Aquaculture Research, 2008, 39, 252^262

doi:10.1111/j.1365-2109.2007.01871.x

A comparison of the expression of immunity-related

rag 1 and ikaros genes with histogenesis of the thymus in Epinephelus malabaricus (Bloch & Schneider) John Han-You Lin1,2,, Han-Tso Lin3,, Carmen Lopez4,Tzong-Yueh Chen1,2, Ming-Shyan Chen1,2 & Huey-Lang Yang1,2 1

Institute of Biotechnology, National Cheng Kung University,Tainan,Taiwan

2

Research Center of Ocean Environment and Technology, National Cheng Kung University,Tainan,Taiwan

3

Department of Biotechnology, School of Health Technology, Ming Chuan University,Taoyuan County,Taiwan

4

Center of Centro de Investigacions Marinas, Conselleria de Pesca, Marisqueoe Acuicultura, Xunta de Galicia, Pontevedra,

Spain Correspondence: H-L Yang, Institute of Biotechnology, National Cheng Kung University, 1, University Road,Tainan 701,Taiwan. E-mail: [email protected] These authors contributed equally to this work.

Abstract Expression of the immunity-related ikaros and rag1 genes during development signals the onset of lymphopoiesis in vertebrates. Partial sequences of ikaros and rag1 in Epinephelus malabaricus were cloned by degenerate primer-mediated reverse transcriptase polymerase chain reaction (RT-PCR). Their expression pro¢les in the thymus, head kidney, trunk kidney, spleen, intestine, brain, liver and pancreas, and the onset of expression at various developmental stages, were examined by RT-PCR using identical primers. Expressions of both ikaros and rag1 genes were detected as early as 3 days post fertilization (dpf). The thymus is thought to be the ¢rst organ of lymphopoiesis to develop in ¢sh; its histogenesis in E. malabaricus was examined and used for comparison. To avoid the potential in£uence of environmental factors on di¡erent hatches, both age and larval morphology were used as indicators of maturation to de¢ne the stage of development. A bud-like thymus was observed at 9 dpf; lymphopoietic cells appeared at 19 dpf; and further development, such as cortex/medulla di¡erentiation and the appearance of lymphocytes, occurred by 26 dpf. Trabecula development was detected at 41dpf. Based on the histological evidence, the lymphoid cell expressed rag1 before the thymus developed, suggesting that extra-thymic lymphopoiesis takes place in E. malabaricus, and that a

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functional immune system might develop early in the metamorphosis stage.

Keywords: Malabar grouper, thymus development, ¢sh immunity, degenerated primer

Introduction The grouper is the most popular food ¢sh among Asian populations. According to an FAO report in 2005, the annual market demand for grouper in 2004 was around 250 000 tonnes, of which 194 000 tonnes came from wild capture and the remaining 55000 tonnes was produced by aquaculture. The Malabar grouper Epinephelus malabaricus (Bloch & Schneider) and the closely related E. coicoides (Hamilton) are among the most economically important ¢sh in Asia and are the main species of grouper cultured in Taiwan. Morphological development of larvae has been reported in some species of grouper (Lin,Yen, Huang, Liu & Lin 1986; Kitajima, Takaya,Tsukashima & Arakawa1991; Kohno, Diani & Supriatna 1993; Masuma,Tezuka & Teruya 1993), but the ontogeny of their lymphoid organs and immune function have been neglected. Grouper hatchery farming in South Asia has a low production rate, largely due to viral nervous necrosis, and this has become a major obstacle for

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Aquaculture Research, 2008, 39, 252^262 Comparison of the expression of immunity related rag 1 and ikaros genes J H-Y Lin et al.

the aquaculture industry. The causative agent, nerve necrosis virus, infects grouper at the larval stage, is lethal to larvae and has been the identi¢ed cause of mass mortalities in grouper hatcheries for at least 10 years, as con¢rmed by the PCR diagnostic method of Chi (Chi, Lo, Chang, Peng, Kou & Chen 1997; Chi, Lo, Lo, Kou & Chen 1997). It is evident that disease control by immunization for grouper larvae is needed, and the study of immune system development during ontogenesis at the larvae stage is important in this context and is essential for the development of an immunization strategy for controlling this early viral infection. Previous studies of the immunogenesis of economically important species include those on Dicentrarchus labrax (L.) (Abelli, Picchietti, Romano, Mastrolia & Scapigliati 1996; Abelli, Baldassini, Meschini & Mastrolia 1998; Santos, Romano, De Sousa, Ellis & Rombout 2000; Scapigliati, Romano, Buonocore, Picchietti, Baldassini, Prugnoli, Galice, Meloni, Secombes, Mazzini & Abelli 2002), E. bruneus, (Bloch) (Kato, Ishimaru, Sawada, Mutsuro, Miyashita, Murata & Kumai 2004), Gadus morhua (L.) (Schroder, Villenat & Jorgensen 1998), Oncorhynchus mykiss (Walbaum) (Hansen & Kaattari 1995), Platichthys £esus (L.) (Pulsford, Tomlinson, Lemaire-Gony & Glynn 1994), Scophthalmus maximus (L.) (Padros & Crespo 1996), Seriola quinqueradiata (Temminck & Schlegel) (Chantanachookhin, Seikai & Tanaka 1991), Pagrus major (Temminck & Schlegel) (Chantanachookhin et al. 1991) and Paralichthys olivaceus (Temminck & Schlegel) (Chantanachookhin et al. 1991). Nevertheless, developmental immunogenesis remains incompletely characterized mainly because there is no consensus on histological and functional indices for de¢ning larval developmental stages in relation to lymphoid organ maturation. The thymus is one of the central immune organs in ¢sh, and developmental studies suggest that it is the ¢rst organ in which lymphopoiesis occurs. Delineation of the inner and outer regions (the cortex and medulla) has been demonstrated histologically (Agustin, Zapata & Varas 1996), but lobules described for D. labrax, G. morhua and S. quinqueradiata have not been found in all species, lessening their value in evaluating histological maturity. Several approaches have been used to study the chronology of development of the ¢sh immune system. Utilizing knock-out technology in ¢sh, the lymphoid genes involved in haematopoiesis and lymphopoiesis were identi¢ed (Trede, Zapata & Zon 2001). The expression of these genes has provided a

useful means to elucidate the developmental stages. These genes include the recombination-activating genes rag1 and rag2, which are responsible for the site-speci¢c V(D)J recombination of immunoglobulin (Ig) in lymphocyte (Hiom, Melek & Gellert 1998; Schatz 2004), and the ikaros, which encodes a transcription factor essential for lymphocyte development (Georgopoulos, Winandy & Avitahl 1997). Although the identities of lymphocytic precursor cells expressing ikaros are not de¢ned, the possibility that ikaros regulates the expression of rag1by binding to its promoter region was demonstrated by Fuller and Storb (1997). The aim of this study was to describe the early development of the adaptive immune system by analysing the expression of rag1 and ikaros genes and to compare this with histological pro¢les of the thymus. To achieve this, we cloned the rag1and ikaros genes of E. malabaricus and examined their expression at various stages of development.

Materials and methods Larva collection Various stages of E. malabaricus larvae were collected from a ¢sh hatchery in southern Taiwan (22123 0 N, 120135 0 E) in August and September 2003. These larvae were kept in a 10 m3 ¢breglass-reinforced plastic tank supplied with ¢ltered aerated seawater at 28^30 1C, and fed with fertilized Crassostrea gigas eggs from 3 to 8 days post fertilization (dpf), with HUFA-enriched rotifers from 5 to 14 dpf (INVE, Dendermonde, Belgium), followed by copepods from14 to 45 dpf and HUFA-enriched Artemia from 20 to 45 dpf. Larvae were collected from the hatchery every 2 days from 1 to 21dpf and every 5 days from 26 to 51dpf. Fish larvae were anaesthetized with 300 mg L
1 MS-222 (ethyl-m-aminobenzoate-methane-sulphonate, Sandoz, Holzkirchen, Germany) and ¢xed in 10 times their volume of 10% formalin in phosphatebu¡ered saline for histological study or with 10 times their volume of RNAlater (Ambion, Austin, TX, USA) for RNA extraction.

Histological analysis Samples of ¢xed larvae were dehydrated and embedded following the procedure of Humason (1972). Sections, 7 mm thick, were mounted on poly-lysinepretreated slides and stained with haematoxylin

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Comparison of the expression of immunity related rag 1 and ikaros genes J H-Y Lin et al. Aquaculture Research, 2008, 39, 252^262

(Harris modi¢ed haematoxylin with acetic acid; Fisher Scienti¢c, Pittsburgh, PA, USA) and eosin Y (10% in ethanol, Electron Microscopy Science, Pittsburgh, PA, USA).

(Clontech; BD Biosciences, San Jose, CA, USA) was used to synthesize the ¢rst strand of cDNA. The experimental procedures followed the manufacturer’s instructions.

RNA extraction and cDNA synthesis

Partial rag1, ikaros and b-actin sequence acquired by degenerated primers of reverse transcriptase polymerase chain reaction (RTPCR)

Three 1-year-old farmed grown groupers (mass 700^ 750 g) were purchased live at a local market. Following deep anaesthetization (600 mg L
1, 2phenoxy-ethanol; Sigma Chemical, St Louis, MO, USA), the thymus, kidney, spleen, intestine, brain, liver and pancreas were removed, diced to cubes of approximately 5 mm3 and stored overnight (16 h) in 10 times the volume of RNAlater (Ambion) at 4 1C. Tissues were frozen by immersion in liquid nitrogen, ground in a mortar and pestle and the RNA was extracted using Trizol reagent (Life Technologies; Invitrogen, Carlsbad, CA, USA). The concentration of extracted RNA was measured at OD260 nm using an Ultrospec 3100 Pro (GE healthcare Bioscience AB, Uppsala, Sweden). The purity of RNA was con¢rmed by an OD260/280 nm ratio of over 1.7 and analysed using agarose gel electrophoresis. The commercial kit of the Turbo Capture 8TM mRNA kit (Qiagen, Valencia, CA, USA) was used for mRNA puri¢cation. The advantage RT-for-PCR Kit

Degenerate sequence primer designations were used in RT-PCR to isolate a partial sequence of rag1, ikaros and b-actin in E. malabaricus. The primers utilized for rag1 cloning (de-rag1F and de-rag1R) have been described previously (Hansen & Kaattari 1995). Primers for ikaros (de-ikarosF and de-ikarosR) and b-actin (de-b-actinF and de-b-actinR) were designed from conserved regions of the gene sequence of related ¢sh species that were downloaded from NCBI GenBank and analysed for alignment using VECTOR NTI 8.0 software (InformMax, Bethesda, MD, USA). The primer sequences, PCR reactive conditions and amplicon size are given in Table 1. Ampli¢cation was carried out in a volume of 25 mL containing 0.5 mM of each primer, 200 mM dNTP and 2 U of Taq enzyme, and 5 mL of cDNA synthesized using RNA prepared from a mixture of thymus and head kidney was added in

Table 1 Primer sequences, ampli¢cation size and PCR reactive conditions Amplicon size (base pairs)

Name

Orient

Primer sequence

de-rag1F

F

5 0 -CAYTGYGAYATHGGNAAYGC-3 0

595

de-rag1R g-rag1F

R F

5 0 -TTRTGNGCRTTCATRAAYTTYTG-3 0 5 0 -GAGTTCTACAAAATCTTCCAGG-3 0

505

g-rag1R de-ikarosF

R F

5 0 -AAGCGCCTGAACAGTTTGTT-3 0 5 0 -GADGAGGSWCARGANATG-3 0

de-ikarosR g-ikarosF

R F

5 0 -ARCCCATGTGRAKRGTRWAC-3 0 5 0 -TGACAAACGTCTCTCAGACCTCTC-3 0

511

g-ikarosR de-bactinF

R F

5 0 -CGTCACCACCTTGAAGCCCTC-3 0 5 0 -AAGATGACHCAGATYATGTTYG-3 0

716

de-bactinR g-actinF

R F

5 0 -CACATCTGCTGGAAGGTSG-3 0 5 0 -GATCATGTTCGAGACCTTCAACAC-3 0

389

g-actinR

R

5 0 -CGATGGTGATGACCTGTCCGTC-3 0

1178

PCR reactive condition 94 1C (5 0 ), followed by 30 cycles of 94 1C (3000 ) 45 1C (3000 ) 72 1C (3000 ) 72 1C, 15 min once for complete then kept 4 1C 94 1C (5 0 ), followed by 25/30 cycles of 94 1C (3000 ) 55 1C (3000 ) 72 1C (3000 ) 72 1C, 15 min once for complete then kept 4 1C 94 1C (5 0 ), followed by 30 cycles of 94 1C (3000 ) 50 1C (3000 ) 72 1C (1 0 ), 72 1C 15 min once for complete then kept 4 1C 94 1C (5 0 ), followed by 35 cycles of 94 1C (3000 ) 55 1C (3000 ) 72 1C (3000 ) 72 1C, 15 min once for complete then kept 4 1C 94 1C (5 0 ), followed by 30 cycles of 94 1C (3000 ) 53 1C (3000 ) 72 1C (1 0 ), 72 1C 15 min once for complete then kept 4 1C 94 1C (5 0 ), followed by 35 cycles of 94 1C (3000 ) 55 1C (3000 ) 72 1C (3000 ), 72 1C, 15 min once for complete then kept 4 1C

The redundance codes for equimolar mixing were: R(A,G), Y(C,T), M(A,C), K(G,T), S(G,C), W(A,T), H(A,T,C), B(G,T,C), V(G,A,C), D(G,A,T), N(A,T,G,C). F, forward; R, reverse; PCR, polymerase chain reaction.

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Aquaculture Research, 2008, 39, 252^262 Comparison of the expression of immunity related rag 1 and ikaros genes J H-Y Lin et al.

the reaction solution as a template (500 mM KCl, 100 mM Tris-HCl, pH 8.4, 1.5 mM MgCl2 and 100 mg mL
1 bovine serum albumin). Agarose gel electrophoresis (1.2%) with 1  TAE bu¡er (40 mM Tris base, 40 mM acetate,1mM EDTA) was used to examine the end product of PCR. The pGEM-Teasy vector system (Promega, Madison,WI, USA) was used to clone the PCR product following the manufacturer’s instructions. A minimum of three clones of each gene product were selected and both directions of the plasmid were sequenced (primers for T7 and SP6 promoters were utilized). The partial sequence has been submitted to GenBank (Accession number of AY551565 for rag1, DQ054836 for ikaros and AY551564 for b-actin).

Rag1, ikaros mRNA expression pro¢les in organs and larvae The speci¢c primers for rag1, ikaros and b-actin (Table1) were used to detect their expression in various organs at di¡erent juvenile developmental stages. To measure the level of mRNA expression of rag1 and ikaros in di¡erent organs at di¡erent larvae stages, a quantitative RT-PCR was performed using a real-time PCR instrument (ABI PRISM 7000 sequence detection system, Applied Biosystem, Warrington, UK). Each reaction mixture contained speci¢c primers for grouper rag1, ikaros and b-actin, a template and was prepared using cDNA dilutions and the SYBR green PCR master mix kit (Applied Biosystem). All reactions were carried out according to the manufacturer’s instructions. The rag1 and ikaros mRNA expression level was determined by normalization against b-actin expression. The primer sequences used in real-time PCR were g-rag1-rt F: 5 0 -TGT GGA GGT GGT GTG TGA G-3 0, g-rag1-rt R: 5 0 -TAT TTG AAG GTA GAG GAG AGA AGG-3 0 for grouper rag1 mRNA detection; g-ikaros-rt F: 5 0 -GCT GCT CTC CAA CTC CAA G-3 0, g-ikaros-rt R: 5 0 -TCT GCT GCT CCT CCT TCA C-3 0 for grouper ikaros mRNA detection; and g-actin-rt F: 5 0 -TGC GTG ACA TCA AGG AGA AG3 0 and g-actin-rt R: 5 0 -AAG GAA TGA AGG CTG GAA GAG-3 0 for grouper b-actin mRNA detection.

Results E. malabaricus larva development The larvae of E. malabaricus from newly hatched to the juvenile stage were examined. Their morphologi-

cal development at 28^30 1C is shown in Fig. 1. Similar to the development of other ¢sh species with pelagic eggs, grouper larvae hatched with a yolk (Fig. 1a), which was exhausted by 5 dpf, interval 2-1 (Fig. 1b). The kite-shaped body form characteristic of grouper larvae was observed at 11dpf, interval 2-3 (Fig. 1c), and a dramatic extension of body length was observed at 19 dpf, interval 3-3 (Fig.1d). At 41dpf of interval 3-6, the morphology of the juvenile had developed, although the ¢rst spine of the dorsal ¢n and the second spines of the ventral ¢ns still extended beyond the ¢nfold (Fig. 1e). At 51dpf, pigmented stripes were visible, and the larvae became juveniles (Fig.1f). The main morphological characteristics are summarized in Table 2 using dpf as an index.

Histological structure during thymus development The ¢rst appearance of the thymus, as a bud-like growth located near the gill cavity (Fig. 2a and b), was observed during the post-larval stage at interval 2-3 (9 dpf, Table 2). This early thymus comprised homogeneous, undi¡erentiated cells with morphology similar to cells in the pharyngeal wall. Heterogeneity of the cells in the thymus was observed in the larvae at interval 3-3 (Table 2). The thymus cell di¡erentiated in di¡erent size and morphology in the centre of the thymus (19 dpf, Fig. 2c and d). These cells had further di¡erentiated by interval 3-4 (26 dpf), to reveal a haematoxylinophilic region composed of lymphopoietic progenitor cells in the cortex, and eosinophilic epidermal reticular cells in the medulla (Fig. 2e and f). The shape of the thymus was elongated at interval 3-5 (31dpf) compared with its bud-shaped appearance at earlier stages. Fish at interval 3-6 (41dpf) contained trabeculae with arterioles (blood^ blood barrier) (Fig. 2g and h).

Characterization of ikaros, rag1 and b-actin partial sequence Partial ikaros, rag1 and b-actin genes of Malabar grouper were obtained by RT-PCR using the degenerated primers described in Table 1. Using the Basic Local Alignment Search Tool, the cloned ikaros DNA sequence was con¢rmed as an isoform type 2 (IK2, exon 3 was alternatively spliced) (Hansen, Strassburger & Du Pasquier 1997). An alignment of amino acid sequences and their

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Comparison of the expression of immunity related rag 1 and ikaros genes J H-Y Lin et al. Aquaculture Research, 2008, 39, 252^262

Figure 1 Developing stages of Epinephelus malabaricus larvae. (a) a newly hatched larva, (b) interval 2-1, (c) interval 2-3, (d) interval 3-3, (e) interval 3-6, (f) juvenile. Scale bar 51mm for (a)^(d), scale bar 51cm for (e) and (f).

corresponding structure with human, mouse and related ¢sh species is shown in Fig. 3a. Similarly, the amino acid alignment of rag1 is shown in Fig. 3b, and the alignment of b-actin is shown in Fig. 3c. A high conservation was observed in rag1, and a nearly identical amino acid sequence of b-actin was revealed in vertebrates from ¢sh to higher forms. Expression of rag1 and ikaros in major organs and at di¡erent stages of larval development A partial 3 0 end of the rag1 gene with 595 bp was isolated by PCR, cloned and sequenced. The primers designed from this sequence (g-rag1F and g-rag1R,Table 1) were used to study the gene expression of rag1. Expression of rag1 was observed in the thymus and head kidney at a high intensity and at a relatively lower intensity in the trunk kidney, spleen and brain (Fig. 4a). There was very low detectable expression in the spleen, intestine, brain or pancreas. The quantita-

256

tive real-time PCR indicated that the rag1 and ikaros mRNA expression level of di¡erent organs during larval development occurred as early as 3 dpf at interval 1-2 (example in Fig. 5a, and data consolidated in Fig. 5b) and continues with the same intensity. The primers of ikaros (g-ikarosR and g-ikarosF) listed in Table 1 were used to study the expression of ikaros. Its expression was detected in immune-related organs such as the thymus, head kidney, trunk kidney, spleen, intestine and, surprisingly, in brain tissue but was very low in the liver and pancreas (Fig. 4a). The quantitative real-time PCR for ikaros is shown in the same results (Fig. 4b). Similar to that of rag1, ikaros expression occurred as early as 3 dpf (interval 1-2 larvae), with expression increasing after15 dpf (Fig.4c). Discussion Length mass and age (dpf) have been used to de¢ne the developmental stages of ¢sh larvae of various

r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 252^262

Aquaculture Research, 2008, 39, 252^262 Comparison of the expression of immunity related rag 1 and ikaros genes J H-Y Lin et al.

Table 2 Developmental stages of Epinephelus malabaricus used for this study Stages

Subdivision

Main characteristics at each stage

Approximately dpf

Prelarval

Interval Interval Interval Interval Interval Interval Interval Interval Interval Interval Interval Interval

Eyes pigmented Pectoral fin present Yolk sac exhausted, Melanophores present Ventral spines project from finfold; spine present 2nd spine of dorsal fin projecting from finfold Preopercular angle spine present 1st spine of dorsal fin present 3rd spine of dorsal fin present Dorsal (XI,15), ventral (I,5), and anal (III,8) fins present Melanophores no longer visible in the gut Exhibits juvenile body shape, but is not pigmented; length of larva up to 2.4 cm Body pigmented, lateral line visible

1 3 5 7 9 13 15 17 21 31 41 51

Postlarval

Metamorphosis

Juvenile

1-1 1-2 2-1 2-2 2-3 3-1 3-2 3-3 3-4 3-5 3-6 4-1

Age of larvae as days post fertilization (dpf) (present study).

species (Chantanachookhin et al. 1991; Padros & Crespo1996; Schroder et al.1998). For the study of the ontogeny of the immune system of E. malabaricus, such measurements might be unreliable. In this study of commercially hatched larvae, the development of E. malabaricus from hatching to juvenile took 35^60 days depending on the season and nutritional conditions. The metamorphosis of grouper larvae correlated with thyroid secretion has been investigated, and has shown that larvae may fail to metamorphose and may eventually die irrespective of their length or mass (Hseu, Chang & Ting 2002). Therefore, morphological characterization of larvae was used in this study as a more reliable means of identifying developmental stage. The histogenesis of Malabar grouper thymus described in this study was similar to that of other marine ¢sh species that lay pelagic eggs (Falk-Petersen 2005). The histological characteristics of thymus in ¢sh are diverse. Higher level histological characteristics, such as lobules, were observed at a late stage of larva development. In S. quinqueradiata, for example, although the thymus^blood barrier was observed at approximately 45 dpf (similar to Malabar grouper in this study), lobules were not seen until the age of 3 months (Chantanachookhin et al.1991). Melanomacrophage centres have been observed in the thymus of 1-year-old E. malabaricus (Lin, Lin & Yang 2004). In the present study, melanomacrophage centres were observed ¢rst in the spleen at interval 3-6 (41dpf, data not shown), but not observed in thymus until the end of the study. The ikaros protein was described as a central regulator of lymphocyte di¡erentiation (Georgopoulos et al. 1997). In mice, the ikaros gene is expressed in

pluripotent haematopoietic stem cells and in all cells of the lymphoid lineage, including mature lymphocytes (Georgopoulos et al. 1997) and some myeloid lineages (Klug, Morrison, Masek, Hahm, Smale & Weissman 1998). The ikaros protein also regulates rag1 gene expression (Fuller & Storb 1997). In the present study, the ikaros was detected in the thymus, head kidney, trunk kidney and spleen. However, it was also expressed at a very low level in the intestine and brain. The expression pro¢le was similar to that described byWillet, Kawasaki, Amemiya, Lin and Steiner (2001). Although the expression is not speci¢c for the lymphocytic lineage, it was noticed that the mRNA ratio of rag1 and ikaros in di¡erent organs was not unique; these two genes were expressed strongly in the thymus and head kidney but ikaros expressed higher in the spleen and intestinal. The ikaros expression level in the intestine was interesting; overall, intestine was believed fare less lymphocytes than other lymphoid tissues tested in this study. Which type of cell was expressed ikaros in intestinal was still unknown and further research was needed. But the ikaros appearance in development could be used as a reference for the onset of lymphopoiesis (Willet et al. 2001). Biologically, the timing of expression in development should be earlier than that of rag1, but in the present study, the appearance of these two genes occurred almost at the same time, which might be due to the sampling timing and the variation of individual samples. The V(D)J recombination of Ig and T cell receptor (TCR) was initiated by expression of the rag1 and rag2 genes (Go¡rini, Algeri, Donnini, Wesolowski-Louvel & Ferrero 1989; Oettinger, Schatz, Gorka & Baltimore 1990; Van Gent, Mcblane, Ramsden, Sadofsky, Hesse & Gellert 1995). Therefore, expression could be applied

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Comparison of the expression of immunity related rag 1 and ikaros genes J H-Y Lin et al. Aquaculture Research, 2008, 39, 252^262

Figure 2 Histology of thymus development in Epinephelus malabaricus. (a) interval 2-3 (sagittal view). The black square outlines the thymus primordium enlarged in (b), (b) Higher magni¢cation of the thymus primordium, (c) interval 3-3 (coronal view). The black square outlines the developing thymus enlarged in (d), (d) Higher magni¢cation of the developing thymus, (e) interval 3-4 (sagittal view). The black square outlines the di¡erentiated thymus enlarged in (f) the medulla (M) and cortex (C), (f) Higher magni¢cation of the di¡erentiated thymus, (g) thymus of interval 3-6 (transverse view) medulla (M), cortex (C), (h) cortex of the thymus of interval 3-6 (transverse view). A trabecula is labelled by the white arrow. Haematoxylin and eosin stain. Scale bar 5100 um.

to monitor the appearance and location of B and T cells throughout the development of the immune system (Oettinger et al. 1990; Greenhalgh, Olesen & Steiner 1993). In the present study, rag1 expressed in the thymus, head kidney and trunk kidney, but not in or-

258

gans unrelated to the immune system. There was almost no expression of rag1 in the spleen of Malabar grouper, similar to the results from studies on zebra¢sh (Willett, Cherry & Steiner 1997) and O. mykiss (Hansen & Kaattari 1995).

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Aquaculture Research, 2008, 39, 252^262 Comparison of the expression of immunity related rag 1 and ikaros genes J H-Y Lin et al.

Figure 3 Amino acid alignment of ikaros, rag1and b-actin. (a) an alignment of ikaros amino acid sequence of Epinephelus malabaricus, human (translated from S80876.1), mouse (L03547.1), Oncorhynchus mykiss (U92201.1), Seriola quinqueradiata (AB060640.1) and D. rerio (AF416372.1). The conserved region is labelled by a black square. (b) An alignment of rag1amino acid sequence of E. malabaricus, human (translated from BC037344.2), mouse (AY241462.1), O. mykiss (U15663.1),Takifugu rubripes (AY700363.1) and D. rerio (U71093.1). The conserved region is labelled by a black block. (c) An alignment of b-actin amino acid sequence of E. malabaricus, human (translated from BC016045.1), mouse (NM_007393.1), E. coicoidase (AY510710.2), O. mykiss (AB196465.1), Takifugu r. (U38848.1), Seriola quinqueradiata (AB179839.1) and Danio rerio (BC045846.1). The conserved region is labelled by a black block.

The onset of expression of rag1during development was indicative of lymphopoiesis initiation in larvae. Extra-thymic lymphopoiesis has been studied in higher vertebrates where B cells are well documented to mature extra-thymically (Ansel & Cyster 2001). In murine studies, a group of T cells with a CD81 mor-

phological phenotype (gd cells), such as an intraepithelial lymphocyte, located in mucus-associated lymphoid tissue have been identi¢ed, and their appearance in development has been proven to be earlier than that of systemic T lymphocytes (withTCR ab types, maturing in the thymus) (Raulet, Garman,

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Comparison of the expression of immunity related rag 1 and ikaros genes J H-Y Lin et al. Aquaculture Research, 2008, 39, 252^262

(a)

M

T

HK

TK

S

IN

B

L

P

500

ikaros

rag1

500

500

β-actin

(b)

1 Relateive mRNA expresion level

rag 1

ikaros

Figure 4 Ikaros and rag1 expression in major organs of Epinephelus malabaricus. The expression in major organs of1-year-old juveniles (T, thymus; HK, head kidney; TK, trunk kidney; S, spleen; IN, intestines; B, brain; L, liver and P, pancreas). Marker of 100 bp (M) is shown to the side, with the location of 500 bp labelled. b-actin expression was detected simultaneously with the control.

0.1

0.01

0.001 T

HK

TK

S

IN

B

L

P

Day post fertilization (dpf) M

1 3

5

7

9 11 13 15 17 19 21 26 31 36 41

500 ikaros 500

rag1

500

β-actin

Saito & Tonegawa 1985; Tanaka, Ohtsuka, Yagita, Shiratori, Omata & Okumura 1995). The B cell is thought to develop after the T cell (Danilova, Hohman, Sacher, Ota, Willett & Steiner 2004; Lam, Dchua, Gong, Lam & Sin 2004), but recently, evidence has been presented that IgL expression could be detected by RT-PCR as early as 3 dpf in zebra¢sh (Lam, Chua, Gong, Wen, Lam & Sin 2002; Danilova et al. 2004), indicating that the rag1 and rag2 genes might be expressed before IgL expression. Use of in situ hybridization may help in identi¢cation of the location of rag1 gene expression cells during early develop-

260

Figure 5 Ikaros and rag1 expression in major organs and developing larvae of Epinephelus malabaricus. Samples extracted from 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 26, 31, 36 and 41 days post fertilization (dpf). Marker of 100 bp (M) is shown to the side, with the b-actin expression as control.

ment, demonstrating that B cells, systemic T cells or peripheral T cells initiate lymphopoiesis. However, our attempt using in situ hybridization did not show any rag1 expressed cells before interval 2-3 (9 dpf, data not shown). A more sensitive method, such as cloned eGPF reporter vectors driven by a rag1 promoter and fertilized egg gene delivery by microinjection (Trede, Langenau, Traver, Look & Zon 2004), could clarify this. In addition to the histological observations and molecular analysis of mRNA expression, results from an experiment on E. coicoides in which protective

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immunity was induced after oral immunization at 18 dpf further support the possibility that the immune system must have some functionality around the time of metamorphosis of larvae (Lin, Lin, Chen & Yang 2007).

Acknowledgments This work was supported by grants from the National Science and Technology Program for Agriculture Biotechnology (94-2317-B-006-005) and (96-2371B-006-001) from the National Science Council, Taiwan.We thank Dr Han Jia Lin, of National Taiwan Ocean University, for his help with the nucleotide sequence analysis. We also deeply appreciate Dr Alan Pike and Ms Kathleen Hills of the Lucidus Consultancy for correcting this manuscript.

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