Molecular Cloning and Functional Characterization of Catalase in Stress Physiology, Innate Immunity, Testicular Development, Metamorphosis, and Cryopreserved Sperm of Pacific Abalone

Catalase is a crucial enzyme of the antioxidant defense system responsible for the maintenance of cellular redox homeostasis. The aim of the present study was to evaluate the molecular regulation of catalase (Hdh-CAT) in stress physiology, innate immunity, testicular development, metamorphosis, and cryopreserved sperm of Pacific abalone. Hdh-CAT gene was cloned from the digestive gland (DG) of Pacific abalone. The 2894 bp sequence of Hdh-CAT had an open reading frame of 1506 bp encoding 501 deduced amino acids. Fluorescence in situ hybridization confirmed Hdh-CAT localization in the digestive tubules of the DG. Hdh-CAT was induced by different types of stress including thermal stress, H2O2 induction, and starvation. Immune challenges with Vibrio, lipopolysaccharides, and polyinosinic–polycytidylic acid sodium salt also upregulated Hdh-CAT mRNA expression and catalase activity. Hdh-CAT responded to cadmium induced-toxicity by increasing mRNA expression and catalase activity. Elevated seasonal temperature also altered Hdh-CAT mRNA expression. Hdh-CAT mRNA expression was relatively higher at the trochophore larvae stage of metamorphosis. Cryopreserved sperm showed significantly lower Hdh-CAT mRNA expression levels compared with fresh sperm. Hdh-CAT mRNA expression showed a relationship with the production of ROS. These results suggest that Hdh-CAT might play a role in stress physiology, innate immunity, testicular development, metamorphosis, and sperm cryo-tolerance of Pacific abalone.


Introduction
Catalase (CAT) is the most frequently used biomarker of oxidative stress in animals of aquatic environments [1,2]. Catalase is a vital antioxidant enzyme that can balance the redox system by regulating antioxidant defenses against oxidative stress [3,4]. This antioxidant enzyme responds to the adverse effects of environmental pollution on organisms [1]. CAT is a part of the reactive oxygen species (ROS) network and plays a central role in balancing hydrogen peroxide (H 2 O 2 ) levels in cells [5][6][7]. The key biological mechanism of CAT is to scavenge the excessive level of ROS by directly breaking down H 2 O 2 into water and oxygen [8][9][10][11]. H 2 O 2 is a highly reactive ROS [12] and a secondary key performer of mitochondrial ROS production [13]. Excessive H 2 O 2 lead to infertility whereas CAT restored fertility [14]. H 2 O 2 challenges induce CAT expression in mollusks [3,15]. Whereas heat stress induces H 2 O 2 production [16,17] that ultimately increases CAT expression [18][19][20]. Heat stress also induces CAT activity and increases its mRNA expression in a marine mollusk, Scapharca subcrenata [21]. Cold stress imbalances CAT activity and expression in scallop, Abalone digestive gland tissues (n = 6) were collected after performing anesthesia with 5% MgCl 2 . Samples were washed with pre-chilled 0.1 M PBS and fixed with 4% paraformaldehyde (PFA) for in situ hybridization.

Preparation of Tissue Sections
Paraformaldehyde-fixed digestive gland samples of Pacific abalone were infiltrated using a 30% sucrose solution. Tissue samples were embedded in optimum cutting temperature compounds (FSC 222, Leica Biosystems, Wetzlar, Germany). Transverse-oriented tissues were then sectioned at 8 µM in thickness using a cryostat device (CM 3050, Leica, Wetzlar, Germany) and mounted on electrostatically charged glass slides (SuperFrost Plus, Radnor, PA, USA). Slides were air-dried for 30 min and stored at −20 • C until use. Consecutive slides were prepared with alternative sections for the hybridization of Hdh-CAT mRNA using sense and antisense riboprobes.

Fluorescence In Situ Hybridization (FISH)
FISH was performed following a previously described protocol [45] and DIG in situ hybridization manual with minor modifications. Briefly, a total of 50 mL hybridization buffer (HB) was prepared using deionized formamide (25 mL), 20X saline sodium citrate (SSC, 12.5 mL), 0.1% Tween-20 (0.5 mL), 1M citric acid (0.46 mL), and DEPC-treated water (11.54 mL). Yeast tRNA was mixed with HB at a ratio of 1:9 to prepare HB mix. Digestive gland tissue sections were prehybridized with HB mix for 2 h at 65 • C, fol-Antioxidants 2023, 12, 109 4 of 22 lowed by hybridization with fluorescein-12-UTP-labeled RNA probe (200 ng/mL, diluted with HB mix) at 65 • C overnight. Samples were subsequently washed with degraded series (75%, 50%, and 25% volume) of hybridization mix with 2X SSC for 10 min each at 65 • C. Samples were then washed with SSC (2X and 0.2X) for 15 min. Then, the tissue sections were sequentially cleaned with degraded series of 0.2X SSC (75%, 50%, and 25% volume) mixed with PBST for 5 min and washed with PBST for 5 min. Tissue sections were then incubated with calf serum (10%) for 1 h at 25 • C and then incorporated with Fab fragments antibody (anti-digoxigenin-fluorescein) for 1 h at 25 • C. Samples were then washed three times (10 min each) with PBST, followed by three times of wash with alkalinetris buffer for 5 min each. Finally, hybridized tissue samples were counterstained and mounted using VECTASHIELD ® antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Fluorescence catalase signals were visualized and captured using a confocal microscope with Airyscan2 (LSM 900, ZEISS, Oberkochen, Germany). Images were processed on ZEISS ZEN 3.2 (Blue 268 edition, Oberkochen, Germany) software.
2.6. Thermal Stress Experiments 2.6.1. Cold Stress of Pacific Abalone at 15 • C Cold temperature stress (15 • C) was carried out by transferring abalone into experimental tanks. Water temperature was reduced from 20 • C to 15 • C with a temperature reduction rate of 1 • C/h until it reached 15 • C. Cold temperature (15 • C) was maintained using an electric cooling unit (SunCool, DA-3000C, DAEIL, Busan, Republic of Korea). Abalone reared at 20 • C were sampled as a control, and the cold stressed at 15 • C were sampled at 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h, respectively. Before sampling, abalone were anesthetized with 5% MgCl 2 . Digestive gland and gill tissue samples (n = 6) were collected and washed with 0.1 M PBS. Hemolymph samples were collected and plasma samples were separated for catalase activity assay. Samples were snap-frozen in LN 2 and stored at −80 • C until tRNA extraction and catalase activity assay.
2.6.2. Heat Stress of Pacific Abalone at 25 • C and 30 • C Abalone were transferred into experimental tanks and acclimatized before conducting the thermal stress experiment. Water temperature was gradually increased from 20 • C to 25 • C and 20 • C to 30 • C at 1 • C/h until it reached 25 • C or 30 • C. Abalone reared at 20 • C were used as control, and the heat-stressed abalone at 25 • C and 25 • C were sampled at 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h. Digestive gland and gill tissue samples (n = 6) were collected and washed with 0.1 M PBS. Hemolymph samples were collected and plasma samples were separated for catalase activity assay. Samples were snap-frozen in LN 2 and stored at −80 • C until tRNA extraction and catalase activity assay.

Seasonal Sample Collection
To detect Hdh-CAT mRNA levels in seasonal samples, abalone were collected in different seasons including winter, summer, autumn, and spring (Wando-gun, Republic of Korea), and transported to Molecular Physiology Laboratory, Chonnam National University. After anesthetizing, digestive gland and gill sample (n = 10) were collected and stored at −80 • C until tRNA extraction.

H 2 O 2 Induction Treatments
Abalone were transferred into experimental tanks and acclimatized one week before the H 2 O 2 induction experiment. Abalone were reared in seawater-supplied tanks with continuous aeration. Abalones (n = 6) were intramuscularly injected with 50 µL (0.3 mg/mL) of H 2 O 2 and an equal volume of PBS was injected into control abalone. Digestive gland and gill samples (n = 6) were collected at 3 h, 6 h, 12 h, 24 h, and 48 h. A blood sample was collected and separated the plasma for catalase activity. Samples were immediately snap-frozen in LN 2 and stored at −80 • C until tRNA extraction and catalase activity assay.

CdCl 2 Exposed Treatments
Acclimatized abalone were transferred to a 50 L aquarium to conduct CdCl 2 (Sigma-Aldrich, St. Louis, MO, USA) exposed treatments of Pacific abalone. The Cd 2+ at concentrations of 1.5 mgL −1 , 3 mgL −1 , 6 mgL −1 , and 12 mgL −1 were selected to conduct this experiment. Abalone were exposed to different concentrations of Cd 2+ and the samples (digestive gland and gill) were collected at 3 h, 6 h, 12 h, 24 h, and 48 h. A blood sample was collected and separated the plasma for catalase activity assay. Samples were immediately snap-frozen in LN 2 and stored at −80 • C until tRNA extraction and catalase activity assay.

Tissue Collection of Starved Pacific Abalone
Experimental abalone were starved for 21 days and re-fed for 7 days. Abalone were reared in the experimental aquarium (50 L). Digestive gland, gill, and hemolymph samples (n = 6) were collected from 7 days (st-7 d), 14 days (st-14 d), 21 days (st-21 d) starved, and 7 days re-fed (ref-7 d) abalones. Samples were immediately snap-frozen in LN 2 and stored at −80 • C until tRNA extraction and catalase activity assay.

Immune Challenges of Pacific Abalone to Check the Hdh-CAT Activity and mRNA Abundance
Lipopolysaccharides from Escherichia coli O55:B5 (LPS, Sigma-Aldrich, Saint Louis, MO, USA), polyinosinic-polycytidylic acid sodium salt (PIC, Sigma-Aldrich, Saint Louis, MO, USA), and Vibrio parahaemolyticus (ATCC 17802, Koram Biotech Corp., Seoul, Republic of Korea) were used to conduct the immune challenge experiments. LPS and PIC were separately injected into the adductor muscle at a concentration of 10 µg/g-BW. V. parahaemolyticus were cultured in Luria-Bertani broth (Becton, Dickinson and Company, Sparks, MD, USA) and injected in the adductor muscle at a previously recommended dose [46]. Control abalone were injected with an equal volume of PBS. Digestive gland, gill, and hemolymph samples (n = 6) were collected 3 h, 6 h, 12 h, 24 h, and 48 h after the injection. Samples were immediately snap-frozen in LN 2 and stored at −80 • C until tRNA extraction and catalase activity assay.

Tissue Collection of Testicular Developmental Stages
Testis tissue samples (n = 10) were collected during gonadal development stages including the degenerative stage (DS), active stage (AS), ripening stage (RS), and spent stage (SS) as described previously [47].

Total RNA Extraction and cDNA Synthesis
Total RNA of all collected samples was extracted using an ISOSPIN Cell & Tissue RNA kit (Nippon Gene, Tokyo, Japan). Total RNA concentration was measured using a spectrophotometer (Nanodrop ACTGene ASP-2680, Piscataway, NJ, USA). cDNA synthesis was accomplished by reverse transcribing the tRNA using a Superscript ® III First-Strand synthesis kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedures. To obtain a partial Hdh-CAT sequence, cDNA synthesized from the digestive gland was reverse transcribed using reverse transcription (RT) primers (Table S1) designed from known catalase sequence of H. discus discus (GenBank accession no. DQ821496.1). Reverse transcription polymerase chain (RT-PCR) reaction mixture (20 µL) was prepared using a 1 µL cDNA template, 1 µL of each sense and antisense primers (20 pmol), 4 µL of HF buffer, 2 µL of dNTP mix, 0.5 µL of DNA polymerase, and 10.5 µL sterile distilled water (dH 2 O). The RT-PCR was conducted in a thermal cycler with the following conditions: an initial denaturation at 94 • C for 3 min; 35 cycles of denaturation at 94 • C for 30 s; annealing at 58 • C for 30 s; and extension at 72 • C for 30 s; with a final dissociation step of 5 min at 72 • C. The PCR products were separated on 1.2% agarose gel and purified using a gel extraction kit (Promega, Madison, WI, USA). Purified DNA was ligated to a pTOP Blunt V2 vector (Enzynomics, Daejeon, Republic of Korea) and transformed into Escherichia coli DHα competent cells (Enzynomics, Daejeon, Republic of Korea). Plasmid DNA was extracted from positive clones using a Hybrid-QTM Plasmid Rapidprep mini kit (GeneAll, Seoul, Republic of Korea). Sequencing was performed using the Macrogen Online Sequencing System (Macrogen, Seoul, Republic of Korea).

Cloning of the Full-Length Hdh-CAT Sequence
To obtain full-length Hdh-CAT sequence, gene-specific primers (GSP) were designed (Table S1) from a partial sequence, including a 15 bp overlap with the 5 end of GSP sequences. Rapid amplification of cDNA ends (RACE) PCR was conducted using 50 µL volume of PCR mixture containing 2.5 µL of first-strand cDNA from the digestive gland, 5 µL of universal primer mix, 25 µL of SeqAMP buffer, 1 µL of SeqAMP DNA polymerase, and 15.5 µL pf PCR grade water. Touchdown PCR was performed with 25 cycles for rapid amplification of 3 cDNA ends (3 -RACE) and 5 -RACE PCR following the manufacturer's protocol. PCR products were purified from 1.2 % agarose gel using a NucleoSpin ® Gel and PCR Clean-up kit (MARCHERY-NAGEL GmbH & Co. KG, Düren, Germany). Purified products were cloned into a linearized pRACE vector (Clontech Laboratories, Inc., Mountain View, CA, USA) and transformed into Stellar competent cells. Plasmid DNA extraction and sequencing were performed as the method described in the "Cloning of partial Hdh-CAT sequence" section. Finally, the sequences were combined by overlapping with the initial cloned fragment to obtain the full-length sequence of Hdh-CAT.

Phylogenetic Analysis
Catalase protein sequences of different organisms were retrieved from the NCBI database and selected to construct the phylogenetic tree. Protein sequences were aligned using the Clustal Omega program. Phylogenetic and molecular evolutionary analyses were performed using MEGA 11 (https://www.megasoftware.net/, accessed on 5 February 2022) with the neighbor-joining algorithm following 1000 bootstrap replicates.

Quantitative Real-Time PCR (qRT-PCR) Analysis
qRT-PCR analysis was performed to quantify Hdh-CAT mRNA expression in different types of tissue samples. qRT-PCR was conducted on a LightCycler ® 96 system (Roche, Grenzach-Wyhlen, Germany) using a 2× qPCRBIO SyGreen Mix Lo-Rox kit (PCR Biosystems, Ltd., London, UK) as described previously [49]. Gene-specific sense and antisense primers (Table S1) were designed to quantify Hdh-CAT mRNA in different tissues. PCR was performed using a 20 µL volume of reaction mix containing 1 µL of cDNA, 1 µL of each sense and antisense primer, 10 µL of SyGreen Mix, and 7 µL of dH 2 O. The following melting temperature was used as default settings: 95 • C for 10 s, 65 • C for 1 min, and 97 • C for 1 min. The relative Hdh-CAT mRNA expression was quantified using the 2 −∆∆ct method [50]. Expression levels were normalized by amplifying a housekeeping Pacific abalone β-actin gene.

Catalase Activity
Catalase activity in hemolymph samples was determined using a catalase colorimetric activity kit (Invitrogen, Frederick, MD, USA) according to the manufacturer's instructions. Catalase concentration (U/mL) at 560 nm was measured (n = 3) using an Epoch TM Microplate Spectrophotometer (Epoch 2, BioTek, Winooski, VT, USA).

Fluorescent Technique to Detect ROS in DG Tissue Samples of Pacific Abalone
Thermal stress (15 • C and 30 • C), H 2 O 2 induced, starved, Cd-exposed (12 mgL −1 ) and V. parahaemolyticus-challenged digestive glands were used to conduct this experiment. Tissue samples were selected based on higher Hdh-CAT mRNA abundance levels (Heat challenged abalone: 48 h; and Cadmium-exposed abalone: 48 h). Reactive oxygen species (ROS: O 2 •− production) were detected using a DHE (dihydroethidium) assay kit (Invitrogen Molecular Probes, Eugene, OR, USA) according to the method described previously [51], with minor modifications. Briefly, digestive gland tissue samples were homogenized and washed using 0.1M PBS. Tissue samples were stained with 10 µM DHE and incubated for 30 min at 10 • C in the dark. DHE-stained cells were visualized under a fluorescence microscope (red filter: 510-560 nm, Eclipse E600, Nikon, Tokyo, Japan) with a 20× objective lens. Gray values of 200 cells in each treatment were measured using ImageJ software version 1.8.0_172 (https://imagej.nih.gov/ij/download.html, accessed on 13 April 2022).

Statistical Analysis
Changes of Hdh-CAT mRNA expression levels were analyzed by GraphPad Prism 9.3.1 software (GraphPad Software, San Diego, CA, USA) following nonparametric one-way analysis of variance (ANOVA). Tukey's post hoc test was performed to calculate statistically significant differences among different experimental tissues of Pacific abalone. Data from qRT-PCR are expressed as the mean ± SD. Differences were considered as significant at p < 0.05. GraphPad Prism 9.3.1 software was used to generate graphs.

Cloning and Bioinformatic Analysis of H. discus hannai Catalase (Hdh-CAT) Sequence
The complete sequence of H. discus hannai catalase was cloned from digestive gland samples by 5 -RACE and 3 -RACE PCR and named Hdh-CAT ( Figure 1A). The fulllength sequence of Hdh-CAT (GenBank: OK042347.1) was 2894 bp in length, including a 148 bp 5 -untranslated region (UTR) and a 1240-bp 3 -UTR with a canonical polyadenylation signal sequence (AATAAA) located 12 bp upstream of the poly-A tail. The open reading frame (ORF) of Hdh-CAT had a length of 1506 bp, encoding 501 deduced amino acids (GenBank: UFT26656.1).
The molecular weight and theoretical isoelectric point (pI) of Hdh-CAT protein were predicted to be 56.46 KDa and 8.81, respectively. Glycine was the most abundant amino acid  Multiple alignments of catalase homologs from selected amino acid sequences of vertebrate and invertebrate are shown in Figure S2. Active site motif and heme-ligand signature motif were well conserved in aligned amino acid sequences of H. discus discus, H. diversicolor, Danio rerio, and Homo sapiens. Multiple alignment revealed 12 NADPH binding site residues (N 145 , H 191 , F 195 , S 198 , R 200 , N 210 , Y 212 , K 234 , V 299 , W 300 , H 302 , and Y 355 ). Hdh-CAT shared the highest sequence identities (99.38%) with CAT of H. discus discus.

Homology Modeling of Hdh-CAT
Three-dimensional structures of Hdh-CAT exhibited four basic domains including an Nterminal domain, an eight-stranded β-barrel domain, a wrapping loop-formed connection domain, and a helical C-terminal domain ( Figure 1B).

Phylogenetic Analysis
A phylogenetic tree was constructed using the neighbor-joining method to show the possible evolutionary linkage of Hdh-CAT with other catalase proteins. The phylogenetic tree showed three major groups where Hdh-CAT was positioned in the mollusk group ( Figure 1C). Hdh-CAT was closely positioned with H. discus discus catalase.

Tissue Distribution Analysis of Hdh-CAT
Hdh-CAT mRNA expression levels in different tissue samples are shown in Figure S3. The expression levels of Hdh-CAT mRNA were significantly higher in the digestive gland (DG) tissue samples than in other examined tissue samples.

Homology Modeling of Hdh-CAT
Three-dimensional structures of Hdh-CAT exhibited four basic domains including an N-terminal domain, an eight-stranded β-barrel domain, a wrapping loop-formed connection domain, and a helical C-terminal domain ( Figure 1B).

Phylogenetic Analysis
A phylogenetic tree was constructed using the neighbor-joining method to show the possible evolutionary linkage of Hdh-CAT with other catalase proteins. The phylogenetic tree showed three major groups where Hdh-CAT was positioned in the mollusk group ( Figure 1C). Hdh-CAT was closely positioned with H. discus discus catalase.

Tissue Distribution Analysis of Hdh-CAT
Hdh-CAT mRNA expression levels in different tissue samples are shown in Figure  S3. The expression levels of Hdh-CAT mRNA were significantly higher in the digestive gland (DG) tissue samples than in other examined tissue samples ( Figure S4).

Hdh-CAT mRNA Expression in Thermal Stressed Tissue Samples of Pacific Abalone
Expression levels of Hdh-CAT mRNA in gill and digestive gland (DG) tissues of thermal-stressed Pacific abalone are shown in Figure 3.    Figure 3D). However, in gill samples, the expression level was neutralized at time points of 48 h to 72 h.

Hdh-CAT mRNA Expression in Heat Stressed (30 • C) Samples
The mRNA expression level of Hdh-CAT in gill samples of heat-stressed Pacific abalone was significantly (p < 0.05) higher at time points of 6 h ( Figure 3E). DG samples showed a significantly (p < 0.05) higher expression level of Hdh-CAT mRNA at time points of 48 h ( Figure 3F).

Hdh-CAT mRNA Expression in Seasonal Samples
Hdh-CAT mRNA expression levels in gill were significantly (p < 0.05) higher in autumn ( Figure 3G) compared to other seasons. Whereas DG samples showed significantly (p < 0.05) higher Hdh-CAT mRNA expression levels in spring ( Figure 3H).

Hdh-CAT mRNA Expression in H 2 O 2 Induced Tissue Samples of Pacific Abalone
The mRNA expression level of Hdh-CAT reached its peak at time points of 24 h in the gill ( Figure 4A), and 12 h in the DG ( Figure 4B) of H 2 O 2 -induced Pacific abalone.

Hdh-CAT mRNA Expression in Heat Stressed (30 °C) Samples
The mRNA expression level of Hdh-CAT in gill samples of heat-stressed Pacific abalone was significantly (p < 0.05) higher at time points of 6 h ( Figure 3E). DG samples showed a significantly (p < 0.05) higher expression level of Hdh-CAT mRNA at time points of 48 h ( Figure 3F).

Hdh-CAT mRNA Expression in Seasonal Samples
Hdh-CAT mRNA expression levels in gill were significantly (p < 0.05) higher in autumn ( Figure 3G) compared to other seasons. Whereas DG samples showed significantly (p < 0.05) higher Hdh-CAT mRNA expression levels in spring ( Figure 3H).

Hdh-CAT mRNA Expression in H2O2 Induced Tissue Samples of Pacific Abalone
The mRNA expression level of Hdh-CAT reached its peak at time points of 24 h in the gill ( Figure 4A), and 12 h in the DG ( Figure 4B) of H2O2-induced Pacific abalone.

Hdh-CAT mRNA Expression in Cdcl2 Exposed Tissues of Pacific Abalone
The expression levels of Hdh-CAT mRNA in different concentrations of CdCl2 exposed tissue samples are presented in Figure 4C,D. Hdh-CAT mRNA expression levels were significantly (p < 0.05) different in different concentrations of CdCl2 exposed gill and DG samples, compared to the control. In gill samples, higher Hdh-CAT mRNA expression levels were shown at the time points of 3 h in the 12 mgL −1 CdCl2, 6 h in the 3 mgL −1 , and 6 mgL −1 CdCl2, respectively ( Figure 4C). DG tissue samples showed significantly higher Hdh-CAT mRNA at the time points of 6 h in the 12 mgL −1 CdCl2-exposed abalone ( Figure  4D).

Hdh-CAT mRNA Expression in CdCl 2 Exposed Tissues of Pacific Abalone
The expression levels of Hdh-CAT mRNA in different concentrations of CdCl 2 exposed tissue samples are presented in Figure 4C,D. Hdh-CAT mRNA expression levels were significantly (p < 0.05) different in different concentrations of CdCl 2 exposed gill and DG samples, compared to the control. In gill samples, higher Hdh-CAT mRNA expression levels were shown at the time points of 3 h in the 12 mgL −1 CdCl 2 , 6 h in the 3 mgL −1 , and 6 mgL −1 CdCl 2 , respectively ( Figure 4C). DG tissue samples showed significantly higher Hdh-CAT mRNA at the time points of 6 h in the 12 mgL −1 CdCl 2 -exposed abalone ( Figure 4D).

Hdh-CAT mRNA Expression in Starved Tissue Samples of Pacific Abalone
Hdh-CAT mRNA expression levels were significantly (p < 0.05) higher in Pacific abalone gill at 14 days after starvation (st-14 d) ( Figure 5A). DG samples showed significantly (p < 0.05) higher Hdh-CAT mRNA expression levels at 21 days after starvation (st-21 d) ( Figure 5A) of Pacific abalone. After re-feeding for seven days, Hdh-CAT mRNA expression levels in DG of starved samples were similar to those in control samples without starvation ( Figure 5B).

Hdh-CAT mRNA Expression in Starved Tissue Samples of Pacific Abalone
Hdh-CAT mRNA expression levels were significantly (p < 0.05) higher in Pacific abalone gill at 14 days after starvation (st-14 d) ( Figure 5A). DG samples showed significantly (p < 0.05) higher Hdh-CAT mRNA expression levels at 21 days after starvation (st-21 d) ( Figure 5A) of Pacific abalone. After re-feeding for seven days, Hdh-CAT mRNA expression levels in DG of starved samples were similar to those in control samples without starvation ( Figure 5B).

Hdh-CAT Expression in Vibrio parahaemolyticus Challenged Samples
The mRNA expression level of Hdh-CAT reached its peak in the gill at 6 h after V. parahaemolyticus challenge ( Figure 6A) and in the DG at 12 h after V. parahaemolyticus challenge ( Figure 6B). Expression levels were stabilized in both tissue samples at 24 h and 48 h after V. parahaemolyticus challenge ( Figure 6A,B).

Hdh-CAT Expression in Vibrio parahaemolyticus Challenged Samples
The mRNA expression level of Hdh-CAT reached its peak in the gill at 6 h after V. parahaemolyticus challenge ( Figure 6A) and in the DG at 12 h after V. parahaemolyticus challenge ( Figure 6B). Expression levels were stabilized in both tissue samples at 24 h and 48 h after V. parahaemolyticus challenge ( Figure 6A,B).

Hdh-CAT Expression in Lipopolysaccharides (LPS) Challenged Samples
The mRNA expression level of Hdh-CAT reached its peak at time points of 24 h in the gill ( Figure 6C), and DG ( Figure 6D) of LPS-challenged Pacific abalone.

Hdh-CAT Expression in Poly I:C Challenged Samples
The mRNA expression level of Hdh-CAT reached its peak at time points of 24 h in the gill ( Figure 6E), and 6 h in the DG ( Figure 6F) of PIC-challenged Pacific abalone.

Hdh-CAT mRNA Expression in Testicular Developmental Stages
Hdh-CAT mRNA expression levels during different testicular developmental stages are given in Figure 7A. Ripening stage showed significantly (p < 0.05) higher Hdh-CAT mRNA expression levels compared to other stages ( Figure 7A).

Hdh-CAT mRNA Expression in Cryopreserved Sperm
Hdh-CAT mRNA expression levels in fresh sperm and different types of cryopreserved sperm samples are shown in Figure 7B. Hdh-CAT mRNA expression levels in cryopreserved sperm samples were significantly lower than those in fresh sperm ( Figure  7B).

Hdh-CAT mRNA Expression in Embryonic and Larval Developmental Stages
Hdh-CAT mRNA expression levels during embryogenesis are shown in Figure 8. Blastula (BL) and trochophore (TRO) larvae showed significantly (p < 0.05) higher Hdh-CAT mRNA expression levels compared to other stages.

Hdh-CAT mRNA Expression in Cryopreserved Sperm
Hdh-CAT mRNA expression levels in fresh sperm and different types of cryopreserved sperm samples are shown in Figure 7B. Hdh-CAT mRNA expression levels in cryopreserved sperm samples were significantly lower than those in fresh sperm ( Figure 7B).

Hdh-CAT mRNA Expression in Embryonic and Larval Developmental Stages
Hdh-CAT mRNA expression levels during embryogenesis are shown in Figure 8. Blastula (BL) and trochophore (TRO) larvae showed significantly (p < 0.05) higher Hdh-CAT mRNA expression levels compared to other stages.

Catalase (CAT) Activity in the Hemolymph
CAT activities in hemolymph samples of cold (15 • C), heat-stressed (25 • C and 30 • C), H 2 O 2 -induced, starved, immune-challenged, and Cd-exposed abalone are summarized in Figure 9. Cold-stressed abalone showed significantly (p < 0.05) higher CAT activity at 6 h after cold stress ( Figure 9A), whereas abalone with heat stress (25 • C and 30 • C) showed higher CAT activities at 48 h after heat stress ( Figure 9B,C). H 2 O 2 -induced abalone showed higher CAT activity at the time point 3 h (Figure 9D), significantly similar to the time point of 6 h post-induction. Starved abalone showed significantly higher CAT activities at st-14 d ( Figure 9E). However, re-fed (ref-7 d) abalone showed similar (p > 0.05) CAT activity to the control. LPS-challenged abalone showed significantly (p < 0.05) higher CAT activity at 12 h after LPS challenge ( Figure 9F). PIC ( Figure 9G) and V. parahaemolyticus ( Figure 9H)challenged abalone showed significantly higher CAT activity at 6 h after challenge. Cd exposure increased CAT activity in a dose-dependent manner ( Figure 9I). After exposure to 6 mgL −1 of Cd, CAT activity was significantly higher at 6 h after exposure ( Figure 9I). cryopreserved sperm samples were significantly lower than those in fresh sperm ( Figure  7B).

Hdh-CAT mRNA Expression in Embryonic and Larval Developmental Stages
Hdh-CAT mRNA expression levels during embryogenesis are shown in Figure 8. Blastula (BL) and trochophore (TRO) larvae showed significantly (p < 0.05) higher Hdh-CAT mRNA expression levels compared to other stages.

Catalase (CAT) Activity in the Hemolymph
CAT activities in hemolymph samples of cold (15 °C), heat-stressed (25 °C and 30 °C), H2O2-induced, starved, immune-challenged, and Cd-exposed abalone are summarized in Figure 9. Cold-stressed abalone showed significantly (p < 0.05) higher CAT activity at 6 h after cold stress ( Figure 9A), whereas abalone with heat stress (25 °C and 30 °C) showed higher CAT activities at 48 h after heat stress ( Figure 9B,C). H2O2-induced abalone showed higher CAT activity at the time point 3 h (Figure 9D), significantly similar to the time point of 6 h post-induction. Starved abalone showed significantly higher CAT activities at st-14 d ( Figure 9E). However, re-fed (ref-7 d) abalone showed similar (p > 0.05) CAT activity to the control. LPS-challenged abalone showed significantly (p < 0.05) higher CAT activity at 12 h after LPS challenge ( Figure 9F). PIC ( Figure 9G) and V. parahaemolyticus ( Figure 9H)challenged abalone showed significantly higher CAT activity at 6 h after challenge. Cd exposure increased CAT activity in a dose-dependent manner ( Figure 9I). After exposure to 6 mgL −1 of Cd, CAT activity was significantly higher at 6 h after exposure ( Figure 9I).

ROS in DG Tissue Samples of Pacific Abalone
The results of ROS production in response to thermal stress (15 • C and 30 • C), starvation, H 2 O 2 induction, Vibrio challenged, and Cd-exposed toxicity are presented in Figure 10. Heatinduced (30 • C) abalone showed higher ROS production at 72 h ( Figure 10A,B). H 2 O 2induced, Vibrio-challenged, and Cd-exposed abalone showed higher ROS production at 48 h ( Figure 10A,B). However, re-feed abalone showed reduced ROS production ( Figure 10A,B).

ROS in DG Tissue Samples of Pacific Abalone
The results of ROS production in response to thermal stress (15 °C and 30 °C), starvation, H2O2 induction, Vibrio challenged, and Cd-exposed toxicity are presented in Figure  10. Heat-induced (30 °C) abalone showed higher ROS production at 72 h ( Figure 10A,B). H2O2-induced, Vibrio-challenged, and Cd-exposed abalone showed higher ROS production at 48 h ( Figure 10A,B). However, re-feed abalone showed reduced ROS production ( Figure 10A,B).

Discussion
The aim of the present study was to isolate catalase and detect its molecular regulation in stress physiology, innate immunity, testicular development, elevated seasonal temperature, metamorphosis, and cryopreserved sperm of Pacific abalone. The full-length cDNA of Pacific abalone catalase (Hdh-CAT) was isolated for the first time, and its expression in digestive gland tissue was characterized. The architecture of Hdh-CAT showed key features of a catalase gene, including a proximal heme-ligand signature motif, a proximal active site signature, and heme-binding site residues. These features are common in most catalases [3].
Fluorescence in situ hybridization confirmed the localization of Hdh-CAT mRNA in the digestive tubules of the digestive gland (DG). Previous studies have reported that CAT is immunolocalized in the digestive tubules of different mollusks including oyster, and mussel [52]. CAT is also immunolocalized in the digestive tubules of crab, and the liver of mullet [52]. Molluscan DG has combined functions of the liver, pancreas, and intestine of vertebrates [53]. The liver is the predominant source of peroxisomes where CAT is exclusively located [54,55]. Tissue distribution analysis also revealed that Hdh-CAT mRNA was highly expressed in DG tissue samples, consistent with findings of CAT in disk abalone [3].
Temperature is a vital abiotic factor that can significantly affect the physiology of marine organisms [21]. In recent decades, seawater temperature has been rising with the acceleration of climate change [21,56]. Temperature changes are known to influence ROS production and activate antioxidant enzymes in mollusk [57]. Marine invertebrates have strong antioxidant defense systems including CAT to normalize ROS accumulation and physiological function [58]. An elevated activity of CAT is essential to counteract thermal stress-induced oxidative damage [59]. In the present study, induced CAT activity and higher Hdh-CAT mRNA expression levels were observed in response to thermal stress. Similar findings have been previously reported for CAT in scallop [4], ark shell [21], Mediterranean mussel [60], and Pacific oyster [61]. The present study also found elevated ROS levels in thermal-stressed abalone, consistent with previous findings of American oyster [31]. It has been reported that both heat and cold stress lead to excessive ROS production [13]. Present findings suggest that thermal stress can increase the CAT activity of Pacific abalone which may have a relationship with ROS production since it has been described that the overproduction of ROS is associated with the dysfunction of the antioxidant defense system. The antioxidant defense system consists of five vital antioxidant genes including catalase, Cu/Zn-superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase, and glutathione reductase [62].
H 2 O 2 is ubiquitously distributed in the surface seawater [63]. H 2 O 2 can affects the survivability, growth, and metabolism of aquatic organisms [64]. In the present study, H 2 O 2injected Pacific abalone showed upregulated Hdh-CAT mRNA expression in gill and DG. Similar phenomena have been reported in H. discus discus [3]. Induced [15]. The fluorescence technique detected higher ROS in DG at 48 h after H 2 O 2 induction, which might be the reason for the lower CAT mRNA abundance and activity. Similar phenomena have been previously reported for CAT in American oyster [31]. Present findings suggest that Hdh-CAT might play a role in the antioxidant defensive mechanism against ROS generation induced by H 2 O 2 . Another antioxidant, SOD, has been previously recommended as an antioxidant defender against H 2 O 2 -induced damage in Pacific abalone [4].
Cd exposure increased Hdh-CAT mRNA expression levels in the gill and DG of Pacific abalone in a dose-dependent manner. Similar findings have been previously reported for CAT in the gill of Pacific oyster after Cd exposure [65]. Higher CAT mRNA expression levels could protect Pacific oyster against Cd exposure-induced oxidative stress [65]. The present study detected higher ROS in Cd-exposed DG using a fluorescent technique, although induced ROS production in Cd-exposed tissue samples of abalone has been previously reported with a colorimetric method [66]. In this study, hemolymph of Cd-exposed abalone showed induced CAT activity. Similar findings have been previously reported for marine invertebrate after exposure to toxic chemicals [67]. Induced H 2 O 2 activity in the hemolymph of Cd-exposed Pacific oyster has been reported previously [65] since CAT is a main scavenger of H 2 O 2 . Taken together, present findings hypothesize that CAT could protect Pacific abalone against ROS induced by exposure to Cd, a heavy metal pollutant.
The present study showed higher Hdh-CAT mRNA expression levels in gill at two weeks and in DG of abalone at three weeks of starved abalone compared to the control. After two weeks of starvation, the gill sample showed gradually decreased Hdh-CAT mRNA expression levels in prolonged periods and re-feeding. However, DG showed gradually increased Hdh-CAT mRNA expression levels until three weeks of starvation. The re-fed sample showed an expression level of Hdh-CAT mRNA similar to the control. Similar phenomena have previously been reported from fish [68,69]. Starvation stimulates oxidative stress-oriented energy deficiencies [68]. When the starvation period is prolonged, cells produce ROS and gradually accumulate. In this circumstance, stored antioxidants cannot be supplied in vivo resulting in gradually decreased antioxidant capacity [23]. The present study also reported similar phenomena in the case of ROS production.
Gill and DG are important immune organs of Pacific abalone [44]. In this study, V. parahaemolyticus influenced the mRNA expression level of Hdh-CAT in gill and DG. Gill showed an earlier response (6 h) than DG (12 h) against Vibrio by showing higher Hdh-CAT mRNA abundance. A possible explanation for this time variation in different tissues might be the species-and tissue-specific nature of pathogen. Vibrio-infected abalone produced a significant amount of ROS; higher CAT mRNA might eliminate the excessive ROS generation. In mollusk, antioxidant response and ROS production show wide variations depending on tissue type, pathogen, and host [28]. Vibrio challenges also induced mRNA expression of catalase in the digestive gland and gill of Mytilus galloprovincialis and H. discus discus [28,70]. The present findings suggest that Hdh-CAT might play a role in the innate immune response of Pacific abalone against V. parahaemolyticus.
Lipopolysaccharide (LPS) is a well-recognized pathogen-associated immune stimulant [44]. In the present study, Hdh-CAT mRNA expression levels were quantified from gill and DG in response to LPS challenge at different time points. Upregulated Hdh-CAT mRNA expression was determined in both the gill and DG of LPS-challenged Pacific abalone. The expression of superoxide dismutase (SOD), an antioxidant gene, has been previously reported to be upregulated in the gill and DG of Pacific abalone [44]. CAT is activated immediately after the activation of SOD in the oxidative defense system. LPS challenge can also upregulate CAT expression level in the hepatopancreases or DG of Scylla paramamosain [71].
Poly I:C (PIC) is a synthetic viral mimic extensively used in viral infection experiments [72]. In the present study, PIC induced Hdh-CAT mRNA expression levels in the gill and DG tissues of Pacific abalone. PIC can also induce mRNA expression of CAT in the liver (DG) of Bostrychus sinensis [73]. Upregulated mRNA expression of CAT in viral challenged gill of H. discus discus has been previously reported [28]. Results of the present study suggest that PIC challenge might activate the immune response in DG more than in gill since DG is a key metabolic organ of mollusk [74].
Sperm have multiple antioxidants including CAT which can be altered during cryopreservation [75]. The present study found that cryopreserved sperm had significantly lower Hdh-CAT mRNA abundance than fresh sperm. Similar findings have been previously reported for cryopreserved rooster sperm [35]. Results of the present study suggest that Hdh-CAT might be considered as a biomarker of cryopreserved sperm of Pacific abalone.
The present study showed higher Hdh-CAT mRNA expression levels in trochophore (TRO) and veliger (VEL) larvae stages of embryogenesis. In abalone, trochophore larvae are considered as hatching steps and veliger larvae are considered as hatched larvae of embryogenesis [76]. Similar findings have been previously observed in Atlantic bluefin tuna [32] and Seabass [77]. The present findings suggest that Hdh-CAT might be involved in hatching succession in the embryogenesis process of Pacific abalone.

Conclusions
Hdh-CAT was for the first time cloned from the digestive gland of Pacific abalone, H. discus hannai. Hdh-CAT was localized in the digestive tubules of the digestive gland. Catalase activity and mRNA expression analysis indicated that Hdh-CAT might regulate the antioxidant defense system against thermal stress, viral infection, bacterial infection, starvation, and cadmium-induced toxicity. Hdh-CAT might have a potential role in testicular development and metamorphosis. Our findings also suggest that Hdh-CAT may have a defensive role against excessive ROS production. Expression levels of Hdh-CAT in cryopreserved sperm suggest that Hdh-CAT might be used as an indicator of cryotolerance of Pacific abalone sperm. Taken together, the present findings suggest that Hdh-CAT might be used as a stress and toxicity indicator.