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Repeated climatic and vegetation changes during the Pleistocene have shaped biodiversity in Northern Europe including Denmark. The Northern Birch Mouse ( Sicista betulina ) was one of the first small rodent species to colonize Denmark after the Late Glacial Maximum. This study analyses complete mitochondrial genomes and two nuclear genes of the Northern Birch Mouse to investigate the phylogeographical pattern in North-western Europe and test whether the species colonized Denmark through several colonization events. The latter was prompt by (i) the present-day distinct northern and southern Danish distribution and (ii) the subfossil record of Northern Birch Mouse, supporting early Weichselian colonization. Samples from Denmark, Norway, Sweden, Russia, Latvia, Estonia, and Slovakia were included. Mitogenomes were obtained from 54 individuals, all representing unique mitogenomes supporting high genetic variation. Bayesian phylogenetic analysis identified two distinct evolutionary linages in Northern Europe diverging within the Elster glaciation period. The results of the two nuclear genomes showed lower genetic differentiation but supported the same evolutionary history. This suggests an allopatric origin of the clades followed by secondary contact. Individuals from southern Denmark were only found in one clade, while individuals from other areas, including northern Denmark, were represented in both clades. Nevertheless, we found no evidence for repeated colonization's explaining the observed fragmented distribution of the species today. The results indicated that the mitogenome pattern of the Northern Birch Mouse population in southern Denmark was either (i) due to the population being founded from northern Denmark, (ii) a result of climatic and anthropogenic effects reducing population size increasing genetic drift or (iii) caused by sampling bias., Competing Interests: The authors declare no competing interests., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Forty strains of Flavobacterium psychrophilum were tested for the production of siderophores using the universal Chrome Azurol S (CAS) assay. The majority of the strains (85%) were CAS positive (CAS+) and some (15%) were CAS negative (CAS-). The cryptic plasmid pCP1 was carried by all positive strains and was lacking from negative strains. While a weak catechol reaction was detectable in CAS+ culture supernatants, the CAS reaction was, to some extent, heat sensitive, questioning whether the positive reaction was caused only by siderophores. The ability to grow in vitro under iron-restricted conditions did not correlate with the CAS reactivity, as growth of both CAS+ and CAS- strains was similarly impaired under iron restriction induced by 2,2 dipyridyl. Suppressed growth under these conditions was restored by addition of FeCl3, haemoglobin and transferrin for both CAS+ and CAS- strains.

The surface of Flavobacterium psychrophilum was examined by electron microscopy to determine if previous findings of haemagglutination positive (HA+) and haemagglutination negative (HA-) abilities could be correlated with expression of pili or of a capsular layer. A thin capsular layer was observed in both HA+ and HA- strains but typical pili were absent. However, long, tubular blebs that released membrane vesicles (MVs) into the supernatant were observed on up to 94% of cells within 1 sample. The surface blebbing was increased for 1 strain following growth on media with restricted iron availability. The MVs had an intact membrane bilayer and were released from blebbing cells of both strains. The protein profiles of MVs, while containing some banding similarity with the profile of outer membrane preparations (OMPs) and of lysed whole cells (WCs), showed several bands that reacted strongly with rabbit anti-whole-cell antisera. Two distinct bands of approximately 62 and 58 kDA were highly expressed in the MVs and not seen in the OMP. MVs contained proteolytic activity towards gelatine but not towards casein and elastin, which were only degraded by live cells. Low molecular weight lipopolysaccharides (LPS) or lipooligosaccharides (LOS) were associated with the MVs. Only the MVs of the HA+ strain possessed haemagglutinin activity. These findings suggest that the F. psychrophilum may, through surface blebbing, release antigenic MVs that contain some proteolytic activity and may aid the bacterium in releasing nutrients from its surrounding environment as well as playing a role in impeding the immune response of its host.

The occurrence of Flavobacterium psychrophilum at four rainbow trout hatcheries was investigated to provide more knowledge about the reservoirs and transmission of this bacterium. Broodstock were sampled at stripping (including both unfertilized and fertilized eggs), and the offspring were then sampled at the eyed egg and fry stages. Water and surface samples (e.g. hatchery trays) were also sampled. Flavobacterium psychrophilum was found in ovarian fluid and milt, indicating that broodstock may serve as a reservoir and are latent carriers of the pathogen. Flavobacterium psychrophilum was not found on or inside eggs, but further egg studies will be necessary to elucidate the possibility of vertical transmission of the pathogen. Flavobacterium psychrophilum was isolated from water samples, but only from water that had been in close contact with farmed rainbow trout or eggs. Flavobacterium psychrophilum isolates were characterized and compared with well-characterized strains, using degradation of elastin, serotype and ribotype profiles. Different ribotypes of F. psychrophilum were found between hatcheries, but a common ribotype A was found at all four hatcheries. Different ribotypes were found in broodstock without clinical disease, whereas only a few ribotypes (mostly ribotype A) were found in diseased fry. The same ribotype A was found in broodstock, in water samples from hatchery trays and in fry, which suggests the possibility of transmission of F. psychrophilum between broodstock and offspring.

Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and Fp(T) exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5 degrees C, compared to that at 15 degrees C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65 degrees C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.

Embryonal-suspensor masses from immature embryos from cones of Sitka spruce (Picea sitchensis (Bong.) Carr.) proliferated on a modified Murashige & Skoog medium with N(6)-benzyl-aminopurine, kinetin, 2,4-dichlorophenoxyacetic acid and an organic nitrogen source. The slimy white embryonal-suspensor masses with proembryos were maintained on a solid proliferation medium with reduced amounts of growth regulators. Transfer of embryonal-suspensor masses to a non-woven polyester carrier with liquid maturation media containing ±2-cis-4-trans-abscisic acid and a reduced amount of inositol and organic nitrogen resulted in synchronized embryo formation. Further development was achieved on a medium without ±2-cis-4-trans-abscisic acid and organic nitrogen. Somatic embryos were successfully transferred ex vitrum.