Evidence of Interleukin Genes in the Sea-Star:

The axial organ of the sea star Asterias rubens is a primitive immune organ the sea star T lymphocytes, when stimulated by various lectins, produce lymphokin-like substances with mitogenic properties as shown in earlier studies; they are correlated with Interleukin genes.


INTRODUCTION
A large number of investigations performed in the last few years, in our Laboratory, have provided evidence that the sea star Asterias rubens (echinoderma) posseses a primitive immune system with cellular and humoral responses functionally similar to those of the immune system of vertebrates.

MATERIALS AND METHODS
Sea stars Asterias rubens were obtained from the Biology Institute (Gothenburg University). Immunizations were performed on 20 animals, in an aquarium with sea water at 10°C by using Horse-Radish Peroxydase (HRP) (Sigma Products) as antigen at a concentration of 1 mg mL −1 . 20 non-injected animals were used as controls. The axial organs were removed; RNA was extracted, using Trizol (Invitrogen) according to manufacturer instructions, from immunized sea stars (HRP) and Controls (C). cDNA was normalized using double strand specific nuclease essentially as described by Zhulidov et al. (2004). cDNA was fragmented using DNA Fragmentase (New England Biolabs), according to the manufacturer's instructions. After ligation of adapters for illumina's GSII sequencing system, the cDNA was sequenced on the illumina GSII platform sequencing 1x 100 bp from one side of the approximately 200 bp fragments. Sequences were assembled using Velvet (Zerbino and Birney, 2008).

RESULTS AND DISCUSSION
We describe first « interleukin genes » then « interleukin receptor genes » as compared to mammals.
One contig (NODE_3558_length_451_cov_24.356985) could be annotated via BLASTX to mouse "Nuclear factor interleukin-3-regulated protein" from the SWISSPROT database, with an e-value of 2.87689e-10. On an aligned Science Publications AJI region of 62 amino acids, 42 positive and 28 identical amino acids were found.

HRP-« Nuclear factor interleukin-3regulated protein »
One contig (NODE_48893_length_689_cov_15.156749) could be annotated via BLASTX to mouse "Nuclear factor interleukin-3-regulated protein" from the SWISSPROT database, with an e-value of 1.49652e-09. On an aligned region of 57 amino acids, 40 positive and 26 identical amino acids were found.
We study now the interleukin 1 and more specially interleukin 1 receptors.

Control-« X-linked Interleukin-1 Receptor Accessory Protein-Like 2 »
One contig (NODE_21299_length_1163_cov_23.764402) could be annotated via BLASTX to mouse "X-linked interleukin-1 receptor accessory protein-like 2" from the SWISSPROT database, with an e-value of 2.64154e-07. On an aligned region of 278 amino acids, 110 positive and 60 identical amino acids were found.

HRP-« X-linked Interleukin-1 Receptor
Accessory Protein-like 2 » One contig (NODE_23687_length_300_cov_7.280000) could be annotated via BLASTX to mouse "X-linked interleukin-1 receptor accessory protein-like 2" from the SWISSPROT database, with an e-value of 7.2295e-05. On an aligned region of 77 amino acids, 38 positive and 27 identical amino acids were found.

Control-« Interleukin-1 Receptor-Associated Kinase 4 »
One contig (NODE_45921_length_271_cov_7.907749) could be annotated via BLASTX to mouse "Interleukin-1 receptor-associated kinase 4" from the SWISSPROT database, with an e-value of 0.000100449. On an aligned region of 96 amino acids, 37 positive and 22 identical amino acids were found.

Control-« Interleukin-17 Receptor B »
One contig (NODE_31878_length_179_cov_15.167598) could be annotated via BLASTX to mouse "Interleukin-17 receptor B" from the SWISSPROT database, with an evalue of 0.000617054. On an aligned region of 42 amino acids, 24 positive and 14 identical amino acids were found.

HRP -« Interleukin-17 Receptor B »
One contig (NODE_46091_length_308_cov_16.399351) could be annotated via BLASTX to mouse "Interleukin-17 receptor B" from the SWISSPROT database, with an e-value of 0.000279731. On an aligned region of 49 amino acids, 28 positive and 16 identical amino acids were found.

As Shown, it Exists Also an Interleukin-17
Receptor A

HRP -« Interleukin-17 receptor A »
One contig NODE_13602_length_446_cov_23.168161) could be annotated via BLASTX to mouse "Interleukin-17 receptor A" from the SWISSPROT database, with an e-value of 0.000671577. On an aligned region of 81 amino acids, 38 positive and 24 identical amino acids were found.

CONCLUSION
The immunization leads to modifications in the case of « Interleukin-1 receptor-associated kinase 4 » and in the case of « Interleukin-17 receptor A », « Interleukin-18 receptor », « Interleukin-12 receptor» where these last appear only, in immunized animals. Furthermore interleukin genes (most of them corresponding to interleukin receptor genes) such as IRPL2, IRAK 4, IL-17RB, IL-17RA, they are present in the sea-star Asterias rubens.
These observations would indicate that certain interleukins and particularly « receptors »recalling mammal ones are present in the immune system of the sea star.
We note specially, in mammals that, the interleukin 17 receptor B is implicated in the immune response by mediating the activation of NF-Kappa B present also in the genome of A. rubens; that the interleukin 17 receptor A belongs to a novel family of inflammatory cytokines. As for the IRAK 4, it is required for various responses induced by Il-1R and toll-like receptor signals (To note that various toll-like receptors have been found in the sea-star genome).
As for the IL6-receptor: it plays an important rôle in the immune response in mice. Embryological, anatomical and biochemical evidences seem to indicate that the « sea-star phylum » is an ancestor of the vertebrates. Since the typical immune system is not present in invertebrates, it can be suggested that it was developed at this point in evolution. We recall that the main acquisition of Echinoderms seems: to be the cellular differentiation in two subpopulations of cells, ancestral to T and B lymphocytes and their interplay with phagocytes resulting in the synthesis of specific humoral primitive antibody (Leclerc, 2012) in correlation with the Complement to be expressed (Leclerc et al., 2013) at the difference of another Echinoderma: the sea-urchin (Hibino et al., 2006;Rast et al., 2006).