Experimental immunology DOI: 10.5114/ceji.2015.52825 Spatio-temporal expression of blunt snout bream (Megalobrama amblycephala) mIgD and its immune response to Aeromonas hydrophila HU XIA, KANG WU, WANJING LIU, WEIMIN WANG, XUEZHEN ZHANG Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Key Laboratory of Health Aquaculture and Product Processing in Dongting Lake Area of Hunan Province, Hunan University of Arts and Science, Hunan Changde, China Abstract The function of IgD in fish and mammals has not been fully understood since its discovery. In this study, we have isolated and characterized the cDNA that encodes membrane-bound form of the im- munoglobulin D heavy chain gene (mIgD) of blunt snout bream (Megalobrama amblycephala) using RT-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA of mIgD consisted of 3313 bp, encoding a putative protein of 943 amino acids. The structure of blunt snout bream mIgD is VDJ-μ1-δ1-δ2-δ3-δ4-δ5-δ6-δ7-TM. Multiple alignment and phylogenetic analyses indicated that blunt snout bream mIgD clusters with the homologues of cyprinid fish and that its highest identity is with that of C. idella (82%). The mIgD expression in early different developmental stages showed that the level of mIgD mRNA decreased dramatically from the unfertilized egg stage to the 32-cell stage, suggesting that mIgD mRNA was maternally transferred. As cell differentiation initially took place in the blastula stage, the mIgD expression increased significantly from the blastula stage to prelarva, which might be attributed to embryonic stem cell differentiation processes. Compared with juvenile fish, the expression and tissue distribution patterns of mIgD in adult individuals exhibited considerable variation. After the injection of Aeromonas hydrophila, mIgD expression was up-regulated in various tissues, reaching the peak expression at 5 d, 14 d or 21 d (depending on the tissue type). The present study provides a theo- retical basis for further research of the teleost immune system. Key words: blunt snout bream (Megalobrama amblycephala), mIgD, cDNA, Aeromonas hydrophila, maternal transfer, spatio-temporal expression. (Centr Eur J Immunol 2015; 40 (2): 132-141) Introduction identified in a large number of species, including Atlantic salmon, Salmo salar [8], Atlantic cod, Gadus morhua L. In all species that possess Igs, the only class of anti- [9], Japanese flounder, Paralichthys olivaceus [10] and body universally found is IgM, which has been well char- grass carp, Ctenopharyngodon idella [11]. The discover- acterized at both the protein and molecular levels and is ies of an IgD-like gene in teleost species have changed the considered to be the predominant teleost serum Ig [1, 2]. evolutionary view, and suggest that the gene existed early However, there are considerably fewer reports about the in vertebrate evolution. teleost homolog(s) of IgD, which was initially discovered The role of IgD in teleost in vertebrate immune sys- in humans from serum of a myeloma patient [3]. For about tems is not fully understood. In channel catfish, transcripts 30 years, IgD was considered to be a relatively recently encoding both membrane and secreted IgD have been evolved class of immunoglobulins as it had been described identified [7]. The IgD heavy chain cDNA clones exist- only in primates and rodents [3-6]. ed only as the membrane form in both Atlantic salmon However, in 1997, a new immunoglobulin H-chain and Atlantic cod [8, 9]. In most species, the IgD-encod- gene with some homology to mammalian IgD was cloned ing gene (Cδ) is located downstream of the IgM-encoding from the channel catfish, Ictalurus punctatus L. [7]. Since gene (Cµ) and is co-expressed with IgM on the surface of then, IgD genes, albeit with some diversity, have been the majority of mature B cells before antigenic stimulation Correspondence: Hu Xia, Hunan University of Arts and Science, Hunan Changde 415000, P.R. China, e-mail: [email protected] 132 Central European Journal of Immunology 2015; 40(2) Spatio-temporal expression of blunt snout bream (Megalobrama amblycephala) mIgD and its immune response to Aeromonas hydrophila [12]. IgD seems to play an important role as an antigen head kidney, trunk kidney, liver, spleen, gill, intestine, receptor optimized for efficient recruitment of B cells into muscle and brain) were immediately collected and frozen antigen-driven responses [8]. in liquid nitrogen, and then stored at –80°C until the RNA The structures of teleost IgD genes are different from was extracted. To determine the expression of the mIgD those of mammals. Human IgD has three constant do- gene in different developmental stages, samples were col- mains, while there are only two constant domains in the lected from unfertilized eggs, zygotes, 2-cell embryos, mouse; further, both human and mouse delta constant re- 4-cell embryos, 32-cell embryos, blastula, gastrula and gions have a flexible hinge region [13]. In contrast, there prelarva. Due to the vast differences in the size amongst is no hinge region and there are seven constant domains the developmental stages, samples totaling ~100 mg from for both catfish and salmon IgD [7, 8]. The initial discov- six parents were pooled for each stage for RNA isolation. ery of IgD in teleosts also found that IgD was a chimeric All samples were flash-frozen in liquid nitrogen immedi- protein containing a Cμ1 domain followed by a number of ately and then stored at –80°C until RNA isolation. All Cδ domains [7]. This chimeric structure was later found in the experimental procedures involving fish were approved grass carp [11], Atlantic salmon [8] and Atlantic cod [9]. by the Institutional Animal Care and Use Committee of Until now, no complete fish IgD heavy chain without Cμ1 Huazhong Agricultural University, Wuhan, China. has been reported. In addition, the structure of the fish IgD gene is different in various species. For instance, a dupli- Challenge experiment cation of domains δ2-δ3-δ4 has been reported in grass carp [11], salmon [8], halibut [14] and catfish [15], but not in For the A. hydrophila challenge experiment in M. am- flounder [10]. Further, in cod, domains δ3-δ6 are absent, blycephala, all the tested fishes (15 ±2 g) were inoculated and there is a tandem duplication of domains δ1 and δ2 [9]. by intraperitoneal injection. The bacteria A. hydrophila So far, the information obtained indicates that teleosts was isolated from diseased M. amblycephala in Dongxi do not share a common IgD structure. To further our under- Lake (Wuhan, China) by our laboratory. A single colony standing of the immune development of teleosts, it is import- was cultured in LB medium at 28°C to mid-logarithmic ant to obtain more information on this gene in additional fish growth. In a pre-challenge experiment prior to the chal- species from different families. Megalobrama amblycephala, lenge trial, the concentration 1 × 107 colony forming units/ commonly known as blunt snout bream or Wuchang fish, ml (CFU/ml) was determined as LD50. The treatment group belongs to Megalobrama, Cyprinidae, Cypriniformes, Acti- was injected with 0.1 ml (1 × 107 CFU/ml) bacterial sus- nopterygii. As a species in Cyprinidae, the largest family of pension per individual, while the control group was in- freshwater fish, M. amblycephala is closely related to the jected with the same volume of phosphate-buffered saline commonly known fish such as zebrafish (Danio rerio) and (PBS, pH 7.2). After the treatment, the fish were returned carp. Although the gene encoding IgD has been identified in to tanks with water temperature of 27 ±0.5°C. Thirty inject- bony fish by database mining, the biological functions of IgD ed individuals (3 pools) from treated and control groups is yet unknown in Megalobrama amblycephala. Therefore, were randomly dissected at 4 h, 1, 3, 5, 14, and 21 d post the aim of the present study was to isolate and to character- ize the IgD gene in blunt snout bream, Megalobrama am- injection. Thirty without injected fishes were sampled as blycephala, which is an economically important freshwater a blank control (0 h). Fish were euthanized by exposure to fish species in the aquaculture industry in China. In addi- 300 mg/l of MS-222 (Sigma, USA) before dissection, and tion, to understand the fish immune system, we examined tissues (including trunk kidney, spleen, gill and liver) were the spatio-temporal expression of mIgD and investigated the sampled, frozen immediately in liquid nitrogen and then immune response of the mIgD gene after Aeromonas hy- stored at –80°C until RNA extraction. drophila infection in M. amblycephala. RNA isolation and cDNA synthesis Material and methods Samples, including different tissues and embryos of different developmental stages, were homogenized using Fish and sampling a mortar and pestle under liquid nitrogen; total RNA was Healthy M. amblycephala of juvenile (body weight: extracted using Trizol reagent (TaKaRa, Japan), according 45-55 g) and adult fish (body weight: 400-500 g) were to the manufacturer’s instructions. Total RNA was treated collected from the fish base of Huazhong Agricultural Uni- with gDNA Eraser (TaKaRa, Japan) to avoid the contam- versity (Wuhan, China). Before experiments, fish were ac- ination of genomic DNA. The quantity and quality of the climatized in quarantine plastic tanks in aerated freshwater RNA was determined using agarose gels and a NanoDrop at 24 ±2°C for two weeks. After acclimation, each fish was 2000 spectrophotometer (Thermo Fisher Scientific, USA). anesthetized with MS-222 (Sigma, USA). To avoid indi- First strand cDNA was synthesized using PrimeScript® vidual differences, tissues were extracted from 30 juvenile RT reagent Kit with gDNA Eraser (TaKaRa, Japan), ac- and 30 adult M. amblycephala. Tissue samples (including cording to the manufacturer’s instructions.