Molecular Immunology 153 (2023) 146–159

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Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm

Tracing the origin of fish immunoglobulins

Serafin Mirete-Bachiller, Francisco Gambon-Deza *
Unidad de Inmunolog´ıa Hospital do Meixoeiro, Vigo, Spain

A B S T R A C T

We have studied the origin of immunoglobulin genes in fish. There are two evolutionary lines of bony fish, Actinopterygii and Sarcopterygii. The former gave rise to
most of the current fish and the latter to the animals that went to land. Non-teleost actinopterygians are significant evolutionary, sharing a common ancestor with
sarcopterygians. There are three different immunoglob- ulin isotypes in ray-finned fish: IgM, IgD and IgT. We deduce that translocon formation in im- munoglobulins
genes occurred already in non-teleost Actinopterygii. We establish a relationship between no teleosts and teleostean fish at the domain level of different immu­
noglobulins. We found two evolutionary lines of immunoglobulin. A line that starts from Immunoglobulin M and another from an ancestral Immunoglobulin W. The
M line is stable, and the W line gives rise to the IgD of the fish. Immunoglobulin T emerges by recombination between both lines.

1. Introduction
The ray-finned fishes represent one of the two lineages of jawed
vertebrates. The early actinoptery- gian fossil record indicates that these
fishes emerged at least mid-Devonian (Pearson and Westoll, 1979). Most
Actinopterygii corresponds to the clade of the teleost. The broad radi­
ation of teleost is explained by fish-specific whole-genome duplication
events (FSGD) and secondary diversifi- cation events (Santini et al.,
2009). The non-teleost actinopterygians belong to a few lineages,
including Polypteriformes (bichirs), Holostei (bowfin and gar), and
Chondrostei (paddlefish and sturgeon) (Sallan, 2014; Friedman, 2015).
These fish are often considered living fossils and share a common
ancestor with lobe-finned fishes (Hurley et al., 2007). The non-teleost
actinopterygians are of interest due to their phylogenetic position.
They preceded the appearance of teleost. The genomes and tran­
scriptomes of species of these fish have recently been obtained. (Bi et al.,
2021). Immunoglobulin genes first appeared in jawed vertebrates 500
million years ago (Flajnik and Kasahara, 2010). Immunoglobulins (Ig)
are glycoprotein complexes that are secreted (antibodies) or bound to
the cell membrane as part of the B cell receptor (BCR). Ig’s molecules are
composed of two heavy chains (IgH) and two light chains (IgL) linked by
disulfide bridges, forming a Y-shaped quaternary structure. The heavy
chain at its N-terminal end has a variable domain (VH) for antigen
recognition. In contrast, the C-terminal has several constant domains
(CH) that mediate effector functions, whereas IgL only has one variable
domain (VL) and one constant domain (CL). The genes for IgH and the
IgL in the jawed vertebrates (except Chondrichthyes) have an organi­
zation, so-called translocon (Flajnik and Kasahara, 2010), in which
multiple variables (V), diversity (D), and joining (J) segments are up­
stream of constant domains (Tonegawa, 1983). Immunoglobulin genes
have been studied in teleost fish, given the commercial interest of many
species and the use of certain species as experimental animals.
Teleost fish have three immunoglobulin isotypes IgM, IgD and IgT.
However, various species lacked immunoglobulin genes(Mir­
ete-Bachiller et al., 2021a; Swann et al., 2020). IgM was cloned and
characterized in Ictalurus punctatus and Gadus morhua (Ghaffari and
Lobb, 1989; Bengte´n et al., 1991). IgM in teleost has the highest con­
centration in serum. The heavy chain of teleost IgM has four constant
domains. Although its structure is highly conserved, there are differ­
ences in the number of domains between the secreted chain, which has
the four constant domains, and the one bound to the membrane, which
has a variable number that goes from three to a single domain (Ross
et al., 1998; Quiniou et al., 2011). Because there is no joining (J) chain,
polymerization takes place via interchain disulfide bonds (Castro and
Flajnik, 2014) putting existing monomers, dimers, trimers, and tetra­
mers (Ye et al., 2010; Su et al., 2019).
IgD in Teleostei was first described in Ictalurus punctatus (Wilson
et al., 1997). It has sub- sequently been described in other bony fishes
(Hordvik et al., 1999; Stenvik and Jørgensen, 2000). Domain analysis of
Salmo salar IgD revealed similarity to shark IgW and IgNAR, suggesting
an early evolutionary origin for IgD (Hordvik et al., 1999). Later it was
established that IgD is as old as IgM and that it is an ortholog of IgW
(Ohta and Flajnik, 2006). Teleost IgD has seven unique constant do­
mains (Gambo´n-Deza et al., 2010), although it lacks a domain capable of
binding to light chains. The Cµ1 exon that contains the cysteine residue
region, which binds to the light chains, is spliced with the first Cδ exon.

* Corresponding author.
E-mail address: fgambon@gmail.com (F. Gambon-Deza).

https://doi.org/10.1016/j.molimm.2022.11.021
Received 5 July 2022; Received in revised form 4 November 2022; Accepted 26 November 2022
Available online 8 December 2022
0161-5890/© 2022 Elsevier Ltd. All rights reserved.
S. Mirete-Bachiller and F. Gambon-Deza
Due to duplication and deletion processes, the number of constant do­
mains between different species varies widely (Parra et al., 2016).
IgT was described in two teleost fish Danio rerio and Oncorhynchus
mykiss (Danilova et al., 2005; Hansen et al., 2005). It was called IgZ or
IgT. However, the current consensus calls it IgT (Dornburg et al., 2021).
It is proved that it is not exclusive to this clade and is also present in fish
holostean (Mirete-Bachiller et al., 2021b). It usually consists of a CH
region encoded by four exons, but there are those with only two and
three exons (Savan et al., 2005; Gambo´n-Deza et al., 2010). It is not
present in all teleosts (Bengte´n et al., 2006; Magada´n-Mompo´ et al.,
2011; Bradshaw and Valenzano, 2020; Mirete-Bachiller et al., 2021b).
Its role in responding to mucosa infection is essential (Zhang et al., 2010;
Ryo et al., 2010).
In teleosts, the genes encoding immunoglobulin heavy chains occur
in a translocon conforma- tion (previously described). In summary, the
genes that encode IgD are located downstream of those of IgM as in the
rest of jawed vertebrates, while those of IgT are located upstream of
both. Thus, the general pattern responds to the following structure Vn-
Dn-Jn-Cτ-Dn-Jn-Cµ-Cδ. How- ever, both the configuration of the IgH
gene locus and the number of genes encoding the different isotypes is
variable among the different species of teleosts. Different studies have
highlighted the latter, in Gasterosteus aculeatusthe genes are organized
into a structure with three repeats of IGHT-IGHM-IGHD separated by
segments including the VH segments, as well as a fourth Cτ gene in the 3‘
end of the locus ((Vn-D-J-Cτ-D3-J4-Cµ-Cδ)3-V6-D-J-Cτ) (Gambo´n-Deza
et al., 2010; Bao et al., 2010). In Ictalurus punctatus, the IgH locus lacks
IgT and presents three repeats of IGHM-IGHD where only one of the IgM
is functional (Bengte´n et al., 2002, 2006). In Salmo salar IgH locus
possesses two parallel IgH isoloci (IGH-A and IGH-B), that are related to
the genomic duplication event in the family Salmonidae (Solem and
Stenvik, 2006). As the number of high-quality genomes increases, the
complexity of the fish IgH locus has been increasing, de- scribing, for
example, a greater number of fish families that lack IgT, different chi­
meras have been described (Buonocore et al., 2020; Bunnoy et al.,
2022). In this article, among other things, we address aspects of the
origin and complexity of the IgH locus in fish.
2. Material and methods
2.1. Sequence data and bioinformatics tools
We looked for immunoglobulin genes from non-teleost actino­
pterygians and teleosts in genome assemblies in webs from the https://
vgp.github.io/ and NCBI repositories. We used the gene- finding pro­
gram CHfinder to investigate immunoglobulin exons from chromosomal
regions. CHfinder is a multiclass neural network machine learning
classifier trained to identify exons coding for the CH domains of fish
immunoglobulins. For the training process, domain sequences of IgM
(CH1, CH2, CH3, and CH4), IgD (CH1, CH1B, CH2, CH3, CH4, CH5, and
CH6), and IgT (CH1, CH2, CH3, and CH4), together with random
background sequences, constituted the training set. The resulting neural
network has an accuracy of 99.99% for identifying Ig domains. The al­
gorithm for identifying these genes was described in a previous work of
our group (Mirete-Bachiller et al., 2021b). CHfinder has been adapted to
the search for immunoglobulin genes in tetrapods and sar- copterygian
fish. In the case of sharks and rays, the different immunoglobulin do­
mains present in them were obtained manually based on the preceding
literature.
2.2. Phylogenetic studies
We used the amino acid sequences of CH exons to obtain a fasta file
with all the sequences.Different programs present in the Galaxy platform
were used to carry out the phylogenetic studies.The se- quences were
aligned by the MAFFT algorithm using the default parameters for gap
opening and extension penalties (1.53 and 0) using the BLOSUM62
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matrix (Galaxy Version 7.221.3) (Katoh and Standley, 2013). The trees
were made with FASTTREE (Galaxy Version 2.1.10 +galaxy1) choos-ing
the protein evolution model WAG+CAT, and we use parameter gamma
(Price et al., 2010). The Figtree program (v1.4.4) was used as a graphical
viewer of phylogenetic trees ().
2.3. Transcriptome studies
For the study of the IgD/T chimaera described in the channidae
family, we used different tran- scriptomes from different tissues of
Channa argus (snakehead). From the project with access num- ber in
NCBI PRJNA299850, we used 6 transcriptomes made on head kidney,
three of which corre- sponded to three control fish (SRR2824352,
SRR2824351,SRR2824334) and three 72 h after infection with Aero­
monas hydrophila (SRR2824332,SRR2824344,SRR2824331). Spleen and
gill transcriptomes were also analyzed (SRR13503249,SRR10895940,
SRR13503245,SRR13503250). Different programs present in the Galaxy
platform were used for the analysis. HISAT2 was used to align tran­
scriptome sequences to sneakhead Chromosome 15 (Kim et al., 2015).
The results ob- tained with HISAT2 in BAM format were filtered using
the option to select only those transcripts that had mapped to the
chromosome (Barnett et al., 2011). To obtain the transcripts potentials
we use Stringtie (Pertea et al., 2015). To visualize the results we used
Genomeview (Abeel et al., 2012).
In an analogous way, the study of the transcriptomes of spleen, gills
and kidneyhead in An- guilla anguilla (eel) (SRR8074177, SRR8074181,
SRR8074185, SRR8074186, SRR8074190) was carried out.
3. Results
3.1. Immunoglobulin genes in non-teleost ray-finned fishes
We looked for immunoglobulin genes using CHfinder in the earliest
Actinopterygii for which reference genome assemblies are available:
Erpetoichthys calabaricus (reedfish), Polypterus sene- galus (gray bichir),
Amia calva (bowfin), Atractosteus spatula (alligator gar), Polyodon spa­
thula (american paddlefish) and Acipenser ruthenus (sterlet sturgeon)
(Rhie et al., 2020; Bi et al., 2021; Du et al., 2020). We have not include
Lepisosteus oculatus (spotted gar) in this analysis because its locus was
previously described (Mirete-Bachiller et al., 2021b). A graphical rep­
resentation of the results obtained with the CHfinder was made for the
CH coding exons of immunoglobulin in these fish (see Fig. 1).
In polypteriformes (reedfish, grey bichir), we found a single gene for
four exon IgM and a multiple-exon IgD. Both Acipenseriformes studied
(American paddlefish and sterlet sturgeon) found two loci located on
different chromosomes with a gene for four exon IgM and a multiple-
exon IgD. Additionally, in the sterlet sturgeon, an IgD is arranged up­
stream of one of the loci. In neither of the two fish did we find the IgW in
Acipenser sinensis (Zhu et al., 2016). In the Holstein (bowfin, alligator
gar), we found IgT, confirming what has already been described.
3.2. Relationship of ray-finned fishes immunoglobulins with other
immunoglobulins
One of the main problems in the phylogenetic study of immuno­
globulins resides in the number and type of domains that compose them.
The IgD in bony fish, IgW and IgNAR in sharks, and the IgW in sar­
copterygian fish have led to the postulation of at least two primitive
immunoglob- ulins IgM and IgD/W (Hordvik et al., 1999; Ohta and
Flajnik, 2006). With the CHfinder program, we obtained 2121 domain
sequences of the immunoglobulin genes. There are domains of im-
munoglobulins from sharks and rays, Actinopterygii, sarcopterygian
fish, and animals that came ashore (amphibians, reptiles). We construct
a phylogenetic tree (see Fig. 2).
The unrooted tree image shows four different clades. Considering
that IgM is the most con- served immunoglobulin in evolution, we take
S. Mirete-Bachiller and F. Gambon-Deza Molecular Immunology 153 (2023) 146–159

Fig. 1. Representation of the coding exons for CHs of immunoglobulins in non-teleost actinopterygians. In green are the exons for IgD, red for IgM and orange for
IgT. It details the location in the genome of the loci for immunoglob- ulin, as well as the number of exons present in each.

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Fig. 1. (continued).

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Fig. 2. A phylogenetic tree was made with the deduced sequences of the coding exons for CHs of the jawed vertebrates. In brown, the domains correspond to the M
lineage, and in blue, those of the W/D lineage, in green the clade consisting only of the CH4 domain of IgT. The objective of the figure is to establish the relationship
existing in origin between the domains of the different immunoglobulins. The CH deducted from each clade is noted.

its domains as a guide (Rast and Litman, 1998). Each clade can be
defined by the CH domains of the IgM present. In this way, we name
each clade CH1M (presence of the CH1 IgM domain), CH2M, CH3M, and
CH4M. This observation suggests that all classes of immunoglobulins
present today come from four ancestral domains., These domains
constituted the first IgM and IgW. Subsequently, the rest of the immu­
noglobulins present today originated (see Fig. 3).
Clade I has two distinct subclades. In the CH1M subclade, we find the
CH1M exon of all the species studied and the CH1T of the fish IgT. The
CH1W subclade is where we find the CH1 exon of the rest of the im­
munoglobulins studied, except Actinopterygii IgD.
Clade II has two subclades. In the CH2M subclade are the CH2M
domains of all classes of immunoglobulins studied. The CH2W subclade
presents the CH2 domains of the rest of the immunoglobulins. IgD of
bony fish instead of CH2D we find CH1D (these results indicate that the
first domain of the IgD corresponds to a CH2D domain). In addition,
existing others CHs suggestive of coming from a common ancestor.
Clade III has two subclades. In the CH3M subclade are the CH3 do­
mains for IgM, IgY, and IgX/As. The CH3W subclade presents different
domains of different immunoglobulins: IgW, IgN, IgD, IgNAR, and the
CH3T of the fish IgT.
In Clade IV, two subclades are identified. In the CH4M subclade, we
find the sequences of the domains CH4M, CH4Y, and CH4X/A. The
CH4W subclade presents different domains of different

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Fig. 3. A schematic representation with the results of the previous phylogenetic tree. Each exon of the Igs studied from different animals is assigned according to
their ancestry to the M lineage or the W lineage. The only Igs of mixed origin examined are IgT, IgA and IgY.
immunoglobulins: IgW, IgN, IgD, and IgNAR.
We observed an extra clade represented only by the sequences of the
CH4T domains of IgT (see Fig. 2). This domain does not root into any
domain of lineage M or W. Our group has previously described this
observation (Mirete-Bachiller et al., 2021b).
Results indicate that at first there were four ancestral domains. The
first immunoglobulins were IgM and IgW. IgM is a highly conserved
antibody, while the immunoglobulins derived from the W lineage have
changed evolution, both in the number of domains and in their combi­
nations according to origin. There is an accelerated dynamic in the
evolution of the W line. This dynamic is manifested by exon duplications
and by the generation of new isotypes. This may explain the emergence
of several subclades (especially in the CH3W and CH4W subclades). In
addition, two new immunoglobulins emerged with the passage of the
animals to the land, IgY and IgA/X. These immunoglobulins arose by
recombination between the M lineage and the W lineage (Gambon- Deza
& Mirete-Bachiller, 2022). Something similar happens with the IgT that
arose with the Neopterygii, although it remains unclear what the origin
of the CH4T domain is. These three immunoglobulins emerged once the
translocon was formed. Until then, there is no evidence of the existence
of recombination processes between the M and W lines.
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Molecular Immunology 153 (2023) 146–159
3.3. Immunoglobulin D in non-teleost ray finned fishes
We studied the immunoglobulin D genes in the earliest Actino­
pterygii. In the Fig. 4 we see the sequence relationships between the
exons of the genes for immunoglobulin D in these fish. IgD gene has
seven unique sequences and duplicates, some of which vary depending
on the species under study. The exons encoding the duplicated domains
are very similar. This fact indicates that the nature of the duplication is a
very recent event. The cladistians fish (reedfish and gray bichir) have at
least one copy of each of the exons. The holosteans fish (bowfin and
alligator gar) have 6 of the 7 exons except exon 2. Acipenseriformes fish
present 4 of the 7 exons and lack exons 2,3 and 6.
We investigate the relationship between IgD exons from the first
Actinopterygii and Teleost IgD exons. We built a phylogenetic tree that,
in addition to the aforementioned exons, including the exons that code
for shark IgW, Latimeria chalumnae (coelacanth) and IgW for three
different species of lungfish, shark IgNAR, and the CH4 exon of shark
IgM was used to root the tree that we obtained. We include these jaw
vertebrates as they are the closest groups in evolutionary terms to
Actinopterygii. In the first place, all the exons of the IgD of the earliest
Actinopterygii have a close correspondence with the exons of the Teleost
IgD. The exons of the IgD of the ray-finned fishes have a common origin
with those of the IgW of the coelacanth. The pairing of domains CH2,
CH5; CH3, CH6, and CH4, CH7 of teleost IgD arose from a duplication
S. Mirete-Bachiller and F. Gambon-Deza
Fig. 4. Phylogenetic tree that establishes relationships between the exons of the immunoglobulin D genes of the oldest fish. The CH deducted from each clade is
noted. To build the tree we use Fasttree, choosing the protein evolution model WAG+CAT and we use Gamma20.
process that has already begun and is observed in non-teleost ray-finned
fishes, this is in line with what has been described by other authors on
teleost IgD (Hordvik et al., 1999; Gambo´n-Deza et al., 2010). Like
teleost, these fish lack exons originating from the primordial CH1 of the
W lineage (see Fig. 5). In addition, the terminal exon (CH7D) of the fish
IgD does not correspond to CH4W as in the rest of the Igs, but a CH2W.
This is another peculiarity of this IgD in addition to the absence of the
aforementioned CH1W.
Recent work described the presence in Dicentrarchus labrax (Euro­
pean sea bass) of an IgD/IgT chimaera and other fish of the moronidae
family. The authors emphasize that this is the product of a genomic
recombination event between the Cδ and Cτ genes after duplication of
the IgH locus, with a δ1/δ2/δ3/δ4/δ5/δ6/τ3/τ4 exon organization. Also
underlining that the expression in different tissues of this chimaera is
greater than that of IgD and, specifically in the skin, (also greater than
that of IgT) (Buonocore et al., 2020). Thanks to the use of CHfinder we
were able to explore the possibility that this chimerism was present in
other fish species. We found a similar chimaera in the genome of two
representatives of the channidae family, Channa argus (Northern
snakehead) and Channa maculata (Blotched snakehead). In Blotched
snakehead the organization of its exons is the following
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Molecular Immunology 153 (2023) 146–159
µ1/δ1/δ2/δ3/δ4/(δ2-δ3-δ4)n/τ3/τ4, and we found it on chromosome 2,
additionally, we found a similar arrangement on a contig unplaced. In
Northern snakehead we found on chromosome 15 two chimaeras with
the same organization and a pseudogene of this chimaera. We believe
that the formation process of this chimaera is analogous to that
described have been lost as well as CH2T, the presence of M1 is
explained by something that we had already underlined in a previous
work of our group, CH1T is evolutionarily related to CH1M and there are
numerous species with identical sequences in these two domains (Mir­
ete-Bachiller et al., 2021b). An interesting point of this finding is that
duplication processes of the IgH locus could be behind the appearance of
recombinant Igs in fish.
3.4. Immunoglobulin T genes
One interesting question is the extra clade of the CH4 of IgT and the
relationship with other immunoglobulin domains. We can speculate that
this exon was incorporated into IgT from a differ- ent gene for immu­
noglobulin heavy chains, like in shark IgNARs. We made different ap­
proaches, blast using the CH4T as a query, we also split the domain into
pieces in case it had been formed by a recombination process with
S. Mirete-Bachiller and F. Gambon-Deza Molecular Immunology 153 (2023) 146–159

Fig. 5. A phylogenetic cladogram made with the sequences of the coding exons for CHs of the non-teleost ray- finned fishes IgD (red), teleosts IgD (green), IgW, IgM,
and IgNAR from sharks (brown), and IgW from Sarcopterygii (black). We use the Ch4 exon from shark IgM to root the cladogram. To build the cladogram we use
Fasttree, choosing the protein evolution model WAG+CAT and we use Gamma20. We identified the 4 primitive exons of the IgW lineage using the previously
deduced normalization scheme (boxes in different colors for each exon).

another exon, none of this brought a result that would support this
speculation. This leads us to consider that this exon, like the rest of the
immunoglobulin exons, arose from a CH4 exon of the W line or the M
line and that this extra clade is due to a process of rapid divergence by
selective pressure. However, we have carried out various phylogenetic
studies and the data obtained are not solid to affirm with certainty that
its origin is in the W line. (Fig. 6).
On the other hand, an interesting finding that we obtained from the
analysis of fish Igs using CHfinder is that, contrary to what was thought
until now, there are IgTs not only with 2, 3, or 4 exons but with multiple
exons. This is analogous to what occurs in fish IgD and, as we can see in
Fig. 2 3, extends to all Igs in which the CH2, CH3, and CH4 exons have
their origin in the W lineage. On the other hand, those Igs whose origin
from these exons is the M lineage Igs with multiple exons have not been
identified (IgM, IgA, and IgY). This data reinforces the idea that the
origin of the CH4 of the IgT could be in the W lineage. This observation
was made between three different species of fish Megalops cyprinoides,
Planiliza haematocheilus and Anguilla anguilla (see Fig. 7).
3.5. IgD/T in the channidae family and multidomain IgT
From the study of Northern Snakehead transcriptomes, we only
found chimaera IgD/T in the headkidney. In the spleen and gills, we did
not obtain mature mRNA molecules from this gene. In three samples

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Fig. 6. Representation of the coding exons for CHs of immunoglobulins in Channa maculata and Channa argus. In green are the exons for IgD, red for IgM and orange
for IgT. It details the location in the genome of the loci for immunoglobulin.

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S. Mirete-Bachiller and F. Gambon-Deza
Fig. 7. Representation of the coding exons for CHs of immunoglobulins in Megalops cyprinoides,Planiliza haema- tocheilus and Anguilla anguilla. In green are the exons
for IgD, red for IgM and orange for IgT. It details the location in the genome of the loci for immunoglobulin.
from the second of the two identified chimaeras, we found mRNA
molecules in both transmembrane and secretory forms. In addition,
some of the transcripts lack the exons that encode the 3 T and 4 T do­
mains, and therefore we would be talking about an IgD. In the mature
mRNA molecules of these chimaeras, we observed the presence of a
small peptide that serves as a link between the 3 T domain and the last
expressed 4D domain. In the gene, this peptide has a separate exon.
Following analysis of the eel transcriptomes, we discovered that only
the spleen contains IgT transcripts that contain multiple immunoglob­
ulin domains for IgT. Only the second IgT of multiple exons (ten) is
expressed. Different transcripts are generated by differential splicing.
However, these transcripts are anomalous in that they have a cap
attached to the J and contain stop codons. This leads us to speculate on
three possible explanations: the transcripts are not productive and do
not generate any type of IgT. The transcripts generate a multidomain IgT
that lacks VDJ, similar to what has already been described for fish IgD.
Another last possibility is that the yield of non-viable transcripts (with
the cap) is much greater than that of viable ones, and therefore we are
unable to visualize them. It is a question that remains open, at the
expense of analyzing the transcriptomes of more fish that present this
particularity and that are available. All this is summarized in a figure
(see fig. reffig:sra2).
4. Discussion
All immunoglobulin heavy chain genes in Actinopterygii and
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Molecular Immunology 153 (2023) 146–159
tetrapods are in the so-called ’translocon configuration’, in which there
are variable (V) genes from many families upstream of many diversity
(D) (for heavy chains) and joining (J) segment (Bengten et al., 2000).
The translocon IgH configuration is already present in the earliest
Actinopterygii. This configuration is an event before and independent of
that occurring in the animals that came ashore. This event explains the
similarities and differences between both configurations. The estab­
lishment of the translocon is thought to have influenced the develop­
ment of recombinant immunoglobulins be- tween the M and W lineages.
(Figs. 8 and 9).
The genomes of non-teleost ray-finned fishes facilitate the study of
the origin of immunoglob- ulins. In this work, we used a machine
learning program (CHfinder) to obtain thousands of Ig exon sequences
from animal species. These sequences allowed us to construct a broad
phylogenetic tree and determine that the exons originated in one of two
primary lineages: the M or W lineage. From this tree, we have also been
able to deduce the appearance of Igs with a mixed lineage of exons. This
phenomenon appears after translocon is evolutive conformed (IgT in
fish, IgA and IgY in land-bound animals (Gambon-Deza and
Mirete-Bachiller, 2022)).
The most exciting findings in fish immunoglobulins have been ob­
tained from the study of IgD and IgT. In the case of IgD, we find that non-
teleost ray-finned fish already have a configuration of 7 exons. Three of
them arose from a duplication process. These findings are consistent
with previous teleost research (Hordvik et al., 1999; Gambo´n-Deza et al.,
2010). In addition, the IgD gene is already located near to IgM gene,
S. Mirete-Bachiller and F. Gambon-Deza Molecular Immunology 153 (2023) 146–159

Fig. 8. Schematic representation of the expression in kidneyhead of the snakehead IgD/T chimera. Boxes in gray represent the expressed immunoglobulin domains,
in pink binding peptide 4D-3 T, in green the segment J, in blue transmembrane domain and in brown cytoplasmic stem.

Fig. 9. Schematic representation of the expression in the spleen of the IgT of multiple domains in eel. Boxes in gray represent the expressed immunoglobulin do­
mains, in black the cap in 5´, in green the segment J, in blue transmembrane domain and in brown cytoplasmic stem.

forming the translocon. Fish IgD requires the CHM1 domain to bind both
VDJ and light chains. Our analysis highlights that fish IgD lacks a CH1W
domain in its structure, the CH1D domain being a CH2W. The terminal
exon corresponds to a CH2W rather than a CH4W or CH4M as in the rest
of the Igs. Although the presence of this terminal exon whose origin is a
CH2W is striking at the genomic level, it is not so much at the tran­
scriptome level. In different species of African lungfish, they found IgW
transcripts where the terminal domain corresponds to the CH2 exon (our
analysis has identified this exon as CH2W) (Zhang et al., 2014). In
Ginglymostoma cirratum (nurse shark), one IgW gene is by itself capable
of producing a wide array of isoforms differing in length and domain
combination. The domain- level truncated isoforms they found have a
CH2W domain as terminal CHW (Zhang et al., 2013). They postulated
that the truncated secreted isoforms had diminished effector functions,
as in duck IgY (Magor et al., 1994). We think that this unique configu­
ration of fish IgD exons is why IgW/D phylogenetic trees with different
groups of animals, fish IgD has yielded a separate clade (Ohta and
Flajnik, 2006). We also describe the presence of an IgD/T chimaera
within the Channidae family, similar to the one previously described in
the Moronidae family. This result leads to the thought that more
chimaeras could be in other families where the IgH locus is duplicated.
IgT is found in Neopterygii, and the early branching fish groups lack
it. It can present up to four different domains. The CH1 domain, its
origin, is in the M lineage, while the rest is in the W lineage. On the other
hand, we have identified several fish genes for IgT with multiple exons
and not only with two, three, or four exons as was believed until now,
similarly to IgD. These findings raise whether IgT evolved before or after
the appearance of Actinopterygii. On the one hand, the need to adopt the
translocon configuration to form recombinant immunoglobulins would
exclude the common ancestor of Sarcopterygii and Actinopterygii,
which does not yet present it, thus limiting the appearance of this IgT to
the appearance of Actinopterygii. The emergence of the Neopterygii
brought with it modifications in the skeleton and better control of
movements through their fins, which allowed the colonization of new
ecological niches (Lopez-Arbarello, 2012). On the other hand, the dis­
covery of IgD/T chimaeras reveals how they emerged as IgTs. We
believe that the common ancestor of Holostean fish and Teleost fish
underwent a duplication of the IgH locus, and like chimaeras, this
duplication generated the IgT gene. This new Ig represented an evolu­
tionary advantage, since the role of IgT has been linked to the specific

156
S. Mirete-Bachiller and F. Gambon-Deza
defence of the mucosa. The latter would explain why CH4T generates a
different clade in the broad phylogenetic tree that we carry out, since it
is the domain closely linked to the function of this immunoglobulin. This
result would also explain why we find it upstream of IgM and not
downstream, as in the case of animals that went to the land where the
genic distance between IgM and IgD is greater and would facilitate
recombination events without the need for locus duplication. In these
animals, the immunoglobulin heavy chain locus is generally unique.
An interesting question is how the organization of the multiple Igs
genes in clusters (elasmo-branch) is passed to translocon (tetrapods and
fish) (Ohta and Flajnik, 2006). The first clues are in three articles
recently published. The genome assemblies of Protopterus annectens
(African lungfish), Neoceratodus forsteri(giant lungfish), and bichir were
generated; the first two are sar- copterygian fishes, and the last one is
one of the first actinopterygian fishes; thus, they share a proxy common
ancestor. They point out that animals have a large genome due to the
expansion of retrotransposons and that these would have contributed to
the genomic rearrangement of these fish, contributing to the appearance
of new genes with additional functions or different regulatory capacities
(Wang et al., 2021; Bi et al., 2021; Meyer et al., 2021). Another
important piece of information comes from another article where they
worked with African lungfish transcriptomes from different species and
described the presence of various immunoglobulins. They deduce that
genomic IgH loci in lungfish show characteristics of cartilaginous fish
and tetrapods, thus pointing out that Lungfish IgH genes are in a
Fig. 10. Schematic representation of the changes that occurred in the genome organization of immunoglobulin genes from elasmobranchs to fish. Elasmobranchs
present multiple loci with gene units composed of one V gene, D and J mini-genes, and a gene for constant chain (can be for IgM or IgW). In Actinopterygii the locus
for the heavy chains is present in the oldest fish. The emergence of IgT occurs with the appearance of the Neopterygii.
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Molecular Immunology 153 (2023) 146–159
transiting form from clusters to a translocon configuration (Zhang et al.,
2014). The IgH loci in Protopterus annectens and Neoceratodus forsteri we
are investigated. We observed a process of pseudogenization of some
IgM and IgW. The latter could be the reason why IgM was not in Lat­
imeria chalumnae (Coelacanth) (Amemiya et al., 2013). Al- though this
pseudogenization process observed in current lungfishes could be
recent, we speculate that these could have been fundamental to move
from the conformation of the IgH locus cluster in sharks to that of
translocon. This work has verified that both reedfish and bichir have an
IgH locus in the translocon configuration. These data lead to the
postulation that the common ancestor of both sarcopterygian and acti­
nopterygian fishes did not yet possess the translocon. Therefore, its
formation occurred at least twice, one with the fish actinopterygians and
another with tetrapods. In the case of fish, a more abrupt event in time
brought this configuration only for the IgH locus and not for the IgL
locus. In animals that went to the land, structural and physiological
modifications required to colonize this medium were greater. Adopting
this configuration for both the IgH locus and the IgL locus required an
intermediate situation we observed in the sarcopterygian. While fish Ig
genes with a recombinant origin arose as a consequence of locus
duplication processes, in the case of animals that went to land, their
appearance was marked by a greater genetic distance between IgM and
IgD that favored recombination processes between these two immuno­
globulins. This distinction indicates that in fish, Cτ is found in the
genome upstream of the IgM, whereas Cυ and Cα are found in
S. Mirete-Bachiller and F. Gambon-Deza
downstream tetrapods (Fig. 10).
In conclusion, there are two evolutionary lines in the immunoglob­
ulin genes. The highly conserved IgM gene line and the IgW gene line
take alternative forms. This last line gives rise to the fish IgD gene. In
fish, the translocon was generated in a different event from the gener­
ation of the translocon in tetrapods. The IgT gene was formed by a
recombination process between the two lines (CH1 was given by CH1M
and the other three come from the W line gene). This recombina- tion
probably occurred as a consequence of the genome duplication that
occurred in the evolution of the Actinopterygii.
CRediT authorship contribution statement
All persons who meet authorship criteria are listed as authors, and all
authors certify that they have participated sufficiently in the work to
take public responsibility for the content, including participation in the
concept, design, analysis, writing, or revision of the manuscript.
Data availability
Data will be made available on request.
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