BALLAST WATER: A POLYPHASIC APPROACH IN THE EVALUATION OF POTENTIAL PHYTOPLANKTONIC BIOINVASION

The main transport vector for exotic-invasive species around the world is ballast water in merchant ships, crossing natural biogeographic barriers. This is one of the main factors responsible for the reduction and homogenization of the global biota. As phytoplankton is the main group transported by these vessels, this study assesses the risk of bioinvasion in the port complex of Itajaí and Navegantes (southern Brazil), using a polyphasic approach, mixing classical taxonomy with molecular biology. Ballast water collections were carried out to analyze the traditional taxonomy and for clonal cultivation in the laboratory. A successful cultivation of 12 local strains and 10-ballast water was successful. In the latter case, some fresh water, contrary to the statement by the commanders of the exchange of water in the deep ocean (> 200 m). Molecular identification was performed by sequencing the complete ITS region, confirming the presence of Thalassiosira minuscula Krasske, 1941, harmful and of public health interest, previously not mentioned for the region. The species Pleurosigma W. Smith, 1852; Asterionellopsis glacialis (Castracane) Round, 1990; Trieres mobiliensis (Bailey) Ashworth and E.C. Theriot, 2013; Thalassiosira minima Gaarder, 1951; Skeletonema pseudocostatum Medlin, 1991; Pectinodesmus holtmannii E. Hegewald, C. Bock and Krienitz, 2013; Neodesmus Hindák, 1976; and Pseudopediastrum boryanum (Turpin) E. Hegewald, 2005, were identified. The results indicate the possibility of growth of the species found in the ballast environment, which may negatively alter the disposal environment. Keywords: exotic species, molecular identification, marine phytoplankton, port complex of Itajaí and Navegantes.

Diatom modulation of select bacteria through use of two unique secondary metabolites

Unicellular eukaryotic phytoplankton, such as diatoms, rely on microbial communities for survival despite lacking specialized compartments to house microbiomes (e.g., animal gut). Microbial communities have been widely shown to benefit from diatom excretions that accumulate within the microenvironment surrounding phytoplankton cells, known as the phycosphere. However, mechanisms that enable diatoms and other unicellular eukaryotes to nurture specific microbiomes by fostering beneficial bacteria and repelling harmful ones are mostly unknown. We hypothesized that diatom exudates may tune microbial communities and employed an integrated multiomics approach using the ubiquitous diatomAsterionellopsis glacialisto reveal how it modulates its naturally associated bacteria. We show thatA. glacialisreprograms its transcriptional and metabolic profiles in response to bacteria to secrete a suite of central metabolites and two unusual secondary metabolites, rosmarinic acid and azelaic acid. While central metabolites are utilized by potential bacterial symbionts and opportunists alike, rosmarinic acid promotes attachment of beneficial bacteria to the diatom and simultaneously suppresses the attachment of opportunists. Similarly, azelaic acid enhances growth of beneficial bacteria while simultaneously inhibiting growth of opportunistic ones. We further show that the bacterial response to azelaic acid is numerically rare but globally distributed in the world’s oceans and taxonomically restricted to a handful of bacterial genera. Our results demonstrate the innate ability of an important unicellular eukaryotic group to modulate select bacteria in their microbial consortia, similar to higher eukaryotes, using unique secondary metabolites that regulate bacterial growth and behavior inversely across different bacterial populations.

Quorum Sensing Regulates ‘swim-or-stick’ Lifestyle in the Phycosphere

Originality-significance statementMotility and biofilm formation are processes regulated by quorum sensing (QS) in bacteria. Both functions are believed to play an important role in interactions between bacteria and phytoplankton. Here, we show that two bacterial symbionts from the microbial community associated with a ubiquitous diatom switch their motile lifestyle to attached cells while an opportunist bacterium from the same community is incapable of attachment, despite possessing the genetic machinery to do so. Further work indicated that the opportunist lacks QS signal synthases while the symbionts produce three QS signals, one of which is mainly responsible for regulating symbiont colonization of the diatom microenvironment. These findings suggest that QS regulates colonization of diatom surfaces and further work on these model systems will inform our understanding of particle aggregation and bacterial attachment to marine snow and how these processes influence the global carbon cycle.SummaryInteractions between phytoplankton and bacteria play major roles in global biogeochemical cycles and oceanic nutrient fluxes. These interactions occur in the microenvironment surrounding phytoplankton cells, known as the phycosphere. Bacteria in the phycosphere use either chemotaxis or attachment to benefit from algal excretions. Both processes are regulated by quorum sensing (QS), a cell-cell signaling mechanism that uses small infochemicals to coordinate bacterial gene expression. However, the role of QS in regulating bacterial attachment in the phycosphere is not clear. Here, we isolated a Sulfitobacter pseudonitzschiae F5 and a Phaeobacter sp. F10 belonging to the marine Roseobacter group and an Alteromonas macleodii F12 belonging to Alteromonadaceae, from the microbial community of the ubiquitous diatom Asterionellopsis glacialis. We show that only the Roseobacter group isolates (diatom symbionts) can attach to diatom transparent exopolymeric particles. Despite all three bacteria possessing genes involved in motility, chemotaxis, and attachment, only S. pseudonitzschiae F5 and Phaeobacter sp. F10 possessed complete QS systems and could synthesize QS signals. Using UHPLC-MS/MS, we identified three QS molecules produced by both bacteria of which only 3-oxo-C16:1-HSL strongly inhibited bacterial motility and stimulated attachment in the phycosphere. These findings suggest that QS signals enable colonization of the phycosphere by algal symbionts.

Diatom Modulation of Microbial Consortia Through Use of Two Unique Secondary Metabolites

Unicellular eukaryotic phytoplankton, such as diatoms, rely on microbial communities for survival despite lacking specialized compartments to house microbiomes (e.g., animal gut). Microbial communities have been widely shown to benefit from diatom excretions that accumulate within the microenvironment surrounding phytoplankton cells, known as the phycosphere. However, mechanisms that enable diatoms and other unicellular eukaryotes to nurture specific microbiomes by fostering beneficial bacteria and repelling harmful ones are mostly unknown. We hypothesized that diatom exudates may attune microbial communities and employed an integrated multi-omics approach using the ubiquitous diatom Asterionellopsis glacialis to reveal how it modulates its naturally associated bacteria. We show that A. glacialis reprograms its transcriptional and metabolic profiles in response to bacteria to secrete a suite of central metabolites and two unusual secondary metabolites, rosmarinic acid and azelaic acid. While central metabolites are utilized by potential bacterial symbionts and opportunists alike, rosmarinic acid promotes attachment of beneficial bacteria to the diatom and simultaneously suppresses the attachment of opportunists. Similarly, azelaic acid enhances growth of beneficial bacteria, while simultaneously inhibiting growth of opportunistic ones. We further show that the bacterial response to azelaic acid is widespread in the world’s oceans and taxonomically restricted to a handful of bacterial genera. Our results demonstrate the innate ability of an important unicellular eukaryotic group to modulate their microbial consortia, similar to higher eukaryotes, using unique secondary metabolites that regulate bacterial growth and behavior inversely in different bacterial populations.

Plankton community of trafficked ports as a baseline reference for Non Indigenous Species arrivals. Case study of the Port of Bar (South Adriatic Sea)

Plankton (ichthyo, zoo and phyto) communities were studied in the temperate shallow waters of the Port of Bar, one of the main cargo ports on the south-eastern Adriatic coast. Sampling was undertaken in February, April, June and October 2015 at 12 stations using the BALMAS Port Baseline Survey protocol. The research was conducted to determine the presence of invasive and potentially toxic plankton species in the port.The most dominant species of ichthyoplankton were eggs and larvae of families Engraulidae, Bothidae and Sparidae with a dominance of Engraulis encrasicolus, Arnoglossus laterna and Diplodus annularis. In addition to ichthyoplankton, sampling of phyto and zooplankton was performed to estimate the abundance and diversity of species.The most numerous zooplankton species throughout investigated period were Penilia avirostris, Euterpina acutifrons, Oithona nana, Acartia clausi, Centropages kroyeri, Paracalanus parvus, Oncaeidae and larvae of Bivalvia. One very unusual occurrence was the spawning of parrotfish Sparisoma cretense (Linnaeus, 1758), a species with Atlantic origin and tropical affinities, whose presence throughout the Mediterranean has shown an increasing trend over the last decade. The most dominant species of phytoplankton were the diatoms Chaetoceros affinis and Chaetoceros spp., Asterionellopsis glacialis, Pseudo-nitzschia spp., Thalassionema nitzschioides, and dinoflagellates Gymnodinium spp. and Prorocentrum triestinum. The potentially toxic species from genus Pseudo- nitzschia reached an abundance of 104 cells L-1. Toxic dinoflagellates Prorocentrum cordatum and P. micans reached values of 103 cells L-1.Although there were no HAOP species found during the survey, presence of several potentially toxic and toxic phytoplankton species whose impact is not sufficiently known indicates the necessity of introduction of regular monitoring activities and definition of preventive protection measures.

Ecophysiological and biochemical variation of the surf zone diatom asterionellopsis glacialis sensu lato from Santa Catarina, Southern Brazil

Patches formed by dense accumulations of diatoms in the surf zone (surf diatoms) are common on sandy beaches with intermediate to dissipative morphodynamic states. Their appearances are correlated with environmental factors such as the passage of cold fronts when onshore winds increase beach hydrodynamics, resuspending epibenthic stocks and accumulating them through the inner surf zone. In Santa Catarina state, Southern Brazil, two beaches are known to have frequent occurrence of accumulations of the surf diatom Asterionellopsis glacialis sensu lato: Rincão Beach (28°50' S) and Navegantes Beach (26°52' S). The high biomass of this alga and its central importance in the trophic structure of the coastal ecosystems suggest studies about its potential applications. In the present study, strains of A. glacialis were isolated, cultured under different conditions and evaluated for ecophysiological aspects: growth rate under different conditions, potential biological activities of exudates, biomass and lipid content, and fatty acid profile. A. glacialis cells in culture showed deformation, which were ameliorated by using agitation and silicon and phosphorus enriched culture media. Exudates of the strains showed no allelopathic effects, although previous studies have indicated activity. Lipid content showed variation depending on the strain and culture media. Values ranged from 9% to 13.6% by dry weight. In all strains saturated fatty acids and polyunsaturated fatty acids were identified. Some hypotheses were proposed to explain the variation of the lipid contents, fatty acid profiles and physiological features between strains of the same species. We believe that the fatty acids profile of this primary producer has important consequences in the sandy beach ecology.

Responses of the diatom <i>Asterionellopsis glacialis</i> to increasing sea water CO₂ concentrations and the effect of turbulence

Emissions of greenhouse gases, such as carbon dioxide (CO2), are lead to increasing global and surface ocean temperatures. At the same time, as CO2 equilibrates between the atmosphere and the surface ocean, it decreases sea water pH. As a result, the changes in physical and chemical properties of the ocean can affect marine primary producers in various ways. A number of researches have addressed the effects of ocean acidification on marine phytoplankton. However, phytoplankton responses to combined effects are still poorly understood. Here, we chose monospecific cultures of the cosmopolitan chain forming diatom Asterionellopsis glacialis (A. glacialis), grown semi-continuously under controlled laboratory conditions, to assess the combined effect of ocean acidification (~ 420 to 2800 µatm) and turbulence. At current CO2 levels, growth rates of A. glacialis increased under enhanced turbulence. This was the result of an optimum shift towards lower CO2 concentrations and accompanied by a prevalence of longer chains (more than 6 cells). For increasing CO2 levels (up to ~ 2800 µatm) and decreased pH values, enhanced turbulence significantly decreased growth rates, chain length and organic matter production of A. glacialis. Thus, our study suggests that, even though A. glacialis benefited from enhanced turbulence, at present carbon dioxide concentration, at higher CO2 levels, turbulence magnified the stress by acidification. If in the future, the ocean surface layer will be more frequently exposed to storm and wind events, then phytoplankton communities might be more sensitive to lower pH, with potential consequences for community composition and productivity.