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Isolation and initial characterisation of complement components C3 and C4 of the nurse shark and the channel catfish.

Complement components C3 and C4 have been isolated from the serum of the nurse shark (Ginglymostoma cirratum) and of the channel catfish (Ictalurus punctatus). As in the higher vertebrates, the fish C4 proteins have three-chain structures while the C3 proteins have two-chain structures. All four proteins have intra-chain thioesters located within their highest molecular mass polypeptides. N-terminal sequence analysis of the polypeptides has confirmed the identity of the proteins. In all cases except the catfish C3 alpha-chain, which appears to have a blocked N-terminus, sequence similarities are apparent in comparisons with the chains of C3 and C4 from higher vertebrates. We have confirmed that the activity/protein previously designated C2n is the nurse shark analogue of mammalian C4. This is the first report of structural evidence for C4 in both the bony and cartilaginous fish.

Amino Acid Sequence↗

Conservation of a dinucleotide simple sequence repeat locus in sharks.

Recent studies indicate that the flanking region and repeat motif structure of conserved microsatellite loci are useful for phylogenetic inference. Most comparative studies of microsatellite loci involve relatively closely related species, however, primarily because primers developed for one species often amplify only related species. We describe an analysis of a microsatellite locus in lamniform sharks that we estimate has been conserved for a billion years. Combined analysis of the flanking sequence and repeat motif structure resulted in a gene tree comparable to those reported from similar analyses of other genes. The conservation of the simple sequence repeat (SSR), and of the sequence flanking the SSR, is explained by a low substitution rate in sharks coupled with the possibility that mutations which interrupt perfect repeats are lost by replication slippage.

Animals↗

Hypermutation in shark immunoglobulin light chain genes results in contiguous substitutions.

Among 631 substitutions present in 90 nurse shark immunoglobulin light chain somatic mutants, 338 constitute 2-4 bp stretches of adjacent changes. An absence of mutations in perinatal sequences and the bias for one mutating V gene in adults suggest that the diversification is antigen dependent. The substitutions shared no patterns, and the absence of donor sequences, including from family members, supports the idea that most changes arose from nontemplated mutation. The tandem mutations as a group are distinguished by consistently fewer transition changes and an A bias. We suggest this is one of several pathways of hypermutation diversifying shark antigen-receptor genes--point mutations, tandem mutations, and mutations with a G-C preference--that coevolved with or preceded gene rearrangement.

Amino Acid Sequence↗

Conformation of immunoglobulin M. I. Characterization of anti-epsilon-1-dimethylamino-5-naphthalenesulfonyl-L-lysine immunoglobulin M antibodies from horse, pig, and shark.

IgM antibodies specific for the fluorophore epsiolon-1-dimethylamino-5-naphthalenesulfonyl-L-lysine(DNS-lysine) were elicited in the horse and nurse shark by immunization with a DNS-lysine streptococcal conjugate; the antibodies were purified by specific adsorption with an immunoadsorbent followed by gel filtration to select the IgM class (molecular weight 900 000). About 90% of the equine anti-DNS was IgM.DNS-Lysine, when bound in the combining sites of a population of these anti-DNS IgM antigodies from horse and nurse shark, as well as from pig, exhibited a marked fluorescence enhancement and shift of the emission spectrum to shorter wavelengths compared with emission in aqueous solution; these results indicate that the environments of the anti-DNS combining sites of this population were relatively hydrophovic. Approximately one-third of the ten possible combining sites in each of these anti-DNS IgM species bound DNS-lysine in this manner with an average intrinsic association constant (Ko) of greater than 10(6) M-1. Small differences were noted in binding behavior among the three species of antibodies. The enzymatic susceptibility of equine IgM was similar to that of human IgM. (Fab')2mu, Fab'mu, and Fabmu fragments were prepared following digestion with pepsin. These fragments could be clearly differentiated on the basis of molecular size. They bound DNS-lysine with the same affinity as intact IgM and the DNS-lysine-fragment complexes exhibited the same spectral properties as the parent IgM. It was concluded that the anti-dNs IgM antibodies from all three species, as well as the enzymatic fragments, were suitable for nanosecond depolarization studies which are reported in the accompanying paper Holowka, D.A., and Cathou, R.E. (1976), Biochemistry, the following papter in this issue.

Animals↗

Observations on metal concentrations in three species of shark (Deania calcea, Centroscymnus crepidater, and Centroscymnus owstoni) from southeastern Australian waters.

Deep sea dog sharks (Deania calcea, Centroscymnus crepidater, and Centroscymnus owstonii) were captured from the waters off western Victoria, Australia, in April and May 1998. The elements As, Ca, Cd, Cu, Fe, Hg, K, Mg, Mn, Na, Sr, and Zn were detected in the muscle tissue. The concentrations of Al, Ba, Be, Cr, Mo, Ni, Pb, and V were below the detection limits of the instrumental techniques employed (ICP-ES and GF-AAS). However, significant concentration differences between species were detected for As, Cd, Hg, and Zn. C. owstoni contained the highest concentrations of each of these elements. The concentrations of Cu, Pb, and Zn in each species were below the maximum levels permitted in food by the Australian Food Standards Code. The maximum permitted concentrations of As and Hg were exceeded in all species, and weekly consumption of 400 g of any of these sharks would result in intake of more than the FAO's provisional tolerable weekly intake. Although C. crepidator and D. calcea have strong relationships between length and mercury concentration, C. owstoni does not. Placing C. crepidator and C. owstoni in the same genus, therefore, is worth re-examination as the mechanisms for controlling mercury in C. owstoni appear to differ from that used by both C. crepidator and D. calcea.

Animals↗

Two new species of Acanthobothrium Beneden, 1849 (Tetraphyllidea: Onchobothriidae) from horn sharks in the Gulf of California, Mexico.

Between 1993 and 1996, 26 individuals of two species of horn sharks were collected from the upper Gulf of California and their spiral intestines examined for onchobothriid tapeworms. Heterodontus francisci was found to host Acanthobothrium puertecitense n. sp. Based on the criteria of Ghoshroy & Caira (2001), this is a Category 4 species (with >15 mm total length, >50 proglottids, >80 testes and a symmetrical ovary). It differs from the four Category 4 species previously reported from either the western Atlantic or eastern Pacific Oceans, including A. bajaense (emend.) which was described from H. francisci, in hook-shape, total length, number of proglottids, number of testes and number of columns of vitelline follicles. H. mexicanus was found to host Acanthobothrium santarosaliense n. sp., which, based on the criteria of Ghoshroy & Caira (2001) is a Category 3 species (with >15 mm total length, >50 proglottids, >80 testes and an asymmetrical ovary). A. santarosaliense differs from the five Category 3 species previously reported from either the western Atlantic or eastern Pacific Oceans in its size, euapolytic nature, lack of dark matrix from the bases of the hooks, hook-shape, and distribution and number of testes. In addition to the difference in ovarian symmetry, A. puertecitense possesses fewer testes, fewer proglottids, is a shorter worm, possesses a more anterior genital pore and vitelline follicles which extend further forward anteriorly than in A. santarosaliense. This represents the first report of onchobothriids from H. mexicanus and the first report of onchobothriids from H. francisci in the Gulf of California. The apparent host-specificity of both new species should be viewed with caution until a greater sample of host individuals of both species can be examined. The fact that both new tapeworm species are known only from the Gulf is also considered to be a preliminary result, given the small sample of host individuals examined from these regions to date. This brings the total number of species of Heterodontus known to host Acanthobothrium to four and the total number of Acanthobothrium species described from heterodontiform sharks to five.

Animals↗

A new species of copepod (Siphonostomatoida: Caligidae) parasitic on the tiger shark Galeocerdo cuvier (Péron & Lesueur) from Western Australian waters.

A new species of sea-louse (Caligidae, Siphonostomatoida), Caligus oculicola n. sp., is described from the eye surface of the tiger shark Galeocerdo cuvier from off the northwestern coast of Australia. This copepod is distinguished from its congeners by a combination of characters that include: (i) a bifid, dentiform process of the maxillule; (ii) a sternal furca with a box longer than wide and diverging, truncate tines; (iii) terminal spines 1 to 3 on the last segment of leg 1 exopod, each with serrate margins and an accessory process (accessory process on the spines extending beyond the tip of the spine itself); and (iv) a two-segmented exopod of leg 4 with an armature formula of I-0; III. This is the first description of a caligid copepod collected from a shark host in Western Australian waters. The host-parasite relationships between Caligus oculicola and its elasmobranch host are discussed.

Animals↗

Rates of mitochondrial DNA evolution in sharks are slow compared with mammals.

The rate of mitochondrial DNA (mtDNA) evolution has been carefully calibrated only in primates. Similarity between the primate calibration and rates estimated for other vertebrates has led to widespread assumption of a constant molecular clock in vertebrates even though this has never been rigorously tested. We report here the examination of mtDNA sequence variation for 13 species of sharks from two orders that are well represented in the fossil record to test the constancy hypothesis. Nucleotide substitution rates in the cytochrome b and cytochrome oxidase I genes in sharks are seven- to eightfold slower than in primates or ungulates. This difference in substitution rate cannot be explained by nucleotide composition bias, codon-usage bias, selection, or choice of genes sequenced, and was confirmed by comparing species recently separated by the rise of the Isthmus of Panama. Such differences in mtDNA substitution rates among taxa indicate that it is inappropriate to use a calibration for one group to estimate divergence times or demographic parameters for another group. High-resolution studies of molecular evolutionary rates require taxon-specific calibrations.

Animals↗

Expanded niche for white sharks.

Until the advent of electronic tagging technology, the inherent difficulty of studying swift and powerful marine animals made ecological information about sharks of the family Lamnidae difficult to obtain. Here we report the tracking of movements of white sharks by using pop-up satellite archival tags, which reveal that their migratory movements, depth and ambient thermal ranges are wider than was previously thought.

Animal Migration↗

Mammal-like muscles power swimming in a cold-water shark.

Effects of temperature on muscle contraction and powering movement are profound, outwardly obvious, and of great consequence to survival. To cope with the effects of environmental temperature fluctuations, endothermic birds and mammals maintain a relatively warm and constant body temperature, whereas most fishes and other vertebrates are ectothermic and conform to their thermal niche, compromising performance at colder temperatures. However, within the fishes the tunas and lamnid sharks deviate from the ectothermic strategy, maintaining elevated core body temperatures that presumably confer physiological advantages for their roles as fast and continuously swimming pelagic predators. Here we show that the salmon shark, a lamnid inhabiting cold, north Pacific waters, has become so specialized for endothermy that its red, aerobic, locomotor muscles, which power continuous swimming, seem mammal-like, functioning only within a markedly elevated temperature range (20-30 degrees C). These muscles are ineffectual if exposed to the cool water temperatures, and when warmed even 10 degrees C above ambient they still produce only 25-50% of the power produced at 26 degrees C. In contrast, the white muscles, powering burst swimming, do not show such a marked thermal dependence and work well across a wide range of temperatures.

Animals↗

Hepatic cholangiocarcinoma and testicular mesothelioma in a wild-caught blue shark, Prionace glauca (L.).

An adult male blue shark, Prionace glauca (L.), caught in July 2000 by a recreational fisherman off Long Island, New York, USA, had a retained fishing hook from a previous capture. The hook penetrated the gastric wall and lacerated the right liver lobe. Macroscopic lesions consisted of transmural gastritis and peritonitis. Alteromonas sp. and Vibrio alginolyticus were isolated from the peritoneal fluid. In addition, a well delineated, sessile mass was found on the otherwise normal serosa of the right testis. Histopathological findings included mesothelial hyperplasia and hypertrophy involving diffusely the gastric, hepatic and parietal serosae, and forming a discrete testicular capsular mass compatible with mesothelioma. In the liver an intrahepatic cholangiocarcinoma, chronic hepatitis, biliary hyperplasia and increased numbers of melanomacrophages were found. In addition organisms compatible with histozoic and coelozoic myxosporeans were found within the skeletal muscle of the abdomen and intrahepatic bile ducts, respectively. This is the third literature report of a liver tumour and the first report of a coelomic mesothelioma from a shark.

Animals↗

Population genetic structure of the lemon shark (Negaprion brevirostris) in the western Atlantic: DNA microsatellite variation.

DNA microsatellite markers were used to characterize the population genetic structure of the lemon shark, Negaprion brevirostris, in the western Atlantic. This study demonstrates for the first time the usefulness of microsatellites to study population genetic structure and mating systems in the Chondricthyes. Lemon sharks (mostly juveniles) were sampled non-destructively from four locations, Gullivan Bay and Marquesas Key in Florida, Bimini, Bahamas, and Atol das Rocas, Brazil. At least 545 individuals were genotyped at each of four dinucleotide loci. The number of alleles per locus ranged from 19 to 43, and expected heterozygosities ranged from 0.69 to 0.90. Relatively little genetic structure was found in the western Atlantic, with small but significant values for estimators of F(ST) and R(ST) among populations, theta (0.016) and rho (0.026), respectively. No sharp discontinuities were found between the Caribbean sites and Brazil, and most alleles were found at all four sites, indicating that gene flow occurs throughout the western Atlantic with no evidence for distinct stocks.

Animals↗

Myoglobin of the shark Heterodontus portusjacksoni: isolation and amino acid sequence.

Myoglobin isolated from red muscle of the shark H. portusjacksoni was purified by ion-exchange chromatography on sulfopropyl-Sephadex and gel-filtration. Amino acid analysis and sequence determination showed 148 amino acid residues. The amino terminal residue is acetylated as shown by mass spectrographic analysis of N-terminal peptides. There is a deletion of four residues at the amino terminal end as well as one residue in the CD interhelical area relative to other myoglobins. The complete amino acid sequence has been determined following digestion with trypsin, chymotrypsin, pepsin and staphylococcal protease. Sequences of the purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed approximately 85 differences from mammalian, monotreme and bird myoglobins. The date of divergence of the shark H. portusjacksoni from these other orders was estimated at 450 +/- 16 million years, based on the number of amino acid differences between species and allowing for multiple mutations during the evolutionary period. This estimate agrees well with similar estimates made using alpha- and beta-globin sequences, in contrast to widely differing estimates of dates of divergence for monotremes using the same three globin chains. Compared with myoglobins from species previously studied, there are many more differences in amino acid sequences, and in many positions residues are found that are more characteristic of alpha- and beta-globins, suggesting a conservation of residues over a long period of evolutionary time. There are fewer stabilizing hydrogen bonds and salt-linkages than in other myoglobins.

Amino Acid Sequence↗

Myoglobins of cartilaginous fishes. II. Isolation and amino acid sequence of myoglobin of the shark Mustelus antarcticus.

Myoglobin isolated from red muscle of the gummy shark M. antarcticus was purified by gel filtration and ion-exchange chromatography on carboxymethyl cellulose in 8 M urea-thiol buffer. Amino acid analysis and sequence determination showed 148 amino acid residues. The amino terminal residue is acetylated as shown by nuclear magnetic resonance and mass spectrographic analysis of an N-terminal peptide. There is a deletion of four residues at the amino terminal end as well as one residue in the CD interhelical area relative to other myoglobins. These overall differences were also found previously in myoglobin of Heterodontus portusjacksoni. The complete amino acid sequence has been determined following digestion with trypsin, chymotrypsin, thermolysin, staphylococcal protease and cyanogen bromide. Sequences of purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed approximately 88 differences from mammalian, monotreme, bird and tuna myoglobins, slightly more than previously reported for H. portusjacksoni usually considered a more primitive animal. There were 24 residues common to both shark myoglobins that were different from those present in other myoglobins. The sequence has been compared to the myoglobin of yellowfin tuna and other myoglobins.

Amino Acid Sequence↗

Production of 19S IgM antibodies with restricted heterogeneity from sharks.

Two of three nurse sharks, Ginglymostoma cirratum, immunized with streptococcal A-variant vaccine, produced over 7 mg/ml serum of 19S IgM antibody to the group-specific carbohydrate. The light chains of this specific antibody from one shark were separated into multiple bands by disc electrophoresis. The light chains from the other antibody-producing animal were much less heterogeneous and gave only one predominant band.

Animals↗

Isolation and characterization of major histocompatibility complex class IIB genes from the nurse shark.

The major histocompatibility complex (MHC) contains a set of linked genes which encode cell surface proteins involved in the binding of small peptide antigens for their subsequent recognition by T lymphocytes. MHC proteins share structural features and the presence and location of polymorphic residues which play a role in the binding of antigens. In order to compare the structure of these molecules and gain insights into their evolution, we have isolated two MHC class IIB genes from the nurse shark, Ginglymostoma cirratum. Two clones, most probably alleles, encode proteins which differ by 13 amino acids located in the putative antigen-binding cleft. The protein structure and the location of polymorphic residues are similar to their mammalian counterparts. Although these genes appear to encode a typical MHC protein, no T-cell-mediated responses have been demonstrated in cartilaginous fish. The nurse shark represents the most phylogenetically primitive organism in which both class IIA [Kasahara, M., Vazquez, M., Sato, K., McKinney, E.C. & Flajnik, M.F. (1992) Proc. Natl. Acad. Sci USA 89, 6688-6692] and class IIB genes, presumably encoding the alpha/beta heterodimer, have been isolated.

Amino Acid Sequence↗

Primordial emergence of the recombination activating gene 1 (RAG1): sequence of the complete shark gene indicates homology to microbial integrases.

The rearrangement of antibody and T-cell receptor gene segments is indispensable to the vertebrate immune response. All extant jawed vertebrates can rearrange these gene segments. This ability is conferred by the recombination activating genes I and II (RAG I and RAG II). To elucidate their origin and function, the cDNA encoding RAG I from a member of the most ancient class of extant gnathostomes, the Carcharhine sharks, was characterized. Homology domains identified within shark RAG I prompted sequence comparison analyses that suggested similarity of the RAG I and II genes, respectively, to the integrase family genes and integration host factor genes of the bacterial site-specific recombination system. Thus, the apparent explosive evolution (or "big bang") of the ancestral immune system may have been initiated by a transfer of microbial site-specific recombinases.

Amino Acid Sequence↗