The immunoglobulins of cartilaginous fishes.

Cartilaginous fishes, comprising the chimeras, sharks, skates, and rays, split from the common ancestor with other jawed vertebrates approx. 450 million years ago. Being the oldest extant taxonomic group to possess an immunoglobulin (Ig)-based adaptive immune system, examination of this group has taught us much about the evolution of adaptive immunity, as well as the conserved and taxon-specific characteristics of Igs. Significant progress has been made analyzing sequences from numerous genomic and transcriptomic data sets. These findings have been supported by additional functional studies characterizing the Igs and humoral response of sharks and their relatives. This review will summarize what we have learned about the genomic organization, protein structure, and in vivo function of these Ig isotypes in cartilaginous fishes and highlight the areas where our knowledge is still lacking.

Comparative study of cartilaginous fish divulges insights into the early evolution of primary, secondary and mucosal lymphoid tissue architecture.

Cartilaginous fish are located at a pivotal point in phylogeny where the adaptive immune system begins to resemble that of other, more-derived jawed vertebrates, including mammals. For this reason, sharks and other cartilaginous fish are ideal models for studying the natural history of immunity. Insights from such studies may include distinguishing the (evolutionarily conserved) fundamental aspects of adaptive immunity from the (more recent) accessory. Some lymphoid tissues of sharks, including the thymus and spleen, resemble those of mammals in both appearance and function. The cartilaginous skeleton of sharks has no bone marrow, which is also absent in bony fish despite calcified bone, but cartilaginous fish have other Leydig's and epigonal organs that function to provide hematopoiesis analogous to mammalian bone marrow. Conserved across all vertebrate phylogeny in some form is gut-associated lymphoid tissues, or GALT, which is seen from agnathans to mammals. Though it takes many forms, from typhlosole in lamprey to Peyer's patches in mammals, the GALT serves as a site of antigen concentration and exposure to lymphocytes in the digestive tract. Though more complex lymphoid organs are not present in agnathans, they have several primitive tissues, such as the thymoid and supraneural body, that appear to serve their variable lymphocyte receptor-based adaptive immune system. There are several similarities between the adaptive immune structures in cartilaginous and bony fish, such as the thymus and spleen, but there are mechanisms employed in bony fish that in some instances bridge their adaptive immune systems to that of tetrapods. This review summarizes what we know of lymphoid tissues in cartilaginous fishes and uses these data to compare primary and secondary tissues in jawless, cartilaginous, and bony fishes to contextualize the early natural history of vertebrate mucosal immune tissues.

Comparative transcriptomics of elasmobranchs and teleosts highlight important processes in adaptive immunity and regional endothermy.

BACKGROUND: Comparative genomic and/or transcriptomic analyses involving elasmobranchs remain limited, with genome level comparisons of the elasmobranch immune system to that of higher vertebrates, non-existent. This paper reports a comparative RNA-seq analysis of heart tissue from seven species, including four elasmobranchs and three teleosts, focusing on immunity, but concomitantly seeking to identify genetic similarities shared by the two lamnid sharks and the single billfish in our study, which could be linked to convergent evolution of regional endothermy. RESULTS: Across seven species, we identified an average of 10,877 Swiss-Prot annotated genes from an average of 32,474 open reading frames within each species' heart transcriptome. About half of these genes were shared between all species while the remainder included functional differences between our groups of interest (elasmobranch vs. teleost and endotherms vs. ectotherms) as revealed by Gene Ontology (GO) and selection analyses. A repeatedly represented functional category, in both the uniquely expressed elasmobranch genes (total of 259) and the elasmobranch GO enrichment results, involved antibody-mediated immunity, either in the recruitment of immune cells (Fc receptors) or in antigen presentation, including such terms as "antigen processing and presentation of exogenous peptide antigen via MHC class II", and such genes as MHC class II, HLA-DPB1. Molecular adaptation analyses identified three genes in elasmobranchs with a history of positive selection, including legumain (LGMN), a gene with roles in both innate and adaptive immunity including producing antigens for presentation by MHC class II. Comparisons between the endothermic and ectothermic species revealed an enrichment of GO terms associated with cardiac muscle contraction in endotherms, with 19 genes expressed solely in endotherms, several of which have significant roles in lipid and fat metabolism. CONCLUSIONS: This collective comparative evidence provides the first multi-taxa transcriptomic-based perspective on differences between elasmobranchs and teleosts, and suggests various unique features associated with the adaptive immune system of elasmobranchs, pointing in particular to the potential importance of MHC Class II. This in turn suggests that expanded comparative work involving additional tissues, as well as genome sequencing of multiple elasmobranch species would be productive in elucidating the regulatory and genome architectural hallmarks of elasmobranchs.

Identification of sturgeon IgD bridges the evolutionary gap between elasmobranchs and teleosts.

IgD has been found in almost all jawed vertebrates, including cartilaginous and teleost fish. However, IgD is missing in acipenseriformes, a branch that is evolutionarily positioned between elasmobranchs and teleost fish. Here, by analyzing transcriptome data, we identified a transcriptionally active IgD-encoding gene in the Siberian sturgeon (Acipenser baerii). Phylogenetic analysis indicated that it is orthologous to mammalian IgD and closely related to the IgD of other fish. The lengths of sturgeon membrane-bound IgD transcripts ranged from 1.2kb to 6.2kb, encoding 3-19 CH domains. As in teleosts, the first CH domain of the sturgeon IgD transcript is also derived from µCH1 by RNA splicing. However, the variable region of the expressed sturgeon IgD shows limited V(D)J usage. In addition to IgD, three IgM variants were also identified in this species, whereas no IgT/Z-encoding genes were observed. This study bridges the gap in Ig evolution between elasmobranchs and teleosts and provides significant insight into the early evolution of immunoglobulins.

Proteomic characterization of the acute-phase response of yellow stingrays Urobatis jamaicensis after injection with a Vibrio anguillarum-ordalii bacterin.

Systemic inflammatory responses of mammals and bony fish are primarily driven by coordinated up-regulation and down-regulation of plasma acute-phase proteins. Although this general principle is believed to be universal among vertebrates, it remains relatively unexplored in elasmobranchs. The objective of this study was to characterize acute changes in the plasma proteome of three yellow stingrays Urobatis jamaicensis following intraperitoneal injection with a commercial Vibrio bacterin. Changes in plasma protein levels were analyzed immediately prior to vaccination (time 0) and at 24 and 72 h post-injection by isobaric tags for relative and absolute quantitation (iTRAQ 4-plex) using shotgun-based nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and de novo peptide sequencing. Pooled 2D-LC-MS/MS and de novo sequencing data revealed differential expression of 156 distinct plasma proteins between time 0 and at least one post-vaccination time point. Using 1.5-fold change in expression as physiologically significant, 14/156 (9.0%) proteins were upregulated in at least one stingray through at least one experimental timepoint. Upregulated proteins included complement factors, Mx-protein, hemopexin, factor X and prothrombin. Seventy-six of 156 (48.7%) proteins were downregulated in the acute-phase response, including transferrin, apolipoprotein B, heparin cofactor 2, alpha2-macroglobulin, and various growth factors. Other differentially upregulated or downregulated proteins included intracellular, cell binding and structural proteins, proteins involved in physiologic processes, and unknown/hypothetical proteins. Selected bioactive factors are discussed for their putative roles in the elasmobranchs acute-phase response. These findings contribute to our understanding of disease processes in elasmobranchs, immunologic phylogeny in vertebrates, and begin the search for potential biomarkers of disease in these ecologically important fish.

Description of an elasmobranch TCR coreceptor: CD8α from Rhinobatos productus.

Cell-mediated immunity plays an essential role for the control and eradication of intracellular pathogens. To learn more about the evolutionary origins of the first signal (Signal 1) for T-cell activation, we cloned CD8α from an elasmobranch, Rhinobatos productus. Similar to full-length CD8α cDNAs from other vertebrates, Rhpr-CD8α (1800bp) encodes a 219 amino acid open reading frame composed of a signal peptide, an extracellular IgSF V domain and a stalk/hinge region followed by a well-conserved transmembrane domain and cytoplasmic tail. Overall, the mature Rhpr-CD8α protein (201 aa) displays ∼ 30% amino acid identity with mammalian CD8α including absolute conservation of cysteine residues involved in the IgSf V domain fold and dimerization of CD8αα and CD8αß. One prominent feature is the absence of the LCK association motif (CXC) that is needed for achieving signal 1 in tetrapods. Both elasmobranch and teleost CD8α protein sequences possess a similar but distinctly different motif (CXH) in the cytoplasmic tail. The overall genomic structure of CD8α has been conserved during the course of vertebrate evolution both for the number of exons and phase of splicing. Finally, quantitative RTPCR demonstrated that elasmobranch CD8α is expressed in lymphoid-rich tissues similar to CD8 in other vertebrates. The results from this study indicate the existence of CD8 prior to the emergence of the gnathostomes (>450 MYA) while providing evidence that the canonical LCK association motif in mammals is likely a derived characteristic of tetrapod CD8α, suggesting potential differences for T-cell education and activation in the various gnathostomes.

Molecular cloning and characterization of CD9 cDNA from cartilaginous fish, red stingray, Dasyatis akajei.

CD9 is a glycoprotein of the transmembrane 4 superfamily (TM4SF) and is involved in various cellular processes. In this study, we describe the isolation of the full-length cDNA encoding for CD9 molecule (daCD9) of red stingray, Dasyatis akajei. This 1252 bp cDNA was isolated from leukocyte cDNA library and contains 681 bp open reading frame encoding 226 amino acid residues. Amino acid sequences analysis and structure prediction display approximately 50% identity to higher vertebrates with the presence of conserved structures, including the four transmembrane domains and certain characteristic residues. Southern blot analysis shows that daCD9 exists as a single copy gene. Northern blot analysis reveals that daCD9 is highly expressed in gill and spleen although its expression can be found in other tissues suggesting daCD9 might play an important role in immune defense in this fish.

Immunological detection of Na(+)/H(+) exchangers in the gills of a hagfish, Myxine glutinosa, an elasmobranch, Raja erinacea, and a teleost, Fundulus heteroclitus.

Na(+)/H(+) exchangers (NHE) are a family of ion exchangers with diverse functions that are well defined in mammals. NHE-1 is expressed in the plasma membrane of most mammalian cells where it regulates intracellular pH, and usually in the basolateral membrane of epithelial cells. It has also been detected in teleost gills where it may participate in systemic pH regulation. NHE-3 is usually expressed in the apical membrane of mammalian epithelial cells where it helps reabsorb Na(+) and HCO(3)(-); it has also been detected in teleost gills. We used Western blotting and heterologous antibodies to screen for expression of NHE-1 and NHE-3 in gills of an agnathan (Myxine glutinosa) and an elasmobranch (Raja erinacea), and NHE-3 in gills of a teleost (Fundulus heteroclitus). Positive NHE-1 bands were detected in gills from the agnathan and elasmobranch. Using the NHE-3 antibody, bands were detected in the gills of the elasmobranch and teleost. These data are some of the first direct evidence of NHEs in the gills of an agnathan and elasmobranch, and confirm the presence of NHEs in the gills of teleosts.

Macrophage-lymphocyte cell clusters in the hypothalamic ventricle of some elasmobranch fish: ultrastructural analysis and possible functional significance.

BACKGROUND: Previous studies have demonstrated the existence of lympho-haemopoietic tissue in the meninges and choroid plexuses of various primitive vertebrates, including the stingray Dasyatis akajei and in early human embryos. In the present study, we extend these results analyzing macrophage-lymphocyte cell clusters found in the floor of the hypothalamic ventricle of several specimens of elasmobranchs. METHODS: After aseptical isolation of the brain from several specimens of smooth dogfish Triakis scyllia, cloudy dogfish Scyliorhinus torazame, gummy shark Mustelus manazo, and stingray Dasyatis akajei their hypothalamic regions were processed routinely by light, scanning, and transmission electron microscopy. RESULTS: The study of serial histological sections demonstrated that the macrophage-lymphocyte cell clusters proceeded from the meningeal lymphohaemopoietic tissue, reaching the ventricular lumen along large blood vessels. In this tissue, macrophages, different sized lymphocytes, lymphoblasts, granulocytes, monocytes, and developing and mature plasma cells were closely packed among a meshwork of fibroblastic reticular cell processes. It never invaded the brain parenchyma. A cell layer of glial elements and a continuous basement membrane interposed between the lymphoid tissue and the neural elements although some macrophages had migrated across the ependymal cell layer. In the ventricular lumen very irregular macrophages with long cell processes and containing abundant engulfed material of unknown origin formed big cell clusters with neighboring lymphocytes, lymphoblasts, and plasma cells, similar to those described during the immune response. Moreover, electron lucent cells which resembled the antigen-presenting cells of higher vertebrates established intimate surface cell contacts with the surrounding lymphocytes. In the third ventricle of several specimens of gummy shark, Mustelus manazo, morphologically similar cell clusters appeared but these were not connected to the meningeal lympho-haemopoietic tissue. No intraventricular cell aggregates were found in the stingray brain. CONCLUSIONS: Although we cannot rule out that these macrophage-lymphocyte cell clusters represent a permanent structure in the elasmobranch brain they rather seem to be only established after specific stimulation for preventing the entrance of noxious, foreign materials into the elasmobranch brain parenchyma.