A diet rich in diatom improves the antibacterial capacity of Pacific oyster Crassostrea gigas by enhancing norepinephrine-regulated immunomodulation 56 ISJ 18: 56-65, 2021 ISSN 1824-307X RESEARCH REPORT A diet rich in diatom improves the antibacterial capacity of Pacific oyster Crassostrea gigas by enhancing norepinephrine-regulated immunomodulation Q Sun1,3,4, Y Zheng1,3,4, X Chen1,3,4, N Kong1,3,4, Y Wang1,3,4, Y Zhang1,3,4, Y Zong1,3,4, Z Liu1,3,4*, L Wang1,2,3,4, L Song 1,2,3,4 1Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian 116023, China 2Functional Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266235, China 3Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China 4Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian 116023, China This is an open access article published under the CC BY license Accepted April 8, 2021 Abstract Microalgae such as dinoflagellate and diatom are the major food source of bivalve species, and sufficient food intake contributes to the immunity and the growth of bivalves. In the present study, a monoamine oxidase gene (named as CgMAO), which is the rate-limiting enzyme of norepinephrine (NE) biosynthesis, was cloned from C. gigas. After the oysters were fed with a diet rich in diatom for 21 and 40 d, the NE contents in oyster serum, as well as the mRNA expression of CgMAO in oyster haemocytes, increased significantly compared with control group. Besides, the mRNA expression of cytokines CgTNF-1 and CgIL17-5 in haemocytes and the activities of immune-related enzymes (SOD and LYZ) in oyster serum also increased significantly after diatom feeding. These results collectively suggested that sufficient microalgae intake might significantly enhance the antibacterial capacity in oyster by prompting the biosynthesis of NE and triggering the subsequent antibacterial processes modulated by NE. Key Words: Crassostrea gigas; antibacterial capacity; microalgae; monoamine oxidase; norepinephrine Introduction Most of the bivalve molluscs live in a microbe-rich environment and are under a persistent threat of infection by resident pathogenic microbes. Due to their filter-feeding habit, they concentrate a rich and diverse bacterial commensal microbiota, composed of various species belonging to different genera like Vibrio, Pseudomonas, Acinetobacter, Photobacterium, Moraxella, Aeromonas, Micrococcus and Bacillus (Kueh and Chan, 1985). Most bacterial diseases of bivalves are caused by a large range of Vibrio species (V. alginolyticus, V. splendidus, and V. anguillarum), Pseudomonas, and Aeromonas (Garnier et al., 2008; Guo and Ford, 2016; Zannella et al., 2017). For example, Brown Ring Disease (BRD) induced by Vibrio tapetis affected both juveniles and adults, killing up to 100 % of clams if the bacterium was able to penetrate soft _________________________________________ Corresponding author: Zhaoqun Liu Dalian Ocean University Dalian 116023, China E-mail: liuzhaoqun@dlou.edu.cn tissues (Allam et al., 2002). In recent years, the excessive host farming densities, as well as a lack of bait algae in seawater, might have contributed to bacterial diseases in marine bivalves. A deep investigation on the interactions between hosts and bacterial pathogens will contribute to the sustainability of bivalve farming industry. The resistance of bivalve molluscs to bacterial challenge can be seen as a sort of stress response which is a set of coordinated physiological reactions enhancing host’s capability for homeostasis maintenance (Ottaviani and Franceschi, 1996). Hormones and neurotransmitters are crucial for the regulation of responses to external and internal stress (Chrousos, 2009). Among the neurotransmitters responsive to stressors, catecholamines (CAs), consisting of dopamine (DA), norepinephrine (NE) and epinephrine (E), play important roles in stimulating responses to a perceived threat (Ottaviani and Franceschi, 1996; Ottaviani, 2011). For example, NE concentration in the haemolymph of scallop Chlamys farreri increased significantly after bacteria challenge, and high concentration of NE repressed the activities of immune-related enzymes in haemolymph (Zhou et al., 2011b; Zhou et al., 2012). Moreover, NE is able 57 to regulate both cellular and humoral immunomodulation in oyster C. gigas through a “nervous-haemocyte” neuroendocrine immunomodulatory axis-like pathway (Liu et al., 2017). Monoamine oxidase (MAO) is the key rate-limiting enzyme of NE metabolism, which catalyzes the oxidative deamination of biogenic and vasoactive amines to their corresponding aldehydes (Tipton et al., 2004). In humans, there are two types of MAO: MAOA and MAOB. MAOA and MAOB are widely distributed in tissues such as neurons, liver, pulmonary vascular endothelium, gastrointestinal tract and blood platelets (Shih and Chen, 2004). The deamination catalyzed by MAO is always associated with the immunomodulation of monoamines and the production of intracellular H2O2, which influences the immune competence of immunocytes ( Shih et al., 1999; Ou et al., 2006; Shih, 2018). Recently, homologues of MAO have been characterized from bivalves. A MAO gene (CfMAO) was cloned from scallop C. farreri, which was widely expressed in tissues including haemocytes, hepatopancreas and adductor muscle. The mRNA expression of CfMAO in scallop haemocytes was activated upon V. anguillarum challenge to modulate the immune response of scallops through the deamination of monoamines such as NE, DA and serotonin (Zhou et al., 2011a). A deeper investigation is expected to reveal the antibacterial mechanism mediated by MAO and the monoamine neurotransmitters. Marine bivalves filter water-suspended particles, such as microalgae, bacteria, organic debris and micro-zooplankton, and the microalgae provide dominant and highly variable nutritional value for the growth and immunity in bivalves (Yang et al., 2017). Cell size, shape and biochemical composition of microalgae determine their nutritive quality and utility as food (Yaakob et al., 2014; Beal et al., 2018). Many studies have shown that the quality of the algal diet affects the growth and development of molluscs such as the great scallop Pecten maximus, C. gigas, O. edulis and R. philippinarum (Sanina et al., 2004). Specifically, diatoms and haptophytes (prymnesiophytes) are nutritious microalgae that are frequently used as feed for oysters (Coutteau, 1992). The prymnesiophytes Isochrysis sp. and P. lutheri are rich sources of docosahexaenoic acid (DHA, 22:6n-3) - comprising 8-10% total fatty acids (Volkman, 1989), while diatoms are a rich source of eicosapentaenoic acid (EPA, 20:5n-3) (Dunstan, 1994). Mixed microalgal diets of prymnesiophytes and diatoms are common in bivalve hatcheries, and considered as highly nutritious in terms of requirements for essential n-3 polyunsaturated fatty acids (PUFAs) (Knuckey, 2002). When the energetic requirements of oysters are not satisfied by microalgae intake, the animals lose weight as they use energy reserves. For instance, poorly fed C. gigas use protein for metabolism, while other species such as O. edulis use lipids. Consumption of tissue reserves eventually result in physiological stress, leaving oysters more susceptible to other stressors, that is to say susceptible to infectious agents (Camargo-Cely and Collin, 2019). And, Delaporte et al. reported that arachidonic acid (ARA, 20:4n-6) supplementation derived from sufficient microalgae uptake led to an increase in hemocyte numbers, phagocytosis, and production of reactive oxygen species by hemocytes of oyster C. gigas (Lawrence, 2006). Therefore, the algal diet is closely correlated to the physiological condition of oysters, and also to their resistance to bacterial infections. The Pacific oyster C. gigas is one of the most important maricultural bivalves and contributes weightily to the aquaculture industry in China. Microalgae, especially dinoflagellates and diatoms are their major food source. However, owing to the intensive mariculture of bivalve molluscs in recent years, the structure of microalgae community in nearshore area has changed dramatically, which in turn severely restricts the shellfish farming industry. In the present study, diets with different proportions of dinoflagellates and diatoms were fed to adult C. gigas, and the antibacterial activities mediated by MAO were explored to (1) illustrate the molecular features of oyster MAO gene; (2) understand the correlations between algal intake and antibacterial capacity in oyster; and (3) investigate if a diet rich in diatoms contributes more to the antibacterial activity than a diet rich in dinoflagellates. Materials and methods Oysters, microalgae feeding and sample collection Oysters Crassostrea gigas (about 3 years old, averaging 150 mm in shell length) were collected from a local oyster farm in Dalian, Liaoning Province, China, and maintained in the aerated seawater at 20 °C for two weeks before processing (Zheng et al., 2020). Oysters were fed with microalgae powder (Prorocentrum micans and Nitzschia closterium f. minutissima, commercially purchased respectively from the Shanghai Guangyu Biotechnology Co., LTD and the Ocean University of China), and the seawater was replaced every day. The current experiment was performed in spring from March to May, which was not the breeding season for C. gigas. The gonad of the oysters in the present study was rarely matured during the feeding procedure. Thus, the current results were believed to be convincing. Oysters in the dinoflagellate dominant group were fed with a mixed diet of 80 % Prorocentrum micans and 20 % Nitzschia closterium f. minutissima, while oysters in the diatom dominant group were fed with a mixed diet of 80 % Nitzschia closterium f. minutissima and 20 % P. micans. All the oysters were fed once a day with a total amount of 10-12 × 1010 cells algae. Oysters without any feeding treatment were employed as the control group. The abundance of microalgae in seawater was measured every day, and each measurement was repeated three times. Nine oysters from each group were sampled randomly at 0, 21 and 40 d. Tissues including hepatopancreas, muscle, labial palp, gonad, gill and mantle were collected for tissue distribution assay, and the haemolymph was centrifuged at 800 xg, 4 °C for 15 min to harvest haemocytes and serum. Samples for quantitative real-time PCR analysis was added with 1 mL TRIzol reagent (Invitrogen) for subsequent RNA extraction and stored immediately at -80 °C. Table 1 Sequences of the primers used in the experiment https://en.wikipedia.org/wiki/Monoamine_oxidase_A https://en.wikipedia.org/wiki/MAOB http://xueshu.baidu.com/s?wd=paperuri%3A%28ac54eaeebee229d2524a14e0485a8a23%29&filter=sc_long_sign&sc_ks_para=q%3D%E5%AF%B9%E5%B0%8F%E6%96%B0%E6%9C%88%E8%8F%B1%E5%BD%A2%E8%97%BB%28Nitzschia%20closterium%20f.minutissima%29%E5%88%86%E7%B1%BB%E5%9C%B0%E4%BD%8D%E7%9A%84%E9%87%8D%E6%96%B0%E8%AE%A4%E8%AF%86&sc_us=18162504800843460590&tn=SE_baiduxueshu_c1gjeupa&ie=utf-8 http://xueshu.baidu.com/s?wd=paperuri%3A%28ac54eaeebee229d2524a14e0485a8a23%29&filter=sc_long_sign&sc_ks_para=q%3D%E5%AF%B9%E5%B0%8F%E6%96%B0%E6%9C%88%E8%8F%B1%E5%BD%A2%E8%97%BB%28Nitzschia%20closterium%20f.minutissima%29%E5%88%86%E7%B1%BB%E5%9C%B0%E4%BD%8D%E7%9A%84%E9%87%8D%E6%96%B0%E8%AE%A4%E8%AF%86&sc_us=18162504800843460590&tn=SE_baiduxueshu_c1gjeupa&ie=utf-8 http://xueshu.baidu.com/s?wd=paperuri%3A%28ac54eaeebee229d2524a14e0485a8a23%29&filter=sc_long_sign&sc_ks_para=q%3D%E5%AF%B9%E5%B0%8F%E6%96%B0%E6%9C%88%E8%8F%B1%E5%BD%A2%E8%97%BB%28Nitzschia%20closterium%20f.minutissima%29%E5%88%86%E7%B1%BB%E5%9C%B0%E4%BD%8D%E7%9A%84%E9%87%8D%E6%96%B0%E8%AE%A4%E8%AF%86&sc_us=18162504800843460590&tn=SE_baiduxueshu_c1gjeupa&ie=utf-8 javascript:; http://xueshu.baidu.com/s?wd=paperuri%3A%28ac54eaeebee229d2524a14e0485a8a23%29&filter=sc_long_sign&sc_ks_para=q%3D%E5%AF%B9%E5%B0%8F%E6%96%B0%E6%9C%88%E8%8F%B1%E5%BD%A2%E8%97%BB%28Nitzschia%20closterium%20f.minutissima%29%E5%88%86%E7%B1%BB%E5%9C%B0%E4%BD%8D%E7%9A%84%E9%87%8D%E6%96%B0%E8%AE%A4%E8%AF%86&sc_us=18162504800843460590&tn=SE_baiduxueshu_c1gjeupa&ie=utf-8 http://xueshu.baidu.com/s?wd=paperuri%3A%28ac54eaeebee229d2524a14e0485a8a23%29&filter=sc_long_sign&sc_ks_para=q%3D%E5%AF%B9%E5%B0%8F%E6%96%B0%E6%9C%88%E8%8F%B1%E5%BD%A2%E8%97%BB%28Nitzschia%20closterium%20f.minutissima%29%E5%88%86%E7%B1%BB%E5%9C%B0%E4%BD%8D%E7%9A%84%E9%87%8D%E6%96%B0%E8%AE%A4%E8%AF%86&sc_us=18162504800843460590&tn=SE_baiduxueshu_c1gjeupa&ie=utf-8 58 Primer Sequence (5’-3’) Sequence information P1 (forward) AGACAACTGATGGAGTGACGGTG Real-time CgMAO primer P2 (reverse) TCCAAAAAGGGGTCTTGTAGTAGC Real-time CgMAO primer P3 (forward) GGTAATAACGAAAGGAAACGAAG Real-time CgDBH primer P4 (reverse) CACCGATAACTTCCCGACAC Real-time CgDBH primer P5 (EF-RTF) AGTCACCAAGGCTGCACAGAAAG Real-time CgEF primer P6 (EF-RTR) TCCGACGTATTTCTTTGCGATGT Real-time CgEF primer P7 (forward) CTTCTCGTCTGCGGCTTCTTT Real-time CgTNF-1 primer P8 (reverse) CAGGGCTGCGGTCTTTCC Real-time CgTNF-1 primer P9 (forward) CGTCCTTGCCTTACTGACTAGA Real-time CgIL17-5primer P10 (reverse) TGTCGTTGTCCTCTACCATGAT Real-time CgIL17-5 primer P11 (forward) AACACATGACTATGGAGGAAGATCAGCT CgMAO specific primer P12 (reverse) GTTTCCACCAATAAAGACACAGTCCC CgMAO specific primer RNA isolation, gene cloning, sequence analysis and quantitative real-time PCR analysis Total RNA was isolated from the oyster haemocytes using TRIzol reagent according to the standard protocol. The synthesis of the first-strand cDNA was carried out with Promega M-MLV RT with oligo (dT)-adaptor priming according to the manufactory's protocol. The full-length cDNA sequence of CgMAO (CGI_10022845) (Boutet et al., 2004) was cloned from the cDNA library using specific primers (Table 1). The sequence analysis was performed as the description of Zhou (Zhou et al., 2011a). The quantitative real-time PCR was carried out on an ABI PRISM7500 Sequence Detection System. CgDBH, CgMAO and the immune-related genes including CgTNF-1 (CGI_10018786) and CgIL17-5 (CGI_10004922) were amplified using the corresponding primers, and a fragment (168 bp) of oyster elongation factor (CgEF, CGI_10012474) was employed as endogenous control (Table 1). Dissociation curve analysis of amplification products was performed to confirm that only one PCR product was amplified and detected. All data were given in terms of relative mRNA expression using the 2−ΔΔCt method (Zhang et al., 2008). Quantification of NE in oyster serum The concentration changes of NE in oyster haemolymph after microalgae fed were determined by norepinephrine ELISA kit (Abnova, KA1891) (Jiang et al., 2014). Briefly, NE was extracted from samples using a cis-diol-specific affinity gel, acylated and then derivatized enzymatically. After the samples were equilibrated, free NE and free NE-antibody complexes were removed by three rounds of washing with wash buffer. The antibody bound to the solid phase was detected by using an anti-rabbit IgG-peroxidase conjugated with TMB as substrate. The product of the enzyme-substrate reaction forms a blue-colored complex. Finally, a stop solution was added to stop the reaction, which would then turn the solution yellow. The reaction was monitored by a microtiter plate reader (BioTek, USA) at 450 nm. Quantification of samples was achieved by comparing their absorbance with a reference curve (n = 6). Measurement of key immune-related enzymes activity The activities of three immune-related enzymes including superoxide dismutase (SOD), catalase (CAT) and lysozyme (LYZ) in the oyster serum were measured by the kits (Nanjing, Jiancheng, A001-1, A007 and A050-1) according to the protocol. Total SOD activity was determined by the hydroxylamine method. One SOD activity unit was defined as the enzyme amount causing 50% inhibition in 1 mL reaction solution. Total CAT activity was determined by using the spectrophotometry to measure yellowish complex compound yielded from the reaction between hydrogen peroxide and ammonium molybdate (Kono, 1978). As for the detection of LYZ activity, 20 μL serum was added to 200 μL bacteria suspension (0.25 mg mL-1 in bacterial buffer supplied from the kit). The mixture was incubated at 37 °C for 15 min, and then bathed in ice for 3 min. The reduction of absorbance at 530 nm was measured at room temperature using a microtiter plate reader (BioTek, USA). The total LYZ activity was measured by comparing the turbidimetry of the samples with that of LYZ standard solution (2.5 mg mL-1, offered by the kit) (Jiang et al., 2013). Antibacterial assay of oyster serum In the antibacterial assay, 50 µL of serum was mixed with 10 µL of alive V. splendidus, and then were pipetted into each well of a flat bottom 96-well plate, incubating at 18 °C for 3 h with gentle agitation. Then, 200 µL of 2216E medium was added into the plate, and the plate was incubated at 28 °C. The OD value of the mixture was continually read using a microtiter plate reader for 24 h at intervals of 30 min. The OD value measured at 600 nm was used to determine the antibacterial ability of serum. The differences were acquired when the absorbance 59 among each group reached the maximum (Zhou et al., 2013). Statistical analysis All the data were given as means ± S.D, and analyzed by Statistical Package for Social Sciences (SPSS) 20. Significant differences between treatments for each assay were tested by one-way analysis of variance (ANOVA), and considered significant at p < 0.05 (a, b, c, etc.). Results Molecular characteristics of CgMAO The nucleotide sequence of CgMAO contained an ORF of 1566 bp, encoding a polypeptide of 521 amino acids with the molecular mass predicted as 58.83 kDa. The theoretical isoelectric point of CgMAO was 6.13. Two FAD building domains, one NAD building domain and one amino oxidase domain (from Leu18 to Leu456) were identified from CgMAO by SMART program analysis. Multiple sequence alignment and phylogenetic analysis The sequence similarity of CgMAO with other MAOs was analyzed by BLAST algorithm (Fig. 1A). It shared 68.8 % similarity with that from Danio rerio, 78.3 % similarity with that from Mizuhopecten yessoensis, 19 % similarity with that from Xenopus tropicalis, 17.7 % similarity with that from Oryzias latipes, 54.5 - 66 % with MAOAs and 66.3 - 67.2 % with MAOBs from vertebrates (Table 2). Specifically, CgMAO exhibited 66.0 % sequence similarity with MAOA from H. sapiens, and 66.9 % sequence similarity with MAOB from H. sapiens. Thus, it was very hard to tell whether CgMAO belonged to MAOA subtype or MAOB subtype, maybe because the MAO gene was the ancestor gene and not fully differentiated. An unrooted phylogenetic tree was constructed using neighbor-joining method with 1000 bootstrap test based on the multiple sequence alignment of CgMAO and MAOs from other vertebrate and invertebrate species. CgMAO was clustered into the invertebrate group with the closest match of scallop M. yessoensis. In the vertebrate group, MAOAs were clustered together and formed a sister branch to the branch of MAOBs (Fig. 1B). Tissue distribution of CgMAO mRNA transcripts The quantitative real-time PCR was performed to investigate the distribution of CgMAO transcripts in different tissues with CgEF as the internal control. The mRNA transcripts of CgMAO were detected in all the tested tissues including haemocytes, gonad, mantle, labial palp, gill, muscle and hepatopancreas (Fig. 1C). It was relatively abundant in hepatopancreas and haemocytes. The highest CgMAO expression level was detected in haemocytes, which was 1029.2-fold (p < 0.05) of that in gill. The expression of CgMAO mRNA in hepatopancreas was 309.5-fold of that in gill (p < 0.05). Fig. 1 (A) Multiple sequences alignment of CgMAO with MAOAs from other species. The black shadow region indicates positions where all sequences shared the same amino acid residue, while the similar amino acids are shaded in grey. Gaps are marked by dashes to improve the alignment. The species and the GenBank accession numbers were as follows: MAOAs from human Homo sapiens (NP 000231), zebra fish Danio rerio (NP_997992), and scallop Mizuhopecten yessoensis (XP_021339456). (B) Consensus neighbor-joining tree based on the sequences of CgMAO and MAOs from other species. The protein sequences used for phylogenetic analysis included MAO from zebra fish Danio rerio (NP_997992), scallop Mizuhopecten yessoensis (XP_021339456), medaka Oryzias latipes (XP_023806277), xenopus Xenopus tropicalis (NP_001120572); MAOAs from human Homo sapiens (NP_000231), cattle Bos taurus (NP_851357), mouse Mus musculus (NP_776101), pig Sus scrofa (NP_001001640), orangutan Pongo abelii (NP_001124913), horse Equus caballu (NP_001075301); and MAOBs from human Homo sapiens (AAB27229), cattle Bos Taurus (AAF23179), mouse Mus musculus (AAI13183), pig Sus scrofa (AAT06259), orangutan Pongo abelii (NP_001124895), horse Equus caballus (NP_001075302) . (C) Tissue distribution of the CgMAO mRNA transcript detected by SYBR Green real-time PCR. CgMAO relative mRNA expression level in gonad, mantle, labial palp, mantle, muscle, haemocytes and hepatopancreas was normalized to that of gill. Each group values were shown as mean ± SD, and bars with different letters were significantly different (p < 0.05) Table 2 The monoamine oxidase (MAOs) from various species javascript:; javascript:; javascript:; https://www.ncbi.nlm.nih.gov/protein/NP_997992.2?report=genbank&log$=prottop&blast_rank=1&RID=F4027JXP014 https://www.ncbi.nlm.nih.gov/protein/XP_021339456.1?report=genbank&log$=prottop&blast_rank=1&RID=F417N35T014 https://www.ncbi.nlm.nih.gov/protein/NP_997992.2?report=genbank&log$=prottop&blast_rank=1&RID=F4027JXP014 javascript:; https://www.ncbi.nlm.nih.gov/protein/XP_021339456.1?report=genbank&log$=prottop&blast_rank=1&RID=F417N35T014 https://www.ncbi.nlm.nih.gov/protein/XP_023806277.1?report=genbank&log$=protalign&blast_rank=1&RID=YJFBK51R013 https://www.ncbi.nlm.nih.gov/protein/NP_001087039.1?report=genbank&log$=prottop&blast_rank=1&RID=F41RN9XG01N 60 Name Organism Accession number Similarity Identity MAOA Homo sapiens NP_000231 66 55.5 MAOA Bos taurus NP_851357 65.8 54.7 MAOA Sus scrofa NP_001001640 65.8 55.3 MAOA Rattus norvegicus NP_387502 65.4 56 MAOA Mus musculus NP_776101 54.5 55.2 MAOB Homo sapiens AAB27229 66.9 55.8 MAOB Bos taurus AAF23179 66.5 55 MAOB Sus scrofa AAT06259 66.3 54.9 MAOB Rattus norvegicus AAA41566 67.2 56.2 MAOB Mus musculus AAI13183 67.4 57 MAO Danio rerio NP_997992 68.8 57.6 Biosynthesis of NE after oysters were fed with different algal diet The concentration of NE in oyster serum kept increasing after algae fed at different time points. At 21 d, NE concentration in diatom dominant group was higher than that of other two groups (p > 0.05), which was 1.21-fold of that in the dinoflagellate dominant group and 1.23-fold of that in the control group (Fig. 2A). At 40 d, NE concentration in either dinoflagellate dominant group or diatom dominant group was higher significantly (p < 0.05) than that in control group. Meanwhile, the mRNA expression of CgDBH and CgMAO, two rate-limiting enzymes for NE biosynthesis, was detected with real-time PCR (Fig. 2B and 2C). After the oysters were fed with microalgae for 21 d, the mRNA expression levels of CgDBH decreased significantly, which were 0.45-fold and 0.5-fold of that in the control group (p < 0.05). At 40 d post feeding, the mRNA expression level of CgDBH in the diatom dominant group was 3.75-fold of that in the control group, which was dramatically higher than that in control group (p < 0.05). Besides, the mRNA expression of CgMAO in the diatom dominant group decreased significantly on both 21 and 40 d post feeding, which was 0.7- and 0.29-fold of that in the control group (p < 0.05). But, once oysters were fed with a diet rich in dinoflagellates for 40 d, the expression of CgMAO was significantly prompted (2.23-fold) comparing with control group (p < 0.05). Changes of CgTNF-1 and CgIL17-5 mRNA expressions in oyster haemocytes after microalgae feeding After the oysters were fed with microalgae for 21 d, the mRNA expression of CgTNF-1 increased significantly, which was 2.43-fold (dinoflagellate dominant group) and 2.20-fold (diatom dominant group) of that in the control group (p < 0.05). At 40 d post feeding, the expression levels in the dinoflagellate dominant group and diatom dominant group were also dramatically higher than that in the control group, which were 3.60-fold and 7.1-fold of that in the control group, respectively (p < 0.05, Fig. 3A). As for CgIL17-5, its expression level increased significantly after oysters were fed with diet rich in diatom for 40 d (p < 0.05) which was 2.0-fold of that in the control group, while its expression level was obviously lower (0.33-fold, p < 0.05) in dinoflagellate dominant group comparing with that in the control group (Fig. 3B). Fig. 2 The norepinephrine (NE) concentration in oyster serum (A), as well as the mRNA expression of CgDBH (B) and CgMAO (C) in haemocytes, after the oysters were feed with diets with different proportion of dinoflagellate and diatom for 21 and 40 d javascript:; javascript:; 61 Fig. 3 The mRNA expression level of CgTNF-1 (A) and CgIL17-5 (B) in haemocytes after the oysters were feed with diets with different proportion of dinoflagellate and diatom for 21 and 40 d The enzyme activity of three key immune-related enzymes including SOD, CAT and LYZ was detected post microalgae feeding (Fig. 4). After the oysters were fed with a diet rich in diatom, the SOD activity increased significantly comparing with the control group (1.14-fold, p < 0.05, Fig. 4A). The CAT activity in the dinoflagellate dominant group increased significantly at 21 d, but was obviously lower than that in the control group at 40 d (p < 0.05, Fig. 4B). As for LYZ, its activity increased significantly in the diatom dominant group at both 21 and 40 d (p < 0.05, Fig. 4C). In the dinoflagellate dominant group, the CAT activity increased significantly at 21 d post feeding, which was 1.32-fold of that in the control group (p < 0.05). Antibacterial activity of oyster serum The antibacterial activity of oyster serum post microalgae feeding was determined. At 40 d after feeding, the antibacterial activity of serum increased significantly (p < 0.05, Fig. 5A) comparing to that in the blank group. Serum in the diatom dominant group exhibited the highest antibacterial activity, whereas the lowest was observed in blank group. The absorbance of bacteria from the blank, dinoflagellate dominant and diatom dominant group was shown in Fig. 5B, when the absorbance difference between the blank and diatom dominant group reached the maximum at 21.5 h after the incubation with the serum. Discussion Bivalve molluscs, such as clams, mussels, oysters and scallops, are relevant bred species, and their global farming maintains a high incremental annual growth rate, representing a considerable proportion of the overall fishery activities. Bivalve molluscs are filter-feeding species. They bio-accumulate large amount of microorganisms, in which bacteria can be a great threat, while microalgae such as dinoflagellate and diatom are their major food source. Also, much previous research has evidenced that sufficient food supply was of great importance to the antibacterial immunity by offering adequate energy for the immune activity. So far, great progress has been made on the study of immune system and energy metabolism in bivalves. But, the comprehensive interpretation of the nutritional immunology in bivalves is still in urgent need. In the present study, correlation between microalgae intake and the antibacterial immune capacity mediated by norepinephrine (NE) in the pacific oyster C. gigas was investigated, aiming to better illustrate how food intake affects the antibacterial immune response in oyster, and what roles the neuroendocrine system play in such process. According to our previous research, the monoamine neurotransmitter NE was able to modulate the antibacterial immunomodulation via a simple “nervous-haemocyte” neuroendocrine-immune axis to promote the synthesis of cytokines and the apoptosis of haemocytes in oyster (Liu et al., 2016a). Monoamine oxidase (MAO) is the crucial enzyme for the metabolism of NE, which catalyzes the oxidative deamination of biogenic and vasoactive amines to their corresponding aldehydes (Slotkin, 1999). Thus, in the present study, the full-length sequence of a previously identified oyster MAO gene (CgMAO) was cloned from C. gigas (Boutet et al., 2004). The nucleotide sequence of CgMAO contained an ORF of 1566 bp, encoding a polypeptide of 521amino acids. There were two FAD building domains, one NAD building domain, and one Amino oxidase domain (from Leu18 to Leu456) in CgMAO. CgMAO shared high sequence similarity with MAOs identified from vertebrate species, and its mRNA transcripts were highly expressed in oyster tissues including hepatopancreas and haemocytes. Results of phylogenetic analysis showed that CgMAO was javascript:; javascript:; javascript:; 62 Fig. 4 The activities of key immune-related enzymes including SOD (A), CAT (B), LYZ (C) in serum after the oysters were feed with diets with different proportion of dinoflagellate and diatom for 21 and 40 d first clustered into the invertebrate group with the closest match of scallop M. yessoensis, and then gathered with MAOs from vertebrate species. These results showed CgMAO was the homologue of MAOs in model creatures, and could perform as the rate-liming enzyme of NE biosynthesis. Besides, the mRNA transcripts of CgMAO were widely expressed in oyster tissues, with the highest expression levels detected in haemocytes and hepatopancreas. Haemocytes are the most significant immunocytes in oysters, and hepatopancreas is the most important immune-related tissues since it accounts for the synthesis of several neurotransmitters such as NE and acetylcholine (ACh) in oyster (Liu et al., 2018). These results indicated that CgMAO might be related to the immune activities in oyster. Dietary conditioning is extremely important for the growth and health of farmed oysters, which basically live on the microalgae in the seawater. In recent years, the oyster farming has suffered severe mortality owing to the biomass shortage and community imbalance of microalgae caused by the increasing farming density (Pernet et al., 2016). Based on our latest research, dinoflagellate and diatom are the major components of the microalgae community in the Bohai Sea and the North Yellow Sea (unpublished data). In this study, diet with different proportion of dinoflagellate and diatom were fed to the oysters for as long as 40 d, and the antibacterial parameters were determined. After oysters were fed with microalgae for 40 d, NE contents in both dinoflagellate dominant group and diatom dominant group were slightly higher than that in the control group. Meanwhile, at 40 d post feeding, the mRNA expression of CgDBH was significantly higher in the diatom dominant group, and the mRNA expression level of CgMAO was also dramatically increased in the dinoflagellate dominant group. DBH is a copper-containing enzyme that uses molecular oxygen and ascorbate to catalyze the addition of a hydroxyl group on the beta-carbon of dopamine to form norepinephrine (Kaufman and Friedman, 1965), while MAO is crucial enzyme for the degradation of NE (Franco et al., 2007; Malagoli et al., 2017). These results indicated that sufficient microalgae supply could promote the biosynthesis of NE, which might then enhance the antibacterial immune process. In order to validate the above hypothesis, a series of parameters related to the antibacterial immunomodulation were determined. After the oysters were fed with microalgae for 40 d, the mRNA expression of CgTNF-1 and CgIL17-5 increased significantly. At 40 d post feeding, the enzyme activities of SOD and LYZ in the diatom dominant group were obviously higher than those in the control group, while the enzyme activity of CAT was significantly higher than control group at 21 d post feeding. In addition, at 40 d after feeding, the antibacterial activity of serum increased significantly, and the diatom dominant group exhibited the highest antibacterial activity. Interestingly, the increase of mRNA expression level of cytokines, as well as the enzyme activity was obviously higher in the diatom dominant group than in the dinoflagellate dominant group. Our previous research demonstrated that NE could regulate the antibacterial activity mediated by oyster haemocytes by binding to the transmembrane receptor CgA1AR-1, and up-regulate the activity of SOD and LYZ in oyster serum (Liu et al., 2016b). CgTNF-1 was reported to be involved in the modulation of immune response including apoptosis and phagocytosis of haemocytes upon LPS stimulation (Sun et al., 2014). Zheng et al. reported that the mRNA transcripts of CgTNF-2 were highly expressed in oyster hemocytes and could be induced by LPS and PGN stimulation. CgTNF-2 exhibited inhibitory effects on the growth of A549 cell, and it could promote the lysozyme activity and synthesis of NO to regulate the antibacterial response in C. gigas (Zheng et al., 2020). Also, CgIL17-5, as an ancient inflammatory cytokine, could not only activate signal transduction for the release of other cytokines, but also mediate the javascript:; javascript:; 63 Fig. 5 The effects of microalgae feeding on the antibacterial activity of oyster larvae. (A) The growth curve for bacteria V. splendidus exposed to oyster serum from the control, dinoflagellate dominant and diatom dominant group at 40 d after feeding. Bacterial growth was recorded as absorbance at 600 nm. (B) The absorbance of bacteria from the control, dinoflagellate dominant and diatom dominant group, when the absorbance different between the control and diatom dominant group reached the maximum. Each group value is shown as mean ± SD, and bars with asterisks are significantly different (p < 0.05) clearance of extracellular bacteria in oysters (Xin et al., 2015). CgIL17-5 was reported to promote nuclear factor NF-кB pathway and mitogen-activated protein kinase (MAPK) pathway to play an important role in antibacterial immunity (Roberts et al., 2008). These results inferred that sufficient microalgae intake could significantly enhance the antibacterial immune response level in oyster by activating NE and cytokine production, elevating the activity of immune-related enzyme, and up-regulating the antibacterial activity of oyster serum. Moreover, oysters fed with a diet rich in diatom exhibited significantly higher antibacterial capacity comparing with those fed with a diet rich in dinoflagellate, which implied that diatom might be the more preferable food for oysters than dinoflagellate. Acknowledgements We are grateful to all the laboratory members for their technical advice and helpful discussions. 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