Searching for a signal: Environmental DNA (eDNA) for the detection of invasive signal crayfish, Pacifastacus leniusculus (Dana, 1852)

Environmental DNA (eDNA) is a rapid, non-invasive method for species detection and distribution using DNA deposited in the environment by target organisms. eDNA has become a recognised and powerful tool for detecting invasive species in a broad range of aquatic ecosystems. We examined the use of eDNA as a tool for detecting the invasive American signal crayfish Pacifastacus leniusculus in Scotland. Species-specific TaqMan probe and primers were designed for P. leniusculus and a robust quantitative PCR (qPCR) assay and DNA extraction protocol were developed. We investigated the detection capability for P. leniusculus from water samples in a controlled laboratory experiment and determined whether crayfish density (low = 1 crayfish 5.5 L-1 or high = 3 crayfish 5.5 L-1) or length of time in tanks (samples taken at 1, 3 and 7 days) influenced DNA detectability. Additionally, the persistence of DNA was investigated after P. leniusculus removal (samples taken at 1, 3 and 7 days post removal). P. leniusculus DNA was consistently detected during the entire 7-day period and higher density tanks yielded stronger positive results with lower Ct values. After removal of P. leniusculus, there was a rapid and continuous decrease in the detectability of DNA. P. leniusculus DNA could only be detected in high density tanks by the end of the 7-day period, while DNA was no longer detectable in low density tanks after 72 hours. Preliminary field experiments sampled water from three sites in winter and five sites in summer. P. leniusculus was known to be present at two of these sites. P. leniusculus was not detected at any site in winter. However, in summer, positive signals were observed at sites with known P. leniusculus, and at sites where P. leniusculus was believed to be present anecdotally, but not confirmed. All sites where crayfish were believed to be absent were negative for eDNA. Therefore, eDNA represents a promising technique to detect and monitor invasive P. leniusculus, although the presence of detectable amounts of eDNA may be season and location dependent, even where invasive crayfish are known to be present

DNA polymorphism underlying allozyme variation at a malic enzyme locus (mMEP-2*) in Atlantic salmon (Salmo salar L.)

A non-synonymous single nucleotide polymorphism (SNP) underlies a diallelic allozyme polymorphism at the mitochondrial NADP-dependent mMEP-2* locus in Atlantic salmon (\textit{Salmo salar} L.). The resultant amino acid substitution, which alters the charge of the allelic products, matches the differential mobility of the two allozyme alleles, while allozyme and SNP assays revealed genotyping concordance in 257 of 258 individuals. A single mismatch, homozygous allozyme vs heterozygote SNP, suggests the presence of a second, less common null allele

Diablo/SMAC: A novel biomarker of pollutant exposure in European flounder (Platichthys flesus)

Diablo (or SMAC) is a protein released from mitochondria following apoptotic stimuli and inhibits the actions of Inhibitors of Apoptosis (IAP) proteins. IAPs regulate the activity of caspases and NFkB, the primary executioners of apoptosis and of inflammation respectively. Thus, Diablo is important for the regulation of cellular responses to damage. In Northern Europe, statutory governmental marine monitoring programs measure various biomarkers in flounder to indicate biological effects of pollutant exposure. More recently transcriptomic techniques have been applied in flounder to gain a more comprehensive understanding of pollutant effects, and to discover novel biomarkers. In most of these studies utilising flounder, Diablo was amongst the most highly increased transcripts identified. The aim of this study was to further examine piscine Diablo, at the gene level and mRNA level, after exposure to prototypical pollutants, and in flounder caught from polluted environments. The results show that two genes encoding Diablo exist in fish species, and in flounder one of these genes is increased in liver after exposure to polyaromatic hydrocarbons and polychlorinated biphenyls, and also in livers from fish living on contaminated estuarine sediments. Therefore, Diablo measurement has potential as a biomarker of pollutant exposure, and could indicate damaging effects of chemical contaminants

Effect of dietary conjugated linoleic acid (CLA) on lipid composition, metabolism and gene expression in Atlantic salmon (Salmo salar) tissues

Dietary conjugated linoleic acid (CLA) affects fat deposition and lipid metabolism in mammals, including livestock. To determine CLA effects in Atlantic salmon (Salmo salar), a major farmed fish species, fish were fed for 12 weeks on diets containing fish oil or fish oil with 2% and 4% CLA supplementation. Fatty acid composition of the tissues showed deposition of CLA with accumulation being 2 to 3 fold higher in muscle than in liver. CLA had no effect on feed conversion efficiency or growth of the fish but there was a decreased lipid content and increased protein content after 4%CLA feeding. Thus, the protein:lipid ratio in whole fish was increased in fish fed 4% CLA and triacylglycerol in liver was decreased. Liver β-oxidation was increased whilst both red muscle β-oxidation capacity and CPT1 activity was decreased by dietary CLA. Liver highly unsaturated fatty acid (HUFA) biosynthetic capacity was increased and the relative proportion of liver HUFA was marginally increased in salmon fed CLA. CLA had no effect on fatty acid Δ6 desaturase mRNA expression, but fatty acid elongase mRNA was increased in liver and intestine. In addition, the relative compositions of unsaturated and monounsaturated fatty acids changed after CLA feeding. CLA had no effect on PPARα or PPARγ expression in liver or intestine, although PPARβ2A expression was reduced in liver at 4% CLA feeding. CLA did not affect hepatic malic enzyme activity. Thus, overall, the effect of dietary CLA was to increase β-oxidation in liver, to reduce levels of total body lipid and liver triacylglycerol, and to affect liver fatty acid composition, with increased elongase expression and HUFA biosynthetic capacity

Functional genomics reveals increases in cholesterol biosynthetic genes and highly unsaturated fatty acid biosynthesis after dietary substitution of fish oil with vegetable oils in Atlantic salmon (Salmo salar)

<p>Abstract</p> <p>Background</p> <p>There is an increasing drive to replace fish oil (FO) in finfish aquaculture diets with vegetable oils (VO), driven by the short supply of FO derived from wild fish stocks. However, little is known of the consequences for fish health after such substitution. The effect of dietary VO on hepatic gene expression, lipid composition and growth was determined in Atlantic salmon (<it>Salmo salar</it>), using a combination of cDNA microarray, lipid, and biochemical analysis. FO was replaced with VO, added to diets as rapeseed (RO), soybean (SO) or linseed (LO) oils.</p> <p>Results</p> <p>Dietary VO had no major effect on growth of the fish, but increased the whole fish protein contents and tended to decrease whole fish lipid content, thus increasing the protein:lipid ratio. Expression levels of genes of the highly unsaturated fatty acid (HUFA) and cholesterol biosynthetic pathways were increased in all vegetable oil diets as was SREBP2, a master transcriptional regulator of these pathways. Other genes whose expression was increased by feeding VO included those of NADPH generation, lipid transport, peroxisomal fatty acid oxidation, a marker of intracellular lipid accumulation, and protein and RNA processing. Consistent with these results, HUFA biosynthesis, hepatic β-oxidation activity and enzymic NADPH production were changed by VO, and there was a trend for increased hepatic lipid in LO and SO diets. Tissue cholesterol levels in VO fed fish were the same as animals fed FO, whereas fatty acid composition of the tissues largely reflected those of the diets and was marked by enrichment of 18 carbon fatty acids and reductions in 20 and 22 carbon HUFA.</p> <p>Conclusion</p> <p>This combined gene expression, compositional and metabolic study demonstrates that major lipid metabolic effects occur after replacing FO with VO in salmon diets. These effects are most likely mediated by SREBP2, which responds to reductions in dietary cholesterol. These changes are sufficient to maintain whole body cholesterol levels but not HUFA levels.</p

Piscine UDP-glucuronosyltransferase 1B

Glucuronidation is an important detoxification pathway for organic pollutants in fish. We report here the isolation and characterisation of UDP-glucuronosyltransferases (UGT) genes from the closely related marine flatfish, plaice (Pleuronectes platessa) and flounder (Platichthys flesus). The deduced amino acid sequences share greater similarity with mammalian UGT1 family genes than UGT2 genes (44-47% and 39-40% amino acid identity respectively) and have been designated UGT1B. Both plaice and flounder UGT1B mRNAs are most highly expressed in liver, but are also expressed in intestine, gill, kidney and adipose tissue to a greater extent than muscle, heart or brain. Plaice UGT1B mRNA was undetectable in gametes or fertilised eggs and there was a large increase in expression between gastrulation and myotome formation after which levels declined some 5-10 fold. Flounder UGT1B mRNA was increased in liver after intraperitoneal injection of Arochlor 1254 or lindane, but not after perflourooctanoic acid or 3-methylcholanthrene. In isolated flounder hepatocytes UGT1B mRNA was increased by benzo(a)pyrene but not by ethynylestradiol. Expression of a cDNA for plaice UGT1B in cos7 cells resulted in higher 1-naphthol conjugation in cell homogenates compared to steroid conjugation, whilst bilirubin and bile acid conjugation undetectable. This indicates that the plaice gene codes for the phenol-conjugating UGT previously purified in our laboratory from this species and that it is likely to play a major role in the detoxification of polyaromatic hydrocarbons in flatfish. Its role in development is unknown. UGT1B genes are also present in pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio) genomes, but that they differ in their genic organisation. Pufferfish possess multiple (repeated) complete UGT1 genes and Southern blots indicate that the homologous plaice UGT1B gene may also be organised in this way. In contrast, zebrafish appear to have two UGT1 loci whose sequences and intron/exon structures are closely related to that of plaice, however, the organisation of these genes is similar to the mammalian UGT1 family since each has multiple repeated exon 1’s which are alternatively spliced to a common set of exons encoding the aglycone binding domain. Taken together with evidence from phylogenetic comparison of fish sequences with UGT1 and UGT2 families in mammals, we suggest these homologous fish UGTs should all be included within the vertebrate UGT1 family and designated as UGT1B

Molecular characterization of a gilthead sea bream (Sparus aurata) muscle tissue cDNA for carnitine palmitoyltransferase 1B (CPT1B)

Understanding the control of piscine fatty acid metabolism is important for determining the nutritional requirements of fish, and hence for the production of optimal aquaculture diets. The regulation and expression of carnitine palmitoyltransferase 1 (CPT1; EC No 2.3.1.21) are critical processes in the control fatty acid metabolism, and here we report a cDNA from gilthead sea bream (Sparus aurata) which encodes a protein with high identity to vertebrate CPT1. This sea bream CPT1 mRNA is predominantly expressed in skeletal and cardiac muscle, with little expression in other tissues. Phylogenetic analysis of other vertebrate CPT1 sequences show that fish genomes contain a single gene related to mammalian CPT1B, and a further two multi-gene families related to mammalian CPT1A. Genes related to mammalian CPT1C are absent in fish. Therefore, based on both functional and evolutionary orthology to mammalian CPT1B, the sea bream CPT1 reported here is a CPT1B isoform. Sea bream CPT1B mRNA expression progressively decreases in heart and muscle up to 12 hours after last feeding, but returns to initial, non-fasted levels after 72 hours. In contrast, in liver non-fasted expression is low, but strongly increases at 24 and 72 hours after last feeding. In white muscle and liver, CPT1B mRNA expression is highly correlated with the expression of peroxisomal proliferator-activated receptor ı (PPARı).Thus fatty acid metabolism by CPT1B and its control by PPARs is similar in fish and mammals, but multiple genes for CPT1A-like proteins in fish also suggest different and more complex pathways of lipid utilisation than in mammals

Development of a minimization instrument for allocation of a hospital-level performance improvement intervention to reduce waiting times in Ontario emergency departments

<p>Abstract</p> <p>Background</p> <p>Rigorous evaluation of an intervention requires that its allocation be unbiased with respect to confounders; this is especially difficult in complex, system-wide healthcare interventions. We developed a short survey instrument to identify factors for a minimization algorithm for the allocation of a hospital-level intervention to reduce emergency department (ED) waiting times in Ontario, Canada.</p> <p>Methods</p> <p>Potential confounders influencing the intervention's success were identified by literature review, and grouped by healthcare setting specific change stages. An international multi-disciplinary (clinical, administrative, decision maker, management) panel evaluated these factors in a two-stage modified-delphi and nominal group process based on four domains: change readiness, evidence base, face validity, and clarity of definition.</p> <p>Results</p> <p>An original set of 33 factors were identified from the literature. The panel reduced the list to 12 in the first round survey. In the second survey, experts scored each factor according to the four domains; summary scores and consensus discussion resulted in the final selection and measurement of four hospital-level factors to be used in the minimization algorithm: improved patient flow as a hospital's leadership priority; physicians' receptiveness to organizational change; efficiency of bed management; and physician incentives supporting the change goal.</p> <p>Conclusion</p> <p>We developed a simple tool designed to gather data from senior hospital administrators on factors likely to affect the success of a hospital patient flow improvement intervention. A minimization algorithm will ensure balanced allocation of the intervention with respect to these factors in study hospitals.</p

Long-chain polyunsaturated fatty acid synthesis in fish: Comparative analysis of Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.) Delta 6 fatty acyl desaturase gene promoters

Fish vary in ability to biosynthesise n-3 long-chain polyunsaturated fatty acids (LC-PUFA), with marine fish such as cod being inefficient in comparison to freshwater and salmonid fish. We investigated differences in the gene promoters of Δ6 fatty acyl desaturase (Δ6 FAD), a critical enzyme in LC-PUFA biosynthesis, in cod and salmon. Progressive deletions and targeted mutations of the promoters were tested for activity in a transfected fish cell line under low or high LC-PUFA treatment, and regions sufficient to direct transcription were identified. Comparison of these regions with sequences of corresponding regions of Δ6 FAD genes from mammals, amphibians and fish indicated a remarkable conservation of binding sites for SREBPs and NF-Y. In addition to these sites, a site was identified in salmon with similarity to that recognised by Sp1 transcription factor, and which was required for full expression of the salmon Δ6 FAD gene. The cod promoter was less active and lacked the Sp1 site. Eicosapentaenoic acid suppressed LC-PUFA synthesis in AS cells and also suppressed activity of the salmon Δ6 FAD promoter although this activity was likely mediated through sites other than Sp1, possibly similar to those recognised by NF-Y and SREBP transcription factors

Passport, a native Tc1 transposon from flatfish, is functionally active in vertebrate cells

The Tc1/mariner family of DNA transposons is widespread across fungal, plant and animal kingdoms, and thought to contribute to the evolution of their host genomes. To date, an active Tc1 transposon has not been identified within the native genome of a vertebrate. We demonstrate that Passport, a native transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells. In transposition assays, we found that the Passport transposon system improved stable cellular transgenesis by 40-fold, has an apparent preference for insertion into genes, and is subject to overproduction inhibition like other Tc1 elements. Passport represents the first vertebrate Tc1 element described as both natively intact and functionally active, and given its restricted phylogenetic distribution, may be contemporaneously active. The Passport transposon system thus complements the available genetic tools for the manipulation of vertebrate genomes, and may provide a unique system for studying the infiltration of vertebrate genomes by Tc1 elements