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Diatoms and bacteria are known for being the first colonizers of submerged surfaces including the skin of marine reptiles. Sea turtle carapace and skin harbor diverse prokaryotic and eukaryotic microbes, including several epizoic diatoms. However, the importance of diatom-bacteria associations is hardly investigated in biofilms associated with animal hosts. This study provides an inventory of diatoms, bacteria and diatom-associated bacteria originating from loggerhead sea turtles using both metabarcoding and culturing approaches. Amplicon sequencing of the carapace and skin samples chloroplast gene rbcL and 16S rRNA gene detected, in total, 634 diatom amplicon sequence variants (ASVs) and 3661 bacterial ASVs, indicating high diversity. Cultures of putative epizoic and non-epizoic diatoms contained 458 bacterial ASVs and their bacterial assemblages reflected those of their host. Diatom strains allowed for enrichment and isolation of bacterial families rarely observed on turtles, such as Marinobacteraceae, Alteromonadaceae and Alcanivoracaceae. When accounting for phylogenetic relationships between bacterial ASVs, we observed that related diatom genera might retain similar microbial taxa in culture, regardless of the turtle's skin or carapace source. These data provide deeper insights into the sea turtle-associated microbial communities, and reveal the potential of epizoic biofilms as a source of novel microbes and possibly important diatom-bacteria associations.

The paraphyletic diatom genus Nitzschia comprises over 1000 morphologically distinct pennate taxa, known from the benthos and plankton of freshwater, brackish and marine environments. The principal diagnostic characters for delimitation of Nitzschia species include valve shape, the position and structure of the raphe, presence/absence and shape of the proximal raphe endings the shape of the terminal raphe fissures, areola structure, and specific morphometric features such as cell size, and stria and fibula density. In this study, we isolated 12 diatom strains into culture from samples collected at the surface or greater depths of the southeastern Adriatic Sea. Morphological analyses included LM, SEM and TEM observations, which, along with specific morphometric features, allowed us to distinguish three new Nitzschia species. These findings were congruent with the results of phylogenetic analyses performed on nuclear-encoded SSU (18S) rDNA and chloroplast-encoded rbcL and psbC genes. One of the new species (Nitzschia dalmatica sp. nov.) formed a lineage within a clade of Bacillariaceae containing members of the Nitzschia sect. Dubiae, which was sister to Psammodictyon. A second lineage was part of a novel clade that is significantly distinct from other Nitzschia species sequenced so far and includes Nitzschia adhaerens sp. nov. and N. cf. adhaerens. A further new species was found, Nitzschia inordinata sp. nov., which appeared as the sister group to the N. adhaerens clade and the conopeoid Nitzschia species in our phylogenetic trees. Our findings contribute to the overall diversity of genus Nitzschia, especially in identifying some deep branches within the Bacillariaceae, and highlight under-scoring of this genus in marine plankton.

Epizoic diatoms form an important part of micro-epibiota of marine vertebrates such as whales and sea turtles. The present study explores and compares the diversity and biogeography of diatom communities growing on the skin and shell of loggerhead sea turtles (Caretta caretta) from four different localities: Adriatic Sea (Croatia), Ionian Sea (Greece), South Africa and Florida Bay (USA) using both light and scanning electron microscopy. We observed almost 400 diatom taxa belonging to more than 100 genera. Diatom communities from Greece and Croatia showed the highest similarity and were statistically different from those recorded from South Africa and Florida. Part of this variation could be attributed to differences in sampling techniques; however, we believe that geography had an important role. In general, contrary to several previous observations from sea turtles, the presumably exclusively epizoic diatoms contributed less than common benthic taxa to the total diatom flora, which might have been related to the loggerhead feeding behavior. Moreover, skin samples differed from carapace samples in having a distinct diatom composition with a higher proportion of the putative true epizoonts. Our results indicate that epizoic diatom communities differ according to loggerhead geographical location and substrate (skin vs. carapace). The relative abundances of common benthic diatoms and putative exclusive epizoic taxa may inform about sea turtle habitat use or behavior though detailed comparisons among different host species have yet to be performed.

The planktonic diatoms belonging to two genera Chaetoceros and Bacteriastrum , included within the family Chaetocerotaceae, are ecologically important as they represent an important component of the phytoplankton in the coastal regions and are often among bloom-forming taxa. We analysed the chaetocerotacean species composition and abundances in the coastal area of northeastern Adriatic in a biweekly study conducted from September 2008 to October 2009 with the aim of investigating seasonal dynamics and species succession on a fine temporal scale and determining the most important ecological factors influencing their distribution. The study identified seven Chaetoceros and three Bacteriastrum species as major phytoplankton components showing the clear annual succession and two types of blooms (one species/multi species) governed by differing ecological conditions. Autumn bloom was composed of 20 chaetocerotacean species with Chaetoceros contortus and Chaetoceros vixvisibilis alternating in dominance. The summer period was characterized by spreading of freshwater from the Po River up to the eastern coast increasing availability of phosphate which triggered the monospecific Chaetoceros vixvisibilis bloom. We explained the chaetocerotacean dominant species succession pattern by the environmental parameters, with the temperature, salinity and phosphate availability as most important factors driving the species seasonality.

Marine picoplankton, unicellular organisms with cell sizes up to 3 μm in diameter, numerically dominate marine ecosystems, encompassing Archaea, Bacteria, Eukarya (protists and fungi) as well as viruses. Autotrophic and heterotrophic picoplankton abundance and community composition with a focus on picoeukaryotes (PEs) were investigated in the winter of 2016 at three stations along a coast-to-offshore transect in the southern Adriatic Sea. Abundances were estimated by flow cytometry, while community composition by Illumina High-Throughput Sequencing (HTS) of 16S and 18S rRNA genes. The photosynthetic picoplankton diversity was also investigated by High-Performance Liquid Chromatography (HPLC) of liposoluble pigments. Heterotrophic bacteria and cyanobacteria (Prochlorococcus and Synechococcus) accounted for up to 7 × 105; 2.3 × 104 and 2.5 × 104 cells mL−1, respectively, while photosynthetic picoeukaryotes peaked with 3 × 103 cells mL−1. Prokaryotes, as revealed by HTS were dominated by Alphaproteobacteria (mainly SAR11, 44.91% of total 16S sequence reads), followed by Gammaproteobacteria (Oceanospirillales and Pseudomonadales, 14.96%), Bacteroidetes (mainly Flavobacteriales, 13%), Cyanobacteria (Prochlorococcus and Synechococcus, 9.52%), Marinimicrobia (SAR406, 7.97%), Deltaproteobacteria (SAR324, 3.83%), Actinobacteria (2.24%) and Chloroflexi (SAR202, 1.90%). Photosynthetic pigment concentrations were very low (12.12 μgL−1 at the most) and taxonomic pigments could be attributed to Prochlorococcus, Synechococcus, Prymnesiophyceae, Bacillariophyceae, Chrysophyceae, and Prasinophyceae. HTS data revealed that PEs were dominated by heterotrophs, such as Syndiniophyceae, parasitic dinoflagellates (79.67% of total 18S sequence reads), Dinophyceae (8.7%) and the radiolarians Collodaria belonging to Sphaerozoidae (22.1%) and Spumellaria (5.0%). On the other hand, photoautotrophs, including Chlorophyta (Mamiellophyceae, Prasinophyceae, Trebouxiophyceae, and Ulvophyceae), Stramenopiles (Bacillariophyta, Chrysophyceae, Dictyochophyceae, Pelagophyceae), photoautotrophic Cryptophyta and some Haptophyta (Prymnesiophyceae), did not exceed 5% of total sequence reads. This study provides the first snapshot of the PEs diversity in oligotrophic euphotic waters of the southern Adriatic Sea, hence setting the stage for large-scale surveying and characterization of the eukaryotic diversity in the entire basin.

Phytoplankton assemblages and pigment distribution were determined in the transitional Boka Kotorska Bay. Samples were collected four times from three stations in the inner part of the Bay between April 2008 and March 2009. Microscopic analysis revealed that the phytoplankton was composed of marine diatoms, dinoflagellates, cryptophytes, chlorophytes and coccolithophorids. Aside from fucoxanthin as the dominant biomarker pigment, alloxanthin, 19′-hexanoyloxyfucoxanthin, peridinin and chlorophyll b provided indicative contributions. Fucoxanthin and alloxanthin showed significant correlation with the phytoplankton chlorophyll a biomass throughout the investigated period. In November 2008, the diatoms were outcompeted by coccolithophorids, which probably efficiently absorbed nutrients during a period of their reduced supply. Due to the reduced nutrient input, in the summer, the phytoplankton community in the Bay was composed mostly of marine dinoflagellates, cryptophytes, and chlorophytes., Prisutnost zajednica fitoplanktona i rasprostranjenost pigmenata utvrđivani su u tranzicijskim vodama Bokokotorskog zaljeva. Uzorci su prikupljeni četiri puta na tri lokaliteta u unutrašnjosti Zaljeva u periodu između travnja 2008. i ožujka 2009. godine. Mikroskopskom analizom utvrđeno je da fitoplankton čine morske dijatomeje, dinoflagelati, kriptofiti, klorofiti i kokolitoforidi. Pored fukoksantina kao dominantnog biomarkerskog pigmenta, bili su zastupljeni i aloksantin, 19′-heksanoiloksifukoksantin, peridinin i klorofil b‏. Fukoksantin i aloksantin su pokazali značajnu korelaciju sa klorofilom a kao indikatorom biomase fitoplanktona tijekom čitavog perioda ispitivanja. U studenom 2008. godine, dijatomeje su nadjačale kokolitoforide, koji su vjerovatno efikasno apsorbirali nutrijente tijekom perioda u kojem je njihova dostupnost bila smanjena. Kao rezultat smanjenog unosa nutrijenata, u ljetnom periodu, zajednicu fitoplanktona u Bokokotorskom zaljevu sačinjavali su uglavnom morski dinoflagelati, kriptofiti i klorofiti.

The diatom genus Entomoneis is known from the benthos and plankton of marine, brackish, and freshwaters. Entomoneis includes diatoms with a bilobate keel elevated above the valve surface, a sigmoid canal raphe, and numerous girdle bands. Owing mostly to the scarcity of molecular data for a diverse set of species, the phylogeny of Entomoneis has not been investigated in depth. The few previous studies that included Entomoneis were focused on broader questions and the available data were from a small number of either unidentified Entomoneis or well-known species (e.g., E. paludosa). Since the first description of new species combining both molecular and morphological characters (E. tenera), we have continued to cultivate and investigate Entomoneis in the plankton of the Adriatic Sea. Combined multigene phylogeny (SSU rDNA sequences, rbcL, and psbC genes) and morphological observations (LM, SEM and TEM) revealed six new Entomoneis species supported by phylogenetic and morphological data: E. pusilla, E. gracilis, E. vilicicii, E. infula, E. adriatica, and E. umbratica. The most important morphological features for species delineation were cell shape, the degree and mode of torsion, valve apices, the appearance and structure of the transition between keel and valve body, the ultrastructure and the shape of the girdle bands, and the arrangement and density of perforations along the valve and valvocopulae. Our results highlight the underappreciated diversity of Entomoneis and call for a more in-depth morphological and molecular investigation of this genus especially in planktonic habitats.

A 275-km-long transversel Northern Adriatic profile from the mouth of the Po River (Italian Adriatic coast) to the Kvarner region (Croatian coastal island area) was investigated in three successive case studies in August 2008, 2009 and 2010. The short Po River pulses in August result in the surface advection of riverine water, nutrients and phytoplankton from the western to the eastern side of the Adriatic. This surface spreading exhibits inter-annual variability depending on the riverine discharge in the preceding period. The Po River discharge pulse in August 2010 in particular resulted in an extraordinary tongue-like advection of riverine water, nutrients, and phytoplankton towards the Eastern Adriatic coast. The phenomenon was detected using both satellite imagery and classical oceanographic measurements. In the advective water, toxic dinoflagellates were most abundant in August 2010, when the influence of the Po was greatest.

The genus Entomoneis Ehrenberg includes diatoms with structurally complex frustules having a bilobate keel elevated above the valve surface and numerous girdle bands. We describe here a new member of the genus, Entomoneis tenera sp. nov. , a minute species found in the plankton of the south-eastern Adriatic Sea. The description and proposed taxonomic affiliation are based on both morphological observations and molecular analyses obtained from three cultivated strains. The cultures were established from plankton net samples taken during BIOTA (Bio-tracing Adriatic Water Masses) cruise conducted in February–March 2015. In addition to characteristic Entomoneis features such as panduriform cells, often twisted around the apical axis and a raphe with simple endings positioned on the sigmoid keel, morphological characteristics of E. tenera are: (1) very small cells, 16–21 μm long and 5–20 μm wide (2) very lightly silicified, delicate frustules without valve striation discernible in light microscopy, (3) broad lanceolate valves with scalpeliform apices (4) a straight to slightly arcuate junction line. Phylogenetic analyses using SSU, rbc L and psb C supported the position of E . tenera within the Entomoneis genus with a clear separation from the other described species.

Increasing pCO2 is hypothesized to induce shifts in plankton communities towards smaller cells, reduced carbon export rates and increased roles of gelatinous zooplankton. Appendicularians, among the most numerous pan-global ‘gelatinous’ zooplankton, continuously produce filter-feeding houses, shortcutting marine food webs by ingesting submicron particles, and their discarded houses contribute significantly to carbon fluxes. We present a first mesocosm-scale study on the effects of temperature, pCO2 and bloom structures on the appendicularian, Oikopleura dioica. There were effects of temperature and nutrients on phytoplankton communities. No shifts in functional phytoplankton groups, nor changes in particle sizes/morphotypes, known to impact appendicularian feeding, were observed under manipulated pCO2 conditions. However, appendicularian abundance was positively correlated with increased pCO2, temperature and nutrient levels, consistent with hypotheses concerning gelatinous zooplankton in future oceans. This suggests appendicularians will play more important roles in marine pelagic communities and vertical carbon transport under projected ocean acidification and elevated temperature scenarios.

The species composition and size-structure of the phytoplankton community in the Boka Kotorska Bay (SE Adriatic Sea) were analysed with respect to abundance and carbon biomass, together with the physico-chemical parameters, with the aim of evaluating the predefined oligo-mesotrophic status of this transitional water ecosystem. Three stations located in the inner part of the Bay were sampled with seasonal frequency in 2008/2009. Picophytoplankton cells were counted using flow cytometry; nanophytoplankton and microphytoplankton were identified and counted by light microscopy. The relative importance of the picoplankton in the Bay, in terms of both abundance and biomass, during all the investigated seasons emphasized their significance in the phytoplankton community. Picocyanobacteria (Synechococcus) constituted a significant part of the summer assemblages with regard to both abundance (up to 3.38×108 cells L−1) and carbon biomass (up to 73% of total phytoplankton carbon). The contribution of the nanophytoplankton was found to be generally low (

The chain-forming diatom Chaetoceros vixvisibilis is one of the most abundant and frequent diatoms in the northeastern Adriatic Sea. This species had not been previously studied by electron microscopy. Its general morphology is similar to those species allocated in the subgenus Hyalochaete: cells and chains (straight, of variable length, usually 4-8 cells per chain, but can be longer) of delicate appearance, valves thinly silicified, with slightly eccentric annulus and costae radiating from it, a single rimoportula present only at terminal valves, long, thin and delicate setae perforated by tiny poroids with no spines, and one plate-like chloroplast per cell. Setae with no spines are not common among members of Hyalochaete (and the whole genus Chaetoceros), except C. socialis. The most characteristic feature is, however, the resting spores commonly found, which also show morphological variability, from solitary to paired, both valves convex to domed and surface smooth or with small granules, and one to four strong spines often branching dichotomically. Abundances of C. vixvisibilis are positively correlated to the Po River inflow. Maximum abundances (> 10 6 cells L -1 ) were found in the period April-July, in the temperature range between 12 and 16°C, salinity between 33 and 38, when total phosphorus concentration is higher than 0.4 μmol L -1 , but occasionally also in nitrogen limited conditions.

Phytoplankton abundance and seasonality in the northeastern Adriatic Sea were directly related to the annual regime of the Po River discharge in the period 2002–2007. In 2005, 2006, and 2007, there was a shift in the timing of the annual Po River maximum discharge and of phytoplankton maxima from spring to autumn. In addition, the eastern coastal area was greatly influenced by oligotrophic karstic rivers and the Eastern Adriatic Current. Dominant phytoplankton taxa were defined on the basis of abundance and frequency of appearance. Such an approach may help in the comparison of phytoplankton communities within different environments. The dominant taxa were the diatoms Cerataulina pelagica, Chaetoceros socialis, Chaetoceros vixvisibilis and Pseudo-nitzschia spp., which appeared at maximum abundances >106 cells l-1 in more than 20% of samples. Among other phytoplankton, the most common was the coccolithophorid Emiliania huxleyi (maximum abundance of 2×105 cells l-1 in 50% of samples), and the dinoflagellate Prorocentrum minimum (maximum abundance of 3×105 cells l-1 in 12% of samples). The present state of the phytoplankton community may provide valuable information for evaluation of possible future environmental changes in the shallow Mediterranean ecosystem.

The Velebit and Pag Channels form a 40 km long and 4-14 km wide system running parallel to the northeastern Adriatic coast. The aim of this study was to investigate phytoplankton abundance, composition and distribution along the system in the autumn period. Sampling was carried out at 8 stations in November 2008. The spatial distribution of physico-chemical parameters indicated the existence of a pronounced horizontal thermohaline and trophic gradient. From the inner part of the Velebit Channel, which is an extension of the karstic Zrmanja River estuary, to the outer end of the Pag Channel, which communicates with the open sea, the temperature and salinity ranged from 11.0 to 16.0 °C and from 23.4 to 38.2, respectively. Nutrient concentrations ranged from 0.001 to 0.1 µmol L-1 for orthophosphates, from 0.85 to 7.0 µmol L-1 for total inorganic nitrogen and from 0.3 to 15.4 µmol L-1 for silicates. Total abundance of phytoplankton decreased from 5.5 x 105 cells L-1 to 8.5 x 104 cells L-1 along the trophic gradient. Diatoms dominated the phytoplankton with maximum abundances from 7.3 x 104 cells L-1 (85% of total phytoplankton) at the most oligotrophic stations up to 9.6 x 105 cells L-1 (96% of total phytoplankton) at the nutrient enriched stations. Statistical analysis on the basis of environmental and biological parameters clustered the stations in three main groups. The first group represented the southern Velebit Channel with higher nutrient levels and phytoplankton dominated by Thalassionema nitzschioides. The second group included stations from the middle Velebit Channel with significantly lower levels of nutrients, and phytoplankton mostly dominated by Bacteriastrum sp. with a contribution by various diatom species. The third group represented the stations from the Pag Channel, and associated with the exclusive domination of Bacteriastrum sp. and a greater contribution of coccolithophorids. This paper also gives the first checklist of species present in the extremely oligotrophic area of the Velebit and Pag Channels. 148 taxa were identified: 91 diatoms, 47 dinoflagellates and 10 other flagellate morphotypes. Detailed electron microscopy analysis revealed three potentially toxic diatoms of Pseudo-nitzschia genus: P. calliantha, P. pseudodelicatissima and P. manii. The results of this work represent a valuable contribution to the knowledge of phytoplankton species composition and distribution relative to prevailing physico-chemical conditions in this poorly investigated transition zone., Velebitski i Paški kanal čine 40 km dug i 4-14 km širok sustav koji se proteže usporedno sa sjeveroistočnom obalom Jadranskog mora. Cilj ovog istraživanja je bio istražiti abundanciju, sastav i raspodjelu fitoplanktona duž sustava u jesenskom razdoblju. Uzorkovanje je obavljeno na 8 postaja u studenom 2008. Prostorna raspodjela fizikalno kemijskih parametara upućivala je na postojanje izrazitog horizontalnog termohalinog i trofičkog gradijenta. Od unutrašnjeg dijela Velebitskog kanala, koji je produžetak estuarija krške rijeke Zrmanje do vanjskog dijela Paškog kanala koji se spaja s otvorenim morem, raspon površinske temperature mora iznosio je 11.0 do 16.0 °C, a saliniteta 23.4 do 38.2. Koncentracije nutrijenata kretale su se u rasponu 0.001 - 0.1 μmol L-1 za fosfate, 0.85 - 7.0 μmol L-1 za otopljeni anorganski dušik i 0.3 - 15.4 μmol L-1 za silikate. Dijatomeje su prevladavale u fitoplanktonu s maksimalnom abundancijom u rasponu od 7.3 x 104 stanica L-1 (85% ukupnog fitoplanktona) na najviše oligotrofnim do 9.6 x 105 stanica L-1 (96% ukupnog fitoplanktona) na postajama s većom koncentracijom nutrijenata. Statistička analiza na osnovi bioloških i fizikalno kemijskih parametara podijelila je postaje u tri glavne grupe. Prva grupa predstavljala je južni dio Velebitskog kanala s većom koncentracijom nutrijenata i prevladavajućom vrstom Thalassionema nitzschioides. Druga grupa uključila je postaje iz središnjeg dijela Velebitskog kanala s nižim razinama nutrijenata i s prevladavajućom dijatomejom Bacteriastrum sp.. Treća grupa uključila je postaje u Paškom kanalu u kojima gotovo isključivo prevladava Bacteriastrum mannii sp. i prisutan je veći udio kokolitoforida. Ovaj rad također donosi prvi popis vrsta fitoplanktona prisutnih u ekstremno oligotrofnom području Velebitskog i Paškog kanala. Identificirano je 148 taksona od čega 91 dijatomeja, 47 dinoflagelata i 10 taksona raznih flagelata. Detaljna analiza pomoću elektronskog mikroskopa otkrila je tri vrste iz potencijalno toksičnog dijatomejskog roda Pseudo-nitzschia: P-n. calliantha, P-n. pseudodelicatissima i P-n. mannii. Rezultati ovog rada predstavljaju značajan doprinos poznavanju sastava i raspodjele fitoplanktona u odnosu na prevladavajuće fizikalno kemijske uvjete u ovom slabo istraženom tranzitnom području.

The population dynamics of Pseudo-nitzschia in relation to environmental factors was investigated from March 2002 to July 2008 in Lim Bay, in the north-eastern Adriatic Sea. Domoic acid was monitored in the breeding population of Mytilus galloprovincialis from 2005 to 2008. The principal-component analysis of environmental parameters showed that the system is mostly temperature driven. The phytoplankton community was mainly composed of diatoms. Pseudo-nitzschia was the dominant diatom, present in 60% of samples, with a maximum (1.6 × 10 6 cells L −1 ) contribution up to 97% of the total diatom abundance. Morphological analysis revealed Pseudo-nitzschia manii and potentially toxic Pseudo-nitzschia pseudodelicatissima , Pseudo-nitzschia pungens , Pseudo-nitzschia fraudulenta and Pseudo-nitzschia calliantha as the dominant species in blooms. Pseudo-nitzschia abundance positively correlated to temperature, phosphate and ammonia in accordance with its maximal abundance in the summer/autumn period when fish farms had a maximum impact on the environment. Domoic acid was detected in M . galloprovincialis in concentrations below regulatory limits, ranging from 0.097 to 0.8721 μg g −1 in five cases from April to October 2005 in Lim Bay, but so far it is not clear which of the species was responsible for DA production. This study is also the first record of P. manii , P pungens and P. fraudulenta species in the Adriatic Sea.

The distribution of phytoplankton and its relation to the hydrographic features in the north-eastern Adriatic was investigated in February 2008. The area ofinterest included a thermohaline gradient in the channel situated between the coast and the islands lying parallel to the coast. The gradient is controlledby the influx of oligotrophic karstic riverine water at the south-eastern end, submarine springs in the middle part, and warmer offshore waters at thenorth-western end of the channel. The change of temperature and salinity in the estuarine transition zone was accompanied by abundant diatoms anddinoflagellates below the halocline, with dominant chain-forming diatoms (Chaetoceros, Bacteriastrum) in abundances reaching 5 ×105cells dm-3. The impact of coastal submarine springs detected by infrared remote sensing resulted in the growth of cyanobacteria in thenitrogen-depleted surface waters. The greater contribution of picoplankton, as well as of nanoplanktonic coccolithophorids and cryptophytes, in the outer channelsystem indicated their preference for oligotrophic conditions. Flow cytometric counts of nanophytoplankton were 10-30 times greater than inverted microscopecounts. Cyanobacteria were about five times more abundant than picoeukaryotes. The study demonstrates how different techniques (remote sensing and in situinvestigations) can be useful in understanding the biological and hydrographic set-up in the specific oligotrophic eastern Adriatic coastal environment.