XUE Ting, LIU Yiping, GE Xuefeng, and LI Chao

Establishment,Characterization and Expression Pattern of a Spleen Cell Line,SMSP,Derived from Turbot()

XUE Ting#, LIU Yiping#, GE Xuefeng, and LI Chao*

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A turbot () cell line named SMSP was obtained from the spleen. The origin of the cells was iden- tified by morphology, chromosome number and COI gene. The optimal basic medium, serum concentration and growth temperature of the cells were detected. SMSP cell line is mainly composed of fibroblast-like cells. Most of the SMSP cells contained 44 chromo- somes, and the sequence of COI gene confirmed that the cells were originated from turbot. The optimal culture conditions were 24℃, DMEM + 10% FBS. The cell line had high transfection efficiency for siRNA and plasmid. After stimulation with lipopolysaccharide (LPS) or poly (I:C), the expressions of immune-related genes such as-β,-12s,-10 and-1β were up-regulated significant- ly in the early stage (< 0.05). This study will provide a model for exploring immune mechanism of turbot against pathogen.

; cell line; spleen; pathogen-associated molecular patterns; immunity

1 Introduction

In recent years, more and more microbiology and immu- nology research began to rely on cell lines (Collet, 2018). In teleost, although a large number of immunologi- cal genes homologous to known mammals have been dis- covered and cloned, the function exploration of these genes is still insufficient (Levraud and Boudinot, 2009). Cell lines have many advantages over living organisms and are more friendly to animals. As an important immune organ of te- leost, spleen is the gathering center of macrophages, T cells, B cells and other immune cells (Bjorgen and Koppang, 2021), which play a critical role in the adaptive immune function. Therefore, isolating of fish spleen cells will be helpful for studying the pathogenic mechanism of pa- thogens and the function of immune-related genes. The spleen cell lines have been established from several te- leosts, such as white sturgeon () (Hedrick, 1991), rainbow trout () (Ganassin and Bols, 1998), Japanese flounder () (Kang, 2003), Asian sea bass () (Parameswaran, 2006), mandarin fish () (Dong, 2008), zebrafish () (Xing, 2009), red spotted grouper () (Huang, 2009), walleye () (Vo, 2015), orange-spotted grouper () (Huang, 2018), and Chinook salmon () (Semple, 2018). However, no spleen cell line from turbot has been described yet.

Turbot () is one of the important mariculture species in China (Xiao, 2008). However, it was often invaded by many pathogens, which cause great economic losses (Xiao, 2008; Wang and Ma, 2019; Xue, 2021). The spleen of the turbot was congested and necrotic after infection with(Pa- dros, 2006).could invade spleen tissue and cause the aggregation of melanomacrophage (Ku- maran, 2010).induced the up-regulation or down-regulation of immunoregulatory genes in the spleen of turbot (Xue, 2021). The inten- sive feeding of turbot also led to the outbreaks of viral di- seases, which cause serious pathological changes in spleen tissue (Brudeseth, 2005). Therefore, it is of great sig- nificance to use spleen to study the mechanism of patho- gen infection.

Spleen cell lines have been used to study the pathogene- sis of pathogens in other teleosts. It has been reported that the spleen cell line GS-1 of orange-spotted grouper was used to study the infection of ranavirus (GIV) and megalo- cytivirus (ISKNV). The diameter of the virus was mea- sured with the aid of this cell line and the temperature at which the virus induced the highest mortality was confirm- ed (Huang, 2018).was successfully culturedusing Asian seabass spleen cell line SISS, which provided anmodel for elucidating its trans- mission mechanism (Praveenraj, 2018). The infec- tion of viral hemorrhagic speticemia virus (VHSV) was studied with the walleye spleen cell line WE-spleen6 (Vo, 2015). RTS11, from the rainbow trout spleen (Ga- nassin and Bols, 1998), has been used to study the patho- genicity of(Rojas., 2010).

In order to construct anmodel for studying pa- thogen infection, the spleen cells were isolated fromand were named SMSP. By optimizing the culture conditions, a high-quality spleen cell line was obtained. The cell source was identified by morphology, chromosome number and COI (cytochrome oxidase subunit 1) gene, and the transfection efficiency and the expression of immune genes stimulated by pathogen-related molecular patterns were also measured. This provides more options for further research on the mechanism of turbot against infections of pathogenic bacteria or viruses.

2 Materials and Methods

2.1 Primary Cell Culture and Subculture of Cells

Turbot weighing 50 g ± 5 g were purchased from a farm in Haiyang, Yantai, China. Before the experiment, turbot were temporarily reared in the laboratory at 24℃ ± 1℃ for 1 week. Healthy turbot were taken to prepare for the pri- mary cell culture. They were anesthetized with MS-222 (A5040, Sigma) for venous blood drawing, followed by dis- section and spleen tissue collection. After washed with PBS, the tissues immersed in DMEM medium (11965092, Gib- co) containing four antibiotics (500 U mL?1penicillin and 500 μg mL?1streptomycin, 1.25 μg mL?1amphotericin B and 250 μg mL?1gentamicin (15140122, R01510, Gibco)) for 2 h. The immersed spleen tissue was cut into pieces of 1 mm3in size and transferred to a 25 cm2flask (Corning, 430168). Excess medium was removed and the flask was placed vertically for 45 minites to allow the tissue to adhere to the bottom of the flask. Then, 5 mL of medium was add- ed, and the flask was placed in an incubator at 24℃. We replaced fresh medium every 3 days until cells covered the bottom of the flask. The spleen cells were cultured in DMEM + HEPES (an effective zwitterionic organic chemi- cal buffer that maintains the pH of DMEM, Sigma, H4034) medium containing 15% fetal bovine serum (FBS) (Gibco, 10099141C), 55 nmol L?1β-mercaptoethanol (Gibco, 219 85023), 1 × non-essential amino acids (Gibco, 11140050), 100 U mL?1penicillin and 100 μg mL?1streptomycin (Gib- co, 15140148).

When the coverage of the migrated cells reached about 80%, the medium was removed, and cells were washed with PBS and then digested with 0.25% trypsin-EDTA (Gibco, 25200056). The cells were passaged at the ratio of 1:2. Af- ter 10 passages, the cells were subcultured every 3 – 4 days at a ratio of 1:3, and the serum concentration gradually de- creased to 10%. Cell morphology was recorded every 5 ge- nerations using the Axio Observer7 microscope (ZEISS, Germany).

2.2 Cryopreservation and Resuscitation

The 30th, 45th and 60th generations of SMSP cells in lo- garithmic growth phase were digested and collected. Cryo- preservation solution was prepared with 10% dimethyl sul- phoxide (MP Biomedicals, 67-68-5) in 20% FBS and 70% medium. The cells were resuspended with cryopreservation solution in 5 × 105cells mL?1and transferred to a cryogenic freezing container (Nalgene, USA) and stored at ?80℃ overnight, and finally placed in liquid nitrogen for long- term storage. Cells were resuscitated after storage for one month. In brief, cells were taken from liquid nitrogen and rapidly thawed at water bath with 37℃. Then they were transferred to a 15 mL centrifuge tube containing 9 mL me- dium and the DMSO was removed by centrifugation. SMSP cells were reuspended with fresh medium and transferred to 25 cm2flask. After 12 hours, photographs were taken to record cell status and the medium was replaced with fresh one.

2.3 Origin Analysis of the Cell Line

The karyotype of SMSP cells was analyzed at the 15th passage. Colchicine at a final concentration of 10 μg mL?1was added to the medium when the SMSP cells were in the logarithmic growth phase. The cells were collected after 12 h of culture. They were absorbed in PBS diluted with ice water (PBS:ice water = 1:5) and then were fixed with a fixative (glacial acetic acid:methanol = 1:3). The fixed cells were sprayed onto the glass slides at high speed, and then stained with Giemsa. The karyotype of the cells was ob- served and photographed with Axio Scope A1 (ZEISS, Ger- many).

The DNA of SMSP cells at passage 50 were extracted and COI (KJ205166.1) primers were designed using Oli- go 7 for PCR amplification. The PCR products were se- quenced and compared with turbot genomic DNA to deter- mine the cell source.

2.4 Optimization of Culture Conditions

The optimal growth conditions including the optimal ba- sal medium, the optimal serum concentration, and the op- timal growth temperature of SMSP were detected. For the detection of the optimal serum concentration, the media with 2%, 5%, 10%, 15% and 20% FBS were prepared, re- spectively. For the detection of the optimal basal medium, DMEM, DMEM: F12 (12634010, Gibco), M-199 (111500 59, Gibco), RPMI-1640 (11875093, Gibco) and L-15 (1141 5064, Gibco) were selected. For the detection of the opti- mal growth temperature, five different incubators with 16, 20, 24, 28 and 32℃ were prepared. The 53rd generation of SMSP cells were digested. The cell concentration was ad- justed and the cells were seeded into 48-well plates with 5 × 104cells per well. Fifteen wells were seeded for each culture condition. The numbers of cells in 3 wells were count- ed every 24 h for each condition. The growth curve was drawn to determine the optimal basal medium, serum con- centration and growth temperature.

2.5 Transfection with FAM-siRNA and EGFP Expression Vector

The 55th generation of SMSP cells were seeded in 24- well plates and transfected when the cell fusion reached 60%. The transfection of FAM-siRNA (GenePharma, Chi- na) and pEGFP-N1 plasmid were performed using trans- fection reagent (TaKaRa, 631450 for siRNA, 631318 for plasmid) according to the instructions. Three parallels were set for each treatment. The results were observed and re- corded under Axio Observer7 microscope 48 h after trans- fection. The transfection efficiency was estimated by com- paring the number of cells under natural light and the num- ber of fluorescing cells under microscope.

2.6 Lipopolysaccharide (LPS) and Poly (I:C) Stimulation

The 60th generation of SMSP cells were seeded into 6- well plates with 2 × 105cells per well and stimulated when the cells fusion reached 80%. The cells were stimulated with 20 μg mL?1LPS (lipopolysaccharide) (Solarbio, China) or 20 μg mL?1poly (I:C) (polyinosinic-polycytidylic acid) (APExBIO, USA) respectively (3 parallels for each treat- ment). The control group was treated with an equal volume of PBS. Cells were collected at 2 h, 6 h, 12 h, and 24 h af- ter stimulation for RNA extraction. Total RNA was isolated using TRIzol reagent (Invitrogen, 15596026) according to the manufacturer. The synthesis of cDNA was carried out using Evo M-MLV RT Kit with gDNA Clean for qPCR kit (AG, China), and real-time fluorescent quantitative PCR (RT-qPCR) was performed using TB Green Premix Ex Taq kit (Takara, Dalian, China) in T100 Thermal Cycler (BIO- RAD, USA). Relative gene expression was calculated by 2?ΔΔCt(Livak and Schmittgen, 2001) and normalized by 18S rRNA gene. All the primers used in this study are listed in Table 1.

Table 1 Primers used in this study

2.7 Statistical Analysis

The data were analyzed by one-way ANOVA followed by a post hoc Duncan’s test using SPSS 20.0 (SPSS, IL, USA) and showed by means ± SD. The< 0.05 was consi- dered as statistically significant differences.

3 Results

3.1 Primary Culture and Subculture

On the 12th day after the spleen tissue was attached to the bottom of the flask, cells started to migrate from the edge of the tissue and eventually formed a monolayer on the flask (Fig.1A). Subsequently, the cells were subcultured every 4 days at the ratio of 1:2. After 10 passages, the cells grew rapidly and could be subcultured in 3 – 4 days with a passage ratio of 1:3. The FBS was reduced gradually from 15% to 10% after 10 generations. The initial adherent cells were composed of epithelial- and fibroblast-like cells. How- ever, after 10 generations, the epithelial-like cells were gra- dually replaced by fibroblast-like cells. Up to now, the cells have been successfully subcultured for 65 passages (Figs. 1B – E).

The cryopreserved SMSP cells at passages 30, 45 and 60 showed > 85% viability after 1 month of storage in liquid nitrogen. As Fig.1F showed, no appreciable change in cell morphology and the growth rate were observed after free- zing and thawing.

3.2 Chromosome and COI Analysis

As shown in Fig.2A, most of the SMSP cells were dip- loid (2N = 4m + 12st + 28t). Count of 100 metaphase spreads of SMSP revealed that the number of chromosomes rang- ing from 25 to 71 and the chromosome number in majority of cells was 44 (Fig.2B). The obtained sequence from PCR was consistent with the turbot COI gene published on the website of NCBI, which confirmed the SMSP cells were originated from turbot (Fig.3).

3.3 Optimal Culture Conditions

The results showed that SMSP grew rapidly in DMEM + HEPES and L-15 medium, and grew slowly or even inhi- bited in other media. The apoptosis of cells gradually occur- red after addition of M199 medium, and all the cells died on the third day (Fig.4A). SMSP could grow in the media with different serum concentrations, and the cell growth rate in- creased with the increase of serum concentration. The num- ber of SMSP was close on the fifth day when the concen- trations of FBS ranged from 5% – 20% (Fig.4B). At a high temperature of 32℃, the cells gradually died. At a tempe- rature of 16 – 28℃, the numbers of cells increased steadily. The highest growth rate of SMSP cells was observed at 24℃, followed by 28℃, 20℃ and 16℃ (Fig.4C).

Fig.1 Photographs of SMSP cells. (A), primary cultured SMSP; (B), 10th passage SMSP; (C), 20th passage SMSP; (D), 30th passage SMSP; (E), 50th passage SMP; (F), resuscitated SMSP. Scale bars, 200 μm.

Fig.2 Karyotype analysis of SMSP cells at passage 15. (A), chromosome number distributions according to count of 100 metaphase spreads of SMSP; (B), Giemsa stain of a metaphase chromosome spread. Scale bars, 5 μm.

Fig.4 Growth curve of SMSP cells at passage 53. (A), effect of basal medium; (B), effects of fetal bovine serum concen- trations; (C), effects of incubation temperatures.

3.4 Transfection Efficiency

To determine the applicability of SMSP for gene func- tion studies, cells were transfected with FAM-siRNA and pEGFP-N1 plasmid. At 48 h post-transfection, strong green fluorescent signals were detected in SMSP cells that trans-fected with FAM-siRNA or pEGFP-N1 plasmid, as shown in Figs.5A – D. The transfection efficiencies of FAM- siRNA and pEGFP-N1 plasmid in SMSP cells were found to be about 50% and 30%, respectively. These results indi- cated that SMSP cells were suitable forgenetic studies.

Fig.5 Detection of fluorescence signals of siRNA derived FAM and plasmid derived EGFP reporter gene in SMSP. (A, B), Fluorescence signals in SMSP cells transfected with FAM-siRNA; (C, D), Fluorescence signals in SMSP cells transfected with pEGFP-N1. NL, nature light. Scale bars, 100 μm.

3.5 Immunological Effects After Stimulation

After stimulation with LPS and poly (I:C), the expres- sions of proinflammatory cytokines-β (tumor necrosis factor β), immune cell regulators-1β (interleukin-1β),-12A (interleukin-12 subunit α),-12B (interleukin-12 subunit β), and-10 in SMSP were detected. All of the de- tected genes were highly expressed in SMSP cells after LPS stimulation (Fig.6A). Most of them were up-regulated with LPS stimulation. After stimulated by poly (I:C), the expres- sions of-β,-1β,-12A,-12B, and-10 also changed in the same way as after LPS stimulation (Fig.6B). These results indicate that SMSP can be used as anmodel to study turbot’s resistance mechanism to pathogens.

Fig.6 Expression of immune-associated genes in SMSP cells after stimulation with LPS or poly (I:C) for 2 h, 6 h, 12 h and 24 h. (A), LPS stimulation; (B), poly (I:C) stimulation.

4 Discussion

Turbot is one of the most important species in marine aquaculture in China, which is often disturbed by various bacteria and viruses. Cell lines are usually derived from a particular tissue or class of cells of a species and, to some extent, have some characteristics of the tissue or class of cells. Therefore, cell lines are widely used in immunology, virology, genetics, toxicology and drug discovery (Maeser, 2021; Biegler, 2022; Fernández-Bravo and Fi- gueras, 2022; Li, 2022; Sathiyanarayanan, 2022). In addition, cell lines are powerful tools for studying the mo- lecular mechanisms of various biological processes (Leong, 1994; Chang, 2021).

Spleen is an important immune organ in teleosts, which contains a large number of macrophages, B cells and T cells (Bjorgen and Koppang, 2021). Spleen cell lines were com- monly used asinfection models for etiological, im- munological, and pharmacological studies. In this study, a cell line was established from the spleen of turbot by ex- plant technique. The method has been successfully used for the culture of a variety of cell lines of teleost (Ruiz-Pala- cios, 2020; Wang, 2020b; Yashwanth, 2020) and it also has been used for the separation of spleen cells of sea bass and zebrafish (Xing, 2009; Huang, 2018). After 12 days of culture, the migration of cells from the edge of the tissue was observed. Up to the 10th gene- ration, fibroblast-like cells gradually predominated. This was consistent with the establishment of most cell lines (Le, 2017; Huang, 2018; Gong, 2022).

SMSP cells grew best at 24℃ in DMEM + HEPES me- dium supplemented with 20% fetal bovine serum. A num- ber of fish cell lines have been successfully established us- ing this medium (Wang, 2010; Zheng, 2012; Gao, 2019). This medium could maintain the pH without CO2incubator. From the test, the growth rate of SMSP in- creased with the increase of serum concentration. How- ever, it was also shown that the growth rate of SMSP cells was not significantly decreased by the supplementation of 10% FBS compared to the supplementation of 20% FBS. Thus, 10% FBS content with a lower cost was ultimately selected as the optimal serum concentration, which is same as most cell lines (Xue, 2018; Zeng, 2022). SMSP can tolerate a wide range of temperatures, similar to most fish cell lines. However, as a cold-water fish-derived (op- timum growth temperature is 13 – 20℃) cell line, the cul- ture temperature of SMSP was lower than that of warm- water fish (Wang, 2010; Xue, 2018; Yang, 2020; Gong, 2022; Zeng, 2022).

The SMSP cells showed a high survival rate after one month of cryopreservation in liquid nitrogen. After resus- citation, the morphology of the SMSP cells did not change significantly, suggesting that the cells could be retained and transported. As the basis of cytogenetics, chromosome num- ber and karyotype are important indicators for species iden- tification (Chennaveeraiah and Patil, 1973). Karyotype ana- lysis showed that 58% of SMSP had 2= 44 diploid chro- mosomes at passage 15. The results indicated that most SMSP cells maintained their ploidy at the 15th passage. Amplification and sequencing of COI gene are often used to determine the origin of cell lines (Wei, 2018; Liu, 2021). The COI sequence of SMSP showed 99.8% homology with the COI sequence of turbot published by NCBI. These results indicated that the SMSP cells were de- rived from turbot. In addition, the SMSP cell line has been cultured for more than 50 passages and should be consi- dered as a permanent cell line (Wang, 2010; Kong, 2021).

Cell lines are importantmodels for gene func- tion research and drug screening studies (Bols, 1991; Gos- wami, 2022), so high transfection efficiencies for siRNA and plasmid are required. In this study, FAM- siRNA and pEGFP-N1 were transfected into SMSP cells to evaluate the cells’ transfection efficiency and the applica- bility for gene function research. The results showed that SMSP exihibited high transfection efficiency for both siRNA and plasmid. Typically, the transfection efficiencies of most fish cell lines are lower than those of mammals (Falco, 2009), hindering the utilization of fish cell lines for gene function studies. Therefore, SMSP with high transfec- tion efficiency will provide a high-quality model to study the function of immune-related genes in turbot, which in- cludes,-1β,-12s and-10.

s are pleiotropic cytokines that are involved in the regulation of adaptive immune responses. In Nile tilapia (),-α and-β participated in adaptive immune regulation by activating caspase-8, there- by promoting apoptosis (Li, 2021).-1β is a key pro- inflammatory cytokine that plays a role in the initiation of inflammatory responses in bacterial and viral infections. The artificially reprogrammed-1β protein could improve the resistance of Sturgeon’s () to bacterial infection (Wang, 2021).-12 was significantly up- regulated in the head kidney of Nile tilapia immunized with a pathogenic bacterial vaccine, indicating that IL-12 was in- volved in the antibacterial immune response (Wang, 2020a).-10 was characterized by a typical inhibitory ac- tivity on the expression of pro-inflammatory cytokines in mandarin fish (Huo, 2019). The changes of the ex- pressions of-β,-1β,-12s and-10 in SMSP cells after stimulation with LPS and poly (I:C) indicated that SMSP cells can be used as a model for studying immune response of turbot after pathogen infection.

In summary, a continuous cultured cell line derived from the spleen of turbot was established. SMSP cells were fi- broblast-like and grew rapidly and stably. With high trans- fection efficiency of plasmid and siRNA, SMSP was an ex- cellentmodel for the study of molecular function. As an immune organ-derived cell line, the expressions of-β,-1β,-12s and-10 in SMSP were up-regu- lated after stimulation with LPS and poly (I:C). Therefore, this cell line might be employed for the study of the patho- genic mechanism of the pathogen and the immune effects of the turbot spleen.

Acknowledgements

This study was supported by the National Natural Sci- ence Foundation of China (No. 31902403), the Young Ex- perts of Taishan Scholars (No. tsqn201909130), the ad- vanced Talents Foundation of QAU grant (No. 663-1120 029), the Shandong Technical System of Fish Industry (No. SDAIT-12-03), and the Breeding Plan of Shandong Provin- cial Qingchuang Research Team (2019), China.

Biegler, M. T., Fedrigo, O., Collier, P., Mountcastle, J., Haase, B., Tilgner, H. U.,., 2022. Induction of an immortalized song- bird cell line allows for gene characterization and knockout by CRISPR-Cas9.,12: 4369, DOI: 10.1038/s41 598-022-07434-7.

Bjorgen, H., and Koppang, E. O., 2021. Anatomy of teleost fish immune structures and organs.,73: 53-63, DOI: 10.1007/s00251-020-01196-0.

Bols, N. C., 1991. Biotechnology and aquaculture: The role of cell cultures.,9: 31-49, DOI: 10.1016/ 0734-9750(91)90403-I.

Brudeseth, B. E., Raynard, R. S., King, J. A., and Evensen, O., 2005. Sequential pathology after experimental infection with marine viral hemorrhagic septicemia virus isolates of low and high virulence in turbot (L).,42: 9-18, DOI: 10.1354/vp.42-1-9.

Chang, K., Han, K., Qiu, W., Hu, Z., Chen, X., Chen, X.,., 2021. Grass carp () interferon regula- tory factor 8 down-regulates interferon1 expression via interac- tion with interferon regulatory factor 2.,137: 202-211, DOI: 10.1016/j.molimm.2021.04.020.

Chennaveeraiah, M. S., and Patil, B. C., 1973. Chromosome num- ber and karyotype study in eight species of crotalaria.,38: 73-79.

Collet, B., Collins, C., and Lester, K., 2018. Engineered cell lines for fish health research.,80: 34-40, DOI: 10.1016/j.dci.2017.01.013.

Dong, C., Weng, S., Shi, X., Xu, X., Shi, N., and He, J., 2008. Development of a mandarin fishfry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV).,135: 273-281, DOI: 10.1016/ j.virusres.2008.04.004.

Falco, A., Encinas, P., Carbajosa, S., Cuesta, A., Chaves-Pozo, E., Tafalla, C.,., 2009. Transfection improvements of fish cell lines by using deacylated polyethylenimine of selected molecu- lar weights.,26: 559-566, DOI: 10.1016/j.fsi.2009.02.013.

Fernández-Bravo, A., and Figueras, M. J., 2022. Immune response of the monocytic cell line THP-1 against sixspp.,13: 875689, DOI: 10.3389/fimmu. 2022.875689.

Ganassin, R. C., and Bols, N. C., 1998. Development of a mono- cyte/macrophage-like cell line, RTS11, from rainbow trout spleen.,8: 457-476, DOI: 10.1006/fsim. 1998.0153.

Gao, Y., Zhou, H., Gao, Z., Jiang, H., Wang, X., Mai, K.,., 2019. Establishment and characterization of a fibroblast-like cell line from the muscle of turbot (L.).,45: 1129-1139, DOI: 10. 1007/s10695-019-00628-3.

Gong, J., Pan, X., Lin, L., Zhu, Y., Yao, J., Wang, C.,., 2022. Establishment and characterization of a spinal cord tissue cell line from mandarin fish,and its susceptibi- lity to several viruses., 45 (10): 1419- 1427, DOI: 10.1111/jfd.13671.

Goswami, M., Yashwanth, B. S., Trudeau, V., and Lakra, W. S., 2022. Role and relevance of fish cell lines in advancedresearch.,49: 2393-2411, DOI: 10.1007/s11033-021-06997-4.

Hedrick, R. P., McDowell, T. S., Rosemark, R., Aronstein, D., and Lannan, C. N., 1991. Two cell lines from white sturgeon.,120: 528-534, DOI: org/10.1577/1548-8659(1991)120<0528:TCLFWS>2.3.CO;2.

Huang, S. M., Kuo, S. T., Kuo, H. C., and Chang, S. K., 2018. Assessment of fish iridoviruses using a novel cell line GS-1, derived from the spleen of orange-spotted grouper(Hamilton) and susceptible to ranavirus and me- galocytivirus.,80: 1766- 1774, DOI: 10.1292/jvms.18-0078.

Huang, X., Huang, Y., Sun, J., Han, X., and Qin, Q., 2009. Cha- racterization of two groupercell lines: Ap- plication to studies of Singapore grouper iridovirus (SGIV) pro- pagation and virus-host interaction.,292: 172-179, DOI: 10.1016/j.aquaculture.2009.04.019.

Huo, H. J., Chen, S. N., Li, L., and Nie, P., 2019. Functional cha- racterization of-10 and its receptor subunits in a perciform fish, the mandarin fish,.,97: 64-75, DOI: 10.1016/j.dci.2019. 03.017.

Kang, M. S., Oh, M. J., Kim, Y. J., Kawai, K., and Jung, S. J., 2003. Establishment and characterization of two new cell lines derived from flounder,(Temminck & Schlegel)., 26: 657-665, DOI: 10. 1046/j.1365-2761.2003.00499.x.

Kong, X., Wang, X., Li, M., Song, W., Huang, K., Zhang, F.,., 2021. Establishment of myoblast cell line and identification of key genes regulating myoblast differentiation in a marine teleost,.,802: 145869, DOI: 10.1016/ j.gene.2021.145869.

Kumaran, S., Deivasigamani, B., Alagappan, K. M., and Sakthi- vel, M., 2010. Infection and immunization trials of Asian sea- bass () against fish pathogen.,31: 539-541.

Le, Y., Li, Y., Jin, Y., Jia, P., Jia, K., and Yi, M., 2017. Estab- lishment and characterization of a brain cell line from sea perch,.–,53: 834-840, DOI: 10.1007/s11626-017-01 85-7.

Leong, C. C., Robinson, B. W., and Garlepp, M. J., 1994. Genera- tion of an antitumour immune response to a murine mesothe- lioma cell line by the transfection of allogeneic MHC genes.,59: 212-216, DOI: 10.1002/ ijc.2910590213.

Levraud, J. P., and Boudinot, P., 2009. The immune system of te- leost fish.(),25: 405-411, DOI: 10.1051/ medsci/2009254405.

Li, K., Qiu, H., Yan, J., Shen, X., Wei, X., Duan, M.,., 2021. The involvement of-α and-β as proinflammatory cy- tokines in lymphocyte-mediated adaptive immunity of Nile ti- lapia by initiating apoptosis.,115: 103884, DOI: 10.1016/j.dci.2020.103884.

Li, Y., Jia, P., Yu, F., Li, W., Mao, C., Yi, M.,., 2022. Estab- lishment and characterization of a liver cell line, ALL, de- rived from yellowfin sea bream,, and its application to fish virology.,45: 141- 151, DOI: 10.1111/jfd.13543.

Liu, Y., Wei, C., Liu, Z., Cao, Z., Sun, Y., Zhou, Y.,., 2021. Establishment of a new fish cell line from the brain of hump- back grouper () and its application in toxi- cology and bacterial susceptibility.,47: 1645-1658, DOI: 10.1007/s10695-021-01006-8.

Maeser, D., Gruener, R. F., and Huang, R. S., 2021. oncoPredict: An R package for predictingor cancer patient drug res- ponse and biomarkers from cell line screening data.,22: bbab260, DOI: 10.1093/bib/bbab260.

Padros, F., Zarza, C., Dopazo, L., Cuadrado, M., and Crespo, S., 2006. Pathology ofinfection in turbot,(L.).,29: 87-94, DOI: 10.1111/j.1365-2761.2006.00685.x.

Parameswaran, V., Shukla, R., Bhonde, R. R., and Sahul Hameed, A. S., 2006. Splenic cell line from sea bass,: Establishment and characterization.,261: 43-53, DOI: 10.1016/j.aquaculture.2006.07.034.

Praveenraj, J., Krishnan, A. N., Jithendran, K. P., Praveena, P. E., and Bhuvaneswari, T., 2018. Experimental infection ofin freshwater fish(Baird and Gi- rard, 1853)–A potential infection model for viral encephalo- pathy and retinopathy.,26: 617-627, DOI: 10.1007/s10499-018-0241-7.

Rojas, V., Galanti, N., Bols, N. C., Jiménez, V., Paredes, R., and Marshall, S. H., 2010. Piscirickettsia salmonis induces apop- tosis in macrophages and monocyte-like cells from rainbow trout., 110: 468-476, DOI: 10.1002/jcb.22560.

Ruiz-Palacios, M., Almeida, M., Martins, M. A., Oliveira, M., Es- teban, M., and Cuesta, A., 2020. Establishment of a brain cell line (FuB-1) from mummichog () and its application to fish virology, immunity and nanoplastics toxico- logy.,708: 134821, DOI: 10. 1016/j.scitotenv.2019.134821.

Sathiyanarayanan, A., Goswami, M., Nagpure, N., Babu, P. G., and Das, D. K., 2022. Development and characterization of a new gill cell line from the striped catfish,(Sauvage, 1878).,48: 367-380, DOI: 10.1007/s10695-022-01053-9.

Semple, S. L., Vo, N. T. K., Poynter, S. J., Li, M., Heath, D. D., DeWitte-Orr, S. J.,., 2018. Extracellular dsRNA induces a type I interferon response mediated via class A scavenger re- ceptors in a novel Chinook salmon derived spleen cell line.,89: 93-101, DOI: 10.1016/j.dci.2018.08.010.

Vo, N. T. K., Bender, A. W., Ammendolia, D. A., Lumsden, J. S., Dixon, B., and Bols, N. C., 2015. Development of a walleye spleen stromal cell line sensitive to viral hemorrhagic septice- mia virus (VHSV IVb) and to protection by synthetic dsRNA.,45: 83-93, DOI: 10.1016/j.fsi. 2015.02.004.

Wang, N., Wang, X. L., Sha, Z. X., Tian, Y. S., and Chen, S. L., 2010. Development and characterization of a new marine fish cell line from turbot ().,36: 1227-1234, DOI: 10.1007/s10695- 010-9402-y.

Wang, Q., Fu, T., Li, X., Luo, Q., Huang, J., Sun, Y.,., 2020a. Cross-immunity in Nile tilapia vaccinated withandvaccines.,97: 382-389, DOI: 10.1016/j.fsi.2019.12.021.

Wang, X., Zhang, R., Liu, L., Ma, G., and Zhu, H., 2021. An-1β homologue induced inflammation and antibacterial im- mune defense in Siberian sturgeon ().,118: 283-293, DOI: 10.1016/j.fsi.2021. 08.030.

Wang, X. A., and Ma, A. J., 2019. Genetic parameters for resis- tance againstin turbot.,42: 713-720, DOI: 10.1111/jfd. 12973.

Wang, Y., Xue, T., Wang, Q., Xia, B., Pan, Q., and Chen, T., 2020b. Virus susceptibility of a new cell line derived from the fin of black carp.,96: 418-426, DOI: 10.1111/jfb.14215.

Wei, S., Yu, Y., and Qin, Q., 2018. Establishment of a new fish cell line from the caudal fin of golden pompanoand its susceptibility to iridovirus.,92: 1675-1686, DOI: 10.1111/jfb.13566.

Xiao, J., Wang, Q., Liu, Q., Wang, X., Liu, H., and Zhang, Y., 2008. Isolation and identification of fish pathogenfrom mariculture in China.,40: 13- 17, DOI: 10.1111/j.1365-2109.2008.02101.x.

Xing, J. G., El-Sweisi, W., Lee, L. E., Collodi, P., Seymour, C., Mothersill, C.,., 2009. Development of a zebrafish spleen cell line, ZSSJ, and its growth arrest by gamma irradiation and capacity to act as feeder cells.–,45: 163-174, DOI: 10.1007/s11626- 008-9159-0.

Xue, T., Liu, Y., Cao, M., Tian, M., Zhang, L., Wang, B.,., 2021. Revealing new landscape of turbot () spleen infected withthrough im- mune related circRNA-miRNA-mRNA axis.(),10: 626, DOI: 10.3390/biology10070626.

Xue, T., Wang, Y. Z., Pan, Q. H., Wang, Q., Yuan, J. F., and Chen, T. S., 2018. Establishment of a cell line from the kidney of black carp and its susceptibility to spring viremia of carp virus.,41: 365-374, DOI: 10.1111/jfd.12736.

Yang, S., Zhao, T., Ma, A., Huang, Z., Liu, Z., Cui, W.,., 2020. Metabolic responses inkidney subject- ed to thermal stress., 103: 37- 46, DOI: 10.1016/j.fsi.2020.04.003.

Yashwanth, B. S., Goswami, M., Kooloth Valappil, R., Thakuria, D., and Chaudhari, A., 2020. Characterization of a new cell line from ornamental fish(Cuvier, 1830) and its susceptibility to nervous necrosis virus.,10: 20051, DOI: 10.1038/s41598-020-76807-7.

Zeng, W., Dong, H., Chen, X., Bergmann, S. M., Yang, Y., Wei, X.,., 2022. Establishment and characterization of a per- manent heart cell line from largemouth bassand its application to fish virology and immunology.,547: 737427, DOI: 10.1016/j.aquaculture.2021. 737427.

Zheng, Y., Wang, N., Xie, M. S., Sha, Z. X., and Chen, S. L., 2012. Establishment and characterization of a new fish cell line from head kidney of half-smooth tongue sole ().,38: 1635-1643, DOI: 10.1007/s10695-012-9660-y.

(August 17, 2022;

October 4, 2022;

February 14, 2023)

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

#The two authors contributed equally to this work.

. E-mail: chaoli@qau.edu.cn

(Edited by Qiu Yantao)