A guide to the use of bioassays in exploration of natural resources.

Bioassays are the main tool to decipher bioactivities from natural resources thus their selection and quality are critical for optimal bioprospecting. They are used both in the early stages of compounds isolation/purification/identification, and in later stages to evaluate their safety and efficacy. In this review, we provide a comprehensive overview of the most common bioassays used in the discovery and development of new bioactive compounds with a focus on marine bioresources. We present a comprehensive list of practical considerations for selecting appropriate bioassays and discuss in detail the bioassays typically used to explore antimicrobial, antibiofilm, cytotoxic, antiviral, antioxidant, and anti-ageing potential. The concept of quality control and bioassay validation are introduced, followed by safety considerations, which are critical to advancing bioactive compounds to a higher stage of development. We conclude by providing an application-oriented view focused on the development of pharmaceuticals, food supplements, and cosmetics, the industrial pipelines where currently known marine natural products hold most potential. We highlight the importance of gaining reliable bioassay results, as these serve as a starting point for application-based development and further testing, as well as for consideration by regulatory authorities.

Timing of puberty in F1-generation hatchery-produced greater amberjack (Seriola dumerili).

We evaluated the onset of puberty of first-generation (F1) hatchery-produced greater amberjack (Seriola dumerili) reared in sea cages for 5 years. Fish were sampled every year in June, at the expected peak of the spawning period in the Mediterranean Sea. No sexual dimorphism in body weight was observed in the study. The ovaries of 1 and 2-year-old (yo) females consisted of primary oocytes only, while at the age of 3-yo early vitellogenic (Vg) oocytes were also identified, but with extensive follicular atresia. At the age of 4-yo, late Vg oocytes were observed, but again extensive follicular atresia characterized the ovaries of 50 % of females. At the age of 5-yo, follicular atresia of Vg oocytes was very limited. In males, gametogenesis was evident already in 1- and 2-yo fish, and 100 % of sampled 3-yo males produced collectable viable sperm. Plasma testosterone (T), 17ß-estradiol (E2), and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) remained similar in 3 - 5-yo females, with T and E2 levels being highest in females in advanced vitellogenesis or with significant follicular atresia, compared to immature females. In males, plasma T declined over the years, while 11-ketotestosterone (11-KT) and 17,20ß-P were highest in 4 and 5-yo males, with spermatozoa motility characteristics being improved from the 4th year onwards. The administration of GnRHa implants to 5-yo fish induced only two spawns, albeit no fertilized eggs were obtained. The results indicate that hatchery-produced greater amberjack males mature well and within the same age observed in the wild, however with smaller gonad size. On the contrary, females mature later than in the wild, also with a smaller gonad size. Spawning in response to GnRHa treatment was not effective, suggesting that Mediterranean hatchery-produced broodstocks may be dysfunctional, and further research is needed to document any improvement as the fish get older, or to determine if the results may be related to the specific stock of fish.

Comparative Chemical Profiling and Antimicrobial/Anticancer Evaluation of Extracts from Farmed versus Wild Agelas oroides and Sarcotragus foetidus Sponges.

Marine sponges are highly efficient in removing organic pollutants and their cultivation, adjacent to fish farms, is increasingly considered as a strategy for improving seawater quality. Moreover, these invertebrates produce a plethora of bioactive metabolites, which could translate into an extra profit for the aquaculture sector. Here, we investigated the chemical profile and bioactivity of two Mediterranean species (i.e., Agelas oroides and Sarcotragus foetidus) and we assessed whether cultivated sponges differed substantially from their wild counterparts. Metabolomic analysis of crude sponge extracts revealed species-specific chemical patterns, with A. oroides and S. foetidus dominated by alkaloids and lipids, respectively. More importantly, farmed and wild explants of each species demonstrated similar chemical fingerprints, with the majority of the metabolites showing modest differences on a sponge mass-normalized basis. Furthermore, farmed sponge extracts presented similar or slightly lower antibacterial activity against methicillin-resistant Staphylococcus aureus, compared to the extracts resulting from wild sponges. Anticancer assays against human colorectal carcinoma cells (HCT-116) revealed marginally active extracts from both wild and farmed S. foetidus populations. Our study highlights that, besides mitigating organic pollution in fish aquaculture, sponge farming can serve as a valuable resource of biomolecules, with promising potential in pharmaceutical and biomedical applications.

Assessing the Toxicity of Lagocephalus sceleratus Pufferfish from the Southeastern Aegean Sea and the Relationship of Tetrodotoxin with Gonadal Hormones.

Given the dramatic increase in the L. sceleratus population in the southeastern Aegean Sea, there is growing interest in assessing the toxicity of this pufferfish and the factors controlling its tetrodotoxin (TTX) content. In the present study, liver, gonads, muscle and skin of 37 L. sceleratus specimens collected during May and June 2021 from the island of Rhodes, Greece, were subjected to multi-analyte profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to quantitate TTX and evaluate whether this biotoxin interrelates with hormones. TTX and its analogues 4-epiTTX, 11-deoxyTTX, 11-norTTX-6-ol, 4,9-anhydroTTX and 5,11/6,11-dideoxyTTX were detected in all tissue types. Liver and gonads were the most toxic tissues, with the highest TTX concentrations being observed in the ovaries of female specimens. Only 22% of the analyzed muscle samples were non-toxic according to the Japanese toxicity threshold (2.2 µg TTX eq g-1), confirming the high poisoning risk from the inadvertent consumption of this species. Four steroid hormones (i.e., cortisol, testosterone, androstenedione and ß-estradiol) and the gonadotropin-releasing hormone (GnRH) were detected in the gonads. Androstenedione dominated in female specimens, while GnRH was more abundant in males. A positive correlation of TTX and its analogues with ß-estradiol was observed. However, a model incorporating sex rather than ß-estradiol as the independent variable proven to be more efficient in predicting TTX concentration, implying that other sex-related characteristics are more important than specific hormone-regulated processes.

Evaluation of Toxicity Equivalency Factors of Tetrodotoxin Analogues with a Neuro-2a Cell-Based Assay and Application to Puffer Fish from Greece.

Tetrodotoxin (TTX) is a potent marine neurotoxin involved in poisoning cases, especially through the consumption of puffer fish. Knowledge of the toxicity equivalency factors (TEFs) of TTX analogues is crucial in monitoring programs to estimate the toxicity of samples analyzed with instrumental analysis methods. In this work, TTX analogues were isolated from the liver of a Lagocephalus sceleratus individual caught on South Crete coasts. A cell-based assay (CBA) for TTXs was optimized and applied to the establishment of the TEFs of 5,11-dideoxyTTX, 11-norTTX-6(S)-ol, 11-deoxyTTX and 5,6,11-trideoxyTTX. Results showed that all TTX analogues were less toxic than the parent TTX, their TEFs being in the range of 0.75-0.011. Then, different tissues of three Lagocephalus sceleratus individuals were analyzed with CBA and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The obtained TEFs were applied to the TTX analogues' concentrations obtained by LC-MS/MS analysis, providing an indication of the overall toxicity of the sample. Information about the TEFs of TTX analogues is valuable for food safety control, allowing the estimation of the risk of fish products to consumers.

Uptake of aquaculture-related dissolved organic pollutants by marine sponges: Kinetics and mechanistic insights from a laboratory study.

Besides the release of organic matter from uneaten feed and fish excreta, a considerable amount of deleterious chemicals may also end up into the marine environment from intensive aquaculture. A fraction of these pollutants remains freely dissolved and pose a threat to marine life due to increased bioavailability. Given the filter-feeding ability of sponges, we investigated the capacity of four ubiquitous Mediterranean species (Agelas oroides, Axinella cannabina, Chondrosia reniformis and Sarcotragus foetidus) in removing aquaculture-related dissolved organic pollutants. These included individual chemicals belonging to antibiotics (i.e., oxytetracycline), antifouling biocides (i.e., diuron and Irgarol 1051) and polycyclic aromatic hydrocarbons (i.e., 2,6-dimethylnapththalene, phenanthrene). The uptake of pollutants was assessed in vitro by exposing small sponge explants to each chemical for a period of 8 h. Additional "cleanup" experiments were performed for complex mixtures mimicking the dissolved organic material encountered in fish farms, such as filtrates of fish feed and excreta. All sponges exhibited a pronounced preference for lipophilic pollutants and a strong positive correlation was revealed between clearance rate and substrate hydrophobicity. Our best filter-feeder (i.e., A. oroides) was able to clear 10.0 ± 1.3 mL of seawater per hour and per gram of sponge, when exposed to 2,6-dimethylnapththalene. Active pumping was found to be the predominant mechanism dictating the assimilation of dissolved pollutants in all sponge species, as it was 3-10 times faster than pollutants' passive adsorption on sponges' pinacoderm. Additionally, the uptaken pollutants were shown to be strongly retained by sponges and they were hardly released back to seawater as a result of desorption or sponge excretory mechanisms. Our study corroborates that sponges are highly efficient in uptaking dissolved organic compounds and it offers new insights into the kinetics and mechanisms ruling this process.

Advanced Methods for Natural Products Discovery: Bioactivity Screening, Dereplication, Metabolomics Profiling, Genomic Sequencing, Databases and Informatic Tools, and Structure Elucidation.

Natural Products (NP) are essential for the discovery of novel drugs and products for numerous biotechnological applications. The NP discovery process is expensive and time-consuming, having as major hurdles dereplication (early identification of known compounds) and structure elucidation, particularly the determination of the absolute configuration of metabolites with stereogenic centers. This review comprehensively focuses on recent technological and instrumental advances, highlighting the development of methods that alleviate these obstacles, paving the way for accelerating NP discovery towards biotechnological applications. Herein, we emphasize the most innovative high-throughput tools and methods for advancing bioactivity screening, NP chemical analysis, dereplication, metabolite profiling, metabolomics, genome sequencing and/or genomics approaches, databases, bioinformatics, chemoinformatics, and three-dimensional NP structure elucidation.

Keeping Lagocephalus sceleratus off the Table: Sources of Variation in the Quantity of TTX, TTX Analogues, and Risk of Tetrodotoxication.

The invasion of the tetrodotoxin (TTX)-bearing silver-cheeked toadfish and potential poisoning due to its consumption (tetrodotoxication) threatens public safety in the Mediterranean Sea. In this study, TTX and TTX analogues of Lagocephalus sceleratus (Gmelin, 1789) were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) in fish collected off the island of Crete (Southern Mediterranean). We tested the synergistic effect of a suite of factors potentially affecting toxins' levels and tetrodotoxication risk using general and generalized linear models, respectively. The type of tissue, geographic origin (Cretan Sea, Libyan Sea), sex, and fish maturity stage were significant predictors of toxin concentrations. Mean TTX was higher in gonads and lower in muscles, higher in the Libyan Sea and in female fish, and lower in juvenile (virgin) fish. The concentration of TTX was also significantly and positively correlated with the concentration of several TTX analogues (4-epiTTX, 4,9-anhydroTTX, 11-deoxyTTX, 5,11/6,11-dideoxyTTX, 5,6,11-trideoxyTTX, 11-norTTX-6-ol). The analysis showed that fish originating from the Libyan Sea had significantly higher probability to cause tetrodotoxication in case of consumption. The variability explained by the models developed in this study was relatively low, indicating that toxin levels are hard to predict and the consumption of L. sceleratus should therefore be avoided.

PepMANDIS: A Peptide Selection Tool for Designing Function-Based Targeted Proteomic Assays in Complex Microbial Systems.

The majority of studies focusing on microbial functioning in various environments are based on DNA or RNA sequencing techniques that have inherent limitations and usually provide a distorted picture about the functional status of the studied system. Untargeted proteomics is better suited for that purpose, but it suffers from low efficiency when applied in complex consortia. In practice, the scanning capabilities of the currently employed LC-MS/MS systems provide limited coverage of key-acting proteins, hardly allowing a semiquantitative assessment of the most abundant ones from most prevalent species. When particular biological processes of high importance are under investigation, the analysis of specific proteins using targeted proteomics is a more appropriate strategy as it offers superior sensitivity and comes with the added benefits of increased throughput, dynamic range and selectivity. However, the development of targeted assays requires a priori knowledge regarding the optimal peptides to be screened for each protein of interest. In complex, multi-species systems, a specific biochemical process may be driven by a large number of homologous proteins having considerable differences in their amino acid sequence, complicating LC-MS/MS detection. To overcome the complexity of such systems, we have developed an automated pipeline that interrogates UniProt database or user-created protein datasets (e.g. from metagenomic studies) to gather homolog proteins with a defined functional role and extract respective peptide sequences, while it computes several protein/peptide properties and relevant statistics to deduce a small list of the most representative, process-specific and LC-MS/MS-amenable peptides for the microbial enzymatic activity of interest.

Histological evaluation of sex differentiation and early sex identification in hatchery-produced greater amberjack (Seriola dumerili) reared in sea cages.

The histological process of gonadal differentiation, together with the endocrine changes of sex steroid hormones and some of their precursors, was studied in hatchery-produced greater amberjack Seriola dumerili from 101 until 408 days post-hatching (dph), with samplings conducted every 50 days. Histological processing showed that sex differentiation began at 101 dph with the formation of the ovarian cavity in females, while the presumptive males did not yet contain any germ cells in their gonad. At 150 dph, we observed the first germ cells in the developing testes. Sex differentiation in almost all sampled individuals was complete at 408 dph. No size dimorphism was observed between the sexes, and the sex ratio was 1:1, suggesting that there was no influence of early rearing in captivity on sex differentiation. Plasma concentrations of adrenosterone (Ad), androstenedione (Δ4), 11-ketotestosterone (11ΚΤ), testosterone (Τ), estradiol (Ε2), progesterone (P4) and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) were measured in males and females with the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) to examine their role in the sex differentiation process. From the seven hormones, the only one that exhibited differences between the sexes was 11-KT and the plasma 11-KT concentration was found to be a useful indication of greater amberjack sex. Variations were observed in the mean values of Ad, Δ4, 11-KT, T, P4 and 17,20ßP over time in one or both sexes, indicating their involvement in the sex differentiation process.

Antibacterial Effects of Essential Oils of Seven Medicinal-Aromatic Plants Against the Fish Pathogen Aeromonas veronii bv. sobria: To Blend or Not to Blend?

Despite progress achieved, there is limited available information about the antibacterial activity of constituents of essential oils (EOs) from different medicinal-aromatic plants (MAPs) against fish pathogens and the complex interactions of blended EOs thereof. The present study aimed to investigate possible synergistic antimicrobial effects of EOs from seven Greek MAPs with strong potential against Aeromonas veronii bv. sobria, a fish pathogen associated with aquaculture disease outbreaks. The main objective was to evaluate whether blending of these EOs can lead to increased antimicrobial activity against the specific microorganism. A total of 127 combinations of EOs were prepared and their effect on A. veronii bv. sobria growth was tested in vitro. We examined both the inhibitory and bactericidal activities of the individual EOs and compared them to those of the blended EOs. The vast majority of the investigated combinations exhibited significant synergistic and additive effects, while antagonistic effects were evident only in a few cases, such as the mixtures containing EOs from rosemary, lemon balm and pennyroyal. The combination of EOs from Greek oregano and wild carrot, as well as the combinations of those two with Spanish oregano or savoury were the most promising ones. Overall, Greek oregano, savoury and Spanish oregano EOs were the most effective ones when applied either in pure form or blended with other EOs.

Genomic adaptation of Pseudomonas strains to acidity and antibiotics in hydrothermal vents at Kolumbo submarine volcano, Greece.

Although the rise of antibiotic and multidrug resistant bacteria is one of the biggest current threats to human health, our understanding of the mechanisms involved in antibiotic resistance selection remains scarce. We performed whole genome sequencing of 21 Pseudomonas strains, previously isolated from an active submarine volcano of Greece, the Kolumbo volcano. Our goal was to identify the genetic basis of the enhanced co-tolerance to antibiotics and acidity of these Pseudomonas strains. Pangenome analysis identified 10,908 Gene Clusters (GCs). It revealed that the numbers of phage-related GCs and sigma factors, which both provide the mechanisms of adaptation to environmental stressors, were much higher in the high tolerant Pseudomonas strains compared to the rest ones. All identified GCs of these strains were associated with antimicrobial and multidrug resistance. The present study provides strong evidence that the CO2-rich seawater of the volcano associated with low pH might be a reservoir of microorganisms carrying multidrug efflux-mediated systems and pumps. We, therefore, suggest further studies of other extreme environments (or ecosystems) and their associated physicochemical parameters (or factors) in the rise of antibiotic resistance.

A Multi-Species Investigation of Sponges' Filtering Activity towards Marine Microalgae.

Chronic discharge of surplus organic matter is a typical side effect of fish aquaculture, occasionally leading to coastal eutrophication and excessive phytoplankton growth. Owing to their innate filter-feeding capacity, marine sponges could mitigate environmental impact under integrated multitrophic aquaculture (IMTA) scenarios. Herein, we investigated the clearance capacity of four ubiquitous Mediterranean sponges (Agelas oroides, Axinella cannabina, Chondrosia reniformis and Sarcotragus foetidus) against three microalgal substrates with different size/motility characteristics: the nanophytoplankton Nannochloropsis sp. (~3.2 µm, nonmotile) and Isochrysis sp. (~3.8 µm, motile), as well as the diatom Phaeodactylum tricornutum (~21.7 µm, nonmotile). In vitro cleaning experiments were conducted using sponge explants in 1 L of natural seawater and applying different microalgal cell concentrations under light/dark conditions. The investigated sponges exhibited a wide range of retention efficiencies for the different phytoplankton cells, with the lowest average values found for A. cannabina (37%) and the highest for A. oroides (70%). The latter could filter up to 14.1 mL seawater per hour and gram of sponge wet weight, by retaining 100% of Isochrysis at a density of 105 cells mL-1, under darkness. Our results highlight differences in filtering capacity among sponge species and preferences for microalgal substrates with distinct size and motility traits.

Grazing effect of flagellates on bacteria in response to phosphate addition in the oligotrophic Cretan Sea, NE Mediterranean.

The planktonic food web in the oligotrophic Mediterranean Sea is dominated by small-sized (<20 µm) microbes, with nanoflagellates being the major bacterial grazers and the main participants in nutrient cycling. Phosphate is a key nutrient in the P-limited Cretan Sea (NE Mediterranean) and P-availability can affect its trophic dynamics. Here, we examined the grazing potential of heterotrophic (HF) and pigmented (PF) nanoflagellates as a response mechanism to phosphate amendment. Flagellate grazing effect on bacteria was quantified in P-amended nutrient-depleted water from the Cretan Sea over the course of 4 days using microcosm experiments. P-addition positively affected HF abundance, while PF abundance remained unchanged. At the community level, P-addition had a negative effect on PF bacterial removal rates. In the control, PF-grazing rate was significantly higher than that of HF throughout the experiment. Pigment analysis showed no changes in phytoplankton community composition as a result of P-addition, indicating that PF grazing rate declined as a physiological response of the cells. The present study emphasizes the dominant grazing role of PF under P-depleted conditions and reveals that during the late stratified season PF respond to P-addition by lowering their grazing rates, enhancing the relative importance of bacterial removal by HF.

Near-equal compressibility of liver oil and seawater minimises buoyancy changes in deep-sea sharks and chimaeras.

Whereas upper ocean pelagic sharks are negatively buoyant and must swim continuously to generate lift from their fins, deep-sea sharks float or swim slowly buoyed up by large volumes of low-density oils in their livers. Investigation of the pressure, volume, temperature (PVT) relationships for liver oils of 10 species of deep-sea Chondrichthyes shows that the density difference between oil and seawater, Δρ, remains almost constant with pressure down to full ocean depth (11 km, 1100 bar), theoretically providing buoyancy far beyond the maximum depth of occurrence (3700 m) of sharks. However, Δρ does change significantly with temperature and we show that the combined effects of pressure and temperature can decrease buoyancy of oil by up to 10% between the surface and 3500 m depth across interfaces between warm southern and cold polar waters in the Rockall Trough in the NE Atlantic. This increases drag more than 10-fold compared with neutral buoyancy during horizontal slow swimming (0.1 m s-1), but the effect becomes negligible at high speeds. Chondrichthyes generally experience positive buoyancy change during ascent and negative buoyancy change during descent, but contrary effects can occur at interfaces between waters of different densities. During normal vertical migrations buoyancy changes are small, increasing slow-speed drag no more than 2- to 3-fold. Equations and tables of density, pressure and temperature are provided for squalene and liver oils of Chimaeriformes (Harriotta raleighana, Chimaera monstrosa, Hydrolagus affinis), Squaliformes (Centrophorus squamosus, Deania calcea, Centroscymnus coelolepis, Centroscyllium fabricii, Etmopterus spinax) and Carcharhiniformes (Apristurus laurussonii, Galeus murinus).

Microbial strains isolated from CO2-venting Kolumbo submarine volcano show enhanced co-tolerance to acidity and antibiotics.

As ocean acidification intensifies, there is growing global concern about the impacts that future pH levels are likely to have on marine life and ecosystems. By analogy, a steep decrease of seawater pH with depth is encountered inside the Kolumbo submarine volcano (northeast Santorini) as a result of natural CO2 venting, making this system ideal for ocean acidification research. Here, we investigated whether the increase of acidity towards deeper layers of Kolumbo crater had any effect on relevant phenotypic traits of bacterial isolates. A total of 31 Pseudomonas strains were isolated from both surface- (SSL) and deep-seawater layers (DSL), with the latter presenting a significantly higher acid tolerance. In particular, the DSL strains were able to cope with H+ levels that were 18 times higher. Similarly, the DSL isolates exhibited a significantly higher tolerance than SSL strains against six commonly used antibiotics and As(III). More importantly, a significant positive correlation was revealed between antibiotics and acid tolerance across the entire set of SSL and DSL isolates. Our findings imply that Pseudomonas species with higher resilience to antibiotics could be favored by the prospect of acidifying oceans. Further studies are required to determine if this feature is universal across marine bacteria and to assess potential ecological impacts.

Comparative Evaluation of Essential Oils from Medicinal-Aromatic Plants of Greece: Chemical Composition, Antioxidant Capacity and Antimicrobial Activity against Bacterial Fish Pathogens.

The administration of antibiotics in aquaculture has raised concern about the impact of their overuse in marine ecosystems, seafood safety and consumers' health. This "green consumerism" has forced researchers to find new alternatives against fish pathogens. The present study focused on 12 Mediterranean medicinal-aromatic plants as potential antimicrobials and antioxidant agents that could be used in fish aquaculture. In vitro assays showed that the essential oils (EOs) from all studied plants had anti-bacterial and antioxidant properties, with their efficacy being dependent on their chemical composition. More specifically, EOs rich in carvacrol, p-cymene and γ-terpinene exhibited not only the strongest inhibitory activity against the growth of bacterial pathogens (inhibitory concentration: 26-88 µg mL-1), but also the greatest total antioxidant capacity (ABTS: 2591-5879 µmole mL-1; CUPRAC: 931-2733 µmole mL-1). These compounds were mainly found in the EOs from Greek oregano (Origanum vulgare subsp. hirtum), Spanish oregano (Thymbra capitata) and savoury (Satureja thymbra) collected from cultivations in Greece. The specific EOs stand out as promising candidates for the treatment of bacterial diseases and oxidative stress in farmed fish. Further in vivo experiments are needed to fully understand the effects of EO dietary supplementation on fish farming processes.

A simple cleanup method for the removal of humic substances from soil protein extracts using aluminum coagulation.

Soil proteomics, the large-scale characterization of the entire protein complement in soils, provides a promising approach for deciphering the role of microbial functioning in terrestrial ecosystems. However, the extraction of soil proteins in sufficient quantities and of adequate purity remains a challenging task mainly due to the co-extraction of interfering humic substances. Up to now, the treatment of soil extracts with liquid phenol has been the "gold standard" for reducing humics, while the NoviPure cleanup kit was recently launched as a non-toxic approach. The present study describes an alternative method for delivering high-purity proteins based on humic coagulation with trivalent aluminum ions (Al3+). Various experimental parameters were optimized individually in order to maximize protein yield and diminish co-extracted humics. The optimized method was applied on a set of soil samples with diverse physicochemical characteristics and a comparison with the other two techniques was conducted. The amount of residual humics resulting from Al3+-based method was 26 and 35% higher than that from phenol treatment and NoviPure Kit, respectively, but these differences were of marginal statistical significance. With regard to extracted proteins, the average yields of the three methods were comparable, without showing any statistically significant differences. Overall, humic coagulation with Al3+ offers comparable cleanup performance in terms of protein yield and purity, but it is less toxic and less complex than the phenol-partitioning method, whereas it is far less expensive than the NoviPure Kit. The new technique is expected to facilitate the implementation of proteomic studies in soils.

Correction to: Microbial community differentiation between active and inactive sulfide chimneys of the Kolumbo submarine volcano, Hellenic Volcanic Arc.

In the original publication there is a mistake in the supplementary material. The correct supplementary material is provided in this correction article.

Isolation and characterization of a novel bacteriophage infecting Vibrio alginolyticus.

Vibrio alginolyticus is a common marine bacterium implicated in disease outbreaks in marine farmed fish and invertebrates. Due to the inappropriate use of antibiotics in aquaculture, alternative therapies have been proposed. One of the most promising options is the use of lytic bacteriophages to control pathogenic bacteria. This work describes the isolation and characterization of a lytic phage (VEN) against a V. alginolyticus strain (V2) isolated from a disease outbreak in common dentex (Dentex dentex) cultured at the Hellenic Centre for Marine Research (HCMR) in Crete, Greece. The bacteriophage is morphologically similar to phages from Podoviridae family and remained stable for 1 year at 4 °C and over 1 h when kept at 50 °C. VEN was able to lyse the host bacteria at several multiplicity of infection (MOI) (0.1-100) in liquid cultures. However, it was unable to infect other V. alginolyticus strains. Its genome consists of 44,603 bp with a GC content of 43.5%, while sequence analysis revealed the presence of 54 potential ORFs with a T7-like genomic organization. Almost 65% of the predicted ORFs presented homology with proteins of the vibriophages Vc1 and phi-A318 infecting Vibrio cyclitrophicus and Vibrio alginolyticus, respectively. Phylogenetic analysis applying the amino acid sequence of the large terminase subunit confirmed the close relationship of these phages. Furthermore, the comparison of the RNA polymerase of these phages revealed that the motifs A, B and C related to the catalytic activity and the recognition loop related to promotor identification were also conserved. VEN has an obligate lytic life cycle demonstrated by experimental data and genomic analysis. These results suggest that VEN may provide a good candidate to control recurrent diseases caused by V. alginolyticus at HCMR.