclopoid copepod and has been recognized as an economically important food source for higher trophic levels (i.e. developing and/or post larvae of crustaceans and fish) in the estuarine and marine environment. Moreover, the copepod has favorable characteristics such as small size, sexual dimorphism, distinctive post-embryonic developmental stages, ease of culturing, and sensitive responses to environmental changes; making P. nana a suitable model species for diverse experimental studies (Lee et al., 2012). In this study, we cloned and characterized the full-length cDNAs of dorsal and dorsal-like genes from the cyclopoid copepod P. nana. Also, we investigated their transcriptional changes at different developmental stages, LPS-exposed conditions, and environmental changes such as different culture densities, salinities, and temperatures. The results of this study are useful to better understand the potential involvement of both genes as immune modulators in copepods. 2. Materials and methods 2.1. Culture and maintenance The cyclopoid copepod P. nana was maintained in a 2 L beaker with the algal diet Tetraselmis suecica (7.8 ± 0.8 μm) in an incubator kept at 25 °C and 15 practical salinity units (psu) under a 16 h light:8 h dark cycle. The culture medium with filtered (1 μm mesh of glass fiber filter) seawater was diluted to 15 psu using distilled water that was changed (100%) using a 50 μm sieve with fresh medium every tenth day. T. suecica were cultivated in 2 L transparent glass bottles with Walne's media (Walne, 1970) and diluted (15 psu) filtered seawater (1 μm mesh of glass fiber filter). The cultures were incubated at 20 °C under a 24 h light photoperiod. The algae culture was fed to copepods during their exponential growth phase.

database to confirm their identities. Both genes were subjected to 5′and 3′-Rapid Amplification of cDNA Ends (RACE) to obtain the remaining parts with 5′- and 3′-RACE system (ver. 2.0 for 5'RACE; ver. E for 3′ RACE; Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Total RNA from approximately 500 adult copepods (both sexes) was isolated with TRIzol® reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) with a tissue grinder and stored at −80 °C until use. Detailed procedure for total RNA extraction is described in Section 2.6. The 5′- and 3′-RACE systems were used to synthesize the single-stranded cDNAs for PCR amplification of the partial cDNA fragments. Genespecific primers (GSPs) were designed for each RACE with partial cDNA sequence based on the manufacture's guideline. The universal primers for each RACE were provided from the kits. The first and nested PCR procedures were carried out with GSP1 and GSP2 primer, respectively (Table S1). A series of RACE were performed under the following conditions: 94 °C/4 min; 40 cycles of 98 °C/25 s, 55 °C/30 s, 72 °C/60 s; and 72 °C/10 min. The final PCR products were isolated from a 1% agarose/TBE gel, cloned into pCR2.1 TA vectors (Invitrogen), and sequenced with an ABI PRISM 3700 DNA analyzer (Bionics Co., Seoul, South Korea). To validate full-length cDNA sequences of dorsal and dorsal-like genes, RT-PCR was employed with two primers: a forward primer containing a start codon, and a reverse primer containing a stop codon. RT-PCR was conducted in a reaction mixture comprising 1 μL of first strand cDNA, 5 μL of 10 × PCR reaction buffer, 1 μL of 10 mM dNTPs, 10 pM concentrations of each primer, and 0.5 μL of NeoTherm™ Taq polymerase (GeneCraft, Köln, Germany). Reaction mixtures were subjected to amplification (1 cycle, 95 °C, 5 min; 30 cycles, 94 °C, 30 s, 55 °C, 30 s, and 72 °C, 30 s; 1 cycle, 72 °C, 7 min) using an iCycler (Bio-Rad, Hercules, CA, USA). The final PCR products were isolated from 1% agarose/Tris-Borate-EDTA (TBE) gels, cloned into pCR2.1 TA vectors (Invitrogen), and sequenced using an ABI PRISM 3700 DNA analyzer (Bionics Co., Seoul, South Korea). All the gene information was registered to the GenBank database, and accession numbers of each gene are listed in Table S1. 2.3. Conserved domain and phylogenetic analysis Conserved domains of dorsal and dorsal-like genes such as the Rel homology domain (RHD) and Ig-like/plexin/transcription factor (IPT) were analyzed through Pfam HMM search (http://pfam.sanger.ac.uk), Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan), and webbased NCBI's Conserved Domain Database (CDD) (Marchler-Bauer et al., 2011). To place dorsal and dorsal-like genes on phylogenetic trees, we performed multiple alignments of these genes using Clustal X software (Ver. 1.83) at the level of deduced amino acid sequences with those of other species. For phylogenetic analysis, we excluded gaps and missing data matrices from the analysis. The generated data matrix was converted to nexus format, and the data matrix was analyzed with Mr. Bayes v3.1.2 program using the general time-reversible (GTR) model. A total of 1000,000 generations were conducted, and the sampling frequency was assigned as every 100 generations. After analysis, the first 10,000 generations were deleted as the burn-in process, and the consensus tree was constructed and then visualized with PHYLIP Tree View software. 2.4. Developmental stage

2.2. Cloning and annotation of dorsal and dorsal-like genes Partial sequences of dorsal and dorsal-like genes were identified in the P. nana RNA-seq database (Lee et al., 2015; number of contigs after trinity assembly, 125,631; length of contigs, 283,955,302 bp; average length, 2260 bp; N50 value, 4178 bp). The cDNA sequences coding for dorsal and dorsal-like genes were subjected to BLAST analysis in the GenBank non-redundant (NR; including all GenBank, EMBL, DDBJ, and PDB sequences except EST, STS, GSS, or HTGS) amino acid sequence

P. nana undergoes anamorphic development with distinct postembryonic developmental stages by molting activity, resulting in naupliar stages (N1–6), copepodid stages (C1–5), and adults (male and female). To prepare different developmental stage samples, entire copepods were separated with three sieves (90, 150, and 200 μm). Of four separated groups (b 90, 90 ~ 150, 150 ~ 200, and N 200 μm), three naupliar stages (N1–2, N3–4, N5–6; 180 individuals were separated into three groups as triplicate for each stage group), four copepod stages



(C1–2, C2–3, C3–4, C4–5; 150 individuals were separated into three groups as triplicate in each stage), and adults (males and females; 120 individuals were separated into three groups as triplicate in each sex) were sampled one by one with two standards (body length and phenotype) under a stereomicroscope (Olympus IX71, Olympus Corporation, Tokyo, Japan). 2.5. Lipopolysaccharide (LPS) exposure LPS was purchased from Sigma (L3024; Sigma-Aldrich, Inc., St. Louis, MO, USA). Lyophilized LPS was dissolved to 10 mg/mL in deionized water. To obtain toxicity values in response to LPS exposure as a preliminary test, ten newly-hatched nauplii (b24 h after hatching) in each of three replicates per concentration were transferred to 12-well tissue culture plates (SPL Life Sciences, Seoul, South Korea) in a 4 mL working volume (30 nauplii in total three replicates). Acute toxicity value of LPS (0, 1, 10, 50, 100, 200, 400, 600, 800, and 1000 μg/L) was determined for 96 h at room temperature (RT). Test solutions were renewed (50% of the working volume) daily, and the green algae T. suecica was added at a density of approximately 6 × 104 cells/mL. No mortality was observed over 96 h of exposure to 1 mg/L LPS except for naturally dying copepods that were occasionally observed 0 or 1 dead individual from 30 copepods in a few LPS-exposed groups of the mortality test. The overall LPS exposure method was followed as reported previously (Kim et al., 2014). Briefly, adult copepods (sex ratio of male and female, 4:6; composition of ovigerous females in total females, 56%) were exposed to 1 mg/L of LPS for 48 h in a 200 mL glass beaker (SPL Labware, Seoul, South Korea). The same volume of deionized water was treated to control group. Three replicates (n = 150 for each replicate) were used for each concentration of the control and LPS-exposed groups. For the time-course experiment, copepods were collected at 0, 1, 3, 6, 9, 12, and 24 h. During the experiment, 50% of culture water was renewed after 24 h and the desired concentrations of LPS were maintained accordingly with a supply of an algal diet of T. suecica every 24 h. Glutathione (GSH) concentration was determined by an enzymatic method with the BIOXYTECH® GSH-420™ kit (OxisResearch®, Portland, OR, USA). After LPS exposure (250, 500, and 1000 μg/L) for 24 h, the copepods (n ≈ 500 for each replicate) were washed in 0.9% NaCl. The rinsed samples were homogenized in trichloroacetic acid at a ratio of 1 to 20 (w/v) with a Teflon homogenizer. The homogenate was centrifuged at 3000 g for 10 min at 4 °C. The upper aqueous layer was collected for the GSH content assay according to the manufacturer's protocol. The GSH content was measured at an absorbance of 420 nm with a spectrophotometer (Ultrospec 2100 pro, Amersham Bioscience, Freiburg, Germany) and the standard curves were generated with GSH equivalents (0, 150, and 350 μM). The total glutathione S-transferase (GST) activity was measured as described by Rhee et al. (2007). After LPS exposure (250, 500, and 1000 μg/L) for 24 h, the copepods (n ≈ 500 for each replicate) were homogenized in cold buffer (0.25 M sucrose, 10 mM Tris, 1 mM EDTA, 0.2 mM DTT and 0.1 mM PMSF, pH 7.4) at a ratio of 1 to 4 (w/v) with a Teflon homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4 °C. The cytosolic fraction containing the enzyme was collected for enzymatic assay with 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The enzymatic assay monitored the conjugation of CDNB and GSH at 340 nm with a spectrophotometer at 25 °C. Total proteins were determined with the Bradford method (Bradford, 1976). The glutathione reductase (GR) activity was measured with an enzymatic method with GR-340™ kits (OxisResearch®, Portland, OR, USA). After LPS exposure (250, 500, and 1000 μg/L) for 24 h, the copepods (n ≈ 500 for each replicate) were homogenized in cold buffer (50 mM Tris–Cl, 5 mM EDTA, and 1 mM 2-mercaptoethanol, pH 7.5) at a ratio of 1 to 4 (w/v) with a Teflon homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4 °C. The upper aqueous layer containing the enzyme was collected for the enzymatic assay according to the

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manufacturer's protocol. The GR activity was then measured at an absorbance of 340 nm with a spectrophotometer at 25 °C. Total proteins were determined with the Bradford method (Bradford, 1976). Overall superoxide dismutase (SOD) enzyme activities were prepared according to our previous study (Kim et al., 2011). The total SOD activities were measured by an enzymatic method with a SOD assay kit (Sigma-Aldrich Chemie, Buchs, Switzerland). After LPS exposure (250, 500, and 1000 μg/L) for 24 h, the copepods (n ≈ 500 for each replicate) were homogenized in ice-cold buffer (0.25 M sucrose, 0.5% triton X-100, pH 7.5) at a ratio of 1 to 4 (w/v) with a Teflon homogenizer. The homogenate was centrifuged at 30,000 g for 30 min at 4 °C. The upper aqueous layer containing the enzyme was collected for the enzymatic assay according to the manufacturer's protocol. The total SOD activities were then measured at an absorbance of 440 nm with a spectrophotometer Thermo™ Varioskan Flash (Thermo Fisher Scientific, Tewksbury, MA, USA) at 25 °C. Enzyme activities were normalized by total protein and represented as % of control. Total proteins were determined with the Bradford method (Bradford, 1976). 2.6. Effect of different culture densities, salinities, and temperatures The overall procedure for different culture densities was followed as reported previously (Lee et al., 2012). Briefly, approximately adult ovigerous females of P. nana (b 48 h after brooding) were cultured at five different densities of 1, 5, 10, 20, or 40 individuals/mL in an incubator (MIR-553, Sanyo, Gunma, Japan) at 25 °C and 15 psu under a 16 h light:8 h dark cycle for 5 days. In each culture density, three replicates were conducted (n = 150 for each replicate). T. suecica of approximately 20,000 cells per female were supplied after the whole culture medium was exchanged with fresh medium every 24 h. During the medium exchange, all adult ovigerous females were separated from newborn nauplii using a 150 μm sieve and restored into the fresh medium, and these nauplii were then counted. For salinity stress, adult copepods (sex ratio of male and female, 4:6; composition of ovigerous females in total females, 56%) were placed in a 200 mL glass beaker (SPL Labware) with various salinities. In each salinity treatment, three replicates were conducted (n = 150 for each replicate). To control salinity, 0.2 μm-filtered artificial seawater (Tetra Marine Salt Pro, Tetra™, Blacksburg, VA, USA) was employed. The copepods were exposed to low (10 psu) or high (20, 25, 32 psu) salinity over their ambient salinity of 15 psu. Samples were taken at 24 h. All experimental treatments were carried out in triplicate. Overall temperature ranges were followed as in our previous study with a minor modification (Rhee et al., 2009). Adult copepods (sex ratio of male and female, 4:6; composition of ovigerous females in total females, 56%) were exposed for 3 h at 15, 20, 25, 30, or 35 °C followed by a recovery period of 1 h. In each temperature exposure, three replicates were conducted (n = 150 for each replicate). The control copepods were maintained at an ambient temperature of 25 °C in a 200 mL glass beaker (SPL Labware). Temperature was controlled in a constant temperature water bath (N-Biotek, Bucheon, South Korea). Each experiment was performed in triplicate. To check whether a temperature change would be stressful to P. nana, mRNA expressions of two heat shock proteins (HQ115579 for Hsp40; HQ115581 for Hsp70) were analyzed in the same temperature conditions, as both Hsp40 and Hsp70 genes showed high sensitivities in response to environmental changes as shown in our previous study (Lee et al., 2012), while other Hsps such as Hsp10, Hsp60, and Hsp90 represented no significant change of expression. 2.7. Total RNA extraction and single-strand cDNA synthesis Entire copepods from each replicate of developmental stage or exposed samples were homogenized in three volumes of TRIzol®


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reagent (Molecular Research Center, Inc.) with a tissue grinder and stored at − 80 °C until use. Total RNA was isolated from the tissues according to the manufacturer's instructions. In brief, the homogenized samples were stored at room temperature (24 °C) for 5 min for phase separation. Chloroform (0.2 volume) was added to the sample, and the tube was vigorously shaken for 15 s. The sample was centrifuged at 12,000 ×g for 15 min at 4 °C. The aqueous phase of the sample was carefully placed into a new tube. Total RNA was precipitated with 0.5 volume of 100% isopropanol. The sample was centrifuged at 12,000 ×g for 10 min at 4 °C. Total RNA was washed with 75% ethanol twice at 4 °C, and the RNA pellet was resuspended with UltraPure™ DEPC-Treated Water (GIBCO® Media Bottles, Carlsbad, CA, USA). Genomic DNA was removed using DNase I (Sigma, St. Louis, MO, USA). The quantity of total RNA was measured at 230, 260, and 280 nm with a spectrophotometer (Ultrospec 2100 pro, Amersham Bioscience, Freiburg, Germany). To check for genomic DNA contamination, we loaded total RNA in a 1% agarose gel that contained ethidium bromide (EtBr) and visualized the gel with a UV transilluminator (Wealtec Corp., Sparks, NV, USA). Also, to verify total RNA quality, we loaded total RNA in a 1% formaldehyde/agarose gel with EtBr staining and checked the integrity and band ratio of 18/28S ribosomal RNAs. A single-strand cDNA was synthesized from total RNA using an oligo(dT)20 primer for reverse transcription (SuperScript™ III RT Kit, Invitrogen) under the following conditions: 65 °C/5 min for denature; place on ice for 1 min; 50 °C/50 min for cDNA synthesis; 85 °C/5 min for termination. To remove RNA, RNase H (Invitrogen) was treated at 37 °C for 20 min.

2.8. Real-time RT-PCR To investigate transcriptional expression patterns of the dorsal and dorsal-like genes, real-time RT-PCR was performed. Each reaction included 1 μL of cDNA and 0.2 μM primer (real-time RT-F/R and 18S rRNA RT-F/R) as shown in Table S1. Primers were designed with dorsal and dorsal-like full-length cDNA sequences using GENRUNNER software (Hastings Software, Inc., Hastings, NY, USA) and confirmed by the Primer 3 program (Whitehead Institute for Biomedical Research, Cambridge, MA, USA). Optimized conditions were transferred according to the following CFX96™ real-time PCR system protocol (Bio-Rad). Reaction conditions were as follows: 95 °C/3 min; 40 cycles of 95 °C/30 s, 55 °C/30 s, and 72 °C/30 s. To confirm the amplification of specific products, cycles were continued in order to check the melting curve under the following conditions: 95 °C/1 min, 55 °C/1 min, and 80 cycles at 55 °C/10 s with 0.5 °C increase per cycle. SYBR® Green (Molecular Probes, Eugene, OR, USA) was used to detect specific amplified products. Amplification and detection of SYBR® Green-labeled products were performed using the CFX96™ real-time PCR system (Bio-Rad). Based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al., 2009), two reference genes were selected from 9 reference candidates (tubulin α; glyceraldehyde 3-phosphate dehydrogenase, gapdh; β-actin; DNA-directed RNA polymerase II subunit RPB2, polr2b; glucose-6-phosphate dehydrogenase, g6pd; hypoxanthine phosphoribosyltransferase 1, hprt1; TATA box binding protein, tbp; elongation factor 1 α, ef1α; 18S ribosomal RNA, 18S rRNA). Data from each experiment were expressed relative to the average ΔCT value of the 18S rRNA and ef1α genes to normalize the expression levels between samples because the expressions of other genes have been shown to be responsive to environmental conditions. Three technical replicates were done in each biological replicate. Data were collected as threshold cycle (C T ) values (i.e. PCR cycle numbers where fluorescence was detected above a threshold and decreased linearly with increasing input target quantity) and used to calculate the ΔCT values of each sample. The fold change in the relative gene expression was calculated by the 2− ΔΔCT method (Livak and Schmittgen, 2001).

2.9. Statistical analysis The SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA) software package was used for statistical analysis. Data are expressed as means ± S.D. Significant differences between observations for developmental stages and exposed groups were analyzed with two-way ANOVA followed by Tukey's test. Any difference showing P b 0.05 was considered significant. 3. Results 3.1. Analysis of cDNA and amino acid sequences of dorsal and dorsal-like genes The full-length cDNA of P. nana dorsal and dorsal-like genes were completely sequenced and deposited in GenBank (Accession Nos. KP258206 for dorsal, KP258207 for dorsal-like). The complete open reading frame (ORF) of P. nana dorsal was 2139 bp in length, encoding a polypeptide of 713 amino acids (Fig. S1). The predicted molecular weight and theoretical isoelectric point (pI) were calculated as 77.9 kDa and 5.54, respectively. The P. nana dorsal protein contained the Rel homology domain (RHD; 76Y–K248) as a characteristic feature of the Rel/dorsal superfamily and also possessed the immunoglobulinlike IPT (Ig-like, Plexins, Transcription factors; 253L–P354) domain. The ORF of the cloned full-length P. nana dorsal-like cDNA was composed of 1758 bp, encoding a putative polypeptide of 586 amino acid residues (Fig. 1). The dorsal-like polypeptide showed a molecular weight of 65.1 kDa with a theoretical pI of 5.38. As shown for the dorsal protein, the two important signature domains, RHD (116I–K283) and IPT (288L– P389), were observed. Both P. nana dorsal and dorsal-like genes possessed the Rel/NF-κB/dorsal signatures (86KGLRFRYECE95 for dorsal and 128KLRFRYECE136 for dorsal-like). Both dorsal and dorsal-like genes were annotated, and their domain structures were analyzed for comparison (Fig. 1). 3.2. Phylogenetic analysis The deduced amino acid sequences of P. nana dorsal and dorsal-like proteins were compared with those of the representative invertebrates and vertebrates from the BLASTX results. The phylogenetic analysis

Fig. 1. Domain analysis with the amino acid sequences of dorsal and dorsal-like genes in the cyclopoid copepod Paracyclopina nana (A) and intertidal copepod Tigriopus japonicus (B). The Rel homology domain (RHD) and the IPP (Ig-like, Plexins, Transcription factors) domain are marked in orange and green color, respectively. The Length of amino acids is drawn to scale. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)



revealed that each protein was closely related to the dorsal and dorsallike proteins of the intertidal copepod T. japonicus (Fig. 2). The phylogenetic topology of both proteins was more closely related to another copepod T. japonicus' dorsal and dorsal-like, but was separated from dorsal or NF-κB proteins of insects and other crustaceans. Although several dorsal isoforms of insects formed a single clade with high similarity in their amino acids, the dorsal and dorsal-like proteins of P. nana did not form a unique clade, and their similarity in amino acid sequences was not high for both positive and identity values (Table 1).

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Table 1 Amino acid similarity of copepods' dorsal and dorsal-like proteins. Abbreviations: Paracyclopina nana, Pn; Tigriopus japonicus, Tj. Positive (%) Pn dorsal Identity (%)

Pn dorsal Pn dorsal-like Tj dorsal Tj dorsal-like

40.23 44.56 37.06

Pn dorsal-like

Tj dorsal

Tj dorsal-like

46.46

52.48 26.92

44.77 55.75 27.34

20.16 47.72

19.85

3.3. Transcriptional expression of dorsal and dorsal-like genes at different developmental stages Transcript levels of the P. nana dorsal and dorsal-like genes were measured in nine distinctive post-embryonic developmental stages comprised by three naupliar stage groups (N1–2, N3–4, N5–6), four copepodid stage groups (C1–2, C2–3, C3–4, C4–5), and an adult stage

(male and female) (Fig. 3A, B). The P. nana dorsal transcript was higher in the early copepodid stage groups (C1–2 and C2–3), while the P. nana dorsal-like transcript was highly expressed in the C2–3 stage group. Although the transcript level of the P. nana dorsal gene was slightly higher in males than females, this difference was not significant. In the case of

Fig. 2. Phylogenetic analysis of the deduced amino acid sequences for P. nana dorsal and dorsal-like genes with those of other species constructed by the Bayesian method. Numbers at branch nodes represent the confidence level of posterior probability. The scale bar represents genetic distance. GenBank accession numbers of Rel/NF-κB proteins were as follows: Aedes aegypti embryonic polarity dorsal (XP_001652840), Anopheles gambiae str. PEST dorsal (XP_310177), Apis mellifera dorsal isoform A (NP_001011577) and isoform B (NP_001164477), Azumapecten farreri Rel/NF-κB (ADD25211), Crassostrea gigas Rel 1 (AAK72690) and Rel 2 (AAK72691), Danio rerio Rel (AAO26402), Daphnia pulex dorsal (EFX79013), Drosophila melanogaster dorsal isoform A (NP_724052), isoform B (AAC35296) and isoform C (NP_724054), Eriocheir sinensis dorsal (AHG95994), Euprymna scolopes Rel/ NF-κB (AAY27981), Fenneropenaeus chinensis dorsal (ACJ36225), Haliotis discus discus Rel/NF-κB (ADI72431), Haliotis diversicolor supertexta Rel/NF-κB (AAW33559), Homo sapiens Rel isoform 1 (NP_002899) and isoform 2 (NP_001278675), Litopenaeus vannamei dorsal (ACZ98167), Mus musculus Rel (NP_033070), Mytilus galloprovincialis Rel/NF-κB (ADM47336), Nasonia vitripennis embryonic polarity protein dorsal (XP_001602675), Oryzias latipes Rel (XP_004076979), Pinctada fucata Rel/NF-κB (ABL63469), Rhodnius prolixus dorsal 1A (ABU96698), 1B (ABU96699) and 1C (ABU96700), Tigriopus japonicus dorsal (AGS12619), and Tribolium castaneum dorsal (NP_001034507).


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Fig. 3. A) Different stages of development of the cyclopoid copepod Paracyclopina nana. Stages 1–6 in the first row are nauplius stages, and nine stages in the second row represent copepodite stages to adults. B) Comparative analysis of the transcriptional abundance of dorsal and dorsal-like genes at various stages of development of P. nana. The values are means of three replicates. Different letters above columns indicate significant differences (P b 0.05).

P. nana dorsal-like, there was a significant difference (P b 0.05) between the levels of adult males and females. 3.4. Transcriptional expression of dorsal and dorsal-like genes in response to environmental conditions No mortality under the experimental concentration of LPS (1 mg/L) was observed over 48 h except for naturally dying copepods. However, significant glutathione (GSH) depletion and induction of enzyme activity of antioxidant proteins such as glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD) were observed in response to different concentrations of LPS at 24 h (Table 2). The transcriptional levels of P. nana dorsal and dorsal-like genes were significantly upregulated in response to LPS exposure after 9 h (dorsal, 2.9 fold; dorsal-like, 3.9 fold; P b 0.05), while their mRNA levels did not change over 6 h (Fig. 4A). Increased transcript levels of both genes were recovered to near initial levels (time zero) at 48 h. Control groups showed no significant change during experiment (Fig. S2). To investigate transcriptional changes of dorsal and dorsal-like genes under environmental stress, P. nana were exposed to different culture densities, salinities, and temperature changes. Previously, nauplii

production of P. nana was shown to reach a maximum density of seven adult females/mL but decreased in density to 10 inds./mL (Lee and Park, 2005). The high culture density (40 inds./mL) had a significant effect on the transcriptional expression of P. nana dorsal (5 fold) and dorsal-like (2.6 fold) genes (Fig. 4B). In particular, mRNA levels of P. nana dorsal were increased by approximately 3.7 fold for 20 inds./mL compared with the 1 ind./mL treatment (P b 0.05). Exposure to different salinities (10, 20, 25, or 30 psu) modulated the transcript levels of P. nana dorsal and dorsal-like genes for 24 h (Fig. 4C). For 10 psu, the level of P. nana dorsal mRNA was slightly Table 2 Changes in total glutathione (GSH) content and enzymatic activity of glutathione Stransferase (GST), glutathione reductase (GR), and superoxide dismutase (SOD) in response to LPS treatment for 24 h. LPS (μ/L)

GSH (% GSH content)

GST (U/mg protein)

GR (U/mg protein)

SOD (U/mg protein)

0 250 500 1000

100 ± 6.8 108 ± 8.7 85 ± 5.5* 82 ± 4.8*

49 ± 4.12 46 ± 3.38 53 ± 4.94 58 ± 3.88*

21 ± 2.36 23 ± 3.11 26 ± 3.59 29 ± 3.47*

18 ± 0.86 17 ± 1.97 21 ± 2.55 25 ± 2.86*



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Fig. 4. A) Time-course effects of LPS (1 mg/L) for 48 h (0, 3, 6, 12, 24, 48, 72, and 96 h) on transcriptional levels of dorsal and dorsal-like genes in P. nana. B) Transcriptional expression profile of dorsal and dorsal-like genes at different culture densities. Transcriptional expression of P. nana dorsal and dorsal-like genes in response to different C) salinities and D) temperatures. Data are means ± S.D. of three replicates of treated copepods. Asterisks (*) indicate P b 0.05.

increased (P N 0.05), while the mRNA level of P. nana dorsal-like was downregulated (2.7 fold; P b 0.05) compared to the control (15 psu). The transcript level of P. nana dorsal was upregulated up to 25 (2.9 fold) and 32 (3.1 fold) psu in a concentration-dependent manner. In the case of dorsal-like, the mRNA levels were upregulated at 20 (2.7 fold; P b 0.05) and 25 (2.8 fold; P b 0.05) psu, but no significant change (P N 0.05) was observed in the 32 psu-exposed P. nana. Upon temperature changes, transcript levels of P. nana dorsal and dorsal-like genes were upregulated at 30 °C (dorsal, 2.1 fold; dorsallike, 2.2 fold; P b 0.05) (Fig. 4D). The transcript levels of both genes at 35 °C were still significantly higher (dorsal, 3.8 fold; dorsal-like, 2.2 fold; P b 0.05) than the ambient culture temperature (25 °C). When P. nana were exposed to low temperatures (15 and 20 °C) compared to the control (25 °C), their transcripts were not significantly changed (P N 0.05). Temperature changes modulated mRNA expression of Hsp40 and Hsp70 (Fig. S3). Hsp70 mRNA expression was significantly induced at 30 (4.6 fold; P b 0.05) and 35 °C (9 fold; P b 0.05), while transcript levels of Hsp40 were elevated at 15 (3.1 fold; P b 0.05) and 35 °C (2.7 fold; P b 0.05). 4. Discussion To examine the transcriptional sensitivity of an immune-relevant response in copepods, dorsal and dorsal-like genes were identified and characterized from the cyclopoid copepod P. nana. According to structural characteristics and phylogenetic relationships, P. nana had the same conserved key transcription factors in innate Toll signaling as shown for the intertidal copepod T. japonicus (Kim et al., 2014). Although the canonical Rel/NF-κB signaling pathway is likely conserved in evolution from invertebrates (except for the nematode Caenorhabditis elegans) to vertebrates, to date, there is still limited

information on the Rel/NF-κB signaling pathway in copepods. To date, no gene annotation for dorsal or dorsal-like genes has been reported in copepods except for T. japonicus (Kim et al., 2014). Extensive transcriptome information of P. nana was recently obtained by a RNAsequence approach (Lee et al., 2015), but no gene for other Rel/NF-κB family members (e.g., Dif or Relish) was observed in both genome and transcriptome databases of P. nana and T. japonicus. We tried to identify any dorsal-like gene with entire amino acid sequences of dorsal protein and Rel homology domain using in silico analyses (i.e. direct mapping to available genome or transcriptome database, BLASTX search at nonredundant (NR) database of GenBank, EMBL, DDBJ, PDB) in other crustaceans including Daphnia, while no gene encoding dorsal-like was found yet. Phylogenetic analysis suggests that this gene is likely to share a common ancestor with invertebrate dorsal. Thus, the annotation of dorsal and dorsal-like genes in P. nana is evidence for a conserved NFκB signaling pathway in copepods and is useful for examining the evolutionary origin of the conserved NF-κB signaling pathway in invertebrates through crustaceans and arthropods. Also, the identification of a dorsal-like gene is an important indicator for copepod-specific gene evolution of the NF-κB signaling pathway, even though more invertebrate dorsal-like genes are not available at the moment. Transcriptional profiles of P. nana dorsal and dorsal-like genes in post-embryogenic developmental stages suggest that each gene would be involved in the general development of copepodite stage. For example, both genes can be modulators in dorsoventral patterning during copepod development, as demonstrated in other invertebrates (Nüsslein-Volhard et al., 1980; Steward, 1987; Roth et al., 1989). Similarly, the dorsal transcript is higher in the early copepodid stages of T. japonicus (Kim et al., 2014), and the Relish transcript is constitutively expressed at most early stages in the mosquito Aedes aegypti (Shin et al., 2002). In Drosophila, the onset of the Toll/Cactus signaling pathway has


8


been accomplished in embryonic and larval stages (Radtke et al., 2005; Qiu et al., 1998). Taken together, P. nana dorsal and dorsal-like genes may be potentially involved in playing a common and/or specialized role in copepod innate immunity during development. To date, diverse aquatic invertebrates including crustaceans have been shown to induce the Rel/NF-κB gene family in response to LPS and/or bacterial challenges (Huang et al., 2010, 2012; Li et al., 2010; Wang et al., 2011; Zhou et al., 2013), although controversial reports have suggested no change in the transcriptional level of Rel/NF-κB members by immune stimulation in abalone and Pacific oyster (Montagnani et al., 2004; Jiang and Wu, 2007; De Zoysa et al., 2010). Particularly, the dorsal transcript is not significantly modulated by different concentrations of LPS or exposure to two Vibrio sp. over time courses in T. japonicus (Kim et al., 2014), while P. nana showed significant induction of dorsal and dorsal-like genes in response to immune stimulation, suggesting that the transcription of both genes was more responsive than that of T. japonicus. Among larger crustaceans, the shrimps Litopenaeus vannamei and Fenneropenaeus chinensis have been extensively studied with respect to the inducible dorsal and NF-κB pathway in response to diverse immune challenges it is conserved in regulating shrimp diseases (Li et al., 2010, 2013; Wang et al., 2011, 2013; Qiu et al., 2014). A dorsal gene from the Chinese mitten crab Eriocheir sinensis was differentially induced by diverse immune modulators (Yu et al., 2013). Although no mortality was observed in the LPS-exposed P. nana, significant oxidative stress was induced by LPS treatment, suggesting that LPS-triggered oxidative stress would induce dorsal expression as suggested previously (Koong et al., 1994; de Martin et al., 1999; Li and Karin, 1999; Morgan and Liu, 2011). Thus, the P. nana dorsal and dorsal-like genes may play a potential role in the innate immune-relevant response to immune challenges (i.e. viral, bacterial, or parasite infections) at the transcriptional level as host defense modulators, although differences in translational regulation, posttranslational modification, and homeostasis maintenance of dorsal protein activity could be of interest for comparison between copepods and more functional studies are required. Despite controversial reports for T. japonicus, the mRNA inducibility of dorsal and dorsal-like genes in copepods may be linked to regulate the expression of other antibacterial regulatory pathways in P. nana. In this study, both genes were significantly modulated by changes in environmental factors, indicating that the transcriptional response of dorsal and dorsal-like genes is very sensitive to environmental stressors. In P. nana, naupliar production decreased with increasing culture density (over 5 inds./mL) due to induction of oxidative stress and the antioxidant defense system (Lee et al., 2012). This crowding effect is considered a strong stressful condition in copepod cultures such as Amphiascoides sp., Centropages typicus, Oithona sp., T. japonicus, and T. fulvus (Walker, 1979; Lazzaretto et al., 1990; Miralto et al., 1996; Kahan et al., 1988; Lipman et al., 2001). Thus, P. nana dorsal and dorsal-like genes may be strongly associated with high density-induced oxidative stress and the antioxidant defense mechanism. Moreover, genes regulated by the Rel/NF-κB superfamily play a major role in regulating the amount of reactive oxygen species (ROS) and oxidative stress that are created by stressful crowding effects (Morgan and Liu, 2011). Upon salinity and heat stress, the transcriptional levels of P. nana dorsal and dorsal-like genes were significantly modulated. These environmental changes strongly induce oxidative stress and stress proteins (e.g., heat shock proteins) in T. japonicus (Seo et al., 2006; Rhee et al., 2009). Salinity fluctuation is considered a modulator of Toll signaling and Rel/NF-κB signaling-involved immune responses in the semiterrestrial and brackish-water crab (Chasmagnathus granulatus), Sydney rock oysters (Saccostrea glomerata), and white shrimp (Litopenaeus vannamei) (Frenkel et al., 2002; Green and Barnes, 2010; Wang and Chen, 2005). At the protein level in a cell, salinity can directly modulate the formation of NF-κB dimerization (Phelps et al., 2000), suggesting that changes in salinity might be transcriptional and/or protein regulators of the Rel/NF-κB superfamily.

Temperature change is a stressful factor for copepods as thermal fluctuation significantly modulates growth, reproduction, post embryonic development, hatching success of embryos, and adult sex ratio of copepods (Lipman et al., 2001; Lee et al., 2003; Devreker et al., 2006; Holste and Peck, 2006; Rhee et al., 2009). For thermal stress in aquatic invertebrates, temperature change significantly regulates innate immune responses including the Toll signaling pathway (Cheng et al., 2004; Hooper et al., 2007). Changes in cell culture temperature modulate the level of NF-κB dimers that further modulate inflammatory responses (Hagiwara et al., 2007). In particular, the mRNA expression of Hsp70 increased significantly at 30 °C and 35 °C in P. nana as shown in the increased expression of the Hsp70 gene in response to elevation of culture temperature in the intertidal copepod T. japonicus (Rhee et al., 2009), suggesting that copepod stress proteins may associate with the Rel/NF-κB signaling pathway, as Hsp70 interacts with the NFκB regulatory complex in response to elevated temperature (Guzhova et al., 1997). Taken together, salinity and temperature changes are obvious stress factors for regulating Rel/NF-κB signaling as shown by the strong modulation of dorsal and dorsal-like genes in P. nana. In conclusion, P. nana dorsal and dorsal-like genes may function in innate immunity and be sensitive in response to immune challenge. Moreover, transcriptional inducibility in response to environmental factors suggests that these dorsal and dorsal-like genes are potentially associated with environmental stress-triggered immune responses and/or mitigation of stressful conditions. Finally, the gene information and their responses will be helpful to better understand fundamental innate immunity in copepods. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.margen.2015.08.002.

Acknowledgments We thank Prof. Hans-U. Dahms for his comments on the revised manuscript and also thank anonymous reviewers' comments on the manuscript. This work was supported by a grant from the National Research Foundation (grant no. 2013010109) funded to Heum Gi Park.

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