Genetic Diversity of IGHM and IGHE in the Leporids Revealed Different Patterns of Diversity in the Two European Rabbit Subspecies (O. cuniculus algirus and O. c. cuniculus)

Simple Summary The study of European rabbit immunoglobulin genes has contributed decisively to the current knowledge on antibody structure and diversification. The European rabbit has also been increasingly used as an animal model for the study of many human diseases, such as syphilis, tuberculosis, and AIDS. As such, the study of its immune system genes is of crucial relevance, but the study of rabbit immunoglobulins has focused only on the IgG and IgA antibodies. In this study, we added to the knowledge of the rabbit immune system by investigating the genetic diversity of two antibodies, IgM and IgE, in wild and domestic rabbits as well as other rabbit close species. With the data obtained in this study, we showed a high similarity between the different rabbit close species studied and we pointed out important genetic differences in the wild and domestic rabbits. Our findings are a valuable tool for the management of rabbit wild populations and domestic breeds and may contribute to the identification of immunoglobulins genetic variants with greater efficiency against pathogens. Abstract The European rabbit (Oryctolagus cuniculus) has been an important model for immunological studies but the study of its immunoglobulins (Ig) has been restricted to its unique IgA and IgG. Here, we studied the genetic diversity of IgM and IgE in several species of leporids and performed population genetics studies on European rabbit wild populations and domestic breeds. The leporids sequencing showed that these Ig are well conserved (98% sequence similarity among leporids), For IgM the Cµ1 and Cµ4 were the most diverse and most conserved domains, respectively, while for IgE the Cε1 was the most diverse domain and Cε2 and Cε3 the most conserved domains. The differences in the pattern of most conserved and most diverse domain between the Ig isotypes are most likely related to each isotype function. The genetic population data showed contrasting results for IgM and IgE. For both Ig, as expected, a greater diversity was observed in the original species range, the Iberian Peninsula. However, unexpectedly the genetic diversity found for IgE in the domestic animals is higher than that for the French wild populations. These results will increase knowledge of the genetic diversity of leporids and wild and domestic rabbit populations and are important tools for the management of wild populations and rabbitries.


Introduction
Immunoglobulins (Igs) are one of the most emblematic components of the jawed vertebrate immune system. These heterodimeric glycoproteins act as a critical part of the immune system by recognizing and binding to antigens and subsequently triggering mechanisms that lead to its destruction [1]. In eutherian mammals, five Ig isotypes have been described, IgM, IgD, IgE, IgG and IgA. Differences between Ig isotypes reside in the constant domains of the heavy chain (CH) and determine that each isotype has specific effector functions [1].
The European rabbit (Oryctolagus cuniculus) has been used as an animal model for diseases and immunological studies (reviewed in [2]). One of the peculiarities of the European rabbit is the unique features of Ig isotypes. As some other mammals the European rabbit has no IgD and no functional IGHD gene has been found in the rabbit germline [3,4]. IgM and IgE are encoded by single genes, IGHM and IGHE, as well as IgG, a surprising fact among mammals that typically have multiple IGHG copies encoding multiple IgG subclasses [4,5]. For IgA, on the other hand, the European rabbit has at least 15 IGHA genes encoding 15 IgA subclasses [6,7] being the most complex IgA system of all studied mammals. These unique aspects may, perhaps, explain that the study of European rabbit Igs has focused on IgG and IgA (e.g., [6][7][8][9][10][11]) forgetting other Ig isotypes. In fact, the information available for European rabbit IgM and IgE is extremely scarce with only a few sequences available in public databases ( Figure 1).
IgM, in its transmembrane form, is the first isotype to be expressed during B-cell development. As such the VH and VL regions associated with this Ig have not undergone much antigenic driven diversification being able to recognize a wider range of antigens than other isotypes, although showing low affinity [1]. In mammals, IgM is secreted in pentamers, in which the IgM monomers are linked by disulfide bonds in the CH4 domain and further bound a J chain. This pentameric form is very effective in opsonizing pathogens and IgM antibodies are also very efficient at fixing complement [12]. Immunoglobulin E (IgE) has the lowest serum concentration and shortest half-life of all Ig subclasses. Despite this, it is a very potent immunoglobulin associated with hypersensitivity, allergic reactions and response to parasitic worm infections [1].
In this study, we extend the knowledge on IgM and IgE isotypes in leporids by sequencing the complete IGHM and IGHE genes for six additional extant leporid genera: Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus. Additionally, we compared the genetic diversity of these Igs between wild rabbit populations of O. c. algirus and wild and domestic populations of O. c. cuniculus.

Material and Methods
For the study of IGHE and IGHM evolution in leporids, we analysed specimens of Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus genera. To assess the genetic diversity of European rabbit we analysed 10 individuals for each of three wild populations: O. c. algirus collected in Portugal and O. c. cuniculus collected in Spain and in France ( Figure 2); as well as 15 domestic animals from 6 breeds: French Lop, Flemish Giant, Argent Champagne, Angora, New Zealand White and Netherland Dwarf. Total genomic DNA was extracted from frozen liver or ear tissue using an EasySpin Genomic DNA Minipreps Tissue Kit (Citomed). No animals were killed for this study. All samples used in this work belong to the Cibio/InBio sample collection and had been previously used in other works [11,29,30]. still unresolved, the widest consensus agrees that the Leporidae first basal group included Pronolagus, and that Romerolagus is at the second basal position [26][27][28].
In this study, we extend the knowledge on IgM and IgE isotypes in leporids by sequencing the complete IGHM and IGHE genes for six additional extant leporid genera: Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus. Additionally, we compared the genetic diversity of these Igs between wild rabbit populations of O. c. algirus and wild and domestic populations of O. c. cuniculus.

Material and Methods
For the study of IGHE and IGHM evolution in leporids, we analysed specimens of Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus genera. To assess the genetic diversity of European rabbit we analysed 10 individuals for each of three wild populations: O. c. algirus collected in Portugal and O. c. cuniculus collected in Spain and in France ( Figure 2); as well as 15 domestic animals from 6 breeds: French Lop, Flemish Giant, Argent Champagne, Angora, New Zealand White and Netherland Dwarf. Total genomic DNA was extracted from frozen liver or ear tissue using an EasySpin Genomic DNA Minipreps Tissue Kit (Citomed). No animals were killed for this study. All samples used in this work belong to the Cibio/InBio sample collection and had been previously used in other works [11,29,30].  PCR amplifications of the four IGHE and IGHM exons was conducted using primers designed on the basis of European rabbit available sequences (GenBank accession number AY386696) [4]. A fragment containing the IGHE CH1 and CH2 domains was amplified using primers Sequences obtained in this study were edited and aligned using CLUSTAL W [31] as implemented in BioEdit software [32] and the amino acid sequences were inferred using the software BioEdit [32]. The obtained sequences were also aligned and compared to European rabbit sequences available in GenBank. Codon numbering is according to the IMGT unique numbering for C-DOMAIN [33].
Sequence nucleotide diversity was estimated using DnaSP version 5.10 [34]. Haplotype reconstruction was obtained for each individual using PHASE version 2.1 [35] as implemented in DnaSP version 5.10 [34]. DnaSP was used to estimate population genetic parameters (S, π), Tajima's D, number of haplotypes, haplotype diversity, variance of haplotype diversity and standard deviation of haplotype diversity.
Sequences obtained in this study were deposited in GenBank (Accession Numbers: MN650919-MN651079).

Leporids IGHE and IGHM
We newly sequenced the IGHM and IGHE genes for six leporid genera: Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus. The gene structure is similar to the European rabbit IGHM and IGHE and we thus inferred the intron-exon organization from published rabbit IGHM and IGHE sequences. Splicing signals are present at the intron boundaries and therefore we assumed that all studied leporids share the same IGHM and IGHE exon organization as the European rabbit.

Cµ1 Domain
The Cµ1 domain is the most varied of leporid IGHM domains with 24 amino acid variable positions, the majority of which involve one substitution and 11 are conservative regarding the amino acid properties. For the Cµ1 domain some genera specific residues are observed: Arg 117 for the European rabbit, Asn 3 and Ala 122 for Pentalagus and Asn 115, Gln 117 and Asn 118 for Romerolagus ( Figure 3).

Cµ2 Domain
The leporid Cµ2 domain is fairly conserved with only 12 amino acid variable positions. The great majority of these polymorphic positions involve one substitution but seven encode for changes in amino acid properties. The European rabbit has specific Ser residues at positions 100, while Lepus have specific Lys 45 residues and Sylvilagus have Ala 90 residues ( Figure 3).

Cµ3 Domain
The variability observed for the leporid Cµ3 domain is similar to that of the Cµ2 domain. Of the 10 variable positions present in the CH3 domain all but one involve one substitution but seven encode for changes in amino acid properties. Interestingly, the European rabbit has two Pro residues at positions 1.

C 2 Domain
In the leporid C 2 domain 16 amino acid variable positions were observed. These involve one substitution but the majority encodes for changes in amino acid properties. Two diagnostic positions were observed for Romerolagus Gln15 and Phe21 (Figure 4).

C 3 Domain
The leporid C 3 domain has 14 amino acid variable positions, two of which involve more than one substitution and five encode for changes in amino acid properties. Two genera specific positions were observed for each of three genera: Lepus has specific Glu84 and Tyr102, Brachylagus has characteristic Asn45.4 and Asn84 and Pentalagus has as distinctive residues Thr85.1 and Thr116 ( Figure 4).

C 4 Domain
Contrarily to that observed for leporid IGHM, the C 4 domain has higher variability than the C 2 and C 3 domains. The C 4 domain has 19 amino acid variable positions, 13 of which involve changes in amino acid properties. Lepus has specific Glu 113 residues, Pentalagus has specific Cys 43 residues and Romerolagus has specific Glu13, Gln 45.2, Lys 45.5, and Gln 101 residues (Figure 4). Less than one quarter of the recovered SNPs involves nonsynonymous substitutions resulting in 7 polymorphic amino acid positions ( Table 2). Unique amino acids were found for O. c. algirus, G at CH1 residue 95, and Spanish wild O. c. cuniculus, I at CH3 residue 103, T and L at CH4 residues 82 and 114, respectively ( Table 2). Table 2. IgM amino acid polymorphic positions for European rabbit populations/domestic animals. The nucleotide variation is shown, dots represent identity. The codon numbering is according to the IMGT unique numbering for the constant domain [33].  (Table 1). Surprisingly, for the Domestic breeds 9 SNPs were observed but only three SNPs were observed for the French O. c. cuniculus (Table 1). The nucleotide diversity values are in agreement with the observed SNPs (Table 1). We recovered 24 haplotypes, two of which are common: one haplotype is shared among all populations (0.322 frequency) and another haplotype is shared between O. c. cuniculus French populations and domestic breed's (0.267 frequency); the remaining haplotypes are rare or uncommon (0.01-0.10 frequencies).

Subspecies Population Domain, Amino Acid Position and Most Common Triplet and Amino Acid
Approximately half of the recovered SNP's involve nonsynonymous substitutions resulting in 12 polymorphic amino acid positions (Table 3). Unique amino acids were found for O. c. algirus: M and F at CH2 residues 81 and 116, respectively; Spanish wild O. c. cuniculus: I and M at CH1 residues 4 and 84, respectively, and K at CH4 residue 82; and for the domestic breeds: K, A and T at CH1 residues 12, 44 and 45, respectively, G at CH2 residue 1.7 and V at CH3 residue 85 (Table 3)  Table 3. IgE amino acid polymorphic positions for European rabbit populations/domestic animals. The nucleotide variation is shown, dots represent identity. The codon numbering is according to the IMGT unique numbering for the constant domain [33].

Subspecies
Population Domain, Amino Acid Position and Most Common Triplet and Amino Acid

Discussion
The study of European rabbit immunoglobulin genes has contributed to elucidating numerous fundamental aspects of antibody structure and diversification mechanisms (reviewed in [36]). The rabbit has also been increasingly used as an animal model to many human diseases [2]. Despite this, the study of rabbit immunoglobulins has focused on the IgG and IgA isotypes. The knowledge on other leporids immune system genes is also very scarce.

IGHM and IGHE Evolution
The results obtained in this study show that throughout Leporid evolution IGHM and IGHE have been fairly conserved (each Ig has 98% sequence similarity among leporids), similarly to what has been found for IGHG [11]. A gradient in IGHM constant domains diversity was observed, with the Cµ1 domain being the most diverse and the Cµ4 the most conserved. This gradient in domain diversity was not observed for IGHE, the C 1 is the most diverse domain but C 4 shows greater diversity than C 2 or C 3 domains, the most conserved domains. For leporid IGHG a gradient in the constant domains diversity was described from the most conserved Cγ1 domain to the most diverse Cγ3 domain [11] and for the European rabbit IgA subclasses the Cα1 domain has twice more variable amino acid sites than Cα2 and Cα3 domains [6,7]. The differences in the pattern of most conserved and most diverse domain between the different Ig isotypes are most likely related to each isotype function. To trigger the effector functions that lead to pathogen elimination Igs must bind to other effector molecules such as Fc receptors and the complement. IgM interacts with three receptors, FcµR, Fcα/µR and pIgR, the binding sites of which have been mapped to the Cµ4 domain [37][38][39] and with the complement which binds to sites in the Cµ3 domain [40][41][42], the domains we found to be the most conserved during leporid IGHM evolution. IgE two receptors, Fc RI and Fc RII, binding sites locate to the C 3 [43,44], again the domain we found to be the most conserved. IgA ligands, both Fc receptors, FcαRI and pIgR, and some pathogen evasion proteins interact with sites on the Cα2 and Cα3 domains (reviewed in [45], the most conserved IgA domains in rabbit IgA subclasses [6,7] and during mammalian evolution [45]. As for rabbit IgG, it's receptor, FcγR, and the complement C1q protein have been shown to bind residues in the Cγ2 domain [46][47][48] a very conserved domain in leporid IgG evolution [11]. Thus, Ig domains which are likely essential for interaction with host ligands have been conserved during leporid evolution. Curiously, for the IgE C 3 domain all but two of the 14 amino acid variable positions we found, including the five positions that encode for changes in amino acid properties, lie in or in direct vicinity of the binding sites to Fc RI and Fc RII. Variation at these codons may prove adaptive by improving the binding of IgE to its Fc receptors. It would be interesting to study the rabbit IgE Fc receptors which may also show specific differences reflecting the host adaptation to different selective pressures imposed by the different environments that each leporid species inhabit.

Wild and Domestic European Rabbits IgM and IgE Diversity
The IP is the region were the European rabbit originated [13]. Later, during the 7 th century the rabbit was domesticated from French wild European rabbit populations [15,16]. Consistent with this, immunological markers genetic diversity has been shown to decrease from the IP to the French wild European rabbit populations and that the domestic animals are, to a great extent, a subset of the diversity found in the French wild O. c. cuniculus [29,30,36,[49][50][51][52]. The results we obtained for IgE show a greater nucleotide and haplotype diversity in the IP O.c. algirus populations, in agreement with the previously described pattern of highest genetic diversity in IP populations. Interestingly, the genetic diversity we found for the domestic animals IgE is higher than that for the French wild O. c. cuniculus (domestic animals nucleotide diversity is twice as high as for French O. c. cuniculus and haplotype diversity is 1,5 times higher for domestic animals, Table 1), similar to what was found for European rabbit IgG [29]. This may reflect changes in allele frequencies caused by the founder effect on the origin of domestic breeds [15]. Striking for IgE is also the fact that despite the highest number of SNPs was found for the wild Iberian populations, the higher number of unique amino acids was found for the domestic animals, again similarly to what was found for IgG [29]. This indicates that the domestic breeds are under a higher selective pressure, possibly due to higher densities leading to exposure to different parasitic worms, leading to the emergence and maintenance of mutations of functional significance. In fact, despite the prophylaxis adopted in rabbitries, the prevalence of parasitic infections in domestic rabbits is high and mixed infestations predominate (approx. 80%; [53,54]) as an excessive stocking density of animals favors invasion mechanisms [54]. On the other hand, the more ancient Iberian European rabbit populations have accumulated a higher number of random mutations. We cannot discard that the higher variability in the domestic rabbits could be associated with a compensatory mechanism already proposed by van der Loo [55]: to compensate the lack of genetic diversity shown by the rabbit domestic breeds for most immunologic genes, some particular genes show a higher diversity when compared to the wild populations.
The results we obtained for IgM, on the other hand, revealed a higher nucleotide and haplotype diversity for the O. c. cuniculus subspecies, particularly for the French populations, and that the domestic animals have the lowest genetic diversity (Table 1). Considering the results previously obtained for most immunological markers [29,30,36,[49][50][51][52] the higher genetic diversity observed for the French O. c. cuniculus was unexpected. Nevertheless, this higher nucleotide and haplotype diversity does not translate into higher number of polymorphic amino acid sites for the French O. c. cuniculus. Noteworthy for IgM is also the fact that only a small proportion of the observed SNP's (7 out of 31 SNPs) involve non-synonymous substitutions, in spite of the observed 31 SNPs only seven polymorphic amino acid positions were detected. IgM is the most ancient Ig isotype being present with the same function in all gnathostomes [56]. As such it is a very conserved gene that most likely suffers stochastic nucleotide substitutions and is subject to strong purifying selection.

Conclusions
Our findings show that throughout Leporid evolution IGHM and IGHE have been fairly conserved. Despite this, variable amino acid residues were identified for IgE which may improve the binding of IgE to its Fc receptors. The observed pattern of genetic diversity of IGHM and IGHE along the wild rabbit populations from Iberia and France is contradictory, being, as expected, higher for the Iberian populations for IGHE but surprisingly higher for French populations for IGHM. This is the first time that these two immunoglobulin isotypes were fully characterized for Leporids and for European rabbit wild populations and domestic breeds. These findings will be useful for the management of rabbit populations and for the understanding of the rabbit adaptive immunity against pathogens.