Mystifying Molecular Structure, Expression and Repertoire Diversity of IgM Heavy Chain Genes (Ighμ) in Clarias Catfish and Hybrids: Two Novel Transcripts in Vertebrates

Two novel immunoglobulin heavy chain (Ighμ) transcripts encoding membrane-bound forms of IgM (mIgM) were discovered in bighead catfish, Clarias macrocephalus. The first transcript contains four constant and two transmembrane domains [Cμ1-Cμ2-Cμ3-Cμ4-TM1-TM2] that have never been reported in teleosts, and the second transcript is an unusual mIgM that has never been identified in any vertebrate [Cμ1-(Cδ2-Cδ3-Cδ4-Cδ5)-Cμ2-Cμ3-TM1-TM2]. Fluorescence in situ hybridization (FISH) in bighead catfish, North African catfish (C. gariepinus) and hybrid catfish revealed a single copy of Ighμ in individual parent catfish, while two gene copies were found in diploid hybrid catfish. Intensive sequence analysis demonstrated multiple distinct structural variabilities in the VH domain in Clarias, and hybrid catfish were defined and used to generate diversity with various mechanisms. Expression analysis of Ighμ in Aeromonas hydrophila infection of the head kidney, peripheral blood leukocytes and spleen revealed significantly higher levels in North African catfish and hybrid catfish than in bighead catfish.


INTRODUCTION
Vertebrate immunoglobulins (Igs) are the hallmark elements in adaptive immune responses to a particular antigen with high discrimination, specificity and long-term memory. Igs are generated by B cells and serve two purposes: 1) cell-surface receptors (membrane-bound forms; mIgs) for signaling and activation of cells and 2) soluble effector molecules (secreted forms; sIgs) for neutralization of microbes and toxins, opsonization (immunophagocytosis), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement activation (immunolysis) (1).
The basic structure of Ig heavy chain (Igh) gene molecules consists of variable (VH) and constant (CH) regions (2). The gene sequence organizations of Igh vary depending on the species (3,4). The immunologic effector functions of the Ig classes are determined by the different constant regions of the CH chain. In most mammals, five classes of Igs are categorized based on different gene sequences; these classes are m, d, g, ϵ, and a, and they correspond to the five major isotypes of Igs, IgM, D, G, E, and A, respectively. In teleost fish, only three Igh isotypes have been identified, namely, IgM, D, and T/Z based on the gene sequences of the isotypes m, d, and t/z, respectively (5)(6)(7). Additionally, distinct Igh classes have been identified in nonmammalian vertebrates, including IgNAR and IgW (in cartilaginous fish) (8), IgO (platypus), IgP (in Pleurodeles waltl) (9), IgX, IgF and IgY (in amphibians) (9,10).
The evolution of Ig molecules in jawed vertebrates diverged from the "multicluster" type to the "translocon" type approximately 470 million years ago (11,12).  (13,14). Interestingly, in catfish groups such as channel catfish (Ictalurus punctatus), the Cz/t genes are not found either in the 3'-region of the VH gene cluster or within the VH gene (15).
The Ighm gene was the first Ig class identified in teleosts and has long been considered the most primitive and most prevalent Ig in fish plasma. It can be expressed as mIg or sIg. Secreted tetrameric IgM represents the main Ig in the serum of teleosts. In teleosts as well as a wide range of mammalian species, molecular characterization of the Ighm gene has revealed that the secreted Ighm transcripts consist of a rearranged VDJ region spliced to the Cm 1 domain, followed by Cm 2 , Cm 3 or Cm 4 (VH-Cm 1 -Cm 2 -Cm 3 -Cm 4 ). Interestingly, the unusual transcript patterns of the Ighm gene indicate that the membrane Ighm transcripts in teleost fish appear to be shorter than those in mammals since the first transmembrane (TM) exon is spliced directly to Cm 1 , Cm 2 or Cm 3 thereby excluding the entire Cm 2 or/and Cm 3 or/and Cm 4 exons (VH-Cm 1 -TM, or VH-Cm 1 -Cm 2 -TM, or VH-Cm 1 -Cm 2 -Cm 3 -TM, respectively). These patterns have been identified and reported in many teleost species (3,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27).
In Thailand, the production of catfish is the second largest fish aquaculture industry, with approximately 159,314 million tons in 2004 (28). Two major species of catfish, bighead catfish (Clarias macrocephalus Gunther, 1864) and North African catfish (C. gariepinus), and their hybrid catfish ("pla-duk-big-uey" in Thai), are economically popular cultured species in southeast Asia, especially in Thailand, Vietnam, Malaysia and Indonesia. Notably, the hybrid catfish (C. macrocephalus × C. gariepinus) demonstrates many commercially desirable dominant characteristics from its parents, such as good quality of meat, rapid growth, better feed conversion, increased survival, resistance to many diseases and tolerance to many environmental conditions. Therefore, bighead catfish has become a remarkable supporter species in catfish production in many counties in southeast Asia.
Most of our current knowledge on the immune systems of catfish comes from other models and economically important fish species, such as zebrafish (Danio rerio) and medaka (Oryzias latipes) (14,(29)(30)(31)(32)(33)(34)(35). In addition, the structural, functional and genetic features of catfish Igs in the genus Clarias are still virtually absent.
The study of molecular Ig genes in catfish could provide a better understanding of their role in immunity, and these genes could be applied as tools for basic research, diagnosis, and therapy in catfish culture farming. Thus far, the study of Ig genes has focused on the molecular structure of Ighm loci in catfish, particularly their splicing patterns, functions, diversification, expression and homogeneity or heterogeneity between species and their hybrids. In the present study, the organization of the Ighm genes was analyzed at the chromosomal level. The obtained data provide new knowledge regarding catfish and may benefit applications in sustainable aquaculture industries.

Animals
Bighead catfish (Clarias macrocephalus Günther, 1864), North African catfish (Clarias gariepinus) and their hybrid catfish (C. macrocephalus × C. gariepinus), 90-120 g in body weight, were obtained from the Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Thailand. The fish were acclimatized in a quarantine tank with aerated freshwater at temperatures between 28 and 31°C for 2 weeks before the start of the experiment. The experimental procedures performed with aquatic animals were carried out in accordance with the Ethical Principles and Guidelines for the Use of Animals National Research Council of Thailand for the care and use of animals for scientific purposes. The protocol was approved by the Animal Ethics Committee, Kasetsart University, Thailand (Ethics ID: ACKU61-FIS-004).

Isolation of Genomic DNA and RNA and cDNA Synthesis
Catfish genomic (g) DNA was isolated from whole blood tissues using a QIAamp DNA Blood and Tissue Mini Kit (QIAamp, CA, USA) according to the manufacturer's protocols.
The PBLs of catfish are target tissues used to isolate total RNA and for cDNA synthesis. Total RNA was analyzed using NucleoZOL ™ reagent (Clontech Laboratories, CA, USA) according to the manufacturer's instructions. The obtained total RNA was then used as templates for first strand cDNA synthesis using the protocol described for the Thermo Scientific RevertAid Reverse Transcriptase Kit (Thermo Fisher Scientific, MA, USA). The products of the gDNAs and first-strand cDNA synthesis were stored at -80°C for further experiments.

Genes of Catfish
To obtain and characterize the Ighm genes of catfish, we used catfish gDNA to amplify the Ighm genes of catfish using forward and reverse specific primers designed from the highly conserved region of the 5′ end of Cm 1 and the 3′UTR of full-length Ighm cDNAs, respectively. The forward primer was Cla_Ighm_Cm 1_ f: 5′-GAACGTCGGTGACCGTAACTTCA-3′ for all Clarias catfish species. The reverse primers were Cla_mac_Ighm_3UTR_r: 5′-GAACACACAAGCATCAGACAGACTG-3′ for big head catfish and hybrid catfish and Cla_gar_Ighm_3UTR_r: 5′-CAAGAACA CACAAGCATCAGACAG-3′ for North African catfish and hybrid catfish.
PCRs were carried out using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocols. The PCR cycling conditions were one cycle of 95°C for 5 min, 30 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 5 min, followed by a final extension at 72°C for 10 min. The PCR products of Ighm genes were cloned and sequenced as described above.

Prediction of Protein Structures of Catfish Ighm Genes
The protein structures of Ighm molecules of catfish were determined to predict the possibility of constant domain structure using the structural bioinformatics web server SWISS-MODEL workspace integrates programs (https:// swissmodel.expasy.org) according to the programs' procedures (36). The amino acids in the constant domain of each Ighm molecule were used to generate the target sequence for protein structure prediction.

Cytogenetic Mapping of Ighm Genes in Catfish
Metaphase chromosomes of catfish were obtained from the head kidney of each catfish species treated with colchicine according to Karami et al. (2015) (37). Prepared tissues were stored in fixative solution at -20°C until use. The chromosomal spreads were analyzed by staining with 15% Giemsa for 45 min and examined under a light microscope (Olympus, MA, USA). The Ighm gene probes for fluorescence in situ hybridization (FISH) used full-length Ighm genes containing the constant domain Cm 1 to the 3′UTR of the Ighm genes of each catfish species. DNA probes were performed according to standard procedures of FISH Tag detection kits (Thermo Fisher Scientific, MA, USA). Metaphase chromosome hybridization was carried out following a previously described method (38). Chromosomes and specific probes were performed with DAPI and Alexa Fluor 488 or 594 dyes following the manufacturer's instructions (Thermo Fisher Scientific, MA, USA). Digital micrographs were recorded and processed using a Nikon/DigitaL Eclipse C2Si confocal microscope (Nikon Instruments Inc., NY, USA).

Diversity Analysis of the Variable Domain of Ighm Genes of Catfish
The 5′ VH region of first-strand cDNAs encoding Ighm genes was synthesized from total RNA of PBLs of each catfish species using a 5′ RACE-Ready cDNA synthesis kit (Clontech, Mountain View, CA, USA), as previously described. PCR was amplified using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) with the 5′ universal primer mix (UPM) and gene-specific primers (GSPs) from the kit and nucleotide sequences located in the 5′ region of the Cm 1 constant domain of Ighm genes (Cla_Ighm_Cm1_r: 5′-GTGCCGCTCGCATCCTTCCAAACG-3′). The PCR cycling conditions were one cycle of 95°C for 5 min, 25 cycles of 95°C for 30 sec, 68°C for 30 sec, and 72°C for 120 sec, followed by a final extension at 72°C for 5 min. The 5′ RACE VH PCR products of Ighm cDNA were ligated to the pJET1.2/blunt cloning vector (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. A different hundred recombinant clones were randomly selected for sequencing by the Macrogen sequencing service (Macrogen Inc., Seoul, South Korea) as previously described.

Analysis of V H , D H and J H Segments
The 5′ VH nucleotide sequences of Ighm genes were trimmed to three separated regions encoding the V H , D H and J H genes using an Ig variable domain sequence analysis tool (NCBI IgBLAST, https://www.ncbi.nlm.nih.gov/igblast/). The nucleotide sequences of each gene region were classified at the family level based on 80% similarity of nucleotide sequences using the matrix global alignment tool (MATGAT 2.0).

Analysis of Complementarity-Determining Regions (CDRs)
CDR nucleotide regions of each variable domain nucleotide sequence were first characterized using IgBLAST (https://www. ncbi.nlm.nih.gov/igblast/). The D H segments were predicted using the VDJsolver 1.0 server (https://services.healthtech.dtu.dk). The CDRs were analyzed for their characterization, including CDR length distribution and amino acid composition in each position, using the High V-QUEST IMGT tool (39).

Analysis of the Variability Plot of the Variable Domains of IgM Molecules
The variability plots of variable domain nucleotide sequences were analyzed using the Shannon (1948) (40) and Kabat and Wu (1971) (41) methods. The variability plots were illustrated using the online bioinformatics software protein variability server (PVS, https://imed.med.ucm.es/PVS/pvs-help.html).
Total RNA and first-strand cDNA synthesis from sixteen organs of catfish were isolated using NucleoZOL ™ reagent (Clontech Laboratories, CA, USA) and a Thermo Scientific RevertAid Reverse Transcriptase kit (Thermo Fisher Scientific, MA, USA) following the manufacturer's instructions.
qRT-PCR cycling conditions were one cycle of 95°C for 5 min, 40 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 90 sec, followed by a final extension at 72°C for 10 min. The expression of the b-actin gene was used as the housekeeping gene to standardize the results by eliminating variation in mRNA and cDNA quantity and quality. The relative expression of the Ighm gene in catfish tissues was calculated using 2 -DDCT analysis according to the protocol by Schmittgen and Livak (2001) (43). The brain RT was used as calibrator. All reactions were done in triplicate.

Statistical Analysis
The relative expression levels of Ighm genes were calculated using b-actin as a reference. All quantitative data are presented as the mean ± standard deviation (SD). Statistical analysis at each time course was performed using SPSS statistics 24.0 software using one-way analysis of variance (ANOVA) and Duncan's new multiple range test (DMRT). The level of statistical significance between species in different organs is indicated as * (P<0.05), ** (P<0.01) and *** (P<0.001) using Student's t-test.

Characterization and Sequence Analysis of cDNA Transcripts Encoding a Constant Region of the Ighm Gene
Through analysis of the cDNA transcripts encoding constant regions of the Ighm genes in bighead catfish, North African catfish and their hybrid catfish, one hundred positive clones of each catfish were first cloned and sequenced to obtain the internal nucleotide sequences of the constant regions of the Ighm genes. A single band of PCR fragment was shown for each catfish species. Sequence analysis indicated no variation in the nucleotide sequences of individual bighead catfish, North African catfish and their hybrid catfish. There were four exons encoding Cm s [Cm 1(partial) -Cm 2 -Cm 3 -Cm 4 (partial) ] of 954 and 949 bp for bighead catfish and North African catfish, respectively ( Figure 1A), compared to the published Ighm sequence of channel catfish (Ictalurus punctatus). In the hybrid, two different nucleotide sequences were observed; each sequence exhibited a 100% match with the Ighm sequence of one of the parents. Among a hundred clones from hybrid catfish, the Ighm nucleotide sequences shared a distribution of 28% and 72% to bighead catfish and North African catfish, respectively ( Figure 1A).
The 5′ and 3′ of the Ighm gene constant regions in each catfish were consequently cloned and sequenced using 5′ and 3′ RACE techniques with specific primers designed from the 5′ end of the Cm 2 exon and 3′ end of Cm 1 for recovering the 5′ and 3′ nucleotide regions of Ighm cDNAs, respectively. The obtained 5′ and 3′ nucleotide sequences of Ighm genes corresponded to the Cm 1 -Cm 2 -Cm 3 and Cm 3 -Cm 4 -TM-polyA tails, respectively ( Figure 1A). No variation difference in the nucleotide sequences was observed in the 5′ Ighm constant region exon in bighead catfish and North African catfish. In addition, the hybrid catfish exhibited two different nucleotide sequences, one from each parent ( Figures 1A, B). There was no variation in the internal nucleotide sequences of the constant regions of the Ighm genes in all studied catfish. ( Figure 1C). However, the sequences of the 3′ constant regions consisted of two major different Ig forms, (mIg and sIg), which are controlled by alternate mRNA processing events in certain species. Up to six nucleotide sequence patterns of 3′ region exons were observed in bighead catfish ( Figure 1D): five patterns for mIg and one pattern for sIg. Among the five patterns of mIg, three corresponded to Cm 1 -Cm 2 -Cm 3 -TM1-TM2 with differences in the polyadenylation signal region (polyA signal: TM-2A, TM-2B and TM-2C, respectively) of the untranslated 3′ sequence, one corresponded to Cm 1 -Cm 2 -Cm 3 -Cm 4 -TM1-TM2 containing the polyA signal TM-2C, and one corresponded to Cm 1 -(non-Ighm exons)-Cm 2 -Cm 3 -TM 1 -TM 2 containing the polyA signal TM-2A. One sIg was encoded by exon Cm 1 -Cm 2 -Cm 3 -Cm 4 and the polyA signal SC-1A ( Figures 1D, E and Supplementary Figures 1-6).
Three nucleotide sequence patterns were observed in North African catfish, and they corresponded to two patterns for mIg and one pattern for sIg ( Figure 1D). Two patterns of mIg corresponded to Cm 1 -Cm 2 -Cm 3 -TM1-TM2 with differences in the polyadenylation signal region (polyA signal: TM-2A and TM-2B, respectively) of the untranslated 3′ sequence. Another pattern corresponded to sIg that contained four exons coding Cm 1 -Cm 2 -Cm 3 -Cm 4 and the polyA signal SC-1A ( Figures 1D, F and Supplementary Figures 7-9).
Two nucleotide sequence patterns of the 3′ region exon corresponding to mIg (Cm 1 -Cm 2 -Cm 3 -TM1-TM2) and sIg (Cm 1 -Cm 2 -Cm 3 -Cm 4 ) were observed in the hybrid. The mIg and sIg nucleotide sequence coding from Cm 1 through polyA tails were absolutely identical to their parent, bighead catfish and African catfish, respectively (Figures 1D, G and Supplementary  Figures 1, 9).

Cytogenetic Mapping of Ighm Genes in Catfish
In situ hybridization with specific probes to the Ighm gene of individual catfish species, bighead catfish and North African catfish revealed that only a single copy of the Ighm gene was found in bighead catfish ( Figure 1H) and North African catfish ( Figure 1I). In addition, a strong hybridization of both the Ighm genes of bighead catfish and North African catfish was represented in their hybrid catfish ( Figure 1J). The expression of Ighm transcripts in the hybrid catfish from the two Ighm genes received from the parents was confirmed.

Amino Acid Sequence Comparison of Ighm Transcripts in Clarias Catfish
The amino acid sequences of Ighm transcripts among bighead catfish, North African catfish and their hybrid catfish were aligned and compared to published complete IgH gene loci of channel catfish (Ictalurus punctatus) and zebrafish (Danio rerio). The aligned sequences are illustrated in Figure 2. The results showed several important features based on the overall relative size of the amino acid sequences of general Ighm transcripts, with 436 and 430 residues for the constant (Cm 1 -Cm 4 ) domain and 44 and 41 residues for the transmembrane domains of bighead catfish and North African catfish, respectively. The mIgM and sIgM forms of the hybrid catfish were similar in size to those of the parents, bighead catfish and North African catfish ( Figure 2). The sequence similarity of the mIgM and sIgM forms of the hybrid catfish were absolutely conserved and similar to those of the parents. These results exhibited highly important significant differences between their phenotypic characterization and relationship to their parent.
The translated amino acid sequences of the individual Cm 1 , Cm 2 , Cm 3 , Cm 4 and transmembrane domains of bighead catfish, North African catfish and their hybrid catfish were used to define significant similarity among their relationships and evolutionary lineage. The amino acid sequences of the Cm 1 and Cm 2 domains of all catfish were similar in size, with 103 and 103 residues, respectively ( Figure 2A). However, the Cm 1 and Cm 2 domains were slightly larger and smaller than the channel catfish Cm 1 and Cm 2 domains (102 and 104 residues, respectively) ( Figure 2A). Additionally, bighead catfish shared amino acid similarity of Cm 1 and Cm 2 to African catfish, the hybrid catfish (mIgM), the hybrid catfish (sIgM), channel catfish and zebrafish with 82. 4 Table 1).
The conservation of the Ighm domain is evident in the catfish Cm 1 , Cm 2 , Cm 3 , and Cm 4 of Ighm structures. The cysteines that likely form the intradomain disulfide bridge (S-S) as well as the tryptophan located within the Cm 1 , Cm 2 , Cm 3 , and Cm 4 regions were absolutely conserved among catfish, channel catfish and zebrafish (Figures 2A-E). The cysteine that likely participates in the disulfide linkage of the catfish chain to the light chain was also conserved in the Cm 1 regions (Figure 2A). Additionally, the predicted results of these protein tertiary structures of Ighm transcripts in bighead catfish, North African catfish and their hybrid catfish are illustrated in Figure 3. The Ighm molecules encoded by general coding exons found in teleost and catfish mIg (Cm 1 -Cm 2 -Cm 3 -TM1-TM2) and sIg (Cm 1 -Cm 2 -Cm 3 -Cm 4 ) exhibited similar predicted structures in all the studied catfish ( Figures 3A-C). Additionally, the predicted results of these protein tertiary structures of Ighm transcripts of unusual splicing patterns generated novel membrane forms of Ighm were larger in size and rather complex compared to other general predicted structures (Figures 3D, E).
The evolutionary relationships of Ighm constant regions among various vertebrate species are illustrated in Figure 4A, and the relationships of teleost Igh constant regions are illustrated in Figure 4B. In this study, the Clarias catfish Ighm constant domains were cladded into the catfish group of vertebrate lineages and classified into the Ighm group among the reported Igh in fish.

Unusual Splicing Patterns Generate Novel Membrane Forms of Ighm Transcripts in Teleosts and Vertebrates
The two novel and unusual splicing patterns of the Ighm membrane form were found in only bighead catfish: one was associated with a new splicing pattern among teleosts, and the other among vertebrates. The constant region of the new Ighm membrane form in teleosts is encoded by four Cm 4 exons and directly spliced into the first transmembrane exons (Cm 1 -Cm 2 -Cm 3 -Cm 4 -TM1-TM2), as reported in other vertebrates, such as tetrapods, birds, sharks, and mammals. However, the castratory event was observed by the lack of a cryptic donor splicing site at the 3′ region of the Cm 4 exon, which is important for splicing to the transmembrane domain to generate the membrane form as reported previously. The predicted crystal protein structure of this molecule is illustrated in Figure 3D. The predicted results of the protein tertiary structures are close to the actual crystal structures of reported Ighm transcripts in bighead catfish ( Figure 3A), North African catfish ( Figure 3B) and their hybrid catfish ( Figure 3C).
The other novel and unusual splicing pattern of the Ighm transcript was notable for vertebrates. The largest molecule of the Ighm membrane-bound form was defined together with the new Ighm splicing pattern among vertebrate animals. The molecular size of the molecule was approximately 92.64 kDa by encoding two major constant IgH loci in teleosts: Cm and non-Cm gene loci. The molecule encoded three Cm s and non-Cm constant exons. The first exon of the 5′N-terminal domain was the Cm 1 exon of the Ighm gene, which was directly spliced to the non-Cm exons (487 residues) ( Figure 2B) and then spliced directly to link the Cm 2 , Cm 3 , TM1 and TM2 exons of the Ighm gene of bighead catfish. The translated amino acid sequences of the Cm exons, including Cm 1 , Cm 2 , Cm 3 , TM1 and TM2, were identical (100%) to the previously described Ighm gene of bighead catfish (Figures 1B, 2). In addition, the encoded non-Cm constant region exons were studied via BLAST and compared to the published complete IgH gene loci of channel catfish and zebrafish. The translated amino acid sequences of the entire non-Cm exons were 57.73 and 34.63% identical to the Ighd gene locus of channel catfish and zebrafish, respectively (Supplementary Table 1). The evolutionary relationship analysis of non-Cm exons of bighead catfish was confirmed; it was grouped into Ighd among other teleosts and shared a branch with the Ighd of the closely related teleost species channel catfish ( Figure 4B). In addition, the nearest neighbors' relationships of presumptive Ighd constant exons were gilthead bream (Sparus aurata, AXL14358), grass carp (Ctenopharyngodon idella, ACV21058) and Wuchang bream (Megalobrama amblycephala, AGR34025). However, the presumptive non-Cm exon sequences clustered within teleost Ighd rather than Ighm. The predicted protein tertiary structure of this molecule was larger in size and rather complex compared to other predicted structures ( Figure 3E).

Genomic Organization of the Ighm Gene Locus
To gain insight into the Ighm locus of Clarias catfish and hybrid catfish, we also defined the complete genomic organization of the gene locus. The corresponding genomic sequences encoding the Ighm constant domain were amplified, cloned, sequenced and compared to the published complete Igh gene locus in channel catfish. Consistent with this finding, only a single copy of the genomic Ighm gene locus was found in bighead catfish and North African catfish ( Figures 1E, F, H, I, and Supplementary  Figures 10, 11). The complete constant region of the Ighm locus, which encodes an Ig m domain, contained approximately 6,793 kb (six exons, five introns and six polyadenylation signals) and 6,660 kb (six exons, five introns and six polyadenylation signals) of bighead catfish and North African catfish, respectively. However, the individual Ighm loci of each bighead catfish and North African catfish were also found in the hybrid catfish, which had the transmembrane and secreted Ighm transcripts, as mentioned before. The nucleotide identity between the two Ighm gene loci in the hybrid catfish was identical to those of the parents, bighead catfish and African catfish ( Figures 1G, J and Supplementary Figures 10, 11).

Diversity Analysis of the Variable Domain (VH) of Ighm Genes
A total of 100 unique nonredundant clones of each catfish were characterized for VH sequences of the Ighm gene. The functional regions of VH encoded by the V H , D H and J H segments were identified, and analysis of framework regions (FWs) and complementarity-determining regions (CDRs) was also performed ( Figure 5A) according to the international immunogenetics (IMGT) information numbering system for Ig structural definition (59).
Based on 80% nucleotide identity, the functional V H segments were classified into 15, 15 and 17 families for bighead catfish, North African catfish and their hybrid catfish, respectively. Between the most 3′ V H segment and the 5′ J H segment, 9, 11 and 13 functional D H segment families were defined in bighead catfish, North African catfish and their hybrid catfish, respectively. Altogether, the functional J H segments of catfish VH have been identified. There were 11, 11 and 8 families of bighead catfish, North African catfish and their hybrid catfish, respectively ( Figure 5B). The comparisons of the V H gene family among catfish in this study and among reported vertebrates are illustrated in Figures 5B, C, respectively. The NCBI accession numbers of VH nucleotide sequences are MN934442-MN934541, MN934542-MN934641 and MN934642-MN934741 for bighead catfish, North African catfish and their hybrid catfish, respectively. In addition, adding P-and N-nucleotides was predominantly observed at the junction of the V and J segments for the generation of V(D)J junctional diversity in bighead catfish, North African catfish and their hybrid catfish ( Supplementary  Figures 12-14).

Analysis of Framework Regions (FWs) and Complementarity-Determining Regions (CDRs)
To determine the extent of diversification in the VH repertoire of the Ighm gene, we calculated the length variation and percent deviations of amino acids in each position of the FWs and CDRs and compared them to those of germline VH genes from the international ImMunoGeneTics information system ® (IMGT ® , http://www.imgt.org) (60).
The FWs of bighead catfish, North African catfish and their hybrid catfish were similar in length and diversity of amino acids. The lengths of amino acids of FW1, FW2, FW3 and FW4 in all catfish were approximately 33, 14, 36 and 14 residues, respectively ( Figure 5D). To examine the degree of sequence variability of the VH repertoire in Clarias catfish, plots of the variability according to the Shannon (1948) (40) and Kabat and Wu (1971) (41) methods were generated. The overall amino acid sequence variability of the VH region was mostly confined to the CDRs and particularly the CDR3s. In addition, the amino acid variability among CDRs (CDR1, CDR2 and CDR3) was highly similar to that of FR1 to FR4 of all studied catfish. Moreover, the comparisons of variability plots of VH sequences among the studied Clarias catfish were closely related in sequence patterns and variability level to each other based on both the Shannon and Kabat and Wu methods ( Figure 5D). Shannon entropy plots of all catfish revealed increased diversity at amino acid positions 31 to 42 for CDR1, 58 to 65 for CDR2 and 102 to 120 for CDR3 ( Figure 5D). They were generally related to the traditional definitions using the Kabat numbering convention ( Figure 5D).
The amino acid length diagrams of CDRs of bighead catfish, North African catfish and their hybrid catfish in Figure 5E indicate that the amino acid length of CDR1 ranged from 3 to 12 residues with an average of 7.6, 7.5 and 6.9 residues, respectively. The most common length was seven for all catfish, with approximately 58 to 71% frequency.
average amino acid lengths of 7.0, 6.9 and 6.8 residues, respectively. Approximately 76 to 79% of nonredundant CDR2s were 7 amino acid residues in length. Shannon entropy plots of amino acid variation of nonredundant CDR1 and CDR2 were mostly similar in diversity to those of the related V H families within the species and among Clarias species. Within 8 residues in length of CDR2 revealed highly conserved residues at position one of the CDR1s ( Figure 5F).

Expression and Tissue Distribution of Ighm Genes
To examine the distribution of Ighm in Clarias catfish, we examined the potential functional coding segment of Ighm, the Cm 2 region, in sixteen tissues of catfish. Ighm was highly expressed in the head kidney, spleen and peripheral blood lymphocytes (PBLs) in all studied catfish ( Figure 6A). The relative expression levels reached approximately 60-, 700-and 600-fold the accustomed expression in head kidney, an important fish immune response organ, of bighead catfish, North African catfish and their hybrid catfish, respectively. However, the lowest expression was also observed in various tissues, including the brain, dendrite, gallbladder, gills, heart, liver, muscle, ovary, skin, stomach, testes and trunk kidney, in all studied catfish (Figures 6A, B). Highly significant expression levels were observed in North African catfish and the hybrid catfish in several tissues, including the gills, head kidney, heart, intestine, liver, muscle, ovary, PBLs, skin, spleen, stomach, and testes, compared to those in bighead catfish ( Figure 6B). No significant difference was observed in tissues of North African catfish and the hybrid catfish. However, the relative expression of Ighm was not significantly different in dendrites, gall bladder or trunk kidney among all studied catfish ( Figure 6B).
Intraperitoneal injection of virulent Aeromonas hydrophila clearly exhibited the pattern of immune responses of catfish, and its significance was defined among the three catfish. In the spleen, the expression of Ighm was moderately increased approximately 200-400-fold after 12-168 hours (hr) of pathogen exposure for all catfish. For the head kidney, the expression of Ighm was strongly increased approximately 200-300-, 850-950-, and 800-950-fold after 12-24 hr. of pathogen exposure for bighead catfish, North African catfish and their hybrid catfish, respectively. In the PBLs, Ighm expression showed significant upregulation of approximately 200-, 450-and 500-fold after 12-24 hr. of exposure for bighead catfish, North African catfish and their hybrid catfish, respectively. In addition, an approximate upregulation of 200-300-fold was also observed in all catfish after 12-24 hr. of exposure ( Figure 6C).

DISCUSSION
The specific features of Ighm constant region transcripts found in bighead catfish, North African catfish and their hybrid catfish conform to the patterns of organization reported previously in other vertebrate and teleost Ighm genes. The Ighm constant regions of the secreted form of Ighm are a typically spliced pattern, as found in other teleosts, which are encoded by four Cm protein domains, Cm 1 , Cm 2 , Cm 3 and Cm 4 , without a transmembrane protein domain at the carboxyl-terminal region (7,61). In addition, the general mRNA splicing patterns to generate the membrane form of Ighm were found in all studied catfish by direct splicing from Cm 3 to the first transmembrane exon with the lack of a cryptic donor splicing site (T/C/G↓GGTAAA), as reported in the 3′ region of the Cm 4 exon in mammalian, shark, and amphibian mIg molecules. In this study, two domains of transmembrane proteins of Ighm were found in all studied catfish as well as in channel catfish and other teleosts (5,14,24,29).
Two unexpected and unusual Ighm constant region transcripts were found in bighead catfish. One is a novel Ighm transcript for teleosts, and the other is a novel transcript for vertebrates. The form is unusual in that the transmembrane domains are directly spliced to exon Cm 4 to create a membrane-bound form of Ighm that did not seem to conform to any patterns of the membrane form of the Ighm structure previously reported in teleosts. The molecule corresponded with Cm 1 -Cm 2 -Cm 3 -Cm 4 -TM1-TM2. Although this molecular pattern feature is typical of most mammals and vertebrates examined previously (62,63), the cryptic donor splicing site at the 3′ region of the Cm 4 exon, the acceptor site of the first transmembrane (TM1) exon (62,64), was not found in the case of this study. In this regard, the presence of the Cm 4 exon between Cm 3 and the first transmembrane domain of the Ighm membrane-bound form produced a unique characteristic of the mIg form as a novel membrane receptor feature of B cells in bighead catfish and teleosts.
Another important Ighm transcript was discovered in bighead catfish, and it is interesting in that the membrane-bound form of Ighm was encoded by two major exon domains, Cm and non-Cm exon domains. The molecular pattern was obviously characterized as Cm 1 -(non-Cm)-Cm 2 -Cm 3 -TM1-TM2. The presence of 487 amino acid residues of the non-Cm exon between the Cm 1 and Cm 2 regions of the typical membranebound form of Ighm was spliced directly to the end of the Cm 1 A B C FIGURE 6 | Representation of the overall relative expression and distribution of Ighm in bighead catfish, North African catfish and their hybrid catfish in sixteen tissues (A). Comparison of the relative expression of Ighm in sixteen different tissues (B). Expression responses of Ighm in immune-related organs, including the spleen, head kidney and PBLs, after intraperitoneal injection of a virulent pathogen, Aeromonas hydrophila, of bighead catfish, North African catfish and their hybrid catfish (C). The relative expression levels of Ighm genes were calculated using b-actin as a reference gene. All quantitative data are presented as the mean ± standard deviation (SD). The levels of statistical significance are indicated by *(P<0.05), **(P<0.01) or ***(P<0.001). The brain RT was used as the calibrator. All experiments are done in triplicate (n = 3). ns, not significant. exon without any additional or lacking amino acids. Thus far, the non-Cm exon was defined compared to the most closely and distinctly related published Igh locus, channel catfish (Ictalurus punctatus) (63,65) and zebrafish (Danio rerio) (13,66). The amino acid similarity of the non-Cm exon was 57.73% and 34.63% to the Ighd gene loci of channel catfish and zebrafish, respectively. It is also characterized as four Ighd constant exons based on the Ighd gene locus of channel catfish, C d2 -C d3 -C d4 -C d5 . There were 79, 220, 94 and 94 amino acid residues, respectively (63,65). In this regard, it is interesting that the 3′ end of non-Cm, C d5, was directly spliced to the Cm 2 -Cm 3 -TM1-TM2 exons with the identical polyadenylation signal at the C-terminus of the described typically membrane-bound form of the Ighm molecule in bighead catfish. The evolutionary relationship analysis of this novel membrane-bound form seemed to confirm the identification by grouping into the Ighd clade among other teleosts and sharing a branch to the Ighd of the closely related teleost species, channel catfish ( Figure 4). However, the presumptive non-Cm exon sequences clustered within the teleost Ighd rather than Ighm gene domains. The new pattern of mRNA splicing of the Ighm membrane formed by two major Ighm and Ighd constant exons could be defined as Cm 1 -Cd2-Cd3-Cd4-Cd5-Cm 2 -Cm 3 -TM1 and TM2. The presence of encoding Ighd constant exons inside the Ighm molecule suggests that this was a novel splicing pattern among vertebrate animals that has never been present in any class of vertebrates thus far (5,9,25,29,66). However, the unusual splicing of the two patterns was not found in either North African catfish or the hybrid catfish. Therefore, it is possible that bighead catfish express Ighm membrane receptors with at least three different mRNA splicing patterns.
Inside the gene organizations of constant Ighm regions, the lengths of the five introns separating these six exons seem to be similar to those of the closely related species channel catfish 42 and quite different from those of other vertebrates. The intron separating Cm 1 and Cm 2 was approximately 1.7 kb for bighead catfish, 1.8 kb for North African catfish and 1.7 kb for channel catfish (63). Xenopus also exhibited a long intron between Cm 1 and Cm 2 of approximately 1.6 kb (67,68). Smaller introns separating Cm 2 to Cm 3 and Cm 3 to Cm 4 of approximately 0.2-0.4 kb and 0.2-0.3 kb were found in bighead catfish and North African catfish, respectively, and were also identified in channel catfish (63). However, a longer intron (approximately 1.4 kb) between exons Cm 2 and Cm 3 was found in the horn shark Heterodontus francisci (69). An extensive distance between Cm 4 and the first transmembrane domain exons of approximately 1-3 kb seems to be observed in bighead catfish, North African catfish and all reported vertebrates, such as mouse, horned shark and channel catfish (63,(67)(68)(69) . The introns separating Cm 1 , Cm 2 , Cm 3 , and Cm 4 in mouse are all less than 0.5 kb (70,71). In addition, a large intron separating TM1 and TM2 exons was also approximately 1.3 kb and 1.2 kb for bighead catfish and North African catfish, respectively, as found in Xenopus (approximately 1.1 kb) and channel catfish (approximately 2.3 kb) (63,67,69) . On the basis of the obtained data, this Ighm gene organization was characteristically generalized to all teleost fish.
The gene localization of the Ighm gene region of the IgH constant region locus of bighead catfish, North African catfish and their hybrid catfish confirms that bighead catfish and North African catfish possess only a single copy of this gene, whereas their hybrid catfish possesses two copies of this gene ( Figure 1J). The finding that the Clarias catfish Ighm gene is most likely found in a single copy in the genome suggests that it may be common to all teleost fish, e.g., in channel catfish (72) and several other teleosts (26,73,74). The obtained Ighm constant gene organization of the hybrid catfish clearly showed that the sequences were identical to those of the parents. The presence of two copies of the Ighm gene in the hybrid catfish may be normal in diploid hybrid animals. In this regard, it is interesting that the Ighm transcripts of the hybrid catfish are processed from different gene loci, and the membrane-bound and secretory forms were made up of the Ighm gene of bighead catfish and North African catfish, respectively. The degree similarity of the amino acid was also identical to those of the parents. This available evidence would seem thus far to support the disease resistance characteristics of North African catfish and the hybrid catfish in several research reports. This suggests that the soluble effector molecules secreted from Ighm of North African catfish and the hybrid catfish may be strongly involved in specific immune responses, such as toxin or microbe neutralization, opsonization (immunophagocytosis), antibody-dependent cellmediated cytotoxicity (ADCC), and complement activation (immunolysis) (1,75).
Furthermore, the ability of Ig to interact and bind antigen was involved in the variable domains of Ighm molecules. Specific pathogens or antigens are recognized via the fragment antigenbinding (Fab) variable region (49). Additional analysis of the VH domain provided extensive insight. In our findings, the overall structures of bighead catfish, North African catfish and their hybrid catfish VH region corresponded to the V H, D H, and J H segments that consist of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, as reported in all vertebrates examined previously (49,76).
Analysis of the V H , D H and J H family classification based on their 80% similarities of amino acids interestingly demonstrated that a large number of V H gene families were defined in all studied catfish, of approximately 15-17 family members from one hundred nonredundant clones. There are similarities in some teleosts and some mammals, e.g., Atlantic salmon, zebrafish, and mice (14-18 families). However, there were also higher levels in the V H gene family than in vertebrates ( Figure 5C) (13,44,58). Much less is known and reported about the D H and J H segments of teleosts than about the V H of teleosts. Bighead catfish, North African catfish and their hybrid catfish process approximately 9, 11, and 13 families and 11, 11, and 8 families for a hundred nonredundant clones of D H and J H segments, respectively, compared to 9 in channel catfish, 4 in mouse, 6 in human, 6 in O. mykiss, and 2 in G. morhua for the J H segment (73). To date, there have been few reports of D H region diversity in vertebrates and teleosts, and significant variability in D H segments can be inferred from the generation of variability in CDR3 defined in the different cDNA clone sequences (49,73). This available evidence indicates that the mechanisms of VH diversity generalization of Ighm are made up of several gene family members that are necessary for generating several distinct VH repertoires in all studied catfish. In addition, junctional diversity occurs at the junction of the V H -D H and D H -J H boundaries. These regions code for CDR3. Diversity is increased by the addition of P-nucleotides and N-nucleotides (49). P-nucleotides are often made-up palindromic sequences and added to the ends of the asymmetrical DNA strand. Moreover, the random nontemplate-encoded addition of 2 to 20 base pairs or N nucleotides changes the amino acid sequence in the hypervariable CDR3. P-and N-nucleotides are principally found in the V H -D H and D H -J H junctions of the assembled heavy chain gene (49,76). Furthermore, it was suggested that inversion (D-D joining), nucleotide deletion, heavy-light chain pairing and somatic hypermutation contribute to diversity in VH antigenic recognition after naive B cells are released to the periphery and encounter antigens (49,73). Based on the findings in the present study, these mechanisms may be used to generate diverse VH repertoires in bighead catfish, North African catfish, and their hybrid catfish.
Analysis of the CDR3s provided strong evidence that the CDR3 of all studied catfish is longer than those of Atlantic cod, trout, frog and mouse but shorter than those of rabbit, human and cattle (13,44,49,58). In addition, overall analysis of the VH domain of Ighm indicated that the amino acid deviation in the CDRs was higher than that in the FWs in all catfish. Notably, these findings generally indicated predominant conservation in the VH repertoire (49,73). However, the calculated amino acid variability and composition of all CDRs in VH interestingly demonstrated that high variability plots and variability in amino acid composition were observed in all CDR1s, CDR2s and CDR3s of all studied catfish compared to those of reported vertebrates (49,77,78). Based on our findings, the potential for increasing the structural variability of the VH domain in Clarias catfish and their hybrids seems to be higher than that of teleosts, particularly given the number of V H and J H family members, long length of CDR3, and high variability in all CDR1s, CDR2s and CDR3s. This may be a general feature of Clarias catfish, which has never been reported thus far.
The functional expression of Ighm in bighead catfish, North African catfish and their hybrid catfish was examined in different tissues. The Ighm gene of all studied catfish and two novel Ighm molecules of bighead catfish were typically expressed in any organ and predominantly expressed in immune-related organs, especially the head kidney, PBLs, spleen and liver tissues, which is in accordance with previous studies (13,79). High expression of the two novel transcripts in immune-involved organs may indicate specific unknown functions that need further investigation.
In our study, significant responses of Ighm expression among Clarias catfish and the hybrid catfish were observed after bacterial exposure. North African catfish and the hybrid catfish exhibited approximately 900-950-fold relative expression levels in head kidney and 400-500-fold in PBLs; these levels are much higher than those of bighead catfish by at least 2-2.5-fold changes. The head kidney in teleosts is considered a primary lymphoid organ similar to the mammalian bone marrow, a major hemopoietic organ and site of Ig and other immune cell production in teleosts (15). A. hydrophila is a significant pathogen of Clarias catfish in Thailand. It is highly pathogenic due to the presence of hemolysin and aerolysin virulence genes, which demonstrate high virulence in Clarias catfish acute septicemia. A virulent strain could cause death within 24 hrs. and resulted in abdominal effusion and varying degrees of internal organ hemorrhage. The precise mechanism by which specific IgM immune responses rapidly increased after 12-24 hrs. post-exposure is still uncertain in the A. hydrophila challenge experiment. It could be suggested in two ways. First, as a characteristic of acute septicemia, the virulent bacteria evenly divide speedily in the blood and are rapidly circulated to the major immune-related tissues such as the head and kidney, activating the chemokine signaling pathway and triggering an inflammatory response, as well as upregulating IgM in the host. Second, prior to conducting the experiment, all studied catfish were exposed to A. hydrophila in their surrounding environment, resulting in a strongly rapid upregulation of IgM specific to A. hydrophila 12-24 hrs. post-exposure.
Unexpectedly, the pattern of Ighm expression in hybrid catfish is consistent with their fatherhood of North African catfish. This evidence may imply a long mysterious question regarding the well-known characteristics of the bacterial resistance of North African catfish and the hybrid catfish, which normally are much higher than those of bighead catfish.
These results indicate that the Ighm transcripts in bighead catfish, North African catfish and the hybrid catfish are typically produced by splicing patterns of mRNA reported in all teleosts. Notably, in bighead catfish, two more unusual spicing Ighm transcripts of membrane-bound forms, 1) Cm 1 -Cm 2 -Cm 3 -Cm 4-TM1-TM2 and 2) Cm 1 -(Cd 2 -Cd 3 -Cd 4 -Cd 5 )-Cm 2 -Cm 3 -TM1-TM2, represent novel patterns of mRNA splicing of the Ighm membrane form for teleosts and vertebrates, respectively. However, the reasons for the addition of Ighd to Ighm molecules are still unknown. All Ighm transcripts were produced from a single gene copy in individual bighead catfish and North African catfish. In addition, the different Ighm transcripts, membrane and secreted forms, of diploid hybrid catfish consisted of two copies of the Ighm gene from the parents, bighead catfish and North African catfish. Extensive insight into the VH domain could generate a number of distinct structural variabilities in the VH domain in Clarias catfish and the hybrid catfish. The overall functional significance of Ighm in hybrid catfish is mostly similar to that in North African catfish.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by The Animal Ethics Committee, Kasetsart University, Thailand (Ethics ID: ACKU61-FIS-004). Written informed consent was obtained from the owners for the participation of their animals in this study.

AUTHOR CONTRIBUTIONS
AB designed and performed all the experiments and wrote the original manuscript. UN-N and PS revised the manuscript. PS designed the experiments and revised the manuscript. All authors listed have read and approved the manuscript for publication.

FUNDING
This work was financially supported by the Thailand Research Fund (TRF) and the Betagro Science Center, Thailand. This study is part of the project entitled "Genetics and Biotechnology for Improvement of Aquatic Animal Production" (contract number DPG5980003) awarded to Professor Uthairat Na-Nakorn under the "Distinguished Research Professor 2016 Award."