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Terrestrial input to marine and freshwater ecosystems colors the water yellow-brown, causing a phenomenon called “brownification”. The effect of brownification on the marine pelagic microbial food web was studied in the oligotrophic eastern Mediterranean in June 2021 by adding HuminFeed in a 15-day mesocosm experiment with 2 treatments: Control (C, no addition) and HuminFeed (HF, single dose of HuminFeed, 2 mg L-1); and 3 replicates per treatment. HuminFeed caused shading, leading to a decrease in the abundance of photo-autotrophic organisms (cyanobacteria Synechococcus and diatoms). Bacteria were positively affected by the HF addition (mainly in terms of production rather than abundance), benefiting either directly from the dissolved organic carbon (DOC) contained in HuminFeed or indirectly from the trophic cascade through the food web. Despite the decrease in HF bacterial abundance during the experiment, an increase in both the high nucleic acid containing bacteria% and heterotrophic bacterial production were observed, suggesting higher activity at the single cell level. In the HF treatment, the increased abundance of dinoflagellates observed could be due to either a dominance of mixotrophic species or a release from predation by copepods. Both ciliates and copepods were severely impacted by HuminFeed, showing lower abundance and distorted forms (ciliates) and reduced reproductive potential (copepods). In conclusion, in the ultraoligotrophic eastern Mediterranean, the simulated brownification negatively affected autotrophs and top predators while benefiting bacteria, thus indicating a shift in the structure of the plankton food web.

In the aquaculture sector, there is an increased interest in developing environmentally friendly alternatives to antibiotics in the treatment and prevention of bacterial infections. This requires an understanding of the effects of different treatments on the fish microbiota as a measure for improving the fish health status. In this study, we focused on the freshwater pathogen Flavobacterium psychrophilum and investigated the effects of antibiotics (florfenicol) and phage therapies on the gut microbiota of healthy and infected rainbow trout fry (1–2 g). Florfenicol-coated feed was administered for 10 days, starting two days after the infection procedure. A two-component mix of phage targeting F. psychrophilum (FpV4 and FPSV-D22) was continuously delivered by feed with a prophylactic period of 12 days. Samples of the distal intestine were collected over time (day -1 and 1, 8, and 33 days post-infection) and analyzed by community analysis targeting the 16S rRNA gene (V3–V4 region). Results showed the dysbiosis effect caused both by the infection and by florfenicol administration. Shifts in the overall composition were detected by β-diversity analysis, and changes in specific populations were observed during taxonomic mapping. Measures of α-diversity were only affected in infected fish (large variation observed 1 and 8 dpi). These community alterations disappeared again when fish recovered from the infection and the antibiotic treatment was terminated (33 dpi). Interestingly, phage addition altered the microbiota of the fish independently of the presence of their target bacterium. The overall gut bacterial community in fish fed phage-treated feed was different from the controls at each time point as revealed by β-diversity analysis. However, it was not possible to identify specific bacterial populations responsible for these changes except for an increase of lactic acid bacteria 33 dpi. Overall, the results indicate that the administered phages might affect the complex network of phage-bacteria interactions in the fish gut. Nevertheless, we did not observe negative effects on fish health or growth, and further studies should be directed in understanding if these changes are beneficial or not for the fish health with an additional focus on the host immune response.

Extracellular enzyme activity is a well-established parameter for evaluating microbial biogeochemical roles in marine ecosystems. The presence and activity of extracellular enzymes in seawater provide insights into the quality and quantity of organic matter being processed by the present microorganisms. A key challenge in our understanding of these processes is to decode the extracellular enzyme repertoire and activities of natural communities at the single-cell level. Current measurements are carried out on bulk or size-fractionated samples capturing activities of mixed populations. This approach – even with size-fractionation – cannot be used to trace enzymes back to their producers, nor distinguish the active microbial members, leading to a disconnect between measured activities and the producer cells. By targeting extracellular enzymes and resolving their activities at the single-cell level, we can investigate underlying phenotypic heterogeneity among clonal or closely related organisms, characterize enzyme kinetics under varying environmental conditions, and resolve spatio-temporal distribution of individual enzyme producers within natural communities. In this perspective piece, we discuss state-of-the-art technologies in the fields of microfluidic droplets and functional screening of prokaryotic cells for measuring enzyme activity in marine seawater samples, one cell at a time. We further elaborate on how this single-cell approach can be used to address research questions that cannot be answered with current methods, as pertinent to the enzymatic degradation of organic matter by marine microorganisms.

Phage-based approaches have gained increasing interest as sustainable alternative strategies to antibiotic treatment or as prophylactic measures against disease outbreaks in aquaculture. The potential of three methods (oral, bath, and injection) for delivering a two-component phage mixture to rainbow trout fry for controlling Flavobacterium psychrophilum infections and reduce fish mortality was investigated using bacteriophages FpV4 and FPSV-D22. For the oral administration experiment, bacteriophages were applied on feed pellets by spraying (1.6 × 108 PFU g–1) or by irreversible immobilization (8.3 × 107 PFU g–1), using the corona discharge technology (Fixed Phage Ltd.). The fish showed normal growth for every group and no mortality was observed prior to infection as well as in control groups during the infection. Constant detection of phages in the intestine (∼103 PFU mg–1) and more sporadic occurrence in kidney, spleen, and brain was observed. When fish were exposed to F. psychrophilum, no significant effect on fish survival, nor a direct impact on the number of phages in the sampled organs, were detected. Similarly, no significant increase in fish survival was detected when phages were delivered by bath (1st and 2nd bath: ∼106 PFU ml–1; 3rd bath: ∼105 PFU ml–1). However, when phages FpV4 and FPSV-D22 (1.7 × 108 PFU fish–1) were administered by intraperitoneal injection 3 days after the bacterial challenge, the final percent survival observed in the group injected with bacteriophages FpV4 and FPSV-D22 (80.0%) was significantly higher than in the control group (56.7%). The work demonstrates the delivery of phages to fish organs by oral administration, but also suggests that higher phage dosages than the tested ones may be needed on feed pellets to offer fish an adequate protection against F. psychrophilum infections.

Flavobacterium psychrophilum causes bacterial cold-water disease (BCWD) in farmed rainbow trout (Oncorhynchus mykiss), with the multilocus sequence typing (MLST) clonal complex (CC) CC-ST10 accounting for the majority of outbreaks globally. The development of alternative strategies to antibiotic treatment of BCWD using bacteriophage-based control of F. psychrophilum, or virulence factors as targets for therapy, requires knowledge of the phage-sensitivity of outbreak strains and of universal traits contributing to their pathogenicity. To examine the association between virulence and both genetic (MLST sequence type (ST) and PCR-serotype) and phenotypic characteristics (adherence, antibiotic resistance, colony spreading motility, hemolytic and proteolytic activity), the median lethal dose (LD50) of 26 geographically disparate F. psychrophilum isolates was determined in rainbow trout. Furthermore, the in vitro sensitivity of the isolates against five bacteriophages was determined by the efficiency of plating (EOP). The tested F. psychrophilum isolates were mainly represented by CC-ST10 genotypes (22 out of 26) and showed up to 3-log differences in LD50 (8.9 × 103 to 3.1 × 106 CFU). No association between MLST ST and virulence was found because of a high variation in LD50 within STs. All identified serotypes (0, 1, and 2) were pathogenic, but ten most virulent isolates belonged to serotype 1 or 2. Isolates of high (LD50 < 105 CFU), moderate (LD50 = 105–106 CFU), and weak (LD50 > 106 CFU) virulence were similar in phenotypic characteristics in vitro. However, the only non-virulent CC-ST10 isolate was deficient in spreading motility and proteolytic activity, indicating that the characteristics are required for pathogenicity in F. psychrophilum. Univariate correlation studies found only non-significant associations between LD50 and the measured phenotypic characteristics, and the multivariable analysis did neither reveal any significant predictors of virulence. The majority of isolates (16 out of 26) were sensitive to at least four bacteriophages, with up to a 6-log variation in the EOP. Most CC-ST10 isolates (16 out of 22) were sensitive to the examined phages, including 5 out of the 7 most virulent isolates represented by prevalent and antibiotic-resistant STs. Our findings suggest that control of BCWD using lytic phages or interventions targeting shared characteristics of pathogenic F. psychrophilum strains should be further explored.

The land-to-ocean flux of organic carbon is increasing in glacierized regions in response to increasing temperatures in the Arctic (Hood et al., 2015). In order to understand the response of the coastal ecosystem metabolism to the organic carbon input it is essential to determine the bioavailability of the different carbon sources in the system. We quantified the bacterial turnover of organic carbon in a high Arctic fjord system (Young Sound, NE Greenland) during the ice-free period (July-October 2014) and assessed the quality and quantity of the 3 major organic carbon sources; (1) local phytoplankton production (2) runoff from land-terminating glaciers and a lowland river and (3) inflow from the ocean shelf. We found that despite relatively low concentrations of DOC in the rivers, the bioavailability of the river–DOC was significantly higher than in the fjord, and characterized by high cell-specific bacterial production and low C:N ratios. In contrast, the DOC source entering via inflow of coastal shelf waters had high DOC concentrations with high C:N and low specific bacterial production. The phytoplankton production in the fjord could not sustain the bacterial carbon demand, but was still the major source of organic carbon for bacterial growth. We assessed the bacterial community composition and found that communities were specific for the different water types i.e., the bacterial community of the coastal inflow water could be traced mainly in the subsurface water, while the glacial river community strongly dominated the surface water in the fjord.

Viruses profoundly influence benthic marine ecosystems by infecting and subsequently killing their prokaryotic hosts, thereby impacting the cycling of carbon and nutrients. Previously conducted studies, based on different methodologies, have provided widely differing estimates of the relevance of viruses on benthic prokaryotes. There has been no attempt so far to compare these independent approaches, including contextual comparisons among different approaches for sample manipulation (i.e., dilution or not of the sediments during incubations), between methods based on epifluorescence microscopy (EFM) or radiotracers, and between the use of different radiotracers. Therefore, it has been difficult to identify the most suitable methodologies and protocols to be used as standard approaches for the quantification of viral infections of prokaryotes. Here, we compared for the first time different methods for determining viral and prokaryotic production rates in marine sediments collected at two benthic sites, differing in depth and environmental conditions. We used a highly replicated experimental design, testing the potential biases associated to the incubation of sediments as diluted or undiluted. In parallel, we also compared EFM counts with the 3H-thymidine incubations for the determination of viral production rates, and the use of 3H-thymidine versus 3H-leucine radiotracers for the determination of prokaryotic production. We show here that, independent from sediment dilution, EFM-based values of viral production ranged from 1.4 to 4.6 × 107 viruses g-1 h-1, and were similar but overall less variable compared to those obtained by the 3H-thymidine method (0.3 to 9.0 × 107 viruses g-1h-1). In addition, the prokaryotic production rates were not affected by sediment dilution, and the use of different radiotracers provided very consistent estimates (10.3-35.1 and 9.3-34.6 ngC g-1h-1 using the 3H-thymidine or 3H-leucine method, respectively). These results indicated that viral lysis was responsible for the abatement of 55-81% of the prokaryotic heterotrophic production, corroborating previous findings of the major role of viruses in benthic deep-sea ecosystems. Moreover, our methodological comparison for the analysis of viral production in marine sediments suggests that microscopy-based approaches are simpler and more cost-effective than those based on radiotracers, also reducing time to results and overcoming issues related to radioactive waste generation.