YANGLANG Arat, WEN Haishen, MAO Xuebin, TIAN Yuan, WANG Lingyu,LI Jinku, QI Xin, SRISAPOOME Prapansak, LI Jifang, and LI Yun

Characterization ofGene Family Members in Spotted Sea Bass () and Their Expression Profiles in Response toInfection

YANGLANG Arat1), 2), WEN Haishen1), MAO Xuebin1), TIAN Yuan1), WANG Lingyu1),LI Jinku1), QI Xin1), SRISAPOOME Prapansak2), LI Jifang1), and LI Yun1), *

1),,266003,2),,,,10900,

Thegene family is a crucial gene cluster that regulates apoptosis which contribute to programmed cell death, cell proliferation and differentiation, and several immune responses. In our study, a complete set of 12 caspase genes were identified in spotted sea bass. These genes were divided into three subfamilies: 2 inflammatory caspases (and), 5 apoptosis initiators (,,,, and), and 5 apoptosis executioners (,,,, and). Their phylogenetic relationships, synteny and gene structures were systematically analyzed. Furthermore, the relative expression profiles of the caspase family members in the liver, intestine, head kidney, and spleen were measured by qPCR after infection with. The results showed that the overall mRNA levels of the caspase genes were dramatically in- creased afterinfection, and the expression patterns varied among genes and tissues. More caspase genes underwent pro- nounced expression changes in the head kidney and spleen than in the liver or intestine, mainly after 48 h of the challenge. Specifically,,,,,,,,, andin the head kidney, and,,, andin the spleen, were the most responsive caspase genes which may contribute significantly to immune regulation in spotted sea bass. Additionally, the apoptosis level in head kidney and spleen after infection were examined using the Caspase assay. Our study provides a systemic overview of the caspase gene family in spotted sea bass afterinfection and lays a foundation for further deciphering the biological roles of these caspase genes.

gene family; spotted sea bass;; gene expression; apoptosis

1 Introduction

The caspase () family is a group of conserved cys- teine-dependent aspartate-specific proteases that have es- sential functions in mediating apoptosis, pyroptosis, ne- croptosis, and inflammatory responses (Man., 2017). The principal morphology of caspases consists of two do- mains: the prodomain and the interleukin-1 beta convert- ing enzyme (ICE) homologues (CASc) domain. The pro- domain is composed of various death domain superfamily members, such as the death domain (DD), death effector do- main (DED), caspase recruitment domain (CARD), and py- rin domain (PYD). The CASc domain consists of the large subunit (p20), small subunit (p10) and proteolytic cleavage site (Takle and Andersen, 2007). Based on their functional and structural similarities, mammalian caspase members can be classified into 3 subfamilies: apoptosis initiators (,,, and), apoptosis executioners (,, and) and inflammatory mediators (,,,, and) (Eckhart, 2008; Spead., 2018; Zeng., 2021). Additional orthologs of caspases, includingto, have been reported in specific mammalian or teleost species (Eckhart., 2008).

Generally, the initiator caspases induce apoptosis through the mitochondrial or Bcl-2-regulated intrinsic pathway, or the death receptor-induced extrinsic pathway (Fan., 2005). During the intrinsic apoptosis pathway, varieties of stimuli such as cellular stresses induce the assembly of apop- tosome complex and activate apoptosis initiators like. Alternatively, apoptosis can be triggered via the extrinsic pathway, which is initiated by ligand binding to death re- ceptors, forming the death-inducing signaling complex (DISC) and then apoptosis initiators such asandare activated (Sakamaki and Satou, 2009; Rami- rez and Salvesen, 2018; Van Opdenbosch and Lamkanfi, 2019). Subsequently, the executioners including,, andare stimulated by the initiator caspases, promoting the process of cell death by driving the execu- tion phase of apoptosis (Slee., 2001). Caspase genes are also involved in pyroptosis cascade, which is a form of the inflammatory programmed cell death pathway. It has been demonstrated that inflammatory caspases regulate in- flammation by mediating the cleavage of gasdermin D di- rectly (,, and), or through the forma- tion of casp-1-containing inflammasome complex, result- ing in pyroptosis cascade (Man., 2017).

In recent decades, although not as well characterized as the mammalian model, caspase genes have been identified in several teleost species, such as,,,, andin striped snakehead () (Kumaresan., 2016),,,, andin tongue sole () (Long and Sun, 2016),,,,,, andin Japanese flounder () (Ku- robe., 2007; Li., 2017, 2019),,,,,, andin puffer fish () (Fu., 2018, 2019, 2020),,, andin large yellow croaker () (Mu., 2010; Li., 2011; Yang., 2021) andandin European sea bass () (Reis., 2007, 2010). No- vel caspase members have been found in zebrafish, includ- ing,,,,,, and(Spead., 2018). Recently, a complete set of 18 casp genes in rain- bow trout were published (Zeng., 2021).

Spotted sea bass,, is an economi- cal marine species that has been found in various Asian countries. In China, the annual production of spotted sea bass exceeds 150000 tons, and spotted sea bass has become one of the most popular marine commercial fishes (Fan., 2019; Tian., 2019). However,, which is a gram-negative bacterium that infects various ma- rine species, causes high mortality rates and results in se- rious economic loss to the spotted sea bass industry (Aus- tin and Zhang, 2006; Tian., 2019; Mao., 2020). In this study, the completefamily gene set was iden- tified in spotted sea bass, the gene structures and phyloge- netic relationships were systematically characterized. The participation of the genes in the immune response was eva- luated by detecting the variations of gene expression afterinfection.

2 Materials and Methods

2.1 Ethics Statement

All fish experiments were conducted in accordance with the guidelines and approval of the respective Animal Re- search and Ethics Committees of Ocean University of Chi- na (permit number: 20141201). The study did not involve endangered or protected species.

2.2 Identification of Caspase Sequences from Spotted Sea Bass

The candidate caspase sequences from spotted sea bass were identified by using the amino acid sequences from human, cattle (), house mouse (), chicken (), zebrafish (), black rock fish () and giant grouper () retrieved from the NCBI protein database (https://www.ncbi.nlm.nih.gov/protein/) and UniProt data- base (https://www.uniprot.org/). TBLASTN with a cutoff e-value of 1e-5 was used to search the reference genome (PRJNA408177) and RNA-Seq databases (PRJNA515783, PRJNA515986). The open reading frame was determined through the ORF finder program (https://www.ncbi.nlm. nih.gov/orffinder/) and verified by Smart BLAST of the NCBI nonredundant database.

2.3 Phylogenetic and Synteny Analysis

The amino acid sequences of caspase genes from spott- ed sea bass and various representative vertebrates were used to construct a phylogenetic tree. Multiple sequence align- ment was performed using ClustalW with default parame- ters. The phylogenetic analysis was conducted through the MEGA7 program (Kumar., 2016) with the neighbor- joining method, as well as through Jones-Taylor-Thornton (JTT) model with 1000 bootstrapped replications. The tree was illustrated with Tree of Life (iTOL, https://itol.embl. de/) version 5.6.3 (Letunic and Bork, 2007).

Synteny analysis was conducted to provide further evi- dence for the gene annotations of caspases from spotted sea bass. Information on neighboring genes of representa- tive teleosts including zebrafish, Nile tilapia and European sea bass was determined from the Genomicus database (https://www.genomicus.bio.ens.psl.eu/genomicus-104.02/ cgi-bin/search.pl) (Louis., 2015). The information of giant grouper was retrieved from NCBI annotation data base (www.ncbi.nlm.nih.gov/genome/browse/#!/proteins/8795/ 519138%7CEpinephelus%20lanceolatus/). The neighbor genes of caspases in spotted sea bass were identified from the reference genome assembly.

2.4 Gene Structure Analyses

The theoretical isoelectric point (pI) and molecular weight (Mw) were predicted with the pI/Mw tool of the online service Expasy (https://web.expasy.org/compute_pi/). The three-dimensional (3D) protein structure of casp genes was predicted by Swiss-Model (https://swissmodel.|expasy.org/) (Waterhouse., 2018) and visualized with the PyMOL Molecular Graphics System software, Version 2.0 (Schr?- dinger, LLC).

2.5 Experimental Fish

Spotted sea bass were obtained from Shuangying Aqua- culture Company in Dongying City, Shandong Province, China. Seventy-five fish (body weight: 60.28 g ± 19.78 g; body length: 13.21 cm ± 1.75 cm) were acclimated in a to- tal of 15 glass tanks (46-L in volume) at a density of 5 fish per tank for 1 week before the formal experiment. The wa- ter salinity, dissolved oxygen, pH and temperature were maintained at 30, 6 – 7 mg L?1, 7.8 and 14 – 15℃, respective- ly. Feed was withheld 72 h before bacterial injection.

2.6 Bacterial Challenge and Experimental Design

.was kindly received from the Fish Immuno-logy Laboratory (School of Marine Science and Engineer- ing, Qingdao Agricultural University, Shandong, China) and confirmed by amplification of thegene, which is considered an effective taxonomic marker to identifyspp., following the procedure from Pang. (2006). Single colony ofwas selected and grown in Luria-Ber- tani (LB) broth supplemented with 1.5% NaCl and incu- bated overnight at 32℃. Bacterial cells were harvested by centrifugation at 5000 r min?1, at 25℃ for 10 min. The bacte- rial pellet was washed two times and diluted with 1.5% NaCl and used as an original stock. Theconcen- tration was determined by measuring the standard curve ge- nerated by the Vibrio TCBS plate count protocol.

At the beginning (0 h), every fish in the tanks 1 – 3 (con- trol group) were intraperitoneally injected with 0.1 mL nor- mal saline solution (0.85%). At the same time, every fish in tanks 4 – 15 (treatment groups) were injected with 0.1 mLsolution at a concentration of 2 × 109CFU mL?1dissolved in 1.5% saline solution. Shortly after injection,three fish in each tank of control group were randomly col-lected and euthanized with MS-222 in a dose of 210 mg L?1(Leary., 2020). At 12 h, 24 h, 48 h, and 72 h after injec- tion, three fish in tanks 4 – 6, 7 – 9, 10 – 12, and 13 – 15 were respectively collected with the same method as described above. Tissue samples, including the head kidney, spleen, liver, and intestine were quickly frozen in liquid nitrogen and stored at ?80℃ before the RNA extraction.

2.7 RNA Extraction and Quantitative Real Time PCR (qPCR) Analysis

Total RNA from each tissue sample was extracted by TRIzol reagent (Invitrogen, CA, USA) according to the ma- nufacturer’s protocol and subsequently reverse transcribed into complementary DNA (cDNA) using a Prime Script RT reagent kit (Takara, Otsu, Japan). The concentration of RNA and cDNA were validated by using a Biodropsis BD-1000 spectrophotometric absorbance machine (Beijing Oriental Science & Technology Development Ltd., Beijing, China).

Primers were designed using the Primer5 software (Palo Alto, CA) according to MIQE guidance (Bustin., 2009), and they are listed in Table 1.

Table1 Primer list for qPCR

The efficiency of each pair of primers was examined with the standard curve using the following formula (Rasmussen, 2001):

The melting temperature (Tm) value for each pair of pri-mers was precisely verified by gradient PCR and interpret- ed with agarose gel electrophoresis. qPCR analysis was per-formed using Applied Biosystems 7300 machines (App- lied Biosystems, CA, USA), and 18S rRNA was set as the internal reference gene (Wang., 2018). All samples, in- cluding 3 biological replications, were repeated in triplicate. The thermocycle program was performed using the follow- ing conditions: 95℃ for 30 s, followed by 40 cycles of 95℃ for 10 s, 60℃ for 30 and 72℃ for 30 s forand. An annealing temperature of 55℃ was used for,,,,,,,,, and. The re- lative expression ratio was calculated using the 2???Ctfor- mula (Schmittgen and Livak, 2008) and the relative expres- sion values of control fish at 0 h was used as the calibrator for each time point. The results are graphically illustrated as the mean ± standard error mean (SEM) using GraphPad Prism version 8.0.2 for Windows (La Jolla, CA, USA).

2.8 Detection of Caspase 3/7 Activity

To analyze the apoptosis level of the infected tissue sam- ples, the Caspase assay was performed using the Caspase 3/7 Activity Apoptosis Assay Kit (Sangon Biotech, Shang- hai, China) according to the manufacturer’s protocol. Brief- ly, 0.2 g tissue sample (head kidney and spleen) was ground into powder with liquid nitrogen, and lysed in the 150 μL lysis buffer at room temperature for 15 min. Then the mix- ture was centrifuged at 800 ×for 10 min, and the superna- tant was transferred into a 96-well plate and incubated at 25℃ for 1 h. Meantime, 3 blank controls were set which contained only Caspase3/7 detection buffer. The fluores- cence value of Caspase3/7 was measured at Ex/Em = 490/ 525 nm by the microplate reader (MD Spectra Max Plus 384).

2.9 Statistical Analyses

The raw data of fold change values at different time courses in each tissue were validated for parametrical sta- tistics. Shapiro-Wilk’s test (> 0.05) (Shapiro and Wilk,1965;Mohd Razali and Yap, 2011) and Levene’s test were used to verify the normality of the distribution and equa- lity of variances in the samples (homogeneity of variance) (> 0.05) (Nordstokke and Zumbo, 2010; Nordstokke., 2011). All normalized data were conducted using analysis of variance (ANOVA). The significant differences between obtained data at each time course were interpreted with post hoc multiple comparisons by Duncan’s multiple range test using SPSS software version 23.0 (SPSS, Chicago, IL) (Walter and Duncan, 1955). Difference was considered Sig- nificant when< 0.05.

3 Results

3.1 Characterization of Caspase Genes in Spotted Sea Bass

In total, 12 caspase genes were identified in spotted sea bass. These genes were further divided into three subfa- milies depending on their deduced functions: inflammatory caspases (and), initiator caspases (,,,, and) and execu- tioner caspases (,,,, and). The number of amino acid residues encoded by spotted sea bass caspase genes ranged from 254 to 539, with the relative molecular weights (Mw) ranging from 29.11 kDa to 60.47 kDa. Detailed information on the pI (isoelectric points), Mw, predicted protein size, chromosome location and GenBank accession number of the caspase genes in spotted sea bass is provided in Table 2.

Table 2 Summary of the characteristics of the caspase gene family identified in spotted sea bass

3.2 Phylogenetic and Syntenic Analyses

Phylogenetic analysis was performed by using the pre- dicted caspase amino acid sequences from spotted sea bass and selected vertebrates. As shown in Fig.1, the caspase genes in spotted sea bass were clustered with correspond- ing teleost counterparts, which are consistent with their an- notations, and categorized into three subfamilies as expected (Fig.1). The results demonstrate that the caspase family was conserved during evolution.

Syntenic analysis was conducted for,,,,, and, which had multiple gene copies or could not be accurately annotated based on the phylogenetic analysis. To further confirm their annotations, the caspase genes and their neighboring genes in spotted sea bass are shown in Fig.2. The results show- ed that for the threegenes (,, and), conserved genomic neighborhoods were found between spotted sea bass, zebrafish, Nile tilapia, and Euro- pean sea bass (Fig.2A). The synteny results ofandwere elucidated in zebrafish and spotted sea bass, which had notably conserved patterns (Fig.2B). Similarly, a highly conserved syntenic block between spotted sea bass and giant grouper was identified in the genomic region sur- rounding(Fig.2C). Additionally, the predict- ed three-dimensional (3D) structures of,,andwere constructed in spotted sea bass, and the results demonstrate that the tertiary structure was highly conserved as illustrated in Fig.3. In this case, all phylogenetic, syntenic and tertiary structure analyses sup- port the accuracy of our annotation and nomenclature ofgenes from spotted sea bass.

3.3 Domain Structure Analysis and Multiple Sequence Alignment of the CASc Catalytic Domain

Thegenes from spotted sea bass were found to con- tain an N-terminal prodomain of varying size and a cataly- tic CASc domain, which was consistent with caspase ortho- logs of mammalian and other vertebrate species (Fig.4). As results showed,,,andof spott- ed sea bass had a caspase-recruitment domain (CARD) in their N-terminal region, whileandhad two death-effector domains (DEDs) instead. The structural fea- tures may facilitate these proteins to combine with diffe- rent adapter proteins and trigger distinct downstream sig- naling pathways. All members in the executioner subgroup, such as,,,and, were characterized by a short prodomain (Fig.4).

As predicted, the CASc domain of spotted sea bass cas- pase genes was composed of two subunits: the large sub- unit (p20), which contained the caspase active site (QACNG, N represents G for, Q for,, and, and R for the other casp genes), and the small subunit (p10) (Fig.5). The sequence alignments of the predicted CASc catalytic domains and the active site (QACNG) are illus- trated in Fig.5.

3.4 Relative Expression Changes of Caspase Genes After the V. harveyi Infection

To investigate the potential involvement of caspase genes from spotted sea bass in innate immunity, the mRNA ex- pression level ofgenes was measured in four classi- cal immune organs, including liver, intestine, head kidney and spleen at 5 time points (0, 12, 24, 48 and 72 h) after theinfection. Our qPCR results show that cas- pase genes exhibited different expression profiles in a time- dependent manner that varied among genes and tissues (Fig.6). Notably, 12 h after injected with, fish clearly exhibited skin ulcer, hemorrhage and caudal fin ero- sion or fin necrosis, which are identified as the important clinical signs caused by the target bacterium (Austin and Zhang, 2006; Tian., 2019; Mao., 2020). However, no mortality was observed in these bacteria injected fish.

Fig.1 Phylogenetic analysis of the caspase gene family in spotted sea bass and selected vertebrate species. The phyloge- netic tree was constructed by using amino acid sequences from spotted sea bass and other selected vertebrates. Those va- lues on each node represented bootstrapping values, and the colors are referred as subfamily classification.

Fig.2 Syntenic analysis of caspase genes in selected vertebrates. (A) casp-3; (B) casp-8; (C) casp-14.

Fig.3 Comparison of three-dimensional structures (3D) of Casp-3 members and Casp-14-like. The caspase active site is enlarged in the middle.

Fig.4 Schematic representation of domain structures of casp genes in spotted sea bass. The functional domains are marked with different color blocks.

In detail, among the inflammatory caspase members,was dramatically upregulated in the head kidney at 48 h (7.07-fold), whilewas highly induced in the head kidney and spleen after 12 h (7.98-fold and 16.11-fold, respectively), and the highest expression va- lues occurred at 48 h (16.51-fold and 19.97-fold, respec- tively) after infection. Then the expressions ofandgenes in these two tissues returned to normal levels at 72 h (Fig.6A). Consistent with our findings, va- rious studies have demonstrated potential functions of te- leostin immune responsive organs by testing its ex- pression variation after bacterial challenge trials. For exam- ple, after challenged with, themRNA level of the striped snakehead () in- creased significantly in the head kidney and trunk kidney but showed a moderate change in the liver and spleen (Ku- maresan., 2016). The expression patterns ofafter infection byshowed high upregu- lation in the head kidney and spleen in tongue sole (Long and Sun, 2016). In addition, themRNA expression in Japanese flounder exhibited significant increases in the head kidney after a challenge of bacteriumand in head kidney macrophages (HKMs) after challenges with LPS and poly (I:C). Further study demonstrated that inflam- matorywas involved in extracellular ATP-mediated immune signaling by interacting with apoptosis-associated speck-like (ASC) protein (Li., 2017).also sig- nificantly contributes to the innate immune system and me- diates the pyroptosis cascade (Winkler and R?sen-Wolff, 2015), which indicates thatmay play a crucial role in the proinflammation of the head kidney after 48 h and may contribute to pyroptosis in spotted sea bass. This hypothe- sis need to be further investigated in the future. However, for, functional studies remain scarce in teleosts, and our findings indicate that this gene may be essential to de- fend against bacterial infection in spotted sea bass.

Fig.5 Schematic of the CASc catalytic domains in conserved regions in the multiple sequence alignment of spotted sea bass caspase gene families. The result was visualized using the DNAMAN software. The active site is labeled with a red box (QACNG). The corresponding sequences of large subunits (p20) and small subunits (p10) of catalytic CASc domain were marked with green and red boxes, respectively. The homology level was highlighted by different colors of shading: black for 100%, 100% < pink < 75% and 75% < blue < 50%.

Fig.6 (A) Expression patterns of caspase genes in the liver, intestine, head kidney, and spleen of spotted sea bass at 0, 12, 24, 48 and 72 h after the V. harveyi infection, and different small letters represent significant differences in different periods within the same tissue (P < 0.05); (B) The relative gene expression changes within each tissue are illustrated in heat map schematic, and the warmer colors indicate the higher expression levels.

It has been demonstrated that the initiator caspases regu- late apoptosis through both intrinsic pathways (and) and extrinsic pathways (and) in high- er vertebrates (Paroni., 2002; McIlwain., 2013). In our results, all caspase genes of apoptosis initiators (,,,, and) were significant- ly differentially expressed (< 0.05) in the intestine, head kidney, and spleen after infection to different extents (Fig. 6A). The mRNA expression levels of the 5 initiator caspase genes significantly increased at 12 h in the intestine (2.40- to 3.73-fold). Moreover, the mRNA expression levels in the head kidney were remarkably induced and achieved the highest values by 48 h (8.24- to 18.02-fold upregulation compared to the control group). Significant variations in the expression levels of the initiator caspases in the spleen (1.59- to 7.47-fold changes) were found at different time points after infection, and the highest expression changes (7.47- fold) was detected forat 48 h (Fig.6A). The induc- tion of initiator caspase gene expression in immune organs or cells by bacterial infection has been illustrated in seve- ral teleosts. For example, the upregulated expression ofandin the kidney and spleen after anin- fection was reported for tongue sole (Long and Sun, 2016). In rainbow trout,andexhibited different expression profiles between the groups challenged withand the control group without infection (Zeng., 2021). Likewise,mRNA levels were stimu- lated with LPS and poly (I:C) treatment in HKMs of Japa- nese flounder (Li., 2019). For European sea bass, which is closely related to spotted sea bass, increased expression ofin the spleen has been observed after assp.(Phdp) infection and is known to trigger the selective apoptosis of macrophages and neutrophils (Reis., 2010). Furthermore,is a crucial initiator caspase and a molecular switch for apop- tosis, pyroptosis and necroptosis. For example,can initiate extrinsic apoptosis and inhibit necroptosis mediated byand(Fritsch., 2019). Moreover, the en- zymatic function ofis necessary to stimulate, induces several key immune responses such as cytokine secretion and trigger the pattern-recognition receptors (PRRs) such as antigen receptors, Fc receptors or Toll-like recep- tors (Su., 2005; Philip., 2016; Fritsch., 2019). Overall, the above experimental evidence supports the in- volvement of teleostgenes in the apoptotic pathway caused by bacterial infection.

For the executioner caspase groups, spotted sea bass con- tained threegenes (,, and), as well asand. As shown in Fig.6A, thegenes in spotted sea bass displayed diverse expres- sion profiles after infection. In the liver, significant mRNA upregulation was detected forand, while downregulation was observed for. The opposite expression patterns suggest a compensatory mechanism forgenes in the liver. Among thegenes, a signi- ficant expression change in the intestine was found for only, and the intestinal mRNA values forandremained constant. All threegenes in the head kidney and spleen showed highly induced expression after the stimulation with bacteria. They tended to increase firstly and then decrease subsequently, except thatshowed increased expression in the spleen until 72 h post-challenge. The highest expression values ofandoccurred in the head kidney at 48 h post-chal- lenge (17.43- and 23.44-fold of those of the control, re- spectively) (Fig.6A). Several teleost species such as the European sea bass, tongue sole, large yellow croaker, and walking catfish () have one unique copy of the, the expression of which was also sig- nificantly increased in different immunity-related tissues af- ter bacterial challenge (Reis., 2007; Li., 2011; Banerjee., 2012; Kumaresan., 2016; Long and Sun, 2016). These expression changes were reasonable becauseis known as the most important effector caspase ac- tivated byandin the apoptotic pathway (Ku- ribayashi., 2006; McComb., 2019; Ponder and Boise, 2019). Similarly, the expression levels ofandgreatly increased in the head kidney and spleen in the-infected fish; notably, the expression values ofat 48 h reached 19.75-fold and 60.09- fold, respectively (Fig.6A). The important roles ofin the apoptotic signaling pathway were reported in seve- ral teleost species such as rainbow trout, walking catfish and Japanese flounder (Laing., 2001; Banerjee., 2012; Li., 2019). However, the specific function ofin the regulation of fish immunity must be further investigated.

In a summary, the overall relative expression levels of caspases were dramatically increased afterchal- lenge, which suggests the essential contribution of the cas- pase family to immune functions againstinfec- tion in spotted sea bass. As illustrated in Fig.6B, more cas- pase genes in the head kidney and spleen were affected by bacterial infection than those in the liver and intestine, and the highest expression variation was observed for two exe- cutioners (and) in the spleen at 48 h (60.09-fold) and 72 h (41.87-fold), respectively. Thus, head kidney and spleen might be the most responsive immune organs andandmight be the most im- portantcaspase genes related toinfection in spot- ted sea bass.

3.5 Changes of Caspase 3/7 Activity After the V. harveyi Infection

Sinceandplay important roles in the ini- tiation process of apoptosis, they have been widely accept- ed as the reliable indicator of apoptosis. To investigate the degree of apoptosis of spotted sea bass afterin- fection, the caspase activity was measured in head kidney and spleen at 3 time points (0, 48 and 72 h). The results show that the caspase activity from spotted sea bass exhibited dif- ferent expression profiles between two tissues (Fig.7). In detail, the fluorescence value of caspase-3/-7 at 72 h was significantly higher than those at 0 h (2.00-fold) and 48 h (1.87-fold), while there was no significant change between the 48 h and 0 h time points, indicating that the high apopto- sis level appeared in the head kidney at 72 h after the in- fection, as shown in Fig.7A. In spleen, the fluorescence value increased firstly and then decreased, exhibiting a higher apoptosis level at 48 h after the infection, as shown in Fig.7B. In general, our results proved that the caspase family was involved in the process of apoptosis, with a cru- cial contribution againstinfection in spotted sea bass.

Fig.7 Fluorescence value of Caspase-3/-7 in the head kidney (A) and spleen (B) of spotted sea bass at 0, 48, 72 h after the V. harveyi infection.

4 Conclusions

In conclusion, we systematically identified a 12-mem- ber caspase gene family in spotted sea bass and charac- terized the expression patterns of these genes underinduction. The confirmed genes in spotted sea bass were significantly homologous with those in other verte- brate species. The relative expression profiles after bacte- rial challenge show that these genes were ubiquitously ex- pressed in crucial immune responsive organs (liver, intes- tine, head kidney, and spleen) in a tissue- and time-depen- dent manner. The tissue-based analysis shows a large re- sponsive signal forgene family members in the head kidney and spleen. The results also indicate that spotted sea bass may present apoptosis signals after 48 h of challenge, and these signals are applicable for maintaining culture sys- tems for disease management and control. Nonetheless, the caspase protein function requires further examination in the future.

Acknowledgements

This research was funded by grants from the National Key R&D Program of China (No. 2020YFD0900204), the National Natural Science Foundation of China (No. 3207 2947), the China Agriculture Research System of MOF and MARA (No. CARS-47), the KU-OUC Dual Master’s Pro- gram and Ocean University of China Scholarship Council. We gratefully acknowledge for thestock from Fish Immunology Laboratory of School of Marine Science and Engineering, Qingdao Agricultural University, Shan- dong, China.

Austin, B., and Zhang, X. H., 2006.: A significant pathogen of marine vertebrates and invertebrates., 43 (2): 119-124, DOI: 10.1111/j.1472-765X. 2006.01989.x.

Banerjee, C., Goswami, R., Verma, G., Datta, M., and Mazumder, S., 2012.induced head kidney macro- phage apoptosis ininvolves the activation of calpain and is caspase-3 mediated., 37 (3-4): 323-333, DOI: 10.1016/j.dci. 2012.02.005.

Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M.,., 2009. The MIQE guidelines: Minimum in formation for publication of quantitative real-time PCR experi- ments., 55 (4): 611-622, DOI: 10.1373/ clinchem.2008.112797.

Eckhart, L., Ballaun, C., Hermann, M., VandeBerg, J. L., Sipos, W., Uthman, A.,., 2008. Identification of novel mamma- lian caspases reveals an important role of gene loss in shaping the human caspase repertoire., 25 (5): 831-841, DOI: 10.1093/molbev/msn012.

Fan, H., Wang, L., Wen, H., Wang, K., Qi, X., Li, J.,., 2019. Genome-wide identification and characterization of toll-like re- ceptor genes in spotted sea bass () and their involvement in the host immune response toinfection., 92: 782-791, DOI: 10.1016/j.fsi.2019.07.010.

Fan, T. J., Han, L. H., Cong, R. S., and Liang, J., 2005. Caspase fa- mily proteases and apoptosis.,37 (11): 719-727, DOI: 10.1111/j.1745-7270.2005.00108.x.

Fritsch, M., Günther, S., Schwarzer, R., Albert, M., Schorn, F., Werthenbach, P.,., 2019. Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis., 575: 1-5, DOI: 10.1038/s41586-019-1770-6.

Fu, S., Ding, M., Liu, H., Wu, L., Li, B., Wang, A.,., 2019. Identification and characterization of caspase-7 in pufferfish () in response to bacterial infection and cell apoptosis., 512: 734268, DOI: 10.1016/j.aquacul ture.2019.734268.

Fu, S., Ding, M., Wang, J., Yin, X., Zhou, E., Kong, L.,., 2020. Identification and functional characterization of three caspases inin response to bacterial infection., 106: 252-262, DOI: 10.1016/j.fsi.2020. 07.047.

Fu, S., Ding, M. M., Yang, Y., Kong, J., Li, Y., Guo, Z.,., 2018. Molecular cloning, characterization and expression analysis of caspase-6 in puffer fish ()., 490: 311-320, DOI: 10.1016/j.aquaculture.2018.03.008.

Kumar, S., Stecher, G., and Tamura, K., 2016. MEGA7: Molecu- lar evolutionary genetics analysis version 7.0 for bigger data- sets., 33 (7): 1870-1874, DOI: 10.1093/molbev/msw054.

Kumaresan, V., Ravichandran, G., Nizam, F., Dhayanithi, N. B., Arasu, M. V., Al-Dhabi, N. A.,., 2016. Multifunctional mur- rel caspase 1, 2, 3, 8 and 9: Conservation, uniqueness and their pathogen-induced expression pattern., 49: 493-504, DOI: 10.1016/j.fsi.2016.01.008.

Kuribayashi, K., Mayes, P. A., and El-Deiry, W. S., 2006. What are caspases 3 and 7 doing upstream of the mitochondria?,5 (7): 763-765, DOI: 10.4161/cbt.5.7.3228.

Kurobe, T., Hirono, I., Kondo, H., Yamashita, M., and Aoki, T., 2007. Molecular cloning, expression, and functional analysis of caspase-10 from Japanese flounder.,23 (6): 1266-1274, DOI: 10.1016/j.fsi. 2007.07.001.

Laing, K. J., Holland, J., Bonilla, S., Cunningham, C., and Secombes, C. J., 2001. Cloning and sequencing of caspase 6 in rainbow trout,, and analysis of its expression un- der conditions known to induce apoptosis.,25 (4): 303-312, DOI: 10.1016/ s0145-305x(00)00061-6.

Leary, S., Underwood, W., Anthony, R., Cartner, S., Grandin, T., Greenacre, C.,., 2020.. American Veterinary Medical As- sociation (AVMA), Schaumburg, 92.

Letunic, I., and Bork, P., 2007. Interactive Tree Of Life (iTOL): An online tool for phylogenetic tree display and annotation., 23 (1): 127-128, DOI: 10.1093/bioinformatics/bt l529.

Li, M., Ding, Y., Mu, Y., Ao, J., and Chen, X., 2011. Molecular cloning and characterization of caspase-3 in large yellow croa- ker ()., 30 (3): 910-916, DOI: 10.1016/j.fsi.2011.01.018.

Li, S., Li, J., Peng, W., Hao, G., and Sun, J., 2019. Characterization of the responses of the caspase 2, 3, 6 and 8 genes to immune challenges and extracellular ATP stimulation in the Japanese flounder ()., 15 (1): 20, DOI: 10.1186/s12917-018-1763-y.

Li, S., Peng, W., Li, J., Hao, G., Geng, X., and Sun, J., 2017. Cha- racterization of Japanese flounder () Cas- pase1 involved in extracellular ATP-mediated immune signal- ing in fish., 67: 536-545, DOI: 10. 1016/j.fsi.2017.06.043.

Long, H., and Sun, L., 2016. Molecular characterization reveals involvement of four caspases in the antibacterial immunity of tongue sole ().,57: 340-349, DOI: 10.1016/j.fsi.2016.08.047.

Louis, A., Nguyen, N. T., Muffato, M., and Roest Crollius, H., 2015. Genomicus update 2015: KaryoView and MatrixView provide a genome-wide perspective to multispecies comparative geno- mics., 43 (Database issue): D682-689, DOI: 10.1093/nar/gku1112.

Man, S. M., Karki, R., and Kanneganti, T. D., 2017. Molecular me- chanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases., 277 (1): 61-75, DOI: 10.1111/imr.12534.

Mao, X., Tian, Y., Wen, H., Liu, Y., Sun, Y., Yanglang, A.,., 2020. Effects ofinfection on serum biochemi- cal parameters and expression profiles of interleukin-17 (IL-17)/ interleukin-17 receptor (IL-17R) genes in spotted sea bass.,110: 103731, DOI: 10.1016/j.dci.2020.103731.

McComb, S., Chan, P. K., Guinot, A., Hartmannsdottir, H., Jenni, S., Dobay, M. P.,., 2019. Efficient apoptosis requires feed- back amplification of upstream apoptotic signals by effector cas- pase-3 or -7., 5 (7): eaau9433, DOI: 10.1126/ sciadv.aau9433.

McIlwain, D. R., Berger, T., and Mak, T. W., 2013. Caspase func- tions in cell death and disease., 5 (4): a008656, DOI: 10.1101/cshperspect.a008656.

Mohd Razali, N., and Yap, B., 2011. Power comparisons of Sha- piro-Wilk, Kolmogorov-Smirnov, Lilliefors and Anderson-Dar- ling tests., 2 (1): 21-33.

Mu, Y., Xiao, X., Zhang, J., Ao, J., and Chen, X., 2010. Molecu- lar cloning and functional characterization of caspase 9 in large yellow croaker ()., 34 (3): 300-307, DOI: 10.1016/j.dci. 2009.10.009.

Nordstokke, D., Zumbo, B., Cairns, S. L., and Saklofske, D., 2011. The operating characteristics of the nonparametric Levene test for equal variances with assessment and evaluation data., 16: 1-8.

Nordstokke, D. W., and Zumbo, B. D. J. P., 2010. A new nonpa- rametric Levene test for equal variances.31 (2): 401-430.

Pang, L., Zhang, X. H., Zhong, Y., Chen, J., Li, Y., and Austin, B., 2006. Identification ofusing PCR amplification of thegene.,43 (3): 249- 255, DOI: 10.1111/j.1472-765X.2006.01962.x.

Paroni, G., Henderson, C., Schneider, C., and Brancolini, C., 2002. Caspase-2 can trigger cytochrome c release and apoptosis from the nucleus., 277 (17): 15147- 15161, DOI: 10.1074/jbc.M112338200.

Philip, N. H., DeLaney, A., Peterson, L. W., Santos-Marrero, M., Grier, J. T., Sun, Y.,., 2016. Activity of uncleaved caspase- 8 controls anti-bacterial immune defense and TLR-induced cy- tokine production independent of cell death.,12 (10): e1005910, DOI: 10.1371/journal.ppat.1005910.

Ponder, K. G., and Boise, L. H., 2019. The prodomain of caspase- 3 regulates its own removal and caspase activation., 5: 56, DOI: 10.1038/s41420-019-0142-1.

Ramirez, M. L. G., and Salvesen, G. S., 2018. A primer on cas- pase mechanisms., 82: 79-85, DOI: 10.1016/j.semcdb.2018.01.002.

Rasmussen, R., 2001. Quantification on the lightcycler. In:. Meuer, S.,., eds., Springer Berlin Heidelberg, Berlin, Heidelberg, 21- 34, DOI: 10.1007/978-3-642-59524-0_3.

Reis, M. I., Costa-Ramos, C., Do Vale, A., and Dos Santos, N. M., 2010. Molecular cloning of sea bass (L.) caspase-8 gene and its involvement inssp.triggered apoptosis.,29 (1): 58-65, DOI: 10.1016/j.fsi.2010.02.016.

Reis, M. I., Nascimento, D. S., Do Vale, A., Silva, M. T., and Dos Santos, N. M., 2007. Molecular cloning and characterisation of sea bass (L.) caspase-3 gene., 44 (5): 774-783, DOI: 10.1016/j.molimm.2006.04. 028.

Sakamaki, K., and Satou, Y., 2009. Caspases: Evolutionary aspects of their functions in vertebrates., 74 (4): 727-753, DOI: 10.1111/j.1095-8649.2009.02184.x.

Schmittgen, T. D., and Livak, K. J. J. N. P., 2008. Analyzing real- time PCR data by the comparative CT method., 3: 1101-1108, DOI: 10.1038/nprot.2008.73.

Shapiro, S. S., and Wilk, M. B., 1965. An analysis of variance test for normality.,52: 591-599, DOI: 10.2307/2333709.

Slee, E. A., Adrain, C., and Martin, S. J., 2001. Executioner cas- pase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis., 276 (10): 7320-7326, DOI: 10.1074/jbc.M008363200.

Spead, O., Verreet, T., Donelson, C. J., and Poulain, F. E., 2018. Characterization of the caspase family in zebrafish., 13 (5): e0197966-e0197966, DOI: 10.1371/journal.pone.0197 966.

Su, H., Bidère, N., Zheng, L., Cubre, A., Sakai, K., Dale, J.,., 2005. Requirement for caspase-8 in NF-kappaB activation by antigen receptor.,307 (5714): 1465-1468, DOI: 10.1126/ science.1104765.

Takle, H., and Andersen, ?., 2007. Caspases and apoptosis in fish., 71 (sc): 326-349, DOI: 10.1111/j.1095- 8649.2007.01665.x.

Tian, Y., Wen, H., Qi, X., Mao, X., Shi, Z., Li, J.,., 2019. Analysis of apolipoprotein multigene family in spotted sea bass () and their expression profiles in res- ponse toinfection., 92: 111-118, DOI: 10.1016/j.fsi.2019.06.005.

Van Opdenbosch, N., and Lamkanfi, M., 2019. Caspases in cell death, inflammation, and disease., 50 (6): 1352-1364, DOI: 10.1016/j.immuni.2019.05.020.

Walter, A., and Duncan, D. B., 1955. Multiple range and multiple test., 11: 1-24.

Wang, H., Wen, H., Li, Y., Zhang, K., and Liu, Y., 2018. Evalu- ation of potential reference genes for quantitative RT-PCR ana- lysis in spotted sea bass () under normal and salinity stress conditions., 6: e5631, DOI: 10.7717/ peerj.5631.

Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R.,., 2018. SWISS-MODEL: Homology mo- delling of protein structures and complexes., 46 (W1): W296-W303, DOI:10.1093/nar/gky427.

Winkler, S., and R?sen-Wolff, A., 2015. Caspase-1: An integral re- gulator of innate immunity.,37 (4): 419-427, DOI: 10.1007/s00281-015-0494-4.

Yang, B., Yin, Z., Gao, S., Chen, M., Xu, D., Mai, K.,., 2021. Characterization of Caspase8 and its role in the regulation of apoptosis-related genes in large yellow croaker ()., 539: 736595, DOI: 10.1016/j.aquaculture. 2021.736595.

Zeng, C., Hou, Z. S., Zhao, H. K., Xin, Y. R., Liu, M. Q., Yang, X. D.,., 2021 Identification and characterization of cas- pases genes in rainbow trout () and their expression profiles afterandinfection., 118: 103987, DOI: 10.1016/j.dci.2020.103987.

(June 30, 2022;

September 1, 2022;

December 20, 2022)

? Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2023

. E-mail: yunli0116@ouc.edu.cn

(Edited by Qiu Yantao)