Identification and genetic characterization of Saprolegnia parasitica, isolated from farmed and wild fish in Finland.

Oomycete infections in farmed fish are one of the most significant disease issues in salmonid aquaculture worldwide. In the present study, Saprolegnia spp. in different farmed fish species in Finland were identified, and the molecular epidemiology of especially Saprolegnia parasitica was examined. We analysed tissue samples from suspected oomycete-infected salmonids of different life stages from a number of fish farms, as well as three wild salmonids. From collected oomycete isolates, the ITS1, 5.8S and ITS2 genomic regions were amplified, analysed phylogenetically and compared with corresponding sequences deposited in GenBank. Of the sequenced isolates, 91% were identified as S. parasitica. Isolates of yolk sac fry were identified as different Saprolegnia spp. Among the isolates from rainbow trout eggs Saprolegnia diclina dominated. In order to determine potential dominating clones among the S. parasitica, isolates were analysed using Multi Locus Sequence Typing (MLST). The results showed that one main clone contained the majority of the isolates. The MLST analysis showed four main sequence types (ST1-ST4) and 13 unique STs. This suggests that the Saprolegnia infections in farmed fish in Finland are not caused by different strains originating in the farm environment. Instead, one main clone of S. parasitica is present in Finnish fish farms.

Estimating genotypic richness and proportion of identical multi-locus genotypes in aquatic microalgal populations.

The majority of microalgal species reproduce asexually, yet population genetic studies rarely find identical multi-locus genotypes (MLG) in microalgal blooms. Instead, population genetic studies identify large genotypic diversity in most microalgal species. This paradox of frequent asexual reproduction but low number of identical genotypes hampers interpretations of microalgal genotypic diversity. We present a computer model for estimating, for the first time, the number of distinct MLGs by simulating microalgal population composition after defined exponential growth periods. The simulations highlighted the effects of initial genotypic diversity, sample size and intraspecific differences in growth rates on the probability of isolating identical genotypes. We estimated the genotypic richness for five natural microalgal species with available high-resolution population genetic data and monitoring-based growth rates, indicating 500 000 to 2 000 000 distinct genotypes for species with few observed clonal replicates (<5%). Furthermore, our simulations indicated high variability in genotypic richness over time and among microalgal species. Genotypic richness was also strongly impacted by intraspecific variability in growth rates. The probability of finding identical MLGs and sampling a representative fraction of genotypes decreased noticeably with smaller sample sizes, challenging the detection of differences in genotypic diversity with typical isolate numbers in the field.

Strain-specific transcriptional responses overshadow salinity effects in a marine diatom sampled along the Baltic Sea salinity cline.

The salinity gradient separating marine and freshwater environments represents a major ecological divide for microbiota, yet the mechanisms by which marine microbes have adapted to and ultimately diversified in freshwater environments are poorly understood. Here, we take advantage of a natural evolutionary experiment: the colonization of the brackish Baltic Sea by the ancestrally marine diatom Skeletonema marinoi. To understand how diatoms respond to low salinity, we characterized transcriptomic responses of acclimated S. marinoi grown in a common garden. Our experiment included eight strains from source populations spanning the Baltic Sea salinity cline. Gene expression analysis revealed that low salinities induced changes in the cellular metabolism of S. marinoi, including upregulation of photosynthesis and storage compound biosynthesis, increased nutrient demand, and a complex response to oxidative stress. However, the strain effect overshadowed the salinity effect, as strains differed significantly in their response, both regarding the strength and the strategy (direction of gene expression) of their response. The high degree of intraspecific variation in gene expression observed here highlights an important but often overlooked source of biological variation associated with how diatoms respond to environmental change.

Seasonal genotype dynamics of a marine dinoflagellate: Pelagic populations are homogeneous and as diverse as benthic seed banks.

Genetic diversity is the basis for evolutionary adaptation and selection under changing environmental conditions. Phytoplankton populations are genotypically diverse, can become genetically differentiated within small spatiotemporal scales and many species form resting stages. Resting stage accumulations in sediments (seed banks) are expected to serve as reservoirs for genetic information, but so far their role in maintaining phytoplankton diversity and in evolution has remained unclear. In this study we used the toxic dinoflagellate Alexandrium ostenfeldii (Dinophyceae) as a model organism to investigate if (i) the benthic seed bank is more diverse than the pelagic population and (ii) the pelagic population is seasonally differentiated. Resting stages (benthic) and plankton (pelagic) samples were collected at a coastal bloom site in the Baltic Sea, followed by cell isolation and genotyping using microsatellite markers (MS) and restriction site associated DNA sequencing (RAD). High clonal diversity (98%-100%) combined with intermediate to low gene diversity (0.58-0.03, depending on the marker) was found. Surprisingly, the benthic and pelagic fractions of the population were equally diverse, and the pelagic fraction was temporally homogeneous, despite seasonal fluctuation of environmental selection pressures. The results of this study suggest that continuous benthic-pelagic coupling, combined with frequent sexual reproduction, as indicated by persistent linkage equilibrium, prevent the dominance of single clonal lineages in a dynamic environment. Both processes harmonize the pelagic with the benthic population and thus prevent seasonal population differentiation. At the same time, frequent sexual reproduction and benthic-pelagic coupling maintain high clonal diversity in both habitats.

Ecologically coherent population structure of uncultivated bacterioplankton.

Bacterioplankton are main drivers of biogeochemical cycles and important components of aquatic food webs. While sequencing-based studies have revealed how bacterioplankton communities are structured in time and space, relatively little is known about intraspecies diversity patterns and their ecological relevance. Here, we use the newly developed software POGENOM (POpulation GENomics from Metagenomes) to investigate genomic diversity and differentiation in metagenome-assembled genomes from the Baltic Sea, and investigate their genomic variation using metagenome data spanning a 1700 km transect and covering seasonal variation at one station. The majority of the investigated species, representing several major bacterioplankton clades, displayed population structures correlating significantly with environmental factors such as salinity and temperature. Population differentiation was more pronounced over spatial than temporal scales. We discovered genes that have undergone adaptation to different salinity regimes, potentially responsible for the populations' existence along with the salinity range. This in turn implies the broad existence of ecotypes that may remain undetected by rRNA gene sequencing. Our findings emphasize the importance of physiological barriers, and highlight the role of adaptive divergence as a structuring mechanism of bacterioplankton species.

Birth mode is associated with earliest strain-conferred gut microbiome functions and immunostimulatory potential.

The rate of caesarean section delivery (CSD) is increasing worldwide. It remains unclear whether disruption of mother-to-neonate transmission of microbiota through CSD occurs and whether it affects human physiology. Here we perform metagenomic analysis of earliest gut microbial community structures and functions. We identify differences in encoded functions between microbiomes of vaginally delivered (VD) and CSD neonates. Several functional pathways are over-represented in VD neonates, including lipopolysaccharide (LPS) biosynthesis. We link these enriched functions to individual-specific strains, which are transmitted from mothers to neonates in case of VD. The stimulation of primary human immune cells with LPS isolated from early stool samples of VD neonates results in higher levels of tumour necrosis factor (TNF-α) and interleukin 18 (IL-18). Accordingly, the observed levels of TNF-α and IL-18 in neonatal blood plasma are higher after VD. Taken together, our results support that CSD disrupts mother-to-neonate transmission of specific microbial strains, linked functional repertoires and immune-stimulatory potential during a critical window for neonatal immune system priming.

A planktonic diatom displays genetic structure over small spatial scales.

Marine planktonic microalgae have potentially global dispersal, yet reduced gene flow has been confirmed repeatedly for several species. Over larger distances (>200 km) geographic isolation and restricted oceanographic connectivity have been recognized as instrumental in driving population divergence. Here we investigated whether similar patterns, that is, structured populations governed by geographic isolation and/or oceanographic connectivity, can be observed at smaller (6-152 km) geographic scales. To test this we established 425 clonal cultures of the planktonic diatom Skeletonema marinoi collected from 11 locations in the Archipelago Sea (northern Baltic Sea). The region is characterized by a complex topography, entailing several mixing regions of which four were included in the sampling area. Using eight microsatellite markers and conventional F-statistics, significant genetic differentiation was observed between several sites. Moreover, Bayesian cluster analysis revealed the co-occurrence of two genetic groups spread throughout the area. However, geographic isolation and oceanographic connectivity could not explain the genetic patterns observed. Our data reveal hierarchical genetic structuring whereby despite high dispersal potential, significantly diverged populations have developed over small spatial scales. Our results suggest that biological characteristics and historical events may be more important in generating barriers to gene flow than physical barriers at small spatial scales.

Spatio-Temporal Interdependence of Bacteria and Phytoplankton during a Baltic Sea Spring Bloom.

In temperate systems, phytoplankton spring blooms deplete inorganic nutrients and are major sources of organic matter for the microbial loop. In response to phytoplankton exudates and environmental factors, heterotrophic microbial communities are highly dynamic and change their abundance and composition both on spatial and temporal scales. Yet, most of our understanding about these processes comes from laboratory model organism studies, mesocosm experiments or single temporal transects. Spatial-temporal studies examining interactions of phytoplankton blooms and bacterioplankton community composition and function, though being highly informative, are scarce. In this study, pelagic microbial community dynamics (bacteria and phytoplankton) and environmental variables were monitored during a spring bloom across the Baltic Proper (two cruises between North Germany to Gulf of Finland). To test to what extent bacterioplankton community composition relates to the spring bloom, we used next generation amplicon sequencing of the 16S rRNA gene, phytoplankton diversity analysis based on microscopy counts and population genotyping of the dominating diatom Skeletonema marinoi. Several phytoplankton bloom related and environmental variables were identified to influence bacterial community composition. Members of Bacteroidetes and Alphaproteobacteria dominated the bacterial community composition but the bacterial groups showed no apparent correlation with direct bloom related variables. The less abundant bacterial phyla Actinobacteria, Planctomycetes, and Verrucomicrobia, on the other hand, were strongly associated with phytoplankton biomass, diatom:dinoflagellate ratio, and colored dissolved organic matter (cDOM). Many bacterial operational taxonomic units (OTUs) showed high niche specificities. For example, particular Bacteroidetes OTUs were associated with two distinct genetic clusters of S. marinoi. Our study revealed the complexity of interactions of bacterial taxa with inter- and intraspecific genetic variation in phytoplankton. Overall, our findings imply that biotic and abiotic factors during spring bloom influence bacterial community dynamics in a hierarchical manner.

Genetic diversity affects ecological performance and stress response of marine diatom populations.

Genetic diversity is considered an important factor, stabilizing ecological functions when organisms are faced with changing environmental conditions. Although well known from terrestrial systems, documentations of this relationship from marine organisms, and particularly planktonic microorganisms, are still limited. Here we experimentally tested the effects of genotypic diversity on ecologically relevant cellular parameters (growth, primary production, particulate organic carbon, particulate organic nitrogen, particulate organic phosphorus and biogenic silica) at optimal and suboptimal salinity conditions in a marine phytoplankton species. Multiple clonal genotyped and phenotypically characterized isolates of the diatom Skeletonema marinoi from the Baltic Sea were grown in monocultures and mixes of 5 and 20 clones at native (5 psu) and reduced (3 psu) salinities and respective parameters were compared. Re-genotyping of 30 individuals from each population at five microsatellite loci at the end of the experiment confirmed maintenance of genotypic richness. Although a diversity effect on growth was not detected, primary production and particulate organic nutrients were positively affected by increased diversity independent of salinity condition. Under salinity stress, highest values of primary production and particulate organic nitrogen content were measured at the high diversity level. The observed diversity effects emphasize the importance of genetic diversity of phytoplankton populations for ecological functions.

Natural co-occurrence of Dinophysis acuminata (Dinoflagellata) and Mesodinium rubrum (Ciliophora) in thin layers in a coastal inlet.

The interdependency of Dinophysis spp., Mesodinium rubrum and Teleaulax spp. has occupied scientists in molecular and ecological domains in recent years. Current knowledge about the predator-prey relationships is based on laboratory investigations. Records on interactions in nature are limited, even though it is known that Dinophysis acuminata and M. rubrum form population maxima in thin layers associated with thermal stratification. We studied the vertical co-occurrence of these taxa in a stratified coastal inlet in Åland, in the Northern Baltic Sea, SW Finland. Vertical profiles were sampled monthly in the summer of 2008 and observations on diurnal migrational patterns of all species were conducted in September 2008. The population maximum of D. acuminata was almost totally confined to thin layers where the depth maximum of M. rubrum was present. However, this pattern was only observed early in the morning or at noon. The population maxima of M. rubrum and Teleaulax spp. overlapped at noon. Dinophysis acuminata and Teleaulax spp. were restricted to the upper 9 m but M. rubrum was found down to 20 m depth. This study offers circumstantial evidence for the interdependency between the three taxa in nature.