The immunoglobulins of carcharhine sharks: a comparison of serological and biochemical properties

The immunoglobulins of three carcharhine sharks were isolated from serum by means of salt precipitation and gel chromatography. The Galapagos shark (Carcharhinus galapagensis), the sandbar shark (Carcharhinus plumbeus) and the tiger shark (Galeocerdo cuvieri) each contained high molecular weight (18S) and low molecular weight (7S) IgM-like molecules as the major serum immunoglobulins. Both within and between species 18S and 7S immunoglobulins closely resemble each other in antigenic character, polypeptide chain composition, chain mass, amino acid composition, carbohydrate content and amino-terminal sequence. These results suggest that the immunoglobulins of carcharhine sharks have undergone little structural divergence during their evolution.     

Reactivities of Shark 19S and 7S IgM Antibodies to <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

LOWER vertebrates such as sharks can synthesize humoral antibodies in response to antigenic stimulation with a wide variety of antigens¹. Physicochemical studies have shown that sharks can synthesize both 19S and 7S immunoglobulins and that these two proteins belong to the same immunoglobulin class, which seems to be structurally homologous to IgM as defined for higher animals. Thus the shark immunoglobulins have been designated 19S IgM and 7S IgM^2–4. Because the predominant immunoglobulin (IgG) of most mammals is absent from sharks, the shark monomeric (7S) IgM might be functionally analogous to IgG. One example of the functional differences between IgM and IgG antibodies is the greater reactivity of the former in agglutination and bactericidal reactions^5,6. We have isolated and characterized functionally the relatively high levels of agglutinating antibodies which the nurse shark, Gingly-mostoma cirratum, synthesizes in response to Salmonella typhimurium “O” antigens.     

Phylogenesis of the general structure of immunoglobulins.

The study of the general structure of macroglobulins and 7S (IgG) immunoglobulins of frog and tortoise by relaxation methods shows the rotational correlation time of 7S immunoglobulins to be 67–68 nsec whereas that of macroglobulins of frog is 135 nsec and of tortoise is 103 nsec. Experimental values of rotational correlation time for 7S immunoglobulins are close to those calculated for the model of this molecule approximated by the rigid rotational ellipsoid and lower than those estimated for macroglobulins. This indicates that frog and tortoise 7S immunoglobulins have a fairly compact general structure with no marked intramolecular rotational freedom for high-molecular fragments, whereas macroglobulins possess it to a limited extent. It is seen from our evidence on the rigidity of 7S immunoglobulins and the limited flexibility of macroglobulins in amphibia and reptiles, as compared to previous data on the limited flexibility of carp macroglobulins and on the pronounced flexibility of IgG and IgM of mammals, that the general structure becomes more flexible on passing from lower vertebrates to representatives of a more recent class of mammals. The reported increased flexibility of immunoglobulins accompanied by the progressive evolution of species is likely to provide one of the first indications of the possible directed selection in the course of molecular evolution.     

Molecular phylogeny of the prickly shark, Echinorhinus cookei, based on a nuclear (18S rRNA) and a mitochondrial (cytochrome b) gene.

The classification of the sharks is unclear. This is particularly true for the superorder Squalomorphii. The relationships between the squalomorphs and other superorders of sharks and the relationships between the different orders within the squalomorphs are a matter of debate. Here, we report a molecular phylogeny for a little known member of this superorder, the genus Echinorhinus. Echinorhinus is most commonly classified in either the family Echinorhinidae (Squaliformes) or the family Squalidae (Squaliformes). However, some authors have suggested a closer relationship to the order Hexanchiformes. In an attempt to shed light on this controversy, we have cloned, sequenced, and compared two genes widely used in molecular phylogeny studies, the cytochrome b and the 18S rRNA from the rare prickly shark, Echinorhinus cookei, and two potential relatives, the spiny dogfish Squalus acanthias (Squaliformes), and the sevengill shark, Notorynchus cepedianus (Hexanchiformes). The sequences of these genes for the prickly shark, the dogfish, and the sevengill shark were found to be equally divergent, suggesting that the prickly shark is no closer to the order Squaliformes than to the order Hexanchiformes.     

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.     

鱼类Fc受体研究

Fc受体是免疫细胞表面一种重要受体分子,通过与免疫球蛋白Fc段结合触发多种生物学功能,是联系体液免疫和细胞免疫的桥梁。部分硬骨鱼中已经发现了Fc受体,在斑马鱼、斑点又尾鲴和鲤鱼中都克隆到了Fc受体的γ亚基,在鲨鱼和大西洋鲑中证明有能够与免疫球蛋白结合的Fc受体存在,并在斑点叉尾鲴、河豚和虹鳟中存在着类似α亚基的Fc受体。对鱼类Fc受体的发现和研究必将为了解鱼类的免疫机制及免疫进化提供重要的资料。     

The structure of immunoglobulin variable regions in the horned shark,Heterodontus francisci

The heavy and light chains of pooled antibodies of the hybodont shark,Heterodontus francisci (horned shark), were subjected to amino acid sequence analysis. Yield determinations showed that more than 90% of the available polypeptides in the respective pools were sequenced. The heavy chains were homogeneous in the initial framework segment and showed a sequence homology of approximately 70% with the corresponding region of the more recently evolved nurse shark and a 45% homology with a human myeloma heavy chain. The light chains were less homogeneous and not identifiable as either kappa or lambda chains as known in higher species. The first half-cystine characteristics of the variable domain intrachain disulfide bridge of immunoglobulins was present in the same position (22 for heavy chains; 23 for light chains) in the horned shark as in mammalian species. The sequence analysis also suggested the presence of a hypervariable region in the horned shark light chains. The combined data imply that the antigen-binding function of immunoglobulins is mediated in much the same manner in this primitive shark as in more recently evolved species, including mammals.     

Structural,antigenic and evolutionary analyses of immunoglobulins and T cell receptors

We have had the pleasure of collaborating with Allen Edmundson for the past 15 years on the structure, binding properties and evolution of immunoglobulins and T cell receptors. Among the most significant contributions of our joint efforts were: (1) the predictive use of structural features of immunoglobulin domains to model the three-dimensional structures of the immunoglobulin domains of human T-cell receptor alpha and beta chains as well as shark light chains and V(H) domains; (2) the finding that normal humans and other vertebrates express autoantibodies against combining site epitopes of their own T cell receptors; (3) the mapping of the peptide autoepitopes recognized in health, autoimmunity and retroviral infection; and (4) the determination that epitope recognition promiscuity is a characteristic property of the combining sites of IgM immunoglobulins ranging from those of sharks to those of humans. We briefly review the salient findings and status of these studies and indicate the future directions that we will pursue in their continuation.     

Effects of the Pleistocene on the mitochondrial population genetic structure and demographic history of the silky shark (<Emphasis Type="Italic">Carcharhinus falciformis</Emphasis>) in the western Atlantic Ocean

The silky shark, Carcharhinus falciformis, is a large-bodied, oceanic-coastal, epipelagic species found worldwide in tropical and subtropical waters. Despite its commercial importance, concerns about overexploitation, and likely ecological significance of this shark as an upper trophic-level predator, understanding of its population dynamics remains unclear for large parts of its distribution. We investigated the genetic diversity, population structure and demographic history of the silky shark along the western Atlantic Ocean based on the use of 707 bp of the mitochondrial DNA control region (mtCR). A total of 211 silky sharks were sampled, originating from five areas along the western Atlantic Ocean. The mitochondrial sequences revealed 40 haplotypes, with overall haplotype and nucleotide diversities of 0.88 (± 0.012) and 0.005 (± 0.003), respectively. The overall population structure was significantly different among the five western Atlantic Ocean regions. Phylogenetic analysis of mtCR sequences from globally sourced silky shark samples revealed two lineages, comprising a western Atlantic lineage and western Atlantic—Indo-Pacific lineage that diverged during the Pleistocene Epoch. In general, tests for the demographic history of silky sharks supported a population expansion for both the global sample set and the two lineages. Although our results showed that silky sharks have high genetic diversity, the current high level of overexploitation of this species requires long-term, scientifically informed management efforts. We recommend that fishery management and conservation plans be done separately for the two western Atlantic matrilineal populations revealed here.     

Microsatellite Analyses of Blacktip Reef Sharks (Carcharhinus melanopterus) in a Fragmented Environment Show Structured Clusters

The population dynamics of shark species are generally poorly described because highly mobile marine life is challenging to investigate. Here we investigate the genetic population structure of the blacktip reef shark (Carcharhinus melanopterus) in French Polynesia. Five demes were sampled from five islands with different inter-island distances (50–1500 km). Whether dispersal occurs between islands frequently enough to prevent moderate genetic structure is unknown. We used 11 microsatellites loci from 165 individuals and a strong genetic structure was found among demes with both F-statistics and Bayesian approaches. This differentiation is correlated with the geographic distance between islands. It is likely that the genetic structure seen is the result of all or some combination of the following: low gene flow, time since divergence, small effective population sizes, and the standard issues with the extent to which mutation models actually fit reality. We suggest low levels of gene flow as at least a partial explanation of the level of genetic structure seen among the sampled blacktip demes. This explanation is consistent with the ecological traits of blacktip reef sharks, and that the suitable habitat for blacktips in French Polynesia is highly fragmented. Evidence for spatial genetic structure of the blacktip demes we studied highlights that similar species may have populations with as yet undetected or underestimated structure. Shark biology and the market for their fins make them highly vulnerable and many species are in rapid decline. Our results add weight to the case that total bans on shark fishing are a better conservation approach for sharks than marine protected area networks.     

Rapid and Simultaneous Identification of Body Parts from the Morphologically Similar Sharks Carcharhinus obscurus and Carcharhinus plumbeus (Carcharhinidae) Using Multiplex PCR

Many commercially exploited carcharhinid sharks are difficult to identify to species owing to extensive morphological similarities. This problem is severely exacerbated when it comes to identifying detached shark fins, and the finless and headless shark carasses typically sold in markets. To assist in the acquisition of urgently needed conservation and management data on shark catch and trade, we have developed a highly streamlined approach based on multiplex polymerase chain reaction (PCR) that uses species-specific primers derived from nuclear ribosomal ITS2 sequences to achieve rapid species identification of shark body parts. Here we demonstrate the utility of this approach for identifying fins and flesh from two globally distributed, morphologically very similar carcharhinid sharks (Carcharhinus obscurus and Carcharhinus plumbeus) intensively targeted in fisheries worldwide, and often confused for each other even as whole animals. The assay is conducted in a 4-primer multiplex format that is structured to simultaneously achieve the following efficiency and cost-reduction objectives: it requires only a single-tube amplification reaction for species diagnosis, it incorporates an internal positive control to allow detection of false-negative results, and it is novel in that it allows species identification even when DNAs from two species are combined in the same tube during the PCR reaction. The latter innovation reduces the required effort for screening a set of unknown samples by 50%. The streamlined approach illustrated here should be amenable for use in a shark conservation and management context where large numbers of samples typically need to be screened; the approach shown may also provide a model for a rapid diagnostic method applicable to species identification in general. Received September 15, 2000; accepted December 15, 2000     

Survey sequencing and comparative analysis of the elephant shark (Callorhinchus milii) genome

Owing to their phylogenetic position, cartilaginous fishes (sharks, rays, skates, and chimaeras) provide a critical reference for our understanding of vertebrate genome evolution. The relatively small genome of the elephant shark, Callorhinchus milii, a chimaera, makes it an attractive model cartilaginous fish genome for whole-genome sequencing and comparative analysis. Here, the authors describe survey sequencing (1.4× coverage) and comparative analysis of the elephant shark genome, one of the first cartilaginous fish genomes to be sequenced to this depth. Repetitive sequences, represented mainly by a novel family of short interspersed element–like and long interspersed element–like sequences, account for about 28% of the elephant shark genome. Fragments of approximately 15,000 elephant shark genes reveal specific examples of genes that have been lost differentially during the evolution of tetrapod and teleost fish lineages. Interestingly, the degree of conserved synteny and conserved sequences between the human and elephant shark genomes are higher than that between human and teleost fish genomes. Elephant shark contains putative four Hox clusters indicating that, unlike teleost fish genomes, the elephant shark genome has not experienced an additional whole-genome duplication. These findings underscore the importance of the elephant shark as a critical reference vertebrate genome for comparative analysis of the human and other vertebrate genomes. This study also demonstrates that a survey-sequencing approach can be applied productively for comparative analysis of distantly related vertebrate genomes.     

Isolation and characterization of polymorphic microsatellite loci in nurse shark (Ginglymostoma cirratum)

We report on the development of nine polymorphic microsatellite loci in nurse shark (Ginglymostoma cirratum) using a combination of enriched and unenriched subgenomic libraries. Based on the small percentage of positive clones in the unenriched library (0.4%) it appears that microsatellites are very scarce in nurse shark genomes. Numbers of alleles at polymorphic loci ranged from two to 15; observed heterozygosity ranged from 0.17 to 0.90. We expect these loci to be useful for studies of breeding structure and paternity.     

The phylogenetic relationships of immunoglobulin allotypes and 7S immunoglobulin isotypes of chickens and other phasianoids (turkey, pheasant, quail)

Pheasants, quail and turkeys from different geographical locations were surveyed for the presence of eight 7S Ig and four IgM chicken allotypes. No IgM and only two 7S Ig allotypes were detected. Chicken 7S Ig allotypic specificity G-1.7 cross-reacted with pheasant and turkey isotypic specificities, and was absent in quail. The other determinant (G-1.9) cross-reacted with an allotype found only in turkeys and golden pheasants. These data suggest that G-1.7 and G-1.9 are probably phylogenetically ancient determinants and that polymorphism of chicken immunoglobulins arose after divergence of chickens from other phasianoid birds. Based on the allotypic and isotypic analysis of the 7S Ig antigenic determinants, turkey 7S Ig was as closely related to chicken 7S Ig as was pheasant 7S Ig. Jungle fowl, the ancestor of chickens, had most of the chicken 7S Ig and IgM allotypes present as polymorphic markers.     

The phylogenetic relationships of immunoglobulin allotypes and 7S immunoglobulin isotypes of chickens and other phasianoids (turkey,pheasant, quail)

Pheasants, quail and turkeys from different geographical locations were surveyed for the presence of eight 7S Ig and four IgM chicken allotypes. No IgM and only two 7S Ig allotypes were detected. Chicken 7S Ig allotypic specificity G-1.7 cross-reacted with pheasant and turkey isotypic specificities, and was absent in quail. The other determinant (G-1.9) cross-reacted with an allotype found only in turkeys and golden pheasants. These data suggest that G-1.7 and G-1.9 are probably phylogenetically ancient determinants and that polymorphism of chicken immunoglobulins arose after divergence of chickens from other phasianoid birds. Based on the allotypic and isotypic analysis of the 7S Ig antigenic determinants, turkey 7S Ig was as closely related to chicken 7S Ig as was pheasant 7S Ig. Jungle fowl, the ancestor of chickens, had most of the chicken 7S Ig and IgM allotypes present as polymorphic markers.     

Highly stable binding proteins derived from the hyperthermophilic Sso7d scaffold

We have shown that highly stable binding proteins for a wide spectrum of targets can be generated through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Sso7d is a small (∼ 7 kDa, 63 amino acids) DNA-binding protein that lacks cysteine residues and has a melting temperature of nearly 100 °C. We generated a library of 10⁸ Sso7d mutants by randomizing 10 amino acid residues on the DNA-binding surface of Sso7d, using yeast surface display. Binding proteins for a diverse set of model targets could be isolated from this library; our chosen targets included a small organic molecule (fluorescein), a 12 amino acid peptide fragment from the C-terminus of β-catenin, the model proteins hen egg lysozyme and streptavidin, and immunoglobulins from chicken and mouse. Without the application of any affinity maturation strategy, the binding proteins isolated had equilibrium dissociation constants in the nanomolar to micromolar range. Further, Sso7d-derived binding proteins could discriminate between closely related immunoglobulins. Mutant proteins based on Sso7d were expressed at high yields in the Escherichia coli cytoplasm. Despite extensive mutagenesis, Sso7d mutants have high thermal stability; five of six mutants analyzed have melting temperatures > 89 °C. They are also resistant to chemical denaturation by guanidine hydrochloride and retain their secondary structure after extended incubation at extreme pH values. Because of their favorable properties, such as ease of recombinant expression, and high thermal, chemical and pH stability, Sso7d-derived binding proteins will have wide applicability in several areas of biotechnology and medicine.     

Movements of Blue Sharks (Prionace glauca) across Their Life History

Spatial structuring and segregation by sex and size is considered to be an intrinsic attribute of shark populations. These spatial patterns remain poorly understood, particularly for oceanic species such as blue shark (Prionace glauca), despite its importance for the management and conservation of this highly migratory species. This study presents the results of a long-term electronic tagging experiment to investigate the migratory patterns of blue shark, to elucidate how these patterns change across its life history and to assess the existence of a nursery area in the central North Atlantic. Blue sharks belonging to different life stages (n = 34) were tracked for periods up to 952 days during which they moved extensively (up to an estimated 28.139 km), occupying large parts of the oceanic basin. Notwithstanding a large individual variability, there were pronounced differences in movements and space use across the species'' life history. The study provides strong evidence for the existence of a discrete central North Atlantic nursery, where juveniles can reside for up to at least 2 years. In contrast with previously described nurseries of coastal and semi-pelagic sharks, this oceanic nursery is comparatively vast and open suggesting that shelter from predators is not its main function. Subsequently, male and female blue sharks spatially segregate. Females engage in seasonal latitudinal migrations until approaching maturity, when they undergo an ontogenic habitat shift towards tropical latitudes. In contrast, juvenile males generally expanded their range southward and apparently displayed a higher degree of behavioural polymorphism. These results provide important insights into the spatial ecology of pelagic sharks, with implications for the sustainable management of this heavily exploited shark, especially in the central North Atlantic where the presence of a nursery and the seasonal overlap and alternation of different life stages coincides with a high fishing mortality.     

Global diversity hotspots and conservation priorities for sharks

Sharks are one of the most threatened groups of marine animals, as high exploitation rates coupled with low resilience to fishing pressure have resulted in population declines worldwide. Designing conservation strategies for this group depends on basic knowledge of the geographic distribution and diversity of known species. So far, this information has been fragmented and incomplete. Here, we have synthesized the first global shark diversity pattern from a new database of published sources, including all 507 species described at present, and have identified hotspots of shark species richness, functional diversity and endemicity from these data. We have evaluated the congruence of these diversity measures and demonstrate their potential use in setting priority areas for shark conservation. Our results show that shark diversity across all species peaks on the continental shelves and at mid-latitudes (30-40 degrees N and S). Global hotspots of species richness, functional diversity and endemicity were found off Japan, Taiwan, the East and West coasts of Australia, Southeast Africa, Southeast Brazil and Southeast USA. Moreover, some areas with low to moderate species richness such as Southern Australia, Angola, North Chile and Western Continental Europe stood out as places of high functional diversity. Finally, species affected by shark finning showed different patterns of diversity, with peaks closer to the Equator and a more oceanic distribution overall. Our results show that the global pattern of shark diversity is uniquely different from land, and other well-studied marine taxa, and may provide guidance for spatial approaches to shark conservation. However, similar to terrestrial ecosystems, protected areas based on hotspots of diversity and endemism alone would provide insufficient means for safeguarding the diverse functional roles that sharks play in marine ecosystems.     

Skeletal anatomy of the Late Cretaceous shark,Squalicorax (Neoselachii: Anacoracidae)

The paleobiology of the Cretaceous neoselachian shark,Squalicorax, has largely been based on isolated teeth. We examined partial and nearly complete skeletons of three species ofSqualicorax, S. falcatus (Aoassiz),S. kaupi (Agassiz), andS. pristodontus (Agassiz), that were collected from the U.S.A. These specimens suggest that the total body length (TL) ofS. falcatus typically measured 1.8–2.0 m, and probably did not exceed 3 m. Moderatesized individuals ofS. kaupi andS. pristodontus perhaps measured about 3 m TL. AlthoughS. pristodontus was the largest form among the three species examined, this taxon possessed a set of large jaws (with large but fewer teeth) relative to its body size compared toS. falcatus orS. kaupi. This suggests that tooth size is not an accurate indicator of the TL if one compares oneSqualicorax species to another. Neurocranial features suggest that the vision ofSqualicorax was not as acute as that of a contemporaneous macrophagous lamniform shark,Cretoxyrhina mantelli (Agassiz) , but olfaction ofSqualicorax may have been better thanC. mantelli. The morphology of placoid scales suggests thatSqualicorax was capable of fast swimming. New skeletal data support the view that the feeding dynamics ofSqualicorax was similar to the modern tiger shark (Galeocerdo Müller & Henle). The present data do not allow for exact ordinal placement, but, contrary to some previous interpretations,Squalicorax can be excluded from the Hexanchiformes and Orectolobiformes. The taxon should more appropriately be placed within the Lamniformes or Carcharhiniformes.      

Shark and ray teeth from the Hauterivian (Lower Cretaceous) of north-east England

Sampling of hiatal horizons within the Hauterivian part of the Speeton Clay Formation of north-east England has produced teeth of several species of sharks and rays, four of which are previously unnamed. One species of shark, Cretorectolobus doylei sp. nov., and two species of rays, Spathobatis rugosus sp. nov. and Dasyatis speetonensis sp. nov., are named, whilst the presence of an indeterminate triakid shark is also noted. Synechodus dubrisiensis (Mackie) is shown to be a senior synonym of S. michaeli Thies. Although the dasyatid ray and triakid shark are by far the oldest representatives of their respective families, the overall composition of the fauna is considered to resemble more closely assemblages known from the Jurassic than those from upper parts of the Cretaceous.