Community composition of arctic root-associated fungi mirrors host plant phylogeny.

The number of plant species regarded as non-mycorrhizal increases at higher latitudes, and several plant species in the High-Arctic Archipelago Svalbard have been reported as non-mycorrhizal. We used the rRNA ITS2 and 18S gene markers to survey which fungi, as well as other micro-eukaryotes, were associated with roots of 31 arctic plant species not usually regarded as mycorrhizal in Svalbard. We assessed to what degree the root-associated fungi showed any host preference and whether the phylogeny of the plant hosts may mirror the composition of root-associated fungi. Fungal communities were largely structured according to host plant identity and to a less extent by environmental factors. We observed a positive relationship between the phylogenetic distance of host plants and the distance of fungal community composition between samples, indicating that the evolutionary history of the host plants plays a major role for which fungi colonize the plant roots. In contrast to the ITS2 marker, the 18S rRNA gene marker showed that chytrid fungi were prevalently associated with plant roots, together with a wide spectrum of amoeba-like protists and nematodes. Our study confirms that arbuscular mycorrhizal (AM) fungi are present also in arctic environments in low abundance.

Salmon gill poxvirus, a recently characterized infectious agent of multifactorial gill disease in freshwater- and seawater-reared Atlantic salmon.

Gill diseases cause considerable losses in Norwegian salmon farming. In 2015, we characterized salmon gill poxvirus (SGPV) and associated gill disease. Using newly developed diagnostic tools, we show here that SGPV infection is more widely distributed than previously assumed. We present seven cases of complex gill disease in Atlantic salmon farmed in seawater and freshwater from different parts of Norway. Apoptosis, the hallmark of acute SGPV infection, was not easily observed in these cases, and qPCR analysis was critical for identification of the presence of SGPV. Several other agents including Costia-like parasites, gill amoebas, Saprolegnia spp. and bacteria were observed. The studied populations experienced significant mortalities, which increased to extreme levels when severe SGPV infections coincided with smoltification. SGPV infection appears to affect the smoltification process directly by affecting the gills and chloride cells in particular. SGPV may be considered a primary pathogen as it was often found prior to identification of complex gill disease. It is hypothesized that SGPV-induced gill damage may impair innate immunity and allow invasion of secondary invaders. The distinct possibility that SGPV has been widely overlooked as a primary pathogen calls for extended use of SGPV qPCR in Atlantic salmon gill health management.

Saprolegnia diclina IIIA and S. parasitica employ different infection strategies when colonizing eggs of Atlantic salmon, Salmo salar L.

Here, we address the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re-emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica).

Co-infection of Nucleospora cyclopteri (Microsporidia) and Kudoa islandica (Myxozoa) in farmed lumpfish, Cyclopterus lumpus L., in Norway: a case report.

This study describes a co-infection of Kudoa islandica (Myxozoa) and Nucleospora cyclopteri (Microsporida) in farmed lumpfish, Cyclopterus lumpus L., in Norway. Several other parasites (Cryptocotyle sp., protozoan ciliates and Gyrodactylus sp.) were also found in gills. In June 2013, the mortality in a farmed lumpfish population increased to 65%. Lumpfish showed erratic swimming behaviour and loss of weight. At necropsy, nodules in the kidney were the only visible lesions. Histologically, all fish showed severe changes with gill inflammation and necrosis in the spleen, kidney and liver. Haemorrhages and necrosis were observed in some hearts. Intracellular microsporidians associated with the lesions were detected in most organs using histological examination and Calcofluor White. Kudoa spores were diagnosed in the skeletal muscle, but no inflammatory response was associated with the presence of the plasmodia. Comparison of 18S ribosomal DNA sequences showed 100% similarity to Kudoa islandica and Nucleospora cyclopteri. Kudoa islandica and N. cyclopteri have previously been described associated with lesions in wild lumpfish in Iceland. In the present case, N. cyclopteri is believed to be the main cause of systemic pathology. This is the first description of K. islandica and N. cyclopteri causing pathology in farmed lumpfish in Norway.

Factors influencing Saprolegnia spp. spore numbers in Norwegian salmon hatcheries.

A quantitative survey of Saprolegnia spp. in the water systems of Norwegian salmon hatcheries was performed. Water samples from 14 salmon hatcheries distributed along the Norwegian coastline were collected during final incubation in the hatcheries. Samples of inlet and effluent water were analyzed to estimate Saprolegnia propagule numbers. Saprolegnia spores were found in all samples at variable abundance. Number of spores retrieved varied from 50 to 3200 L(-1) in inlet water and from 30 to >5000 L(-1) in effluent water. A significant elevation of spore levels in effluent water compared to inlet water was detected. The estimated spore levels were related to recorded managerial and environmental parameters, and the number of spores in inlet water and temperature was the factor having most influence on the spore concentration in the incubation units (effluent water). Further, the relative impact of spore concentration on hatching rates was investigated by correlation analysis. From this was found that even high spore counts did not impact significantly on hatching success.

Saprolegnia species in Norwegian salmon hatcheries: field survey identifies S. diclina sub-clade IIIB as the dominating taxon.

Saprolegnia isolates within the recognized clades encompassing the taxa S. parasitica and S. diclina act as opportunist and aggressive pathogens to both fish and their eggs. They are responsible for significant economic losses in aquaculture, particularly in salmonid hatcheries. However, the identity, distribution and pathogenic significance of involved species often remain unexplored. In this study, 89 Saprolegnia isolates were recovered from water, eggs and salmon tissue samples that originated from salmon (Salmo salar) hatcheries along the coast of Norway. The cultures were characterized morphologically and molecularly in order to provide an overview of the species composition of Saprolegnia spp. present in Norwegian salmon hatcheries. We demonstrate that S. diclina clearly dominated and contributed to 79% of the recovered isolates. Parsimony analyses of the nuclear ribosomal internal transcribed spacer (ITS) region split these isolates into 2 strongly supported sub-clades, S. diclina sub-clade IIIA and IIIB, where sub-clade IIIB accounted for 66% of all isolates. A minor portion of the isolates constituted other taxa that were either conspecific or showed strong affinity to S. parasitica, S. ferax, S. hypogyna and Scoliolegnia asterophora. The unique sub-clade IIIB of S. diclina was most prevalent in water and salmon eggs, while S. parasitica isolates were more frequently isolated from post hatching stages. The study demonstrated that morphological criteria in many cases were insufficient for species delimitation due to lack of sexual structures or incoherent morphological expression of such features within the tested replicates.

In vitro passages impact on virulence of Saprolegnia parasitica to Atlantic salmon, Salmo salar L. parr.

The effect of serial in vitro subculturing on three pathogenic strains of Saprolegnia parasitica was investigated. The isolates were passed through Atlantic salmon, Salmo salar L. parr, and then re-isolated as single spore colonies. All strains caused infection. The isolate obtained from diseased fish served as a virulent reference culture and was designated 'AP' ('activated through passage'). Successive subculturing was made by obtaining an inoculum from AP to produce the 2nd subculture and then passaged to the 3rd subculture (from the 2nd), until the 15th passage was obtained. Spores used to produce storage cultures were collected at passages 5, 10 and 15. The different passages of each strain were used to artificially infect Atlantic salmon parr. Morphological characterization of growth patterns was performed to observe differences occurring due to serial in vitro subculturing. Two of the strains declined in virulence after 15 successive in vitro subcultures, whereas one did not. This study is the first to investigate attenuation of virulence in Saprolegnia and whether or not isolates of S. parasitica should be passed through the fish host prior to challenge experiments. It reveals that some strains degenerate more rapidly than others when subjected to successive in vitro subculturing on glucose-yeast extract.

Lysosomes as a possible target of enniatin B-induced toxicity in Caco-2 cells.

Enniatins are cyclic hexadepsipeptidic mycotoxins with ionophoric, antibiotic, and insecticidal activity. Enniatin B (EnnB), the most important analogue, is produced by many Fusarium species and is a common contaminant in grain-based foods. The compound's cytotoxic potential has been shown in different experiments; however, the mode of action has not been detailed so far. In the present study, several mutually confirmative experiments have been performed indicating that EnnB-initiated cytotoxicity could be connected with lysosomal membrane permeabilization (LMP). Lysosomal functionality, as assessed by the Neutral Red assay, was already affected after 3 h of toxin exposure. After 24 h, cell proliferation was decreased, and there was indication for a cell cycle arrest in the G(2)/M phase leading to the initiation of apoptosis or necrosis. Intracellular ROS-production was observed. However, antioxidants did not alter the observed EnnB-induced loss of lysosomal functionality leading to the conclusion that ROS was not an initial factor but one produced later in the event cascade. The collected data suggested that lysosomal destabilization is an upstream event in EnnB-initiated cytotoxicity followed by a certain extent of translocation of cathepsins into the cytosol, which was observed using immunological and proteomic methods. It appeared that cell death induced by EnnB was delayed and occurred not as a massive lysosomal breakdown but was probably progressing and leading to partial and selective LMP, starting a nonapoptotic cell death pathway with morphological features that had been previously considered as necrotic. The molecular mechanism of EnnB-triggered lysosomal destabilization, and the cellular processes leading to mitochondrial permeabilization and cell death are still unknown. They may, however, be connected to the compound's ionophoric properties.

Pathogenicity of Saprolegnia spp. to Atlantic salmon, Salmo salar L., eggs.

Live and dead Atlantic salmon eyed eggs were challenged with eight different Saprolegnia isolates, selected because of their varied origins, known morphological characteristics and growth/germination pattern. Some isolates were also tested for pathogenicity to Atlantic salmon parr. Challenge of eggs was performed by exposure to spores in suspension or by co-incubation of live eggs with infected dead eggs. The phenotypic characteristics of the isolates were evaluated in relation to their observed pathogenicity from the challenge experiment, to identify possible virulence factors leading to egg-infection by Saprolegnia. The results from the experiments confirm that live eggs are refractory to infection with Saprolegnia spores in suspension and that an infection of live eggs can only occur from an infection nucleus represented by dead eggs or debris. It was observed that strains pathogenic to salmon parr were not particularly infective towards eggs, and the isolates that gave the highest infection rates to eggs were species considered to be saprotrophs.

Vaccine-associated systemic Rhodococcus erythropolis infection in farmed atlantic salmon Salmo salar.

In 7 instances between 2000 and 2003, clinical investigation of populations of fresh- and seawater-reared, vaccinated, Atlantic salmon Salmo salar suffering total losses of between 0.1 and 35 % revealed infection with a Gram-positive rod-shaped bacterium. The isolations were geographically widespread, occurring in both Norway and Scotland. In all cases, a Gram-positive bacterium, subsequently identified as Rhodococcus erythropolis, was isolated in pure culture. Infections, although systemic, were focused within the peritoneal cavity. While initial attempts to reproduce the disease by intraperitoneal injection of unvaccinated Atlantic salmon failed, Koch's postulates were subsequently fulfilled in fish vaccinated with a commercially available oil-adjuvanted vaccine.

Stabilization of an active harmonically mode-locked fiber laser using two-photon absorption.

Two-photon absorption provided by a semiconductor mirror structure is shown to reduce amplitude fluctuations significantly in a harmonically mo e-locked fiber ring laser. Pulse dropouts are eliminated in a laser that produces picosecond pulses at a repetition rate of 2 GHz.

[Plague in southern Netherlands during the Middle Ages and modern times. Complaint about the status of the disease in its socioeconomic context]. / Pest in de Zuidelijke Nederlanden tijdens de Middeleeuwen en de Moderne Tijden. Een status quaestionis over de ziekte in haar sociaal-economische context.

In this contribution the latest insights with regard to the demographic impact of plague in the Netherlands are discussed, although is remains difficult to clearly distinguish this factor from other causes of mortality. When, how and why did the plague reappear in Europe after several centuries of absence to become endemic for the next three centuries? When and why did it disappear in Western Europe in the seventeenth century? The first epidemics of plague probably were not as catastrophic in the Netherlands as they were in many other parts of Europe, which is remarkable since the Netherlands was, together with Northern Italy, the most populous region of Europe. The explanation is to be found in the general socioeconomic context, that was in many regards better than in the neighbouring regions. The 'crisis of the late Middle Ages' was not as deep in the Netherlands as elsewhere: a relationship demography--economy is therefore probable. Nevertheless, mortality was high, partly because of plague, but also because of other diseases--for which the common term pestilentia was in use. Some unique statistical data for Flanders illustrate this mortality from the late Middle Ages onward. The succession of mortality leading to a high average mortality rate was more important than accidental mortality, that could have a spectacular but often not long-lasting impact.