the innate immune system provides a first line of defense against various 
 microorganisms and is essential for the control of pathogenic infections .
the 
 adaptive immune system has evolved to provide a more versatile means of defense 
 which prepares increased protection against subsequent reinfection with pathogen . 

although the innate immune system can not always recognize or eliminate infectious 
 pathogens , play a crucial part in the initiation and subsequent direction of 
 adaptive immune responses , as well as participating in the removal of pathogens that 
 have been targeted by an adaptive immune response .
moreover , because there is delay 
 before the initial adaptive immune response takes effect , the innate immune response 
 has a critical role in controlling infections during this period .
the innate immune system is mediated by pattern recognition receptors ( prrs ) that 
 primitive part of the immune system through pathogen - associated molecular patterns 
 ( pamps ) ( magnadttir , 2006 ) .
lectins belong to 
 the prr class and a group of sugar binding proteins that recognize the exposed 
 carbohydrates on the cell surface of potential pathogenic microbes , and then 
 agglutinate various cells by binding to cell - surface glycoconjugates ( saraiva et al . ,
lectins act as a 
 mediator of self and non - self - recognition and play a significant role in cellular 
 functions , including cell communication , agglutination , proliferation , opsonization , 
 phagocytosis , signal transduction , metastasis and apoptosis ( wassaman et al . , 1986 ; sharon 
 & lis , 2004 ) .
a fish lily - type lectin sequence was identified in snakehead murrel ( channa 
 striata ) , large yellow croaker ( larimichthys crocea ) , 
 spotnape ponyfish ( leiognathus nuchalis ) , bartail flathead 
 ( platycephalus indicus ) , turbot ( scophthalmus 
 maximus ) , rock bream ( oplegnathus fasciatus ) , 
 orange - spotted grouper ( epinephelus coioides ) ( arasu et al . , 2013 ; tasumi et al . ,
several articles were published 
 that lectins are involved in the immune response against pathogen infection ( choi et al . , 2015 ; kim et al . , 2011 ; park et al . , 
 2012 ; thulasitha et al . ,
however , these studies are limited to information that the lectin is involved in the 
 innate immune response .
the rock bream ( oplegnathus fasciatus ) is a commercially important 
 marine fish and it has a high economic value in the south korea . although the recent 
 rapid development of the rock bream farming , the spread of bacterial and viral 
 diseases causing enormous economic loss of the aquaculture industry .
rock bream 
 iridovirus ( rbiv ) is one of the major pathogens in rock bream aquaculture and 
 identified as the most important pathogen infecting rock bream in the last decade 
 ( inouye et al .
however , there 
 is little available information based on temporally integrated observations in the 
 immune responses against virus infection .
therefore , research on immunity - related 
 genes in rock bream is necessary to cope with widespread disease infection . in the previous
, ofltl-1 has been named as rbltl and reported that changes of 
 expression in the ontogeny , spatial and pathogen infection response ( park et al . , 2016 ) .
although the 
 cdna sequence of the ofltl-2 and -3 has been reported , no relevant study on 
 the in vivo function of ofltls has been conducted . in this study
, 
 we investigated that domain analysis and sequence alignment , expression changes in 
 the developmental stage , tissue - specific distribution and expression profile in the 
 immediate and long - lasting immune response against rbiv infection .
the amino acids are numbered along the right margin as well the top of the 
 sequences .
the genbank accession numbers of lily type lectins are gu565039 
 ( ofltl-2 ) and gu565040 ( ofltl-3 ) , respectively .
the cdna and amino acid sequences of ofltls were analyzed using the basic local 
 alignment search tool ( blast ) at the national center for biotechnology 
 information website ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) and the expert 
 protein analysis system ( http://www.expasy.org ) , as reported previously ( wilkins et al . , 1999 ) .
the brood stock of rock bream ( 5 years old ) were reared in the genetics and 
 breeding research center of the national institute of fisheries science ( nifs , 
 geoje , republic of korea ) with aerated seawater and natural photoperiod in a 
 30-ton tank .
when the water temperature was 23.01.0 , photoperiod maintained on 
 a 15h light : 9h dark to induce the maturation of gonads .
after spawned 
 naturally , the fertilized eggs in the same clutch were collected by a blotting 
 silk net and incubated with weak aeration and fresh seawater at 23.50.5. after 
 25 h , the larvae hatched and were placed in a 15-ton tank at a density of 46 
 larvae / l .
larvae were fed with the l - type rotifer brachionus 
 plicatilis from 3 to 20 days after hatching ( dah ) and 
 artemia nauplii from 16 to 30 dah , and then gradually 
 switched to extruded pellet food beginning at 25 dah . during the rearing time , 
 the rock bream larvae and juveniles were maintained in the conditions as below : 
 water temperature ( 24.01.0 ) and salinity ( 32.50.5% ) . experimental analysis for tissue distribution including brain , eye , gill , 
 intestine , kidney , liver , skin , spleen and stomach samples were dissected from 
 ten healthy rock breams ( total length of approximately 10 cm , 5 - 6 months old ) 
 and immediately frozen in liquid nitrogen , followed by storage in a -80 freezer 
 until use .
the rock breams were anaesthetized prior to experiments involving 
 tissue collection and pathogen injection , and samples were collected under 
 aseptic conditions .
all fish were acclimatized to experimental condition for 1 week 
 before processing . for the rbiv challenge experiment ,
the rock breams were divided randomly into two 
 groups : a control group and a challenged group .
the control and challenged fish 
 were injected with 100 l of phosphate buffered saline ( pbs ) or a rbiv 
 suspension ( 10 tcid50 virus/ fish ) , respectively ( umasuthan et al . , 2013 ) .
the temperature to 
 which the experimental fish were subjected was controlled at 20 using a 
 re - circulation system , without flow and feeding . challenged fish collected under 
 aseptic conditions at 0 , 3 , 6 , 12 , 24 and 72 hours post - injection and pooled 
 together in equal amounts and frozen in liquid nitrogen .
total rna was extracted from the ground fish with tri solution ( bsk - bio co. ) as 
 described in the manufacturer 's protocol .
the total rna was treated with dnase - i 
 ( sigmaaldrich ) to remove genomic dna contamination .
the concentration of total 
 rna was quantified using spectrophotometrically ( biotek , gen 5.2 ) and rna 
 quality was assessed via electrophoresis in 1% agarose gels .
cdna was 
 synthesized with suprimescript rt premix ( 2x ) ( gennet bio ) using an oligo ( dt ) 
 primer . to synthesize cdna ,
the reverse transcription reaction was conducted as 
 follows : a mixture containing 1 g of total rna , the oligo ( dt ) primer and 
 rnase - free dh2o was held at 65 for 5 min , then placed on ice for 5 
 min .
collect the contents of the tube by brief centrifugation and add 10 l 2x 
 suprimescript rt premix , and then incubates at 60 min at 50. the final 
 inactivation reaction was carried out for and 10 min at 70. specific primers 
 for rock bream lectins and -actin were designed using the primer 3 program 
 ( table 1 ) .
expression analysis of rock breams lectins was conducted using quantitative 
 real - time pcr with specific primers , and the mrna levels of -actin were used as 
 an internal control .
quantitative real - time pcr was conducted using the abi 7500 
 real - time detection system ( applied biosystems ) according to the manufacturer s 
 instructions .
the final reaction volume contained 10 l of fast sybr green pcr 
 master mix ( applied biosystems ) , 100 ng of cdna , 0.3 l of each of the forward 
 and reverse primers . the amplification procedure consisted of an initial 
 denaturation step for 20 seconds at 95 , then 40 cycles of 3 seconds at 95 and 
 30 seconds at 60 , followed by a final dissociation stage .
dissociation curve 
 analysis of the amplification products was performed after quantitative 
 real - time pcr to confirm the specificity of the pcr products .
the relative expression ratio of the target gene versus the 
 -actin gene was calculated using the 2ct method ( pfaffl , 2001 ) .
all samples were analyzed 
 with three duplicates , and all data are presented in terms of relative mrna 
 levels , expressed as the meanse ( n=3 ) .
statistical analyses 
 were performed with spss 17.0 software ( spss inc . ) and the data were subjected 
 to one - way analysis of variance ( anova ) .
differences were considered significant 
 at p<0.05 ( * ) and extremely significant at 
 p<0.01 ( * * ) .
ofltls amino acid sequence do not have a signal peptide nor a trans - membrane 
 region , but contains bulb - type mannose binding lectin(b - lectin ) domain between 3 
 and 112 aa ( total of 110 aa sequence ) that typical features for its fundamental 
 structure .
a long dimerization interface site is also available within the 
 b - lectin domain profile between aa 4 and 112 ( total of 109 aa ) .
the deduced 
 amino acid sequence of rock bream lily type lectin-2 shared 69.2 % identity with 
 ofltl-3 .
ofltls two qxdxnxvxy motifs were completely conserved , but third 
 d - mannose binding domain lack in the rock bream . to determine whether a role of rock bream lily - type lectins in the early 
 developmental stage , temporal expression analysis was conducted using the 
 samples from immediately after hatching to 60 days larvae and juveniles rock 
 bream fish . for a quantitative analysis ,
quantitative real - time pcr was carried 
 out using gene - specific primers designed from the ofltl-2 and 3 coding sequence . 

expression of ofltl-3 was maintained the basal level without substantially 
 increased until 9 dah , and it began to increase from 10 dah and rapidly was 
 increased up to 60 dah ( fig .
the 
 overall temporal expression pattern of ofltl-2 is similar to ofltl-3 , although 
 tran sient expression decreased in the early stage that is different with 
 ofltl-3 ( fig .
ofltls 
 are not to expression from the early development , is an increased expression 
 after immune - related organs is fully formed . to investigate the expression pattern of ofltls
according to the 
 developmental stage , using the sample from fertilization up to 60 days 
 larvae and juveniles rock bream fish . after removing seawater from the 
 sample , soaked in trizol solution and homogenized to extract total rna . 

( c ) the development scheme of the immune system and 
 expression profile of ofltls in the rock bream early developmental 
 stages .
light gray bar shows the bottleneck when many larvae die ( 12 to 
 16 dah ) . to investigate the role of the rock bream lectins in vivo ,
relative mrna expression of each 
 tissue was calculated using rock bream -actin as a reference gene and the 
 result was further compared with the lowest expression tissue to determine the 
 relative tissue - specific expression profile ( set as 1 ) .
ofltl2 and 3 were 
 predominantly expressed in the liver and skin that the highest expression in the 
 skin and the following order was liver . also , the expression of ltls was almost 
 at basal level except the liver and skin tissue , although the difference between 
 the high - order depending on the gene ( fig .
the spatial distribution of ( a ) ofltl-2 , ( b ) ofltl-3 in various tissues 
 of healthy rock bream fish .
the transcript level was determined via 
 quantitative realtime pcr , and rock bream -actin was chosen as an 
 internal reference gene .
an asterisk indicates a statistically 
 significant difference * * p<0.01 ) compared with 
 lowest expression the tissue ( set as 1 ,   black box ) .
the results are reported as the mean standard deviation ( sd ) 
 of triplicates .
significance was analyzed via one - way analysis of 
 variance ( anova ) using the spss 17.0 program . to investigate the function of ofltls in the immune response to a viral 
 infection
, the temporal patterns of ofltls mrna expression were examined using 
 the whole body of rock bream fish infected with rbiv .
expression patterns and 
 timing are preferentially confirmed with rbiv - induced marker genes to verify a 
 successful challenge experiment
. the mrna level of cathepsin h expression was 
 high in both the early ( 3h ) and later stages ( 24h ) ; these results are consistent 
 with previous findings ( kim et al . , 2013 ) . 

4 , although expression of 
 ofltl-2 was weak in the early response period , it gradually increased until 24 
 hours post - infection and then dramatically increased to 72 hours . while , 
 expression of ofltl-3 maintaining almost the basal levels until 12 hours 
 post - infection and it was exponentially in infected 24 hours post - infection , and 
 then increased expression of ofltl-3 was reduced to 1/9 at 72 hours after 
 infection .
the ex - pression patterns of osltls were determined in the whole body ( a ) 
 ofltl-2 and ( b ) ofltl-3 through quantitative real - time pcr .
cathepsin h 
 was used as markers to ensure a successful rsiv infection expe - riment 
 ( inset in fig .
the samples 
 were analyzed at 0 , 3 , 6 , 12 , 24 and 72 hours post - injection .
the 
 expression of -actin was used as internal control for quantitative 
 real - time pcr , and each experiment was performed in triplicate .
an 
 asterisk indicates a statistically significant difference ( * 
 p<0.05 ; * * p<0.01 ) compared 
 with 0 h ( set as 1 ) .
significance was analyzed via one - way 
 analysis of variance ( anova ) using the spss 17.0 program .
in contrast to higher vertebrates , fish is living in aquatic environments from early 
 embryonic stages of life , which are an ideal medium for microorganism growth 
 compared to air and every moment is constantly exposed to a pathogen attack .
the 
 bony fishes are derived from one of the earliest divergent vertebrate lineages to 
 have both innate and acquired immune systems .
however , the adaptive immune response 
 did not clearly reveal in fish and it is expected not sophisticated like mammals . 
 therefore , the innate immune system is the only defense weapons of invertebrates and 
 a fundamental defense mechanism of fish and more dependent their innate immune 
 systems for survival and the resistance to the pathogens .  
lectins play important roles in the recognition and elimination of pathogens via the 
 innate immune system .
researches on the immunological functions of lectin are 
 concentrated in the innate immune response of higher vertebrates .
however , our 
 results seem to be involved both in the innate and adaptive immune response ; 
 lily - type lectins are expressed after completing immune system during early 
 developmental stage and preferentially increased in the second half reaction in a 
 viral challenge , these results are related to the adaptive immune response . on the 
 other side , ofltls
are expressed in the innate immune response related tissue . 
 because fish have not sophisticated and specialized the adaptive immune systems
, the 
 innate immunity - related genes will be able to engage in the long - lasting immune 
 responses to infection .
research findings are not infrequently has been reported 
 that the early and late results are quite cross .
therefore , outside the framework 
 defined in the higher vertebrates various studies will be needed . in the development of the immune system rock bream
, spleen anlage was observed 
 between the swim bladder and the intestine at 5 dah , and the thymus was formed as a 
 paired structure under the pharyngeal epithelium above the gill arch at 10 dah .
the 
 order of the immune organs becoming lymphoid was the pronephric kidney ( 10 dah ) , 
 thymus ( 15 dah ) and spleen ( 21 dah ) ( zhizhong et 
 al . , 2013 ) .
ofltls were hardly expressed in the initial developmental 
 phase and increased from 10 dah .
thereafter , expression of ofltls was proportionate 
 to development of rock bream immune organ .
it can be inferred that they will 
 participate in the immune response after the immune system is fully established , not 
 involved from the initial development period .
fish are permanently exposed to various external hazards that aerobic and anaerobic 
 bacteria , viruses , parasites , marine pollutants . to protect fish from pathogenic 
 microorganisms , mucus acts as a physical , chemical and biological barrier between 
 the fish and the environment which first site of interaction with pathogens .
the 
 mucus composition is very complex and includes immuneglobulins , agglutinins , 
 lectins , lysins and lysozymes that are anti - microbial factor and is secreted from 
 the fish skin .
these factors are very important recognize the microorganisms 
 invasion and to protect the fish from invading pathogens ( lee et al . , 2015 ; suzukiet al . , 
 2003
the liver is unique anatomical and immunological organ : the typical function of the 
 liver is carbohydrate , protein and lipid metabolism , bile secretion , antioxidation 
 and detoxification . as well as , the liver play a critical roles against invading 
 pathogen in the innate immune responses of fish that particularly enriched 
 macrophages and lymphocytes compared with other organs and modulated of liver injury 
 and recruitment of circulating lymphocytes ( racanelli & rehermann , 2006 ; castro 
 et al . , 2014 ) .
although it does not clearly separated innate and acquired immune response dedicated 
 organ in fish , liver and skin are primarily involved in many innate immune response . 
 in this study ,
these 
 results are to be expected that ofltls contribute to the innate immune response 
 against viral infection , but the transcripts were increased to more intensively in 
 the period of acquired immune of rbiv challenge experiments . for this reason
, the 
 further expression analysis is needed using a virus - infected liver and skin tissue 
 of the fish because it uses the rsv infection whole fish in this experiment .
 abstractlectins belong to the pattern - recognition receptors ( prrs ) class and play 
 important roles in the recognition and elimination of pathogens via the innate 
 immune system . 
 recently , it was reported that lily - type lectin-1 is involved 
 when a pathogen attacks in the early immune response of fish . however 
 , this 
 study is limited to information that the lectin is involved in the innate immune 
 response against viral infection . in the present study , 
 the lily - type lectin-2 
 and -3 of oplegnathus fasciatus ( ofltl-2 and 3 ) have been 
 presented to be included b - lectin domain and two d - mannose binding sites in the 
 amino acid sequence that an important feature for the fundamental structure . to 
 investigate the functional properties of ofltls 
 , the tissue distribution in the 
 healthy rock bream and temporal expression during early developmental stage 
 analysis are performed using quantitative real - time pcr . ofltl-2 and 3 
 are 
 predominantly expressed in the liver and skin , but rarely expressed in other 
 organ . 
 also , the transcripts of ofltls are not expressed during the early 
 developmental stage but its transcripts are increased after immune - related 
 organs which are fully formed . in the challenge experiment with rbiv ( rock bream 
 iridovirus ) , 
 the expression of ofltls was increased much more strongly in the 
 late response than the early , unlike previously known . 
 these results suggest 
 that ofltls are specifically expressed in the immune - related tissues when those 
 organs are fully formed and it can be inferred that the more intensively 
 involved in the second half to the virus infection . 