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.     

Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.     

CD4+CD25+ cells controlling a pathogenic CD4 response inhibit cytokine differentiation, CXCR-3 expression, and tissue invasion

It is well established that CD4(+)CD25(+) regulatory T cells (Tregs) inhibit autoimmune pathology. However, precisely how the behavior of disease-inducing T cells is altered by Tregs remains unclear. In this study we use a TCR transgenic model of diabetes to pinpoint how pathogenic CD4 T cells are modified by Tregs in vivo. We show that although Tregs only modestly inhibit CD4 cell expansion, they potently suppress tissue infiltration. This is associated with a failure of CD4 cells to differentiate into effector cells and to up-regulate the IFN-gamma-dependent chemokine receptor CXCR-3, which confers the ability to respond to pancreatic islet-derived CXCL10. Our data support a model in which Tregs permit T cell activation, yet prohibit T cell differentiation and migration into Ag-bearing tissues.     

Identification of quantitative trait loci (QTL) for oil and protein contents and their relationships with other seed quality traits in Brassica juncea

A detailed RFLP-genomic map was used to study the genetics of oil, seed and meal protein and sum of oil and seed/meal protein contents in a recombinant doubled-haploid population developed by crossing black- and yellow-seeded Brassica juncea lines. Two yellow seed color genes (SC-B4, SC-A6) and one QTL for erucic acid content (E_1b) showed pleiotropic effect for oil, protein and sum of oil and seed/meal protein contents. Six (O-A1, O-A6, O-A9, O-B3, O-B4, O-B5) and five (SP-A1, SP-A9, SP-B4, SP-B6, SP-C) QTLs were significant for oil and seed protein contents, respectively. Tight linkage of three of these QTLs (SP-A1, SP-A9, SP-B4, O-A1, O-A9, O-B4), with opposite effects, poses challenge to the plant breeders for simultaneous improvement of negatively correlated (r = −0.7**) oil and seed protein contents. However, one QTL for oil content (O-B3) and two for seed protein content (SP-B6, SP-C) were found to be unlinked, which offer the possibility for simultaneous improvement of these two traits. QTLs significant for meal protein (MP-A1, MP-A6, MP-A9, MP-B5, MP-B6) were significant at least for oil, seed protein or sum of oil and seed/meal protein contents (T-A6, T-A7, T-B4, T-B5). Sum of oil and seed protein contents and sum of oil and meal protein contents had a perfect correlation, as well as same epistatic interactions and QTLs with similar additive effect. This indicates that protein in seed or meal has practically the same meaning for breeding purposes. Epistatic interactions were significant for the quality traits, and their linkage reflected association among the traits.     

New cytogenetic information on Allium iranicum (Alliaceae) from Iran

Karyotype analysis and chromosome behaviour in tetraploid Allium iranicum is reported. The somatic karyotype 2n = 32, consists of 12 pairs of metacentric chromosomes, two pairs of submetacentric chromosomes and two pairs of submetacentric satellite chromosomes. Chromosome complement follows two sets of 16 pairs of homologous chromosomes. A detailed analysis of Pachytene, Diplotene and Metaphase I of meiosis in pollen mother cells in this taxon showed that the most common chromosome configurations were bivalents at all subphases mentioned. It is concluded that A. iranicum is most likely a natural allotetraploid and certainly differs from related species A. ampeloprasum, A. commutatum and A. porrum.     

Efficient large-scale protein sequence comparison and gene matching to identify orthologs and co-orthologs

Broadly, computational approaches for ortholog assignment is a three steps process: (i) identify all putative homologs between the genomes, (ii) identify gene anchors and (iii) link anchors to identify best gene matches given their order and context. In this article, we engineer two methods to improve two important aspects of this pipeline [specifically steps (ii) and (iii)]. First, computing sequence similarity data [step (i)] is a computationally intensive task for large sequence sets, creating a bottleneck in the ortholog assignment pipeline. We have designed a fast and highly scalable sort-join method (afree) based on k-mer counts to rapidly compare all pairs of sequences in a large protein sequence set to identify putative homologs. Second, availability of complex genomes containing large gene families with prevalence of complex evolutionary events, such as duplications, has made the task of assigning orthologs and co-orthologs difficult. Here, we have developed an iterative graph matching strategy where at each iteration the best gene assignments are identified resulting in a set of orthologs and co-orthologs. We find that the afree algorithm is faster than existing methods and maintains high accuracy in identifying similar genes. The iterative graph matching strategy also showed high accuracy in identifying complex gene relationships. Standalone afree available from http://vbc.med.monash.edu.au/～kmahmood/afree. EGM2, complete ortholog assignment pipeline (including afree and the iterative graph matching method) available from http://vbc.med.monash.edu.au/～kmahmood/EGM2.     

Metabolism of chlorobiphenyls by a variant biphenyl dioxygenase exhibiting enhanced activity toward dibenzofuran

The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) catalyzes the dihydroxylation of biphenyl and of several polychlorinated biphenyls (PCBs) but it poorly oxidizes dibenzofuran. In this work we showed that BphAE(RR41), a variant which was previously found to metabolize dibenzofuran more efficiently than its parent BphAE(LB400), metabolized a broader range of PCBs than BphAE(LB400). Hence, BphAE(RR41) was able to metabolize 2,6,2',6'-, 3,4,3',5'- and 2,4,3',4'-tetrachlorobiphenyl that BphAE(LB400) is unable to metabolize. BphAE(RR41) was obtained by changing Thr335Phe336Asn338Ile341Leu409 of BphAE(LB400) to Ala335Met336Gln338Val341Phe409. Site-directed mutagenesis was used to create combinations of each substitution, in order to assess their individual contributions. Data show that the same Asn338Glu/Leu409Phe substitution that enhanced the ability to metabolize dibenzofuran resulted in a broadening of the PCB substrates range of the enzyme. The role of these substitutions on regiospecificities toward selected PCBs is also discussed.