Identification of genetic markers associated with Gyrodactylus salaris resistance in Atlantic salmon Salmo salar

Gyrodactylus salaris Malmberg, 1957 is a freshwater monogenean ectoparasite of salmonids, first recorded in Norway in 1975 and responsible for extensive epizootics in wild Atlantic salmon Salmo salar L. The susceptibility of different populations of Atlantic salmon to G. salaris infection differs markedly, with fish from the Baltic being characterised as relatively resistant whereas those from Norway or Scotland are known to be (extremely) susceptible. Resistance to Gyrodactylus infection in salmonids has been found to be heritable and a polygenic mechanism of control has been hypothesised. The current study utilises a 'Quantitative trait loci' (QTL) screening approach in order to identify molecular markers linked to QTL influencing G. salaris resistance in B1 backcrosses of Baltic and Scottish salmon. Infection patterns in these fish exhibited 3 distinct types; susceptible (exponential parasite growth), responding (parasite load builds before dropping) and resistant (parasite load never increases). B1 backcross fish were screened at 39 microsatellite markers and single marker-trait associations were examined using general linear modelling. We identified 10 genomic regions associated with heterogeneity in both innate and acquired resistance, explaining up to 27.3% of the total variation in parasite loads. We found that both innate and acquired parasite resistance in Atlantic salmon are under polygenic control, and that salmon would be well suited to a selection programme designed to quickly increase resistance to G. salaris in wild or farmed stocks.     

Atlantic salmon eggs favour sperm in competition that have similar major histocompatibility alleles

Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran in vitro fertilization competitions that mimicked the gametic microenvironment, and replicated a paired-male experimental design that allowed us to compare differences in sperm competition success among males when their sperm compete for eggs from females that were genetically either similar or dissimilar at the MH class I locus. Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm-egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation.     

Mass-spectrometric characterization of two posttranslational modifications of cysteine dioxygenase

Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields “mature” and “immature” forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry. Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier. Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation, they may actually arise via nonenzymatic, nonphysiological processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Simplified cysteine dioxygenase activity assay allows simultaneous quantitation of both substrate and product

A high-performance liquid chromatography (HPLC) method for enzyme activity assays using a hydrophilic interaction liquid chromatography (HILIC) column in combination with an evaporative light scattering detector was developed. The method was used to measure the activity of the non-heme mono-iron enzyme cysteine dioxygenase. The substrate cysteine and the product cysteine sulfinic acid are very weak chromophores, making direct ultraviolet (UV) detection without derivatization rather insensitive; moreover, derivatization of cysteine is often not efficient. Using the system described, underivatized substrate and product in samples from cysteine dioxygenase activity assays could be separated and analyzed. Furthermore, it was possible to quantify cysteic acid, the noncatalytic oxidation product of cysteine sulfinic acid. Acetone was used both to stop the enzymatic reaction by protein precipitation and as an organic mobile phase, making sample preparation very easy and the assay highly reproducible.     

Radiosynthesis and biodistribution of a prosthetic group (¹⁸F-FENMA) conjugated to cyclic RGD peptides

We have recently reported a new N-methylaminooxy-based prosthetic group for the site-selective introduction of 1?F-fluorine under mild acidic aqueous conditions into model peptides functionalized with a Michael acceptor moiety. To further investigate the utility of this methodology, the radiosynthesis of two cyclic RGD peptides was carried out, and in vivo biodistribution and microPET studies were performed in tumor-bearing mice. A cyclic RGD peptide was functionalized with the Michael acceptors trans-β-nitrostyrene carboxylic acid and 3-vinylsulfonylpropionic acid. Radiolabeling was then performed with the prosthetic group O-(2-(2-[1?F]fluoroethoxy)ethyl)-N-methylhydroxylamine (1?F-FENMA) yielding the 1?F-conjugates in moderate yields (8.5-12%). Biodistribution, blocking, and microPET imaging studies were performed in a mouse xenograft model. The vinylsulfonyl-modified conjugate demonstrated good in vitro plasma stability. Biodistribution and microPET studies revealed excellent tumor uptake with low background in key organs and renal elimination as the predominant route of excretion. Blocking studies with coinjected nonlabeled RGD peptide confirmed the in vivo specificity for the integrin α(v)β?. On the other hand, 1?F-FENMA-nitrostyrene-RGD, although stable at conjugation pH 5, was found to rapidly degrade at physiological pH through loss of the 1?F-prosthetic group.     

Habitat use and diet of sympatric Arctic charr (<Emphasis Type="Italic">Salvelinus alpinus</Emphasis>) and whitefish (<Emphasis Type="Italic">Coregonus lavaretus</Emphasis>) in five lakes in southern Norway: not only interspecific population dominance?

Stable coexistence of Arctic charr and whitefish does occur in a number of native lake fish communities in Scandinavia. Even so, whitefish introductions into Arctic charr lakes have resulted in serious decline and possibly local extinction of Arctic charr. In this article, we analyze the habitat use and diet of the two species in five Norwegian lakes differing in basin shape and environmental conditions. In two of the lakes, both species are native, and appear to live in a relatively stable coexistence. Here, whitefish mainly occupy the littoral and upper pelagic zone, while Arctic charr live in the deeper habitats. Diets are generally quite different in terms of the zooplankton species eaten. In the three other lakes, either whitefish or both species have been introduced. In the shallowest lake, habitat segregation is similar to that seen in the pristine lakes, although Arctic charr appears to be on the brink of extinction. In the remaining two lakes, however, Arctic charr dominates, and occurs in higher numbers than whitefish in all the habitats. Our observations indicate that coexistence of the two species in oligotrophic and relatively pristine lakes requires an extensive profundal zone to serve as a refugium for Arctic charr. If the littoral zone is rendered inaccessible or unprofitable for whitefish due to dominance of a third competitor or predator, or as a result of lake regulation, then Arctic charr may be the dominant species.     

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.     

Thermal Behaviour of Honeybees During Aggressive Interactions

We report here on the interrelationship of aggressive behaviour and thermoregulation in honeybees. Body temperature measurements were carried out without behavioural disturbance by infrared thermography. Guard bees, foragers, drones, and queens involved in aggressive interactions were always endothermic, i.e. had their flight muscles activated. Guards made differential use of their endothermic capacity. Mean thorax temperature was 34.2–35.1°C during examination of bees but higher during fights with wasps (37°C) or attack of humans (38.6°C). They usually cooled down when examining bees whereas examinees often heated up during prolonged interceptions (maximum >47°C). Guards neither adjusted their thorax temperature (and thus flight muscle function and agility) to that of examined workers, nor to that of drones, which were 2–7°C warmer. Guards examined cool bees (<33°C) longer than warmer ones, supporting the hypothesis that heating of examinees facilitates odour identification by guards, probably because of vapour pressure increase of semiochemicals with temperature. Guards in the core of aggressive balls clinged to the attacked insects to fix them and kill them by heat (maximum 46.5°C). Bees in the outer cluster layers resembled normal guards behaviourally and thermally. They served as active core insulators by heating up to 43.9°C. While balled wasps were cooler (maximum 42.5°C) than clinging guards balled bees behaved like examinees with maximum temperatures of 46.6°C, which further supports the hypothesis that the examinees heat up to facilitate odour identification.