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Fish red blood cells express immune genes and responses

2018-03-07YaweiShenDanWangJinliangZhaoXiaowuChen

Aquaculture and Fisheries 2018年1期

Yawei Shen,Dan Wang,Jinliang Zhao,Xiaowu Chen,*

aNational Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306,China

bShanghai Engineering Research Center of Aquaculture,Shanghai Ocean University,Shanghai 201306,China

cShanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding,Shanghai Ocean University,Shanghai 201306,China

1.Introduction

In vertebrates,the most abundant cell type in circulation are the erythrocytes or red blood cells(RBC),that are nucleated cells in the majority of vertebrates with the exception of mammals.All nucleated non-mammalian RBC contain organelles in the cytoplasm(Davinia&Mackenzie,2011)and show different maturation stages including changes in the cytoplasmatic shape,staining,nuclear size and chromatin density.RBC total RNA(tRNA)and organelles content follow an inverse relationship with cellular age and young RBC possess a higher concentration of tRNA while aged cells display loss of cellular organelles including ribosomes and mitochondria(Lund,Phillips,Moyes,&Tufts,2000).The white blood cells(WBCs)are the immune system circulatory cells and participate in both innate and acquired immune responses by expressing cell-specific immune-relevant genes(Abbas et al.,2005).Extensive studies in fish characterized the immune modulatory mechanisms of different WBCs types(Magnad'ottir,2006;Zapata,Diez,Cejalvo,Gutierrez-de Frias,Cortes,2006),however,immune-relevant genes can also be expressed in RBCs and still serve functions associated with the immune response.In circulation the major role of RBCs is the transport of gases to cells and tissues,however they seemed to serve other function than oxygen delivery(Morera&MacKenzie,2011).Progress has been achieved on the characterization of RBCs immune function,from identifying RBC immune adherence function(Fearon,1980;Nelson,1953)to the“red cell immune system”hypothesis which claims that RBCs are as important as WBCs in immune defense(Siegel,Liu,&Gleicher,1981).In fish,some genes including enolase,myxovirus resistance and type I interferon(IFN)genes that are characteristic of WBC are also expressed in RBCs and changes of the gene expression profile with external stimuli has been observed for fish and chicken RBC(Passantino et al.,2007;St Paul,Paolucci,Barjesteh,Wood,&Sharif,2013).several Studies expression of immune-relevant genes have also shown that RBCs constitutively express toll-like receptors(TLRs)in the Atlantic salmon(Salmo salar)and rainbow trout(Oncorhynchus mykiss)(Morera et al.,2011;Workenhe et al.,2008).

An alternative strategy to characterize the function of blood cells is to investigate their entire gene expression profile rather than individual transcripts.Transcriptomic analysis of human RBCs or WBCs have been conducted(Abbas et al.,2005;Kohane&Valtchinov,2012)and characterization of the mammalian RBCs at different developmental stages(erythroblasts,reticulocytes and mature)indicated a wide spectrum of expressed genes associated with the immune defense(Goh,2007;Kabanova et al.,2009;Sennikov et al,2004).Across vertebrates many viral,prokaryotic and eukaryotic pathogens directly target RBCs and a significant number of associated pathologies have been described(Lewis,Hori,Rise,Walsh,&Currie,2010;Morera et al.,2011).In contrast,fewer studies have described the expression profile of blood cells in nonmammalian vertebrates.In zebra fish comparative analyses evidenced that there is a higher gene turnover of transmembrane proteins in the Natural Killer(NK)cells in relation to T when compared to mammals(Carmona et al.,2017).In the rainbow trout(O.mykiss)the genes that are differential expressed in RBCs under different temperatures are involved in stress response,immune response and apoptosis(Lewis et al.,2010;Morera et al.,2011).Major phenotypic changes also occur in fish RBCs when infected with Pseudorabies(PRV)and PRV infection of salmon RBC was found to activate innate antiviral immunity but to suppress other gene expression programs(Dahle et al.,2015).Nucleated RBC from fish and birds express and regulate specific pattern recognition receptor(PRR)and are capable of specific pathogen associated molecular pattern(PAMP)detection that is central to innate immune response.Thus nucleated RBC from non-mammalian vertebrates seem to play a key role in the immune response(Morera et al.,2011)but insufficient systematic and comprehensive data is available.Next-generation sequencing(NGS)is a robust technology to study gene expression due to its high-throughput and accuracy(Metzker,2010).In the present study we applied NGS technology to characterize the expression profile of RBCs and WBCs of the teleost fish Nile tilapia(Oreochromis niloticus).Tilapia is an important economic and commercial teleost fish species and availability of a sequenced and annotated nuclear genome facilitates transcript mapping and annotation(Brawand et al.,2014).The present study elucidates on the expression profile of tilapia blood cells and provides new insights into the evolution of RBC immune function across vertebrates.

2.Materials and methods

2.1.Fish and blood cells purification

Experiments with fish were performed in agreement with the guidelines for the care and use of animals for scientific purposes approved by the Committee on the Ethics of Animal Experiments of Shanghai Ocean University.Fish were sacrificed,and all efforts were exerted to minimize suffering.Clove oil(30-40 mg/L)was used for anesthesia.Nile tilapia(O.niloticus)were maintained in outdoor breeding pond of 5m wide and 10m long at the Xinchang Aquafarm of Shanghai Ocean University.The strain “New GIFT”has been selected for more than 20 years.Blood samples were collected from the caudal vein of six fish with a heparinized syringe and RBCs and WBCs were separated by gradient centrifugations.Anticoagulant blood was carefully layered over the separation medium(1.080g/mL)(Beijing Propbs Biotechnology company,China)and centrifuged at 400×g and 4°C for 20 min.RBCs were collected from the bottom and WBCs that are localized in the middle were treated with RBC lysis solution(Beijing Propbs Biotechnology Company,China)to eliminate potential RBCs before collection.RBCs and WBCs samples were washed twice with 1×PBS and stored at-80°C for RNA isolation.

2.2.Cells staining and microscopy

Smears of separated RBCs and WBCs samples were Giemsastained for standard light microscopy.The purity of separated RBCs and WBCs were examined by counting 10 fields of smears based on cell morphology.For fluorescent staining of mitochondria and lysosomes,live RBCs were immediately washed with 1×PBS after separation and incubated with MitoTracker Red CMXRos and LysoTracker®Green DND-26(Invitrogen,USA)in 200nmol/L Dulbecco's Modified Eagle Medium at 28°C for 10 min.RBCs were washed with 1×PBS and used to prepare smears.All microscope observations and photographs were carried out using a Nikon Eclipse Ni-E microscope.

2.3.Polyinosinic:Polycytidylic acid[Poly(I:C)]challenge

Fish of 100±10g body weight were acclimatized under a standard photoperiod of 12h light/12h dark for a period of 2weeksat 28°C and were fed with fish bait daily at 5%body weight.Healthy fish were randomly selected and distributed in two groups(n=5 individuals each group).The fish of the control group were intraperitoneally injected with sterile 1×PBS and the experimental fish group was injected with poly(I:C)(0.5μg/g body weight)(Sigma-Aldrich)dissolved in sterile 1×PBS.Poly(I:C)is an immunostimulant and was used in the form of its sodium salt to simulate a viral infection.After 24h post injection,blood samples from the control and experimental groups were collected and RBCs and WBCs were isolated as described above and subsequently used for RNA isolation and for Real-Time Quantitative Reverse Transcription PCR(qRT-PCR)analysis.

2.4.RNA isolation and RNA-Seq

tRNA was extracted from RBCs and WBCs of six fish using TRIzol reagent according to the manufacturer's instructions(Invitrogen,USA)and RNA quantity and quality were determined using the NanoDrop Spectrophotometer 2000 and 2100 Bioanalyzer(Agilent,USA).Two micrograms of tRNA from RBCs and WBCs per fish were mixed and pools of control and experimental fishes were used to produce two libraries for sequencing.tRNA samples were treated with DNase I(TaKaRa)to eliminate contaminating genomic DNA and Poly(A)mRNAs was purified using Dynabeads Oligo(dT)25(Life Technologies,USA)and subsequently broken into short fragments for the synthesis of first-and second-strand cDNAusing the UltraTMRNA Library Prep Kit(NEB,USA).End-repaired fragments were linked with adapters and purified to obtain cDNAs of desirable lengths,followed by PCR amplification and purification using the AMPure XP beads.After PicoGreen staining and fluorospectrophotometry checking,10 ng of cDNA libraries were used for cluster generation with TruSeq PE Cluster Kit(Illumine,USA)and sequencing analysis on an Illumina HiSeq2500 platform.

2.5.Transcriptome data analysis

Raw reads of 150 bp in length were filtered into clean reads using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).Reads of adaptor sequences,low-quality bases and read lengths<40 bp were removed.Clean reads of RBCs and WBCs samples were mixed and mapped against the reference O.niloticus genome using Tophat and Bowtie2 software(Trapnell,Pachter,&Salzberg,2009)and successfully mapped reads were used for quantification analysis.Values of reads per kilobase of exon model per million mapped reads(RPKM)were calculated to normalize transcript expression abundancE(Mortazavi,Williams,McCue,Schaeffer,&Wold,2008).The RPKM values were used to compare differences of transcript expression levels between RBCs and WBCs using the DEGseq package based on the MA plot-based according to the random sampling model(Wang,Feng,Wang,Wang,&Zhang,2010a).The Benjamini-Hochberg method was used to determine the threshold of the q value in multiple tests.False discovery rate(FDR)<10-3was used to identify differentially expressed transcripts between two samples.Gene ontology(GO)was performed based on the blast results output obtained from the Swissprot database(blastp,E<10-5)(Ashburner et al.,2000)combined with Blast2go software(http://www.blast2go.org/)(Conesa,Gotz,Garcia-Gomez,Terol,Talon,Robles,2005).All transcripts were submitted to the KOG(eukaryotic orthologous groups)database to determine the KOG term(Tatusov et al.,2003)and protein-coding sequences were analyzed against the Kyoto Encyclopedia of Genes and Genomes database for bidirectional(blastp,E<10-3)to assign KO numbers with tools KAAS(http://www.genome.jp/tools/kaas/help.html)(Kanehisa,Goto,Furumichi,Tanabe,&Hirakawa,2010).Metabolic pathways were generated according to the KO assignment.

2.6.Real-Time Quantitative Reverse Transcription PCR

Equal amounts of 2μg total RNA from tilapia RBCs and WBCs was used for cDNA synthesis following the manufacturer's instruction of PrimeScript™RT reagent Kit with the gDNA Eraser Kit(TaKaRa).cDNAs were diluted 1:10 with sterile water and qRT-PCR reactions were run with three replicates for a 20μL volume reaction containing 2×SYBR Master Mix,SYBR Green(Roche,USA)and 1μL of the diluted cDNA for 40 cycles of 95°C for 5s and 60°C for 34 s on the Applied Biosystems 7500RT-PCRsystem.Following threshold-dependent cycling,melting was performed from 60°C to 95°C at 0.1°C/s melt rates with a smooth curve setting averaging 1 point.Melting peaks were visualized by plotting the absolute value of the first derivative against the temperature.The melting temperature(Tm)was defined as the peak of the curve;if the highest point was a plateau,then the midpoint was identified as the Tm.Gene specific primers were synthesized by Sangon Biotech(Table S1).To determine the amplification efficiency of each gene specific primer,standard curves were generated using ten-fold dilutions of the cDNA in triplicates for five consecutive 10 times dilution.The relative expression software tool was used to calculate gene expression fold change(Pfaffl,Horgan,&Dempfle,2002).Data was analyzed using the Applied Biosystems 7500 system software version 1.2.3.

2.7.Statistical analysis

Statistical analysis was estimated with unpaired sample t-test using the Statistical Package for Social Sciences(SPSS 19.0)for Windows.Significant differences were estimated when P<0.05.Error bars represent±S.D.of three independent experiments.

3.Results

3.1.Isolation and staining of tilapia RBCs and WBCs

Giemsa staining of the smears of separated cells revealed distinct morphologies between tilapia WBCs and RBCs.WBCs displayed heterogeneity in staining,with different sizes and shapes of the whole cells and their nuclei(Fig.1A),whereas RBCs were more homogeneous and exhibit an oval shape and prominent nuclei residing in the center of the cells(Fig.1B).Cell counting based on distinct morphologies revealed that the contaminating RBCs in WBCs were less than 5%,and separated RBCs similarly contained few contaminating WBCs(less than 0.1%).Staining with fluorescent dyes MitoTracker Red CMXRos and LysoTracker®Green DND-26 identify numerous mitochondria(Fig.1C)and lysosomes(Fig.1D)in RBCs.Thus,gradient centrifugation provides pure RBCs and RBCs satisfactory for gene expression analysis using RNA-seq and other experiments.In addition,tilapia RBCs possess not only prominent nuclei but also mitochondria and lysosomes necessary for metabolism.

3.2.Sequencing and bioinformatics analysis

Two cDNA libraries from RBCs and WBCs were sequenced separately and in total,13.94 and 14.15 million raw reads with 150 bp in length were generated,respectively.After trimming 14.06(99.35%)and 13.25(95.03%)million high-quality paired-end reads for WBCs and RBCs were obtained(Table 1).Combined transcriptomes were mapped against the tilapia reference genome and 20.876 transcripts were annotated and 17.768 transcripts were GO annotated and subdivided into 629 GO terms..GO classification of the individual tilapia WBCs and RBCs libraries show differences at first-level and up to 64.67%,21.98%,and 13.25%of the terms were assigned to molecular function,cellular component and biological process of RBCs,whereas for WBCs were 45.08%,33.80%and 21.12%,respectively(Fig.2A).

The KEGG pathway approach was adopted to determine biological functions,gene interactions and metabolic pathways.In total,5805 proteins from two libraries with KO number were assigned to 32 s-level KEGG pathways(Fig.2B).KEGG analysis revealed that the term “signal transduction”assigned the most number of transcripts(n=1657),followed by “immune system”(n=809),which included 16 pathways,namely:Chemokine signaling pathway,Platelet activation,Leukocyte transendothelial migration,Tcell receptor signaling pathway,Fc gamma R-mediated phagocytosis,Naturalkiller cell-mediated cytotoxicity,TLR signaling pathway,B cell receptor signaling pathway,Fc epsilon RI signaling pathway,Complement and coagulation cascades,Antigen processing and presentation,RIG-I-like receptor signaling pathway,NOD-like receptor signaling pathway,Hematopoietic cell lineage,Cytosolic DNA-sensing pathway and Intestinal immune network for IgA production.A total of 101 proteins of two libraries belonging to the TLR signaling pathway were expressed in blood cells(Fig.2C).Taken individually,the KEGG classification of tilapia WBCs and RBCs showed that signal transduction,endocrine system and immune system are the three most represented categories in both RBCs and WBCs.A total of 1339 and 1535 terms were assigned to the immune system in RBCs and WBCs,respectively.

COG is a database where orthologous gene products are classified and 11542 transcripts were assigned into 25 KOG categories.The most represented group was the cluster for“signal transduction mechanisms”(n=1705)and was followed by “general function prediction only”(n=1508).In total,134 transcripts were annotated within the “defense mechanisms”category(Fig.S1).

3.3.Differential expressed genes

Fig.1.Tilapia blood cells.

Table 1 Statistics for the FastQC of reads from tilapia WBCs and RBCs.

In total,20876 transcripts were found to be expressed in the tilapia blood cell transcriptome,of which the great majority(16231)are identical between the RBCs and WBCs libraries.In general,17.248 genes are expressed in RBCs(reads number≥1)and plasma ferritin,hemoglobin alpha and beta and beta-2-microglobulin were highly expressed in RBCs.1017 and 3628 transcripts were specifically expressed in tilapia RBCs and WBCs,respectively(Fig.3A).Among the 16231 of the co-expressed transcripts,3251 were differentially expressed(FDR<0.001)between the two cell types and 707(21.75%)were highly expressed in RBCs and 2544(78.25%)were most abundant in WBCs(Fig.3B).Importantly,WBCs and RBCs possess 809 transcripts assigned to 16 immune system pathways,such as RIG-I-like receptor signaling pathway,NOD-like receptor signaling pathway and TLR signaling pathway.There are 21 and 163 transcripts were expressed specifically in tilapia RBCs and WBCs respectively,and 23 and 233 transcripts were highly expressed in RBCs and WBCs respectively.We focused on the TLR signaling pathway and a total of 101 TLR pathway genes were found in tilapia blood cells,whereas 8 were specifically found in RBCs and 2 RBC and 26 WBC genes were highly expressed.

3.4.Tilapia RBCs express immune genes and responses

Seven TLR pathway genes were selected for validation:irf1-5,irf9 and tlr3.The relative expression levels of the 7 genes were comparable in the blood transcriptome(Fig.4A).The expression levels were quantified by real-time PCR on cDNA and confirmed a similar pattern to their relative abundance(Fig.4B).To characterize the immune response of RBCs,tilapias were intraperitoneally injected with poly(I:C)and blood samples collected 24 h post injection.The WBCs and RBCs were analyzed and the expression levels of the 7 candidate immune genes were quantified by qRTPCR.All genes were up-regulated in poly(I:C)-challenged WBCs when compared to control(Fig.5A)of which 5 genes were also significantly up-regulated in RBCs(Fig.5B).Therefore,both WBCs and RBCs express similar immune responses genes.

4.Discussion

Current data on the immune system is mostly available from mice and humans.However,characterization of the fish immune response could provide novel insights to understand the function and evolution of the immune system and immune defense in vertebrates(Lieschke&Trede,2009).The Nile tilapia is an economically important fish species in Aquaculture and is the most studied cichlid specie being a popular model organism in physiological(Eppler et al.,2007;Kandiel,El-Asely,Radwan,&Abbass,2014),sex developmental(Li et al.,2015)and immunological(Subramaniam et al.,2016;Wang et al.,2016)research with available annotated genome data.

Fig.2.Classification of all genes from tilapia blood cells.

The transcriptome of immune organs including spleen and kidney of fish have been studied(Xiang,He,Dong,Zhang,&Shao,2010;Zhang,et al.,2013),however description of the expression profile of blood cells which play a key role in immune defense is currently lacking.In both human and fish,blood cells are an effective prognostic marker for disease diagnosis as WBCs,including neutrophils,eosinophils,basophils,lymphocytes and monocytes participate in both innate and adaptive immunity defense in vertebrates(Mohr&Liew,2007).However,there are increasing evidences that suggest that RBCs,the most common type of blood cells present in circulation,mayalso play a key role in several aspects of the immune system.In mammals,RBCs extrude their nucleus prior entering the circulation,but microarray DNA analysis showed that 107 out of 1019 genes expressed in RBCs are associated with the immune response(Kabanova,Kleinbongard,Volkmer,Andree,Kelm,Jax,2009).Mature RBCs of non-mammals possess nuclei throughout their life(Claver&Quaglia,2009;Finstad et al.,2014)and organelles essential for protein synthesis,such as ribosomes,endoplasmic reticulum,Golgi apparatus and mitochondria have been observed in the RBCs of rainbow trout and salmon(Boutilier&Ferguson,1989;Kanehisa,Goto,Furumichi,Tanabe,Hirakawa,2010;Lund,Phillips,Moyes,Tufts,2000;Morera et al.,2011).In the present study, fluorescent staining detected the presence of mitochondria and lysosome in tilapia RBCs.Mitochondria are regarded as power stations and are responsible for energy supply of the cell.The lysosome is involved in multiple processes,including cell signaling,secretory process and polymer degradation and it could contain more than 60 enzymes and serve as an important defensive mechanism in cells(Settembre,Fraldi,Medina,&Ballabio,2013).Comprehensive gene expression analysis of fish RBCs will provide understanding about their function.

RBCs are the most common type of blood cells whose main function is to deliver oxygen to body tissues.Gas delivery-related proteins,such as hemoglobin,are highly expressed in RBCs.In most vertebrates,hemoglobin is the iron-containing oxygentransport metalloprotein in the RBCs.In mammals,hemoglobin makes up 35%of the total protein content(Weed,Reed,&Berg,1963).Hemoglobin is also found outside RBCs and their progenitor lines and include the dopaminergic neurons in the substantia nigra,macrophages,alveolarcells and mesangial cells in the kidney.In these tissues,hemoglobin serves a non-oxygen-carrying function;it also acts as an antioxidant and a regulator of iron metabolism(Biagioli et al.,2009).In the present study,hemoglobin alpha and beta were the most abundant transcripts found in tilapia RBC transcriptome.The RPKM value for hemoglobin alpha and beta is up to 1.38 million,which accounts for about four- fifths of all transcripts expressed and suggests that oxygen delivery remains the main function of RBCs in both human and fish.Other genes were also found to be highly expressed in tilapia RBCs.Such as plasma ferritin,is an indirect marker of the total amount of iron stored in the body;hence,serum ferritin is used as a diagnostic gene marker for iron-deficiency anemia in mammals(Ponka,Beaumont,&Richardson,1998;Wang,Knovich,Coffman,Torti,&Torti,2010b).A study also showed that this protein is involved in fish immune response(Neves,Wilson,&Rodrigues,2009).Our results indicate that beta-2-microglobulin(β2m),a cell membrane protein,is highly expressed in tilapia RBCs.In general,the β2m was barely expressed in RBCs from rabbit,rat and human,but a report on rat RBCs showed that 103molecules of β2m exist in a single cell(BjÖrck,ÅkerstrÖm,&Berggård,1979).β2m is also known as a component of MHC class I molecules.Mouse models deficient in β2m gene demonstrate that β2m is necessary for the cell surface expression of MHC class I and for the stability of the peptide binding groove(Hoglund et al.,1998).The predicted protein secondary structure of both carp and tilapia orthologues is almost identical to the corresponding regions of the mammalian β2m protein(Dixon,Stet,van Erp,&Pohajdak,1993),indicating that this is a conserved immune gene across vertebrates.The existence of respiration-irrelevant genes with comparatively high expression imply that RBCs in tilapia may also participate in multiple organismal processes,including immune response and similar findings were also described in rainbow trout and chicken(Morera et al.,2011;Paolucci,Barjesteh,Wood,Sharif,2013).

Fig.3.Gene expression profile in tilapia RBCs and WBCs.

Fig.4.Expression of immunity genes in tilapia blood cells.

Fig.5.Expression of immune genes after poly(I:C)challenge.

In vertebrates,RBCs are morphologically variable cells and gene expression patterns vary distinctly among organisms.Tilapia RBC transcripts were mapped for KEGG pathway and sixteen immune pathways were found,including chemokine signaling pathway and leukocyte transendothelial migration.Chemokines are small chemoattractant peptides that provide directional cues for cell trafficking and are vital in the host response.The chemokine signal is transduced by the chemokine receptors expressed on immune cells.After receptor activation,the G-protein activates diverse downstream signaling pathways that generate various responses,including chemotaxis,degranulation,release of superoxide anions and changes in the avidity of integrins(Murdoch&Finn,2000).The leukocyte transendothelial migration pathway is the second-most abundant gene category in RBCs.Neither the innate nor adaptive immune system “responds”unless leukocytes cross the blood vessels.This process involves the rapid and transient delivery of soluble elements to the site of injury and prolonged delivery of leukocytes.The regulation of the leukocyte recruitment steps of capture,rolling,activation and adhesion are crucial for the inflammatory response(Muller,2011).In our study,we focused on the TLR signaling pathway that plays a crucial role in the innate immune system by recognizing pathogen-associated molecular patterns of various microbes.TLRs signal through the recruitment of specific adaptor molecules,triggers the enrollment of adaptors and signaling transduction,but eventually activate serine/threonine protein kinases,which phosphorylate and translocate the IFN regulatory factor(irf)in the nucleus to transcribe IFN-α and-β and other IFN-induced genes (Fitzgerald et al.,2003; Sharma et al.,2003).TLR signaling plays important roles in many aspects of the innate immune response to pathogens.After treatment with poly(I:C)immunostimulants, five genes were found significantly up-regulated in tilapia RBCs when compared to control.In WBCs,all candidate seven genes were up-regulated after treatment.Tilapia irf3 function has been studied extensively,and it is suggested to be an indispensable regulator for the induction of IFN against viral infection(REF).Following viral infection,pattern recognition receptors(PRRs)initiate signaling transduction and activate TBK1 kinase to phosphorylate several serine and threonine residues in the C-terminal serine-rich region of mammalian IRF3(Gu,Wei,Tang,Chen,&Zhao,2016;Tamura,Yanai,Savitsky,&Taniguchi,2008).In overall,809 immune genes were categorized after KEGG analysis and system analysis revealed that the quantity and expression level of their majority are of lower expression level in RBCs than in WBCs.According to our data,RBCs apart for their role as oxygen-delivery vectors they are also important assistant components of the blood cell immune response in fish.Further studies to characterize the immune role of RBCs are necessary to better elucidate on their role in immune defense in vertebrates.

Acknowledgements

This work was supported by China Agriculture Research System(CARS-46);Shanghai Collaborate Innovation Center for Aquatic Animal Genetics and Breeding(ZF1206)and by the Open Fund of Key Laboratory of Experimental Marine Biology from the Chinese Academy of Sciences(No.KF2017NO2).

Appendix A.Supplementary data

Supplementary data related to this article can be found at https://doi.org/10.1016/j.aaf.2018.01.001.

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