BACKGROUND
the solute carrier family  <dig>  is a gene family of divalent metal transporters, composed by two functional paralogs, slc11a <dig> and slc11a <dig>  the first member of the slc <dig> family, slc11a <dig>  also known as the natural resistance-associated macrophage protein  <dig> , is a divalent cation/proton transporter, which has been proposed to function as either a symporter  <cit>  or an antiporter  <cit> .
its expression is almost exclusively restricted to the membrane of late endosomes and lysosomes of immune cells of myeloid lineages   <cit>  and to neuronal cells  <cit> .
slc11a <dig> was first found to play a crucial role in the defense against several unrelated pathogens in mice, such as mycobacteria, leishmania and salmonella  <cit> , and several studies have shown that polymorphisms in slc11a <dig> are involved in many infectious  <cit>  and autoimmune  <cit>  diseases in humans.
however, the resistance mechanisms attributed to slc11a <dig> are still not fully understood  <cit> .
the second member of the slc <dig> family, slc11a <dig>  is also referred to as natural resistance-associated macrophage protein  <dig> , divalent cation transporter  <dig>  or divalent metal transporter  <dig> .
slc11a <dig> is a divalent cation/proton symporter  <cit> , with a ubiquitous expression  <cit> .
it is known to take up iron from the intestinal brush border in mammals and has been linked to transferrin-dependent iron transport from acidified endosomes to the cytosol in many different tissues  <cit> .
polymorphisms in slc11a <dig> are known to underline microcytic anemia in mice and rats, resulting from an impairment of iron recycling and intestinal absorption  <cit> .
slc <dig> homologs have been found in many distant evolutionarily related groups, such as humans  <cit> , mice  <cit> , rats  <cit> , birds  <cit> , fishes  <cit> , insects  <cit> , nematodes  <cit> , plants  <cit> , yeast  <cit>  and even bacteria  <cit> .
complete slc <dig> mrna coding sequences for teleost fishes have been published in the last few years.
single genes were described in carp   <cit> , channel catfish   <cit> , zebrafish   <cit> , striped bass   <cit> , japanese flounder   <cit> , turbot   <cit>  and red sea bream   <cit> , while two copies have been described in rainbow trout   <cit>  and fugu   <cit> , with evidence from other teleosts available in various genome databases.
most animal studies, particularly in teleost fishes, are focused on gene isolation and constitutive expression analysis, with little information on evolutionary and functional aspects.
furthermore, a complex picture emerges from the comparison of phylogenetic and expression studies between fishes and mammals  <cit> .
in fact, little is known about the structure and function of slc <dig> in lower vertebrates, although some studies provide evidence for a role of teleost slc11a <dig> orthologs in the nutritive metal uptake in the intestine  <cit> , and also an involvement in the response to bacterial infection.
it has been shown that slc <dig> mrna levels are elevated in response to lipopolysaccharide  and edwardsiella ictaluri  <cit>  in channel catfish, to vibrio angillarum in turbot  <cit>  and red sea bream  <cit>  and to mycobacterium in striped bass  <cit> .
however no research has yet elucidated this complex pattern of phylogenetic relationships and functional roles of teleost and mammalian slc <dig> genes and no explanations are provided as to why it seems that a homolog of the mammalian slc11a <dig> is performing the functions attributed to slc11a <dig>
a complement of two slc <dig> genes is shared between mammals and teleosts.
whilst it is known that mammalian slc11a <dig> and slc11a <dig> have likely resulted from genome duplications in early vertebrate ancestry   <cit> , the potential role of the teleost fish-specific genome duplication   <cit>  in the evolutionary history of this gene family has not been considered.
a comprehensive synteny study could thus help to improve our understanding of the evolution and functional specialization of these genes in teleost fish.
european sea bass  was selected as the teleost model for this study due to the growing amount of data on its immune system  <cit> , the possibility of making use of its partially sequenced genome  <cit>  and our previous experience with sea bass models of infection and iron modulation  <cit> .
sea bass is also an important marine aquaculture species in europe, afflicted by several diseases such as pasteurellosis and vibriosis.
since the early 1980s, its production has risen considerably  <cit>  evolving from extensive culture units to semi-intensive or intensive systems.
this massive fish concentration leads to an increase in organismal stress and as a consequence fish defenses get compromised, making them more susceptible to pathogen attack.
isolation and characterization of the slc <dig> gene in sea bass, as a candidate gene for host defense to infection with pathogens, may be of great benefit to better understand its role in the immune system and to the selection of disease resistant stocks  <cit> .
moreover, sea bass is part of the acanthopterygii superorder, which includes stickleback, tetraodon and fugu, organisms that have their genome fully sequenced, making possible a number of comparative genetic studies.
the aims of this study were to identify and characterize the sea bass slc <dig> homologs, clarify their evolutionary history and to determine their functional roles, in particular those related with the host iron metabolism and resistance to infection.
we evaluated the modulation of slc <dig> gene expression in sea bass upon iron modulation  or exposure to photobacterium damselae spp.
piscicida, in in vivo or in vitro experimental models.
we expect that this approach should provide an insight on the evolutionary history of the slc <dig> genes in the vertebrata subphylum.
RESULTS
southern blot
in order to determine the number of copies of slc <dig> genes in the sea bass genome, a southern blot analysis was performed .
after independent digestion of  <dig> μg of genomic dna with ecori or hindii and hybridization with a slc <dig> dig-labeled probe, different hybridization bands were visible.
no uncut products were observed.
whether digested with ecori or hindii, two different hybridization bands were visible, suggesting the existence of two copies of the slc <dig> genes in the sea bass genome.
sea bass slc <dig> transcripts
five different sea bass slc <dig> transcripts were obtained by primer walking and 5'/3' race with liver, spleen and intestine cdna and analysis of whole-genome shotgun contigs .
one transcript was named slc11a2-α and the other four transcripts were named slc11a2-β <dig> to β <dig>  since they were found to share  <dig> bp of their coding region , with the differences between them limited to the 5' and 3' endings .
sizes of the untranslated regions and coding regions, as well as starting and ending exons for each transcript.
slc11a2-β <dig> and slc11a2-β <dig> transcripts are of the same length regarding the coding region, although they differ in the first  <dig> bp, since they result from the alternative usage of either exon 1a  or exon 1b , both with  <dig> bp.
they also have in common the final exon , and the same 3' utr .
slc11a2-β <dig> and slc11a2-β <dig> transcripts are also of the same length and also differ in the first  <dig> bp, due to alternative usage of either exon 1a  or exon 1b .
both are slightly larger than slc11a2-β <dig> and slc11a2-β <dig>  since exon  <dig> presents an alternative splice site, losing the final  <dig> bp  and being partially substituted by the  <dig> bp of exon  <dig> .
slc11a2-β <dig> and slc11a2-β <dig> also share the same 3' utr, albeit different and smaller than the 3' utr for slc11a2-β <dig> and slc11a2-β <dig> .
genomic organization
the genomic organization of sea bass slc11a2-α and slc11a2-β was analyzed and compared with those of other fishes, amphibians and mammals .
exon/intron boundaries were determined by comparison of cdna, genomic dna and putative amino acid sequences, splice-site consensus matching and analysis of whole-genome shotgun contigs .
comparison of genomic dna sequences with the previously obtained cdna sequences showed that slc11a2-α consists of  <dig> exons and  <dig> introns, with a single initiator methionine and stop codon.
the genomic interval from the initiator methionine to the stop codon is  <dig> bp.
comparison of genomic dna sequences with the cdna sequences of the four slc11a2-β transcripts showed that those four transcripts result not from four different slc11a2-β genes, but rather from the alternative splicing of two 5' exons and two 3' exons of a single slc11a2-β gene , similar to what happens with human slc11a <dig>  <cit> .
the slc11a2-β gene comprises a total of  <dig> exons, with two initiator methionines and two stop codons.
differences in the 5'-termini are generated by alternative promoters with subsequent, mutually exclusive splicing of the respective first exons to exon  <dig>  whereas differences in the 3'-termini are due to alternative splicing of exon  <dig>  which presents an alternative 5' donor site .
in two isoforms, this leads to the reading of exon  <dig> and in the other two isoforms to the partial reading of exon  <dig> and also exon  <dig> .
the genomic interval from the first initiator methionine in exon 1a to the second stop codon in exon  <dig> is  <dig> bp.
for both α and β genes, exons 2- <dig> present a high homology with the equivalent exons from other fish and mammals, whereas exons in the n- and c-terminus are variable in size and sequence.
intron sizes, much like in fugu and tetraodon, are reduced when compared with mammalian homologs.
sea bass slc11a2-α and slc11a2-β present a compaction factor of  <dig> × and  <dig> × to human slc11a <dig> and  <dig> × and  <dig> × to human slc11a <dig>  respectively.
no clear sequences matching the iron responsive element  motifs, commonly found in mammalian slc11a <dig>  were identified in any of the 3' regions of either slc11a2-α or slc11a2-β.
however, one ire motif was found in the 5' region of slc11a2-β , with an upstream distance to the start codon of  <dig> bp, using regrna  <cit>  and corroborated manually.
full-length genomic sequences of sea bass slc11a2-α and slc11a2-β were deposited in genbank with accession numbers hq <dig>  and hq <dig> .
structure analysis of sea bass slc <dig> putative proteins
to predict the functionality of sea bass slc <dig> putative proteins, we compared them with other known slc <dig> proteins, and analyzed them using several bioinformatics tools , searching for characteristic features of this protein family.
the complete open reading frame of sea bass slc11a2-α comprises a single transcript of  <dig> nucleotides, encoding a putative protein of  <dig> residues , with a predicted molecular mass of  <dig>  kda.
the complete open reading frame of sea bass slc11a2-β comprises  <dig> different transcripts, encoding  <dig> putative proteins  that result from the alternative usage of two 5' exons encoding distinct n-termini and the alternative splicing of two 3' exons encoding distinct c-termini of the proteins.
two transcripts of  <dig> nucleotides, named slc11a2-β <dig>  and slc11a2-β <dig> , and two transcripts of  <dig> nucleotides, named slc11a2-β <dig>  and slc11a2-β <dig> , encode putative proteins of  <dig> and  <dig> residues, with predicted molecular masses of  <dig>  and  <dig>  kda, respectively.
the  <dig> amino acid discrepancy in length results not from the substitution of exon  <dig> for exon  <dig>  but rather from an alternative splice site in exon  <dig>  only the final  <dig> amino acids encoded by exon  <dig>  are replaced by the  <dig> amino acids encoded by exon  <dig> .
both exon 1a and exon 1b have the same size and encode the same number of amino acids.
comparison with other teleost and mammalian species slc <dig> proteins showed that the signature features of the slc <dig> family  <cit>  can also be found in the sea bass : twelve putative transmembrane domains , a conserved transport motif , cysteine residues in loops  <dig>   <dig> and  <dig>  other motifs observed in previously described slc <dig> proteins, including some exclusive to teleost fish, are also present in sea bass.
two n-linked glycosylation sites , located in loop  <dig>  are present in all slc11a2-β forms, but only one is present in slc11a2-α loop  <dig>  with another located in the c terminus.
a conserved protein kinase c phosphorylation site  was also found immediately before tmd <dig>  and one other was only found to be shared by sea bass slc11a2-α and fugu slc11a-α, in loop  <dig>  a third protein kinase c phosphorylation site was found to be present in the 5' cytoplasmatic extremity of sea bass slc11a2-β <dig> and slc11a2-β <dig>  striped bass slc <dig>  fugu slc11a2-α and trout slc11a2-α and β. six, five and four casein kinase ii phosphorylation sites  were found in slc11a2-β, slc11a2-β and slc11a2-α, respectively.
one tyrosine kinase phosphorylation site -d/e-xx-y) in loop  <dig> was found to be conserved in teleosts, with the exception of fugu slc11a-α.
one tyrosine based sorting signal  in the n terminus was also found to be conserved in teleosts, with the exception of the alpha forms of trout, sea bass and fugu.
several n-myristoylation sites  were also found to be conserved among all slc11a2-β forms , but one site was only found to be present in the 3' cytoplasmatic extremity of sea bass slc11a1-β, as well as striped bass slc <dig> and rainbow trout slc11a2-β .
all phylogenetic analysis, performed with the maximum-likelihood, maximum-parsimony, neighbor-joining and bayesian inference methods were found to be consensual, placing sea bass slc11a2-β clustered with other fishes slc11a2-β and slc <dig>  and sea bass slc11a2-α clustered with other fishes slc11a2-α, with the exception of trout slc11a2-α.
as with all other teleost fish slc <dig> homologs described so far, sea bass slc <dig> homologs are also closer to mammalian slc11a <dig> than to slc11a <dig>
paralogy and synteny
to determine the evolutionary history of the slc <dig> gene family in vertebrates, we explored in detail the genomic "context" of both human slc11a <dig> and slc11a <dig> paralogs.
other authors  <cit>  have previously suggested that slc11a <dig> and slc11a <dig> genes might have originated from the duplications of the genome in vertebrate ancestry, the so-called 2r, given their location to the hox chromosomes in hsa <dig> and hsa <dig> .
we find strong support for this hypothesis.
within a 1mb window in the vicinity of the slc11a <dig> and slc11a <dig> genes, various gene families have paralog members mapping to expected hox chromosome regions.
that is the case of tmbim <dig>  which maps close to the slc11a <dig> paralog in hsa <dig>  while faim <dig> maps to hsa <dig>  tns <dig> maps to the left end of the window of the slc11a <dig> gene, and has paralogs mapping to the three other human hox genome locations.
in the case of the slc11a <dig> gene,  <dig> gene families show a consistent duplication pattern, with paralogs mapping to expected regions of hox-linked paralogy, namely hsa2q .
in the analysed teleosts a similar slc <dig> gene complement is also found .
however, the precise orthology/paralogy relationships to the mammalian counterparts are yet to be firmly established.
while the phylogeny clearly indicates that both fish genes strongly group with tetrapod slc11a <dig> , other features  make the orthology assignment more contentious  <cit> .
syntenic data can be a powerful tool to facilitate the finding of orthologs and in the identification of duplication processes  <cit> .
the abundance of genome data from teleosts allows us the use of mapping data to clarify the phylogenetic findings.
we have analysed the genome locations and gene environment of the teleost slc <dig> genes in zebrafish , fugu , tetraodon , medaka  and stickleback  .
we find a strong degree of conservation between the gene arrangements of various fish species.
the vicinity of the teleost slc <dig> loci have their human ortholog equivalents mapping to the slc11a <dig> chromosome, hsa12q.
thus, we conclude that in agreement with the phylogenetic data, the genomic location of the teleost genes indicates that they are both slc11a2-like.
furthermore, we observe that the mechanistic origin of the slc <dig> genes is likely linked to the fish 3r genome duplication  <cit> .
we find in the vicinity of both fish isoforms, gene families with duplicates mapping to both slc <dig> fish chromosomal regions.
for example, we noticed that agap <dig> gene family in medaka has one member mapping to chromosome  <dig> , while a second isoform maps in chromosome  <dig> with the second isoform of teleost slc <dig>  the phylogenies of the genes families with duplicated members show that the duplication timing dates back to the origin of the teleosts .
thus, we can safely conclude that the teleost genes are of the slc11a <dig> type , and resulted from the fish specific genome duplication.
constitutive expression of slc11a2-α and slc11a2-β in sea bass tissues
to gain some insight into sea bass slc <dig> basic functions, mrnas constitutive expression of both forms was determined in relevant tissues.
constitutive expressions of slc11a2-α and slc11a2-β  were evaluated by real-time pcr in several tissues, namely liver, spleen, head/trunk kidney, gill, brain, stomach, pyloric ceca, anterior/mid/posterior sections of the intestine and rectum .
the liver was found to be the organ with the highest overall expression of the slc11a <dig> genes, followed by the mid and posterior portions of the intestine, stomach and spleen.
regarding the contribution of each slc11a <dig> form in the different tissues, there is a clear predominance of slc11a2-β in the liver, spleen, head kidney, rectum, gill and brain and a predominance of slc11a2-α in the stomach, pyloric ceca and all portions of the intestine.
constitutive expression of the four isoforms of slc11a2-β by semi-quantitative rt-pcr
in order to assess the relative contribution of each β isoform to the overall slc11a2-β constitutive expression, a more thorough analysis was performed.
relative constitutive expressions of the four isoforms of sea bass slc11a2-β were determined by semi-quantitative pcr, since the distance between alternative 5' and 3' exons is too great to adequately use real-time pcr .
the four isoforms were named β <dig> , β <dig> , β <dig>  and β <dig> .
in most of the tested tissues, there is a predominance of the slc11a2-β <dig> form, followed by slc11a2-β <dig>  slc11a2-β <dig> has a lower constitutive expression in most tissues, but presents significant expression in the head kidney, stomach and rectum, whereas slc11a2-β <dig> is the form with the lowest expression, although it represents 26% of the overall expression of slc11a2-β in the gill.
in situ hybridization
an in situ hybridization was performed in several tissues to identify the subcellular distribution of sea bass slc <dig> mrnas, to obtain further information useful in establishing parallelisms with their mammalian homologous counterparts, and to reiterate the results observed in the constitutive expression analysis.
in the liver , slc11a2-α mrna is very scarce, being detected in few hepatocytes whereas slc11a2-β in abundantly slc11a2-α mrna is scarce, being only conspicuous in the melanomacrophage centers.
slc11a2-β on the other hand is abundant not only in the melanomacrophage centers but also in the spleen's white pulp.
in the head kidney , both slc11a2-α and slc11a2-β have a similar distribution, being abundant in the melanomacrophage centers and the surrounding lymphomyeloid tissue, and to a much lesser degree in the hematopoietic tissue.
in the intestine, slc11a2-α and slc11a2-β mrna present similar patterns of distribution in the anterior  and mid section .
slc11a2-α is found to be mostly concentrated in the apical  membrane of the enterocytes, whereas slc11a2-β presents a more homogeneous distribution, not only in enterocytes but also in other intestinal cells.
however, in the posterior intestine , both forms of slc11a <dig> seem to be accumulated in or around the goblet cells, with limited presence in the enterocytes.
in all tissues, hybridization with sense probes  for either slc11a2-α or slc11a2-β produced no significant staining.
hematological parameters and tissue iron content in the in vivo experimental models
several hematological and iron parameters were measured to validate the models of in vivo experimental iron modulation or infection with photobacterium damselae spp.
piscicida.
during the experimental infection, mortality was monitored and found to be high, but with fish surviving past the final experimental time point, indicating a chronic infection .
in the iron overloaded animals, no changes were observed in hematocrit , and a small but significant increase was observed in the rbc count during the course of the experiment .
increases in serum iron and transferrin saturation  were also observed, the highest levels at  <dig> days after overload, with decreases during the course of the experiment and recovery to control levels at  <dig> days.
liver iron levels were increased , with the maximum value at  <dig> days post-modulation and a decrease towards  <dig> days, but still over  <dig> times higher than the control animals.
in the iron deficient animals, significant decreases of all hematological parameters were observed during the course of the experiment, with values remaining below the control levels .
liver iron levels did not differ significantly from control animals during the experimental iron deficiency .
significant and steady decreases of all hematological parameters were observed during the course of the infection .
liver iron levels increased slightly but not significantly from control animals .
slc11a <dig> expression in the in vivo models of iron modulation and infection
in order to determine the potential involvement of sea bass slc11a2-α and slc11a2-β in the iron metabolism and immune response, we evaluated their expression under conditions of iron modulation  or bacterial infection.
in the experimental iron modulation model, slc11a2-α and slc11a2-β expressions were initially evaluated in the liver, spleen, head kidney and intestine,  <dig> days after iron modulation .
in iron deficiency, there was a significant increase of slc11a2-α expression in the intestine, with no major changes in the other tested organs.
no significant changes were observed in slc11a2-β expression.
in iron overload, no significant changes in slc11a2-α were observed, whereas slc11a2-β expression was found to be increased in the liver.
slc11a2-α and slc11a2-β expressions were subsequently evaluated in the liver at  <dig>   <dig> and  <dig> days after iron modulation .
as before, no significant changes were observed in slc11a2-α expression to either condition in the liver, whereas slc11a2-β was found t be significantly up-regulated at  <dig> days post iron overload , decreasing during the course of the experiment to near control values, but still over 2-fold higher.
expression of slc11a2-α and slc11a2-β was also evaluated  <dig> days after iron modulation in several sections of the digestive tract , in order to identify the portion of the intestine responsible for the response of slc11a2-α.
significant increases of slc11a2-α expression were observed in the mid and posterior sections of the intestine, with no significant changes in the anterior section or rectum.
in the experimental model of infection, slc11a2-α and slc11a2-β expressions were measured in the liver, spleen and head kidney by real-time pcr,  <dig>   <dig>   <dig> and  <dig> h after infection.
in the liver , a steady increase in slc11a2-β expression was observed during the course of the infection, with the maximum value at  <dig> h post-infection , followed by a recovery to control levels at  <dig> h. in the spleen , an increase of slc11a2-β was also observed during the course of the infection, accompanied by a recovery to control levels at  <dig> h. in the head kidney , a significant increase of slc11a2-β was observed  <dig> h after infection and was maintained during its course.
no significant changes were observed in slc11a2-α expression in any of the tested organs.
slc11a <dig> expression in the in vitro models of iron overload and infection
we investigated the role of slc11a2-α and slc11a2-β in the iron metabolism and immune response at a cellular level, by using leukocytes from spleen, one of the most important erythropoietic and immune organs in fish.
in the iron overload in vitro model, expression of slc11a2-α and slc11a2-β in leukocytes was measured by real-time pcr at  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> hours after the addition of ferric ammonium citrate .
no significant changes were observed in slc11a2-α during the course of the experimental iron overload.
slc11a2-β, on the other hand, increased significantly from  <dig> h post-infection , reaching the maximum increase at  <dig> h  and decreased at  <dig> h, returning to control levels at  <dig> h.
in the in vitro infection model with heat-inactivated p. damselae, expression of slc11a2-α and slc11a2-β in leukocytes was also measured by real-time pcr at  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> hours after infection .
no significant changes were observed in slc11a2-α during the course of infection.
slc11a2-β, on the other hand, increased significantly at  <dig> h post-infection , followed by a decrease at  <dig> h  and returned to control levels at  <dig> h.
discussion
in the present study, we set out to analyse the evolutionary and functional patterns of slc <dig> genes in vertebrates, in particular teleosts.
we have used the european sea bass  as a model and have successfully isolated two slc <dig> genes, named slc11a2-α and slc11a2-β.
a single transcript was found to be produced, for the slc11a2-α gene, whereas slc11a2-β can produce up to four transcripts.
the four β transcripts are the result of alternative exon splicing in the 5' and 3' ends, in a similar fashion to what has been described for human slc11a <dig>  <cit> .
but unlike human slc11a <dig>  where the differences in the 3' end result from alternate exon usage  <cit> , in sea bass the differences result from an alternative splice site in exon  <dig>  thus, only the second half of exon  <dig> is replaced by exon  <dig>
neither sea bass slc11a2-α nor slc11a2-β were found to present a potential iron responsive regulatory protein binding site  in the 3' utr, a feature that in contrast is present in mammalian slc11a <dig> genes  <cit>  and has also been described in some teleost fish, such as fugu  <cit>  and common carp  <cit> .
it is, however, absent in other teleosts, such as turbot  <cit> , channel catfish  <cit>  and japanese flounder  <cit> .
in the slc <dig> gene family, ires have only been described in the 3'-utr but unexpectedly, an ire motif was found in the 5' region of slc11a2-β, albeit not in the 5'-utr itself but rather  <dig> bp upstream the start of the 5'-utr.
the significance of this finding is still unknown, although as with other 5' ires, it may be involved in translation repression  <cit> .
the single sea bass slc11a2-α transcript encodes for a single functional putative protein, whereas the four slc11a2-β transcripts encode for four putative proteins.
the four β proteins differ in the n and c termini, as a result of the encoding by the two alternative exons  <dig> and the alternative splice site in exon  <dig>  since both n and c termini are cytoplasmic, they may be involved in post-transcriptional regulatory processes and in the subcellular localization of these proteins, as occurs in mammalian slc11a <dig>  <cit> .
all sea bass slc11a <dig> proteins present the characteristic features of the slc <dig> gene family:  <dig> transmembrane  domains, a consensus transport motif , several glycosylation and phosphorylation sites and conserved cysteine residues.
multiple alignments clearly show that these features are highly conserved and are very similar to those observed not only in other teleost fish, but also in mammals.
however, there are some disparities in the features found in the 5' and 3' endings, namely some n-myristoylation and phosphorylation sites that are not present in all sea bass slc11a2-β forms, and which may also contribute to a differential post-transcriptional regulation and subcellular localization.
in all the tree models, produced by maximum-likelihood, neighbour-joining, maximum parsimony or bayesian inference, phylogenetic analysis places all sea bass slc <dig> proteins clustered with other teleost fish slc <dig> proteins, which in turn cluster with mammalian slc11a <dig> proteins, clearly separated from slc11a <dig>  among teleost proteins, there are two clear separate clusters.
one cluster encompasses sea bass slc11a2-β protein as well as all other slc <dig> and slc11a2-β proteins.
the other cluster includes sea bass slc11a2-α and all other α proteins, with the exception of rainbow trout slc11a2-α, suggesting that rainbow trout slc11a2-α may have a different evolutionary origin than other slc11a2-α proteins.
there are some explanations that could account for the existence of two closely related slc11a <dig> genes in sea bass, which are in agreement with what has already been proposed for other teleost fish, such as fugu.
the current evolutionary scenario proposes that slc <dig> paralogs appeared through the 2r genome duplication  <cit> , and functionally diverged.
this is supported by the close linkage to hoxc and hoxd which are assumed to be duplicate loci.
in addition, the teleost fish paralogs are more similar to mammalian slc11a <dig> and may result from a third, fish specific, genome duplication  <cit> .
the synteny analysis performed in the current study strongly supports the latter, suggesting that the ancestor of all vertebrates had a single slc <dig> gene that quadrupled as part of the 2r genome duplications.
two of the new genes were lost, while two were maintained in the ancestor of tetrapods and teleosts.
in teleosts the slc11a <dig> clade expanded as the result of 3r to originate two isoforms, slc11a2-α and slc11a2-β, while the slc11a <dig> ortholog was lost .
unlike other teleosts  <cit> , trout's slc11a <dig> homologs do not seem to result from 3r but rather a salmonid specific tetraploidization  <cit> .
the overall constitutive expression pattern of sea bass slc <dig> transcripts resembles that of the mammalian slc11a <dig> gene  <cit> , but also of slc11a <dig> genes described in other teleost fishes  <cit> .
slc11a2-α was found to be ubiquitously expressed, but with a higher expression along the digestive tract, with a particularly high incidence in the mid and posterior sections of the intestine.
this already gives some indications for its possible involvement in intestinal iron absorption in sea bass, since the slc11a <dig> mediated uptake of iron and other food derived metals in teleosts occurs predominantly in the mid and posterior regions of the intestine, as previously described  <cit> .
slc11a2-β was also found to be ubiquitously expressed, although unlike slc11a2-α, being abundant in the liver and in varying levels in all other tested tissues.
no clear pattern was observed regarding the distribution of the four isoforms of slc11a2-β, although a prevalence of the β <dig> and β <dig> forms in most tested tissues was evident.
further analysis is needed to determine the exact function and contribution of each isoform.
it is possible that they function in different subcellular compartments, with some of the forms more relevant in the immune response, while others may play a more prominent role in overall iron metabolism.
the in situ hybridization results in the liver, spleen, head kidney and sections of the intestine mostly reflect the measured constitutive expression.
in the liver and spleen there is a clear prevalence of slc11a2-β, whereas in the head kidney although the constitutive expression indicates a prevalence of slc11a2-β, there seems to be an almost even distribution of both forms.
note that in the spleen and kidney slc11a2-β mrna is clearly visible in the white pulp and lymphomyeloid tissue, respectively, which are both areas rich in b- and t-lymphocytes and actively involved in the immune response  <cit> .
in the intestine, the in situ results show that slc11a2-α and slc11a2-β are expressed in different compartments of the enterocytes, with slc11a2-α mrna mostly concentrated close to the brush border of the apical pole, whereas slc11a2-β is spread all over the cells, not only in the enterocytes but also in other intestinal cells.
this observation provides further evidence for a sub-functionalization of slc11a <dig> homologs in sea bass, with slc11a2-α localization resembling that of the mammalian slc11a <dig> ire-positive isoforms and slc11a2-β with the more general distribution of mammalian slc11a <dig> ire-negative isoforms  <cit> .
an issue that remains puzzling is the distribution of slc11a <dig> isoforms in the posterior intestine, with both forms concentrated in or around the goblet cells, the intestinal glandular cells that secret mucin  <cit> .
in mammals, there are conflicting reports on the presence of slc11a <dig> in the goblet cells, with some reporting its absence  <cit>  while others report its presence  <cit> , suggesting that, along with mobilferrin, slc11a <dig> facilitates iron uptake.
to better understand the role of sea bass slc11a <dig> genes, in vivo and in vitro experimental models of iron modulation and infection were created.
haematological and iron parameters, and expression levels of slc11a2-α and slc11a2-β were evaluated under those experimental conditions.
in the experimentally iron deficient animals, with decreased haematological parameters, slc11a2-α expression was significantly up-regulated in the intestine, whereas slc11a2-β did not respond in any of the tested tissues.
further characterization of the slc11a2-α response to iron deficiency in the intestine showed that the up-regulation was mostly confined to the mid and posterior portions of the intestine.
this is in accordance with previous findings that intestinal metal uptake in teleost fish occurs mainly in the posterior portion of the intestine  <cit> , unlike mammals, where uptake occurs primarily in the duodenum  <cit> .
these results further reinforce the idea that sea bass slc11a2-α seems to be mainly involved in iron uptake by the enterocytes.
in the experimentally iron overloaded animals, steady increases of the hematocrit and red blood cell counts were observed, as well as high levels of serum iron and transferrin saturation in the first days of the experiment.
also, increased levels of iron were observed in the liver, further confirming the iron overload status.
slc11a2-α expression did not change significantly in response to iron overload in any of the tested tissues and slc11a2-β was up-regulated only in the liver.
together with the increased liver iron content, this is likely associated with the need to cope with the excess of iron introduced into the system, in order to prevent its toxic effects.
in normal conditions, iron in the blood is bound to transferrin, and transferrin-iron complexes are internalized by a transferrin receptor-mediated endocytic pathway.
through acidification of the endosome, iron is released from transferrin and slc11a <dig> is recruited to the endosome membrane, where it exports iron to the cytoplasm.
iron is then used in cellular processes or stored in ferritin molecules.
when more iron is introduced into the system, an increased expression of these genes could be expected.
we have already demonstrated that during iron overload there is an increased expression of ferritin, although not accompanied by an up-regulation of transferrin, most likely due the already high levels of constitutive expression in the liver  <cit> .
in the in vivo experimental infection with live p. damselae, a significant and continuous decrease of all haematological parameters was observed, suggesting a condition that is commonly known as anaemia of inflammation or chronic disease.
this response was first described in humans  <cit>  and later reported for teleost fish, namely sea bass  <cit>  and nile tilapia  <cit> .
during bacterial infection, inflammatory cytokines, such as il- <dig>  prompt the liver to increase the production of the iron metabolism regulatory peptide hepcidin, causing the internalization of ferroportin and thus preventing cellular iron export  <cit> .
the iron retention leads to impairment in erythropoiesis, due to the lack of iron availability.
overall, these changes contribute to limiting the availability of iron for pathogen growth.
we have previously shown that hepcidin  <cit>  and ferritin  <cit>  levels are increased during bacterial infection in sea bass, thus confirming the presence of this mechanism in teleosts.
the bacterial challenge significantly up-regulated the expression of slc11a2-β in the liver, spleen and head kidney, in a time-dependent fashion, but produced no significant changes in slc11a2-α expression.
a similar up-regulation was observed in slc11a <dig> homologs of other teleost fish in response to v. anguillarum  <cit> , mycobacterium  <cit> , e. ictaluri  <cit>  and lps  <cit> .
further analysis will be required in order to determine whether in this case slc11a2-β is acting as the mammalian counterpart of slc11a <dig>  being recruited to the membrane of pathogen-containing phagosomes, or of slc11a <dig>  being recruited to the membrane of holotransferrin-positive recycling endosomes involved in iron uptake from the extracellular environment.
since there are no slc11a <dig> homologs in sea bass, and there are  <dig> different proteins encoded by slc11a2-β, it is tempting to propose that slc11a2-β is performing both slc11a <dig> and slc11a <dig> mammalian functions.
in the in vitro iron overload of spleen-isolated leukocytes, slc11a2-β was found to be up-regulated in a time-dependent fashion, whereas no significant changes in slc11a2-α expression were observed.
the increase in slc11a2-β expression is most likely due to the increase in iron accumulation in the macrophages, to cope with the excess iron introduced in the medium, which could become toxic for the cells.
the in vitro experimental infection with heat-inactivated p. damselae, in spleen-isolated leukocytes, produced the same effect as that observed in vivo, with an up-regulation of slc11a2-β and no changes in slc11a2-α expression.
not much information is available on the expression of slc <dig> homologs in leukocytes, but it is known that mammalian slc11a <dig> expression is stimulated by m. avium in peritoneal macrophages  <cit>  and by lps and ifn-γ in raw <dig>  macrophages  <cit> .
mammalian slc11a <dig> expression is also stimulated by m. avium in peritoneal macrophages, although differentially from slc11a <dig>  <cit> .
also, catfish slc <dig> was found to be stimulated in monocytes by lps  <cit> .
as for the in vivo experimental infection, it has yet to be determined whether slc11a2-β is functioning as slc11a <dig> or slc11a <dig>  but since both forms are known to be required for an efficient erythrophagocytosis and iron recycling in macrophages  <cit> , it is likely that it is performing both functions.
expression studies of the four slc11a2-β forms and subcellular localization in the leukocytes could provide some insight on this matter.
CONCLUSIONS
we have successfully isolated two slc <dig> paralogs in sea bass, that we named slc11a2-α and slc11a2-β.
we have demonstrated that they have a role not only in iron metabolism but also in the response to bacterial infection.
the clarification of the evolutionary scenario along with the functional data suggests a curious and complex pattern of sub-functionalization  <cit>  and paralog functional equivalence, a process previously named as synfunctionalization  <cit> : the fish specific paralog slc11a2-α retained in part the original mammalian slc11a <dig> function, whereas the slc11a2-β isoform not only retained the original slc11a <dig> function but also acquired the slc11a <dig> function after the loss of this isoform in teleosts .

 the solute carrier family is a gene family of divalent metal transporters, composed by two functional paralogs, slc11a and slc11a the first member of the slc family, slc11a also known as the natural resistance-associated macrophage protein, is a divalent cation/proton transporter, which has been proposed to function as either a symporter or an antiporter. its expression is almost exclusively restricted to the membrane of late endosomes and lysosomes of immune cells of myeloid lineages and to neuronal cells. slc11a was first found to play a crucial role in the defense against several unrelated pathogens in mice, such as mycobacteria, leishmania and salmonella, and several studies have shown that polymorphisms in slc11a are involved in many infectious and autoimmune diseases in humans. however, the resistance mechanisms attributed to slc11a are still not fully understood. the second member of the slc family, slc11a is also referred to as natural resistance-associated macrophage protein, divalent cation transporter or divalent metal transporter. slc11a is a divalent cation/proton symporter, with a ubiquitous expression. it is known to take up iron from the intestinal brush border in mammals and has been linked to transferrin-dependent iron transport from acidified endosomes to the cytosol in many different tissues. polymorphisms in slc11a are known to underline microcytic anemia in mice and rats, resulting from an impairment of iron recycling and intestinal absorption. slc homologs have been found in many distant evolutionarily related groups, such as humans, mice, rats, birds, fishes, insects, nematodes, plants, yeast and even bacteria. complete slc mrna coding sequences for teleost fishes have been published in the last few years. single genes were described in carp, channel catfish, zebrafish, striped bass, japanese flounder, turbot and red sea bream, while two copies have been described in rainbow trout and fugu, with evidence from other teleosts available in various genome databases. most animal studies, particularly in teleost fishes, are focused on gene isolation and constitutive expression analysis, with little information on evolutionary and functional aspects. furthermore, a complex picture emerges from the comparison of phylogenetic and expression studies between fishes and mammals. in fact, little is known about the structure and function of slc in lower vertebrates, although some studies provide evidence for a role of teleost slc11a orthologs in the nutritive metal uptake in the intestine, and also an involvement in the response to bacterial infection. it has been shown that slc mrna levels are elevated in response to lipopolysaccharide and edwardsiella ictaluri in channel catfish, to vibrio angillarum in turbot and red sea bream and to mycobacterium in striped bass. however no research has yet elucidated this complex pattern of phylogenetic relationships and functional roles of teleost and mammalian slc genes and no explanations are provided as to why it seems that a homolog of the mammalian slc11a is performing the functions attributed to slc11a a complement of two slc genes is shared between mammals and teleosts. whilst it is known that mammalian slc11a and slc11a have likely resulted from genome duplications in early vertebrate ancestry, the potential role of the teleost fish-specific genome duplication in the evolutionary history of this gene family has not been considered. a comprehensive synteny study could thus help to improve our understanding of the evolution and functional specialization of these genes in teleost fish. european sea bass was selected as the teleost model for this study due to the growing amount of data on its immune system, the possibility of making use of its partially sequenced genome and our previous experience with sea bass models of infection and iron modulation. sea bass is also an important marine aquaculture species in europe, afflicted by several diseases such as pasteurellosis and vibriosis. since the early 1980s, its production has risen considerably evolving from extensive culture units to semi-intensive or intensive systems. this massive fish concentration leads to an increase in organismal stress and as a consequence fish defenses get compromised, making them more susceptible to pathogen attack. isolation and characterization of the slc gene in sea bass, as a candidate gene for host defense to infection with pathogens, may be of great benefit to better understand its role in the immune system and to the selection of disease resistant stocks. moreover, sea bass is part of the acanthopterygii superorder, which includes stickleback, tetraodon and fugu, organisms that have their genome fully sequenced, making possible a number of comparative genetic studies. the aims of this study were to identify and characterize the sea bass slc homologs, clarify their evolutionary history and to determine their functional roles, in particular those related with the host iron metabolism and resistance to infection. we evaluated the modulation of slc gene expression in sea bass upon iron modulation or exposure to photobacterium damselae spp. piscicida, in in vivo or in vitro experimental models. we expect that this approach should provide an insight on the evolutionary history of the slc genes in the vertebrata subphylum.  southern blot in order to determine the number of copies of slc genes in the sea bass genome, a southern blot analysis was performed. after independent digestion of μg of genomic dna with ecori or hindii and hybridization with a slc dig-labeled probe, different hybridization bands were visible. no uncut products were observed. whether digested with ecori or hindii, two different hybridization bands were visible, suggesting the existence of two copies of the slc genes in the sea bass genome. sea bass slc transcripts five different sea bass slc transcripts were obtained by primer walking and 5'/3' race with liver, spleen and intestine cdna and analysis of whole-genome shotgun contigs. one transcript was named slc11a2-α and the other four transcripts were named slc11a2-β to β since they were found to share bp of their coding region, with the differences between them limited to the 5' and 3' endings. sizes of the untranslated regions and coding regions, as well as starting and ending exons for each transcript. slc11a2-β and slc11a2-β transcripts are of the same length regarding the coding region, although they differ in the first bp, since they result from the alternative usage of either exon 1a or exon 1b, both with bp. they also have in common the final exon, and the same 3' utr. slc11a2-β and slc11a2-β transcripts are also of the same length and also differ in the first bp, due to alternative usage of either exon 1a or exon 1b. both are slightly larger than slc11a2-β and slc11a2-β since exon presents an alternative splice site, losing the final bp and being partially substituted by the bp of exon. slc11a2-β and slc11a2-β also share the same 3' utr, albeit different and smaller than the 3' utr for slc11a2-β and slc11a2-β. genomic organization the genomic organization of sea bass slc11a2-α and slc11a2-β was analyzed and compared with those of other fishes, amphibians and mammals. exon/intron boundaries were determined by comparison of cdna, genomic dna and putative amino acid sequences, splice-site consensus matching and analysis of whole-genome shotgun contigs. comparison of genomic dna sequences with the previously obtained cdna sequences showed that slc11a2-α consists of exons and introns, with a single initiator methionine and stop codon. the genomic interval from the initiator methionine to the stop codon is bp. comparison of genomic dna sequences with the cdna sequences of the four slc11a2-β transcripts showed that those four transcripts result not from four different slc11a2-β genes, but rather from the alternative splicing of two 5' exons and two 3' exons of a single slc11a2-β gene, similar to what happens with human slc11a. the slc11a2-β gene comprises a total of exons, with two initiator methionines and two stop codons. differences in the 5'-termini are generated by alternative promoters with subsequent, mutually exclusive splicing of the respective first exons to exon whereas differences in the 3'-termini are due to alternative splicing of exon which presents an alternative 5' donor site. in two isoforms, this leads to the reading of exon and in the other two isoforms to the partial reading of exon and also exon. the genomic interval from the first initiator methionine in exon 1a to the second stop codon in exon is bp. for both α and β genes, exons 2- present a high homology with the equivalent exons from other fish and mammals, whereas exons in the n- and c-terminus are variable in size and sequence. intron sizes, much like in fugu and tetraodon, are reduced when compared with mammalian homologs. sea bass slc11a2-α and slc11a2-β present a compaction factor of × and × to human slc11a and × and × to human slc11a respectively. no clear sequences matching the iron responsive element motifs, commonly found in mammalian slc11a were identified in any of the 3' regions of either slc11a2-α or slc11a2-β. however, one ire motif was found in the 5' region of slc11a2-β, with an upstream distance to the start codon of bp, using regrna and corroborated manually. full-length genomic sequences of sea bass slc11a2-α and slc11a2-β were deposited in genbank with accession numbers hq and hq. structure analysis of sea bass slc putative proteins to predict the functionality of sea bass slc putative proteins, we compared them with other known slc proteins, and analyzed them using several bioinformatics tools, searching for characteristic features of this protein family. the complete open reading frame of sea bass slc11a2-α comprises a single transcript of nucleotides, encoding a putative protein of residues, with a predicted molecular mass of kda. the complete open reading frame of sea bass slc11a2-β comprises different transcripts, encoding putative proteins that result from the alternative usage of two 5' exons encoding distinct n-termini and the alternative splicing of two 3' exons encoding distinct c-termini of the proteins. two transcripts of nucleotides, named slc11a2-β and slc11a2-β, and two transcripts of nucleotides, named slc11a2-β and slc11a2-β, encode putative proteins of and residues, with predicted molecular masses of and kda, respectively. the amino acid discrepancy in length results not from the substitution of exon for exon but rather from an alternative splice site in exon only the final amino acids encoded by exon are replaced by the amino acids encoded by exon. both exon 1a and exon 1b have the same size and encode the same number of amino acids. comparison with other teleost and mammalian species slc proteins showed that the signature features of the slc family can also be found in the sea bass: twelve putative transmembrane domains, a conserved transport motif, cysteine residues in loops and other motifs observed in previously described slc proteins, including some exclusive to teleost fish, are also present in sea bass. two n-linked glycosylation sites, located in loop are present in all slc11a2-β forms, but only one is present in slc11a2-α loop with another located in the c terminus. a conserved protein kinase c phosphorylation site was also found immediately before tmd and one other was only found to be shared by sea bass slc11a2-α and fugu slc11a-α, in loop a third protein kinase c phosphorylation site was found to be present in the 5' cytoplasmatic extremity of sea bass slc11a2-β and slc11a2-β striped bass slc fugu slc11a2-α and trout slc11a2-α and β. six, five and four casein kinase ii phosphorylation sites were found in slc11a2-β, slc11a2-β and slc11a2-α, respectively. one tyrosine kinase phosphorylation site -d/e-xx-y) in loop was found to be conserved in teleosts, with the exception of fugu slc11a-α. one tyrosine based sorting signal in the n terminus was also found to be conserved in teleosts, with the exception of the alpha forms of trout, sea bass and fugu. several n-myristoylation sites were also found to be conserved among all slc11a2-β forms, but one site was only found to be present in the 3' cytoplasmatic extremity of sea bass slc11a1-β, as well as striped bass slc and rainbow trout slc11a2-β. all phylogenetic analysis, performed with the maximum-likelihood, maximum-parsimony, neighbor-joining and bayesian inference methods were found to be consensual, placing sea bass slc11a2-β clustered with other fishes slc11a2-β and slc and sea bass slc11a2-α clustered with other fishes slc11a2-α, with the exception of trout slc11a2-α. as with all other teleost fish slc homologs described so far, sea bass slc homologs are also closer to mammalian slc11a than to slc11a paralogy and synteny to determine the evolutionary history of the slc gene family in vertebrates, we explored in detail the genomic "context" of both human slc11a and slc11a paralogs. other authors have previously suggested that slc11a and slc11a genes might have originated from the duplications of the genome in vertebrate ancestry, the so-called 2r, given their location to the hox chromosomes in hsa and hsa. we find strong support for this hypothesis. within a 1mb window in the vicinity of the slc11a and slc11a genes, various gene families have paralog members mapping to expected hox chromosome regions. that is the case of tmbim which maps close to the slc11a paralog in hsa while faim maps to hsa tns maps to the left end of the window of the slc11a gene, and has paralogs mapping to the three other human hox genome locations. in the case of the slc11a gene, gene families show a consistent duplication pattern, with paralogs mapping to expected regions of hox-linked paralogy, namely hsa2q. in the analysed teleosts a similar slc gene complement is also found. however, the precise orthology/paralogy relationships to the mammalian counterparts are yet to be firmly established. while the phylogeny clearly indicates that both fish genes strongly group with tetrapod slc11a, other features make the orthology assignment more contentious. syntenic data can be a powerful tool to facilitate the finding of orthologs and in the identification of duplication processes. the abundance of genome data from teleosts allows us the use of mapping data to clarify the phylogenetic findings. we have analysed the genome locations and gene environment of the teleost slc genes in zebrafish, fugu, tetraodon, medaka and stickleback. we find a strong degree of conservation between the gene arrangements of various fish species. the vicinity of the teleost slc loci have their human ortholog equivalents mapping to the slc11a chromosome, hsa12q. thus, we conclude that in agreement with the phylogenetic data, the genomic location of the teleost genes indicates that they are both slc11a2-like. furthermore, we observe that the mechanistic origin of the slc genes is likely linked to the fish 3r genome duplication. we find in the vicinity of both fish isoforms, gene families with duplicates mapping to both slc fish chromosomal regions. for example, we noticed that agap gene family in medaka has one member mapping to chromosome, while a second isoform maps in chromosome with the second isoform of teleost slc the phylogenies of the genes families with duplicated members show that the duplication timing dates back to the origin of the teleosts. thus, we can safely conclude that the teleost genes are of the slc11a type, and resulted from the fish specific genome duplication. constitutive expression of slc11a2-α and slc11a2-β in sea bass tissues to gain some insight into sea bass slc basic functions, mrnas constitutive expression of both forms was determined in relevant tissues. constitutive expressions of slc11a2-α and slc11a2-β were evaluated by real-time pcr in several tissues, namely liver, spleen, head/trunk kidney, gill, brain, stomach, pyloric ceca, anterior/mid/posterior sections of the intestine and rectum. the liver was found to be the organ with the highest overall expression of the slc11a genes, followed by the mid and posterior portions of the intestine, stomach and spleen. regarding the contribution of each slc11a form in the different tissues, there is a clear predominance of slc11a2-β in the liver, spleen, head kidney, rectum, gill and brain and a predominance of slc11a2-α in the stomach, pyloric ceca and all portions of the intestine. constitutive expression of the four isoforms of slc11a2-β by semi-quantitative rt-pcr in order to assess the relative contribution of each β isoform to the overall slc11a2-β constitutive expression, a more thorough analysis was performed. relative constitutive expressions of the four isoforms of sea bass slc11a2-β were determined by semi-quantitative pcr, since the distance between alternative 5' and 3' exons is too great to adequately use real-time pcr. the four isoforms were named β, β, β and β. in most of the tested tissues, there is a predominance of the slc11a2-β form, followed by slc11a2-β slc11a2-β has a lower constitutive expression in most tissues, but presents significant expression in the head kidney, stomach and rectum, whereas slc11a2-β is the form with the lowest expression, although it represents 26% of the overall expression of slc11a2-β in the gill. in situ hybridization an in situ hybridization was performed in several tissues to identify the subcellular distribution of sea bass slc mrnas, to obtain further information useful in establishing parallelisms with their mammalian homologous counterparts, and to reiterate the results observed in the constitutive expression analysis. in the liver, slc11a2-α mrna is very scarce, being detected in few hepatocytes whereas slc11a2-β in abundantly slc11a2-α mrna is scarce, being only conspicuous in the melanomacrophage centers. slc11a2-β on the other hand is abundant not only in the melanomacrophage centers but also in the spleen's white pulp. in the head kidney, both slc11a2-α and slc11a2-β have a similar distribution, being abundant in the melanomacrophage centers and the surrounding lymphomyeloid tissue, and to a much lesser degree in the hematopoietic tissue. in the intestine, slc11a2-α and slc11a2-β mrna present similar patterns of distribution in the anterior and mid section. slc11a2-α is found to be mostly concentrated in the apical membrane of the enterocytes, whereas slc11a2-β presents a more homogeneous distribution, not only in enterocytes but also in other intestinal cells. however, in the posterior intestine, both forms of slc11a seem to be accumulated in or around the goblet cells, with limited presence in the enterocytes. in all tissues, hybridization with sense probes for either slc11a2-α or slc11a2-β produced no significant staining. hematological parameters and tissue iron content in the in vivo experimental models several hematological and iron parameters were measured to validate the models of in vivo experimental iron modulation or infection with photobacterium damselae spp. piscicida. during the experimental infection, mortality was monitored and found to be high, but with fish surviving past the final experimental time point, indicating a chronic infection. in the iron overloaded animals, no changes were observed in hematocrit, and a small but significant increase was observed in the rbc count during the course of the experiment. increases in serum iron and transferrin saturation were also observed, the highest levels at days after overload, with decreases during the course of the experiment and recovery to control levels at days. liver iron levels were increased, with the maximum value at days post-modulation and a decrease towards days, but still over times higher than the control animals. in the iron deficient animals, significant decreases of all hematological parameters were observed during the course of the experiment, with values remaining below the control levels. liver iron levels did not differ significantly from control animals during the experimental iron deficiency. significant and steady decreases of all hematological parameters were observed during the course of the infection. liver iron levels increased slightly but not significantly from control animals. slc11a expression in the in vivo models of iron modulation and infection in order to determine the potential involvement of sea bass slc11a2-α and slc11a2-β in the iron metabolism and immune response, we evaluated their expression under conditions of iron modulation or bacterial infection. in the experimental iron modulation model, slc11a2-α and slc11a2-β expressions were initially evaluated in the liver, spleen, head kidney and intestine, days after iron modulation. in iron deficiency, there was a significant increase of slc11a2-α expression in the intestine, with no major changes in the other tested organs. no significant changes were observed in slc11a2-β expression. in iron overload, no significant changes in slc11a2-α were observed, whereas slc11a2-β expression was found to be increased in the liver. slc11a2-α and slc11a2-β expressions were subsequently evaluated in the liver at and days after iron modulation. as before, no significant changes were observed in slc11a2-α expression to either condition in the liver, whereas slc11a2-β was found t be significantly up-regulated at days post iron overload, decreasing during the course of the experiment to near control values, but still over 2-fold higher. expression of slc11a2-α and slc11a2-β was also evaluated days after iron modulation in several sections of the digestive tract, in order to identify the portion of the intestine responsible for the response of slc11a2-α. significant increases of slc11a2-α expression were observed in the mid and posterior sections of the intestine, with no significant changes in the anterior section or rectum. in the experimental model of infection, slc11a2-α and slc11a2-β expressions were measured in the liver, spleen and head kidney by real-time pcr, and h after infection. in the liver, a steady increase in slc11a2-β expression was observed during the course of the infection, with the maximum value at h post-infection, followed by a recovery to control levels at h. in the spleen, an increase of slc11a2-β was also observed during the course of the infection, accompanied by a recovery to control levels at h. in the head kidney, a significant increase of slc11a2-β was observed h after infection and was maintained during its course. no significant changes were observed in slc11a2-α expression in any of the tested organs. slc11a expression in the in vitro models of iron overload and infection we investigated the role of slc11a2-α and slc11a2-β in the iron metabolism and immune response at a cellular level, by using leukocytes from spleen, one of the most important erythropoietic and immune organs in fish. in the iron overload in vitro model, expression of slc11a2-α and slc11a2-β in leukocytes was measured by real-time pcr at and hours after the addition of ferric ammonium citrate. no significant changes were observed in slc11a2-α during the course of the experimental iron overload. slc11a2-β, on the other hand, increased significantly from h post-infection, reaching the maximum increase at h and decreased at h, returning to control levels at h. in the in vitro infection model with heat-inactivated p. damselae, expression of slc11a2-α and slc11a2-β in leukocytes was also measured by real-time pcr at and hours after infection. no significant changes were observed in slc11a2-α during the course of infection. slc11a2-β, on the other hand, increased significantly at h post-infection, followed by a decrease at h and returned to control levels at h. discussion in the present study, we set out to analyse the evolutionary and functional patterns of slc genes in vertebrates, in particular teleosts. we have used the european sea bass as a model and have successfully isolated two slc genes, named slc11a2-α and slc11a2-β. a single transcript was found to be produced, for the slc11a2-α gene, whereas slc11a2-β can produce up to four transcripts. the four β transcripts are the result of alternative exon splicing in the 5' and 3' ends, in a similar fashion to what has been described for human slc11a. but unlike human slc11a where the differences in the 3' end result from alternate exon usage, in sea bass the differences result from an alternative splice site in exon thus, only the second half of exon is replaced by exon neither sea bass slc11a2-α nor slc11a2-β were found to present a potential iron responsive regulatory protein binding site in the 3' utr, a feature that in contrast is present in mammalian slc11a genes and has also been described in some teleost fish, such as fugu and common carp. it is, however, absent in other teleosts, such as turbot, channel catfish and japanese flounder. in the slc gene family, ires have only been described in the 3'-utr but unexpectedly, an ire motif was found in the 5' region of slc11a2-β, albeit not in the 5'-utr itself but rather bp upstream the start of the 5'-utr. the significance of this finding is still unknown, although as with other 5' ires, it may be involved in translation repression. the single sea bass slc11a2-α transcript encodes for a single functional putative protein, whereas the four slc11a2-β transcripts encode for four putative proteins. the four β proteins differ in the n and c termini, as a result of the encoding by the two alternative exons and the alternative splice site in exon since both n and c termini are cytoplasmic, they may be involved in post-transcriptional regulatory processes and in the subcellular localization of these proteins, as occurs in mammalian slc11a. all sea bass slc11a proteins present the characteristic features of the slc gene family: transmembrane domains, a consensus transport motif, several glycosylation and phosphorylation sites and conserved cysteine residues. multiple alignments clearly show that these features are highly conserved and are very similar to those observed not only in other teleost fish, but also in mammals. however, there are some disparities in the features found in the 5' and 3' endings, namely some n-myristoylation and phosphorylation sites that are not present in all sea bass slc11a2-β forms, and which may also contribute to a differential post-transcriptional regulation and subcellular localization. in all the tree models, produced by maximum-likelihood, neighbour-joining, maximum parsimony or bayesian inference, phylogenetic analysis places all sea bass slc proteins clustered with other teleost fish slc proteins, which in turn cluster with mammalian slc11a proteins, clearly separated from slc11a among teleost proteins, there are two clear separate clusters. one cluster encompasses sea bass slc11a2-β protein as well as all other slc and slc11a2-β proteins. the other cluster includes sea bass slc11a2-α and all other α proteins, with the exception of rainbow trout slc11a2-α, suggesting that rainbow trout slc11a2-α may have a different evolutionary origin than other slc11a2-α proteins. there are some explanations that could account for the existence of two closely related slc11a genes in sea bass, which are in agreement with what has already been proposed for other teleost fish, such as fugu. the current evolutionary scenario proposes that slc paralogs appeared through the 2r genome duplication, and functionally diverged. this is supported by the close linkage to hoxc and hoxd which are assumed to be duplicate loci. in addition, the teleost fish paralogs are more similar to mammalian slc11a and may result from a third, fish specific, genome duplication. the synteny analysis performed in the current study strongly supports the latter, suggesting that the ancestor of all vertebrates had a single slc gene that quadrupled as part of the 2r genome duplications. two of the new genes were lost, while two were maintained in the ancestor of tetrapods and teleosts. in teleosts the slc11a clade expanded as the result of 3r to originate two isoforms, slc11a2-α and slc11a2-β, while the slc11a ortholog was lost. unlike other teleosts, trout's slc11a homologs do not seem to result from 3r but rather a salmonid specific tetraploidization. the overall constitutive expression pattern of sea bass slc transcripts resembles that of the mammalian slc11a gene, but also of slc11a genes described in other teleost fishes. slc11a2-α was found to be ubiquitously expressed, but with a higher expression along the digestive tract, with a particularly high incidence in the mid and posterior sections of the intestine. this already gives some indications for its possible involvement in intestinal iron absorption in sea bass, since the slc11a mediated uptake of iron and other food derived metals in teleosts occurs predominantly in the mid and posterior regions of the intestine, as previously described. slc11a2-β was also found to be ubiquitously expressed, although unlike slc11a2-α, being abundant in the liver and in varying levels in all other tested tissues. no clear pattern was observed regarding the distribution of the four isoforms of slc11a2-β, although a prevalence of the β and β forms in most tested tissues was evident. further analysis is needed to determine the exact function and contribution of each isoform. it is possible that they function in different subcellular compartments, with some of the forms more relevant in the immune response, while others may play a more prominent role in overall iron metabolism. the in situ hybridization results in the liver, spleen, head kidney and sections of the intestine mostly reflect the measured constitutive expression. in the liver and spleen there is a clear prevalence of slc11a2-β, whereas in the head kidney although the constitutive expression indicates a prevalence of slc11a2-β, there seems to be an almost even distribution of both forms. note that in the spleen and kidney slc11a2-β mrna is clearly visible in the white pulp and lymphomyeloid tissue, respectively, which are both areas rich in b- and t-lymphocytes and actively involved in the immune response. in the intestine, the in situ results show that slc11a2-α and slc11a2-β are expressed in different compartments of the enterocytes, with slc11a2-α mrna mostly concentrated close to the brush border of the apical pole, whereas slc11a2-β is spread all over the cells, not only in the enterocytes but also in other intestinal cells. this observation provides further evidence for a sub-functionalization of slc11a homologs in sea bass, with slc11a2-α localization resembling that of the mammalian slc11a ire-positive isoforms and slc11a2-β with the more general distribution of mammalian slc11a ire-negative isoforms. an issue that remains puzzling is the distribution of slc11a isoforms in the posterior intestine, with both forms concentrated in or around the goblet cells, the intestinal glandular cells that secret mucin. in mammals, there are conflicting reports on the presence of slc11a in the goblet cells, with some reporting its absence while others report its presence, suggesting that, along with mobilferrin, slc11a facilitates iron uptake. to better understand the role of sea bass slc11a genes, in vivo and in vitro experimental models of iron modulation and infection were created. haematological and iron parameters, and expression levels of slc11a2-α and slc11a2-β were evaluated under those experimental conditions. in the experimentally iron deficient animals, with decreased haematological parameters, slc11a2-α expression was significantly up-regulated in the intestine, whereas slc11a2-β did not respond in any of the tested tissues. further characterization of the slc11a2-α response to iron deficiency in the intestine showed that the up-regulation was mostly confined to the mid and posterior portions of the intestine. this is in accordance with previous findings that intestinal metal uptake in teleost fish occurs mainly in the posterior portion of the intestine, unlike mammals, where uptake occurs primarily in the duodenum. these results further reinforce the idea that sea bass slc11a2-α seems to be mainly involved in iron uptake by the enterocytes. in the experimentally iron overloaded animals, steady increases of the hematocrit and red blood cell counts were observed, as well as high levels of serum iron and transferrin saturation in the first days of the experiment. also, increased levels of iron were observed in the liver, further confirming the iron overload status. slc11a2-α expression did not change significantly in response to iron overload in any of the tested tissues and slc11a2-β was up-regulated only in the liver. together with the increased liver iron content, this is likely associated with the need to cope with the excess of iron introduced into the system, in order to prevent its toxic effects. in normal conditions, iron in the blood is bound to transferrin, and transferrin-iron complexes are internalized by a transferrin receptor-mediated endocytic pathway. through acidification of the endosome, iron is released from transferrin and slc11a is recruited to the endosome membrane, where it exports iron to the cytoplasm. iron is then used in cellular processes or stored in ferritin molecules. when more iron is introduced into the system, an increased expression of these genes could be expected. we have already demonstrated that during iron overload there is an increased expression of ferritin, although not accompanied by an up-regulation of transferrin, most likely due the already high levels of constitutive expression in the liver. in the in vivo experimental infection with live p. damselae, a significant and continuous decrease of all haematological parameters was observed, suggesting a condition that is commonly known as anaemia of inflammation or chronic disease. this response was first described in humans and later reported for teleost fish, namely sea bass and nile tilapia. during bacterial infection, inflammatory cytokines, such as il- prompt the liver to increase the production of the iron metabolism regulatory peptide hepcidin, causing the internalization of ferroportin and thus preventing cellular iron export. the iron retention leads to impairment in erythropoiesis, due to the lack of iron availability. overall, these changes contribute to limiting the availability of iron for pathogen growth. we have previously shown that hepcidin and ferritin levels are increased during bacterial infection in sea bass, thus confirming the presence of this mechanism in teleosts. the bacterial challenge significantly up-regulated the expression of slc11a2-β in the liver, spleen and head kidney, in a time-dependent fashion, but produced no significant changes in slc11a2-α expression. a similar up-regulation was observed in slc11a homologs of other teleost fish in response to v. anguillarum, mycobacterium, e. ictaluri and lps. further analysis will be required in order to determine whether in this case slc11a2-β is acting as the mammalian counterpart of slc11a being recruited to the membrane of pathogen-containing phagosomes, or of slc11a being recruited to the membrane of holotransferrin-positive recycling endosomes involved in iron uptake from the extracellular environment. since there are no slc11a homologs in sea bass, and there are different proteins encoded by slc11a2-β, it is tempting to propose that slc11a2-β is performing both slc11a and slc11a mammalian functions. in the in vitro iron overload of spleen-isolated leukocytes, slc11a2-β was found to be up-regulated in a time-dependent fashion, whereas no significant changes in slc11a2-α expression were observed. the increase in slc11a2-β expression is most likely due to the increase in iron accumulation in the macrophages, to cope with the excess iron introduced in the medium, which could become toxic for the cells. the in vitro experimental infection with heat-inactivated p. damselae, in spleen-isolated leukocytes, produced the same effect as that observed in vivo, with an up-regulation of slc11a2-β and no changes in slc11a2-α expression. not much information is available on the expression of slc homologs in leukocytes, but it is known that mammalian slc11a expression is stimulated by m. avium in peritoneal macrophages and by lps and ifn-γ in raw macrophages. mammalian slc11a expression is also stimulated by m. avium in peritoneal macrophages, although differentially from slc11a. also, catfish slc was found to be stimulated in monocytes by lps. as for the in vivo experimental infection, it has yet to be determined whether slc11a2-β is functioning as slc11a or slc11a but since both forms are known to be required for an efficient erythrophagocytosis and iron recycling in macrophages, it is likely that it is performing both functions. expression studies of the four slc11a2-β forms and subcellular localization in the leukocytes could provide some insight on this matter.  we have successfully isolated two slc paralogs in sea bass, that we named slc11a2-α and slc11a2-β. we have demonstrated that they have a role not only in iron metabolism but also in the response to bacterial infection. the clarification of the evolutionary scenario along with the functional data suggests a curious and complex pattern of sub-functionalization and paralog functional equivalence, a process previously named as synfunctionalization: the fish specific paralog slc11a2-α retained in part the original mammalian slc11a function, whereas the slc11a2-β isoform not only retained the original slc11a function but also acquired the slc11a function after the loss of this isoform in teleosts. 
both members of the slc gene family have been clearly identified in tetrapods; however slc11a has never been documented in teleost fish and is believed to have been lost in this lineage during early vertebrate evolution. our data suggests that despite the absence of slc11a its functions have been undertaken by one of the slc11a duplicated paralogs in teleost fish in a case of synfunctionalization, being involved in both iron metabolism and response to bacterial infection. induction of slc11a2-α and slc11a2-β in sea bass, upon iron modulation or exposure to photobacterium damselae spp. two different slc genes were isolated in the european sea bass, and named slc11a2-α and slc11a2-β, since both were found to be evolutionary closer to tetrapods slc11a through phylogenetic analysis and comparative genomics. piscicida, was evaluated in in vivo or in vitro experimental models. overall, slc11a2-α was found to respond only to iron deficiency in the intestine, whereas slc11a2-β was found to respond to iron overload and bacterial infection in several tissues and also in the leukocytes.
 the slc11a1/nramp and slc11a2/nramp genes belong to the slc11/nramp family of transmembrane divalent metal transporters, with slc11a being associated with resistance to pathogens and slc11a involved in intestinal iron uptake and transferrin-bound iron transport. both members of the slc gene family have been clearly identified in tetrapods; however slc11a has never been documented in teleost fish and is believed to have been lost in this lineage during early vertebrate evolution. in the present work we characterized the slc genes in teleosts and evaluated if the roles attributed to mammalian slc genes are assured by other fish specific slc gene members.  two different slc genes were isolated in the european sea bass, and named slc11a2-α and slc11a2-β, since both were found to be evolutionary closer to tetrapods slc11a through phylogenetic analysis and comparative genomics. induction of slc11a2-α and slc11a2-β in sea bass, upon iron modulation or exposure to photobacterium damselae spp. piscicida, was evaluated in in vivo or in vitro experimental models. overall, slc11a2-α was found to respond only to iron deficiency in the intestine, whereas slc11a2-β was found to respond to iron overload and bacterial infection in several tissues and also in the leukocytes.  our data suggests that despite the absence of slc11a its functions have been undertaken by one of the slc11a duplicated paralogs in teleost fish in a case of synfunctionalization, being involved in both iron metabolism and response to bacterial infection. this study provides, to our knowledge, the first example of this type of sub-functionalization in iron metabolism genes, illustrating how conserving the various functions of the slc gene family is of crucial evolutionary importance. 