Recombinant shark natural antibodies to thyroglobulin

As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.     

Genetic estimates of contemporary effective population size: to what time periods do the estimates apply?

Although most genetic estimates of contemporary effective population size (Ne) are based on models that assume Ne is constant, in real populations Ne changes (often dramatically) over time, and estimates (Ne) will be influenced by Ne in specific generations. In such cases, it is important to properly match Ne to the appropriate time periods (for example, in computing Ne/N ratios). Here I consider this problem for semelparous species with two life histories (discrete generations and variable age at maturity--the 'salmon' model), for two different sampling plans, and for estimators based on single samples (linkage disequilibrium, heterozygote excess) and two samples (temporal method). Results include the following. Discrete generations: (i) Temporal samples from generations 0 and t estimate the harmonic mean Ne in generations 0 through t - 1 but do not provide information about Ne in generation t; (ii) Single samples provide an estimate of Ne in the parental generation, not the generation sampled; (iii) single-sample and temporal estimates never provide information about Ne in exactly the same generations; (iv) Recent bottlenecks can downwardly bias estimates based on linkage disequilibrium for several generations. Salmon model: (i) A pair of single-cohort (typically juvenile) samples from years 0 and t provide a temporal estimate of the harmonic mean of the effective numbers of breeders in the two parental years (N b(0) and N b(t)), but adult samples are more difficult to interpret because they are influenced by Nb in a number of previous years; (ii) For single-cohort samples, both one-sample and temporal methods provide estimates of Nb in the same years (contrast with results for discrete generation model); (iii) Residual linkage disequilibrium associated with past population size will not affect single-sample estimates of Nb as much as in the discrete generation model because the disequilibrium diffuses among different years of breeders. These results lead to some general conclusions about genetic estimates of Ne in iteroparous species with overlapping generations and identify areas in need of further research.     

Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific Oysters, Crassostrea gigas

Despite some 26 published reports addressing oyster sperm cryopreservation, systematic factor optimization is lacking, and sperm cryopreservation has not yet found application in aquaculture on a commercial scale. In this study, the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization of shipped sperm samples from diploid oysters. Evaluation of cooling rates revealed an optimal rate of 5 degrees C/min to -30 degrees C followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations suggested that a low concentration (2%) of polyethylene glycol (FW 200) was effective in retaining post-thaw motility and fertilizing capability when combined with permeating cryoprotetcants such as dimethyl sulfoxide (DMSO), methanol (MeOH), and propylene glycol (P-glycol). However, polyethylene glycol alone was not as effective as MeOH, DMSO, and P-glycol when using the same methods. The highest post-thaw motility (70%) and percent fertilization (98%) were obtained for samples cryopreserved with 6% MeOH. However, this does not exclude other cryoprotectants such as DMSO or P-glycol identified as effective agents in other studies. There was no significant difference in post-thaw motility between straw sizes of 0.25- and 0.5-ml. Equilibration time (exposure to cryoprotectant) of 60 min could be beneficial when the cryoprotectant concentration is low and solution is added in a step-wise fashion at low temperature. Differences in post-thaw sperm quality (e.g., motility or percent fertilization) among individual males were evident in this research. As a consequence, a generalized classification describing males with different tolerances (broad, intermediate, and narrow) to cryopreservation was developed. This classification could be applied to strain or species differences in tolerances to the cryopreservation process. The present study demonstrated that oyster sperm could be collected and shipped chilled to another facility for cryopreservation, and that it could be shipped back to the hatchery for fertilization performed at a production scale yielding live larvae with >90% fertilization. Given the existence of facilities for commercial-scale cryopreservation of dairy bull sperm, the methods developed in the present study for oysters provide a template for the potential commercialization of cryopreserved sperm in aquatic species.     

Global-scale genetic identification of hammerhead sharks: Application to assessment of the international fin trade and law enforcement

The future status of sharks is an issue of widespread conservation concern due to declines in many species in the face of high levels of exploitation to satisfy market demands for products, especially fins. Substantial declines in the large-bodied hammerhead sharks, Sphyrna lewini, S. mokarran and S. zygaena, even in regions where some management occurs, indicate that informed conservation measures are warranted for these circumglobally distributed species. Despite the importance of assessing shark catch and trade on a species-specific basis to detect potential overexploitation of individual species, achieving this goal for hammerheads has proven elusive due to difficulties in identification of their products. Here, we present the development and application of a diagnostic, streamlined, five-primer multiplex polymerase chain reaction assay utilizing species-specific primers based on nuclear ribosomal ITS2 for the three hammerhead species throughout their global distribution. Application of this assay to investigations of the fin market confirmed the presence of hammerhead fins in the international trade. A study of the world’s largest fin market in Hong Kong revealed a high concordance between specific Chinese-name trade categories and fins from these three species (“Bai Chun” with S. lewini, “Gui Chun” with S. zygaena and “Gu Pian” with S.␣mokarran), and clear species preferences. This concordance information allows the use of market records for monitoring species-specific trends in trade and exploitation rates. The assay is also proving useful for identification of shark body parts in U.S. fisheries law-enforcement activities. Screening of morphologically identified “ S. lewini” from globally distributed areas using this assay with subsequent whole ITS2 sequencing suggests a cryptic species closely related to S. lewini occurs off the SE USA coast.     

Genetic profiling reveals illegal international trade in fins of the great white shark, <Emphasis Type="Italic">Carcharodon carcharias</Emphasis>

Great white sharks are protected by national legislation in several countries, making this species the most widely protected elasmobranch in the world. Although the market demand for shark fins in general has continued to grow, the value and extent of utilization of white shark fins in trade has been controversial. We combine law enforcement with genetic profiling to demonstrate that illegal trade in fins of this species is occurring in the contemporary international market. Furthermore, we document the presence of fins from very young white sharks in the trade, suggesting a multiple-use market (food to trophies) exists for fins of this species. The presence of small fins in the trade contradicts the view that white shark fins have market value only as large display trophies, and not as food. Our findings indicate that effective conservation of protected shark species will require international management regimes that include monitoring of the shark fishery and trade on a species-specific basis.     

Effects of shark hepatic stimulator substance on the function and antioxidant capacity of liver mitochondria in an animal model of acute liver injury

This study was carried out to investigate whether shark hepatic stimulator substance （HSS） can prevent acute liver injury and affect mitochondrial function and antioxidant defenses in a rat model of thioacetamide （TAA）-induced liver injury. The acute liver injury was induced by two intraperitoneal injections of TAA （400 mg/kg） in a 24 h interval. In the TAA plus shark HSS group, rats were treated with shark HSS （80 mg/kg） 1 h prior to each TAA injection. In this group, serum liver enzyme activities were significantly lower than those in the TAA group. The mitochondrial respiratory control ratio was improved, and the mitochondrial respiratory enzyme activities were increased in the TAA plus shark HSS group. The mitochondrial antioxidant enzyme activities and glutathione level were higher in the TAA plus shark HSS group than in the TAA group. These results suggest that the protective effect of shark HSS against TAA-induced acute liver injury may be a result of the restoration of the mitochondrial respiratory function and antioxidant defenses and decreased oxygen stress.     

Life-history divergence in Chinook salmon: historic contingency and parallel evolution

By jointly considering patterns of genetic and life-history diversity in over 100 populations of Chinook salmon from California to British Columbia, we demonstrate the importance of two different mechanisms for life-history evolution. Mapping adult run timing (the life-history trait most commonly used to characterize salmon populations) onto a tree based on the genetic data shows that the same run-time phenotypes exist in many different genetic lineages. In a hierarchical gene diversity analysis, differences among major geographic and ecological provinces explained the majority (62%) of the overall G(ST), whereas run-time differences explained only 10%. Collectively, these results indicate that run-timing diversity has developed independently by a process of parallel evolution in many different coastal areas. However, genetic differences between coastal populations with different run timing from the same basin are very modest (G(ST) < 0.02), indicating that evolutionary divergence of this trait linked to reproductive isolation has not led to parallel speciation, probably because of ongoing gene flow. A strikingly different pattern is seen in the interior Columbia River Basin, where run timing and other correlated life-history traits map cleanly onto two divergent genetic lineages (G(ST) approximately 0.15), indicating that some patterns of life-history diversity have a much older origin. Indeed, genetic data indicate that in the interior Columbia Basin, the two divergent lineages behave essentially as separate biological species, showing little evidence of genetic contact in spite of the fact that they comigrate through large areas of the river and ocean and in some locations spawn in nearly adjacent areas.     

Conservation Genetics of the East Pacific Green Turtle (Chelonia mydas) in Michoacan, Mexico

The main continental nesting rookeries of the east Pacific green turtle (EPGT), Chelonia mydas, on the Michoacan (Mexico) coast suffered drastic population declines following intense exploitation in the 1960s--1970s with annual abundance of nesting females plummeting from about 25,000 to an average of about 1400 between 1982 and 2001. Analyses of data from three nDNA microsatellite loci and 400 bp mtDNA control region sequences from a total of 123 nesting females sampled from four Michoacan rookeries found no evidence of population sub-structuring. The recent order of magnitude reduction in the population size shows no apparent impact on genetic diversity in either control region sequences (overall h = 0.48; pi = 0.0036) or microsatellite loci (overall Na = 20.8; Hexp = 0.895). Our estimates of annual effective female population size (Nef; from theta = 2Nemicron) of 1.9-2.3 x 10(3), in spite of being an order of magnitude below historical records, appear to be sufficient to allow recovery of this population without significant loss of genetic diversity. These findings highlight the importance of continued conservation to reverse the decline of this population before it becomes vulnerable to genetic erosion.     

Isolation and characterization of microsatellite DNA markers in the Pacific abalone,Haliotis discus hannai

We developed 17 new microsatellite markers in Haliotis discus hannai. All loci were found to be polymorphic with an average of 13.1 alleles per locus (range 3–28). The mean observed and expected heterozygosities were 0.77 (range 0.17–1.00) and 0.79 (range 0.42–0.96), respectively. Six loci deviated significantly from Hardy–Weinberg proportions, and thus should be used with caution. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for studies of trait mapping, kinship and population genetics.