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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.

11 pages, 3 figures, 1 table, supplementary information https://doi.org/10.1038/s41598-022-19064-0.-- Data availability: DNA sequence data generated for this study are published on the NCBI GenBank online sequence depository under the accession numbers listed in Table S1. Additional micrographs and cleaned voucher material from the sequenced cultures are available from lead author MPA, Our understanding of the importance of microbiomes on large aquatic animals—such as whales, sea turtles and manatees—has advanced considerably in recent years. The latest observations indicate that epibiotic diatom communities constitute diverse, polyphyletic, and compositionally stable assemblages that include both putatively obligate epizoic and generalist species. Here, we outline a successful approach to culture putatively obligate epizoic diatoms without their hosts. That some taxa can be cultured independently from their epizoic habitat raises several questions about the nature of the interaction between these animals and their epibionts. This insight allows us to propose further applications and research avenues in this growing area of study. Analyzing the DNA sequences of these cultured strains, we found that several unique diatom taxa have evolved independently to occupy epibiotic habitats. We created a library of reference sequence data for use in metabarcoding surveys of sea turtle and manatee microbiomes that will further facilitate the use of environmental DNA for studying host specificity in epizoic diatoms and the utility of diatoms as indicators of host ecology and health. We encourage the interdisciplinary community working with marine megafauna to consider including diatom sampling and diatom analysis into their routine practices, Financial support for sequencing and SEM comes from the Jane and the Roland Blumberg Centennial Professorship in Molecular Evolution at UT Austin and the US Department of Defense (grant number W911NF-17-2-0091). Sampling in South Africa was done with partial financial support from The Systematics Association (UK) through the Systematics Research Fund Award granted to RM (2017 and 2020). Work in the Adriatic Sea was supported by Croatian Science Foundation, project UIP-05-2017-5635 (TurtleBIOME). KF has been fully supported by the “Young researchers' career development project – training of doctoral students” of the CSF funded by the EU from the European Social Fund. NJR was funded by the Spanish government (AEI) through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Background: Our understanding of the importance of microbiomes on large aquatic animals—such as whales, sea turtles and manatees—has advanced considerably in recent years. Recent activity describing the epizoic diatoms growing on marine vertebrates suggests that these epibiotic diatom communities constitute diverse, polyphyletic, and compositionally stable assemblages that include both putatively obligate epizoic and generalist species. Here, we outline a successful attempt to culture putatively obligate epizoic diatoms without their hosts and propose further applications and research avenues in this growing area of study. Results: We cultured cells of epizoic diatoms from multiple host species sampled in the wild and captivity. Analyzing the DNA sequences of these cultures, we found that several unique diatom taxa have independently evolved to occupy in epibiotic habitats. We created a library of reference sequence data for use in metabarcoding surveys of sea turtle and manatee microbiomes that will further facilitate the use of environmental DNA for studying host specificity in epizoic diatoms and the utility of diatoms as indicators of host ecology and health. Conclusions: Our discovery that epizoic diatoms can be cultured independently from their hosts raises several questions about the nature of the interaction between these diatom species and their hosts. We encourage the interdisciplinary community working with marine megafauna to consider including diatom sampling and diatom analysis into their routine practices.

Diatoms are unicellular, photoautotrophic eukaryotic microorganisms, often forming colonies and can be found in most aquatic and moist terrestrial habitats. All known diatoms today have specific golden-brown pigment fucoxanthin that masks chlorophylls in diatom plastid, but one genus represents an exception, having the additional specific pigment marennine due to whom the cells appear blue. Blue diatoms from genus Haslea cause a global phenomenon of ˝greening˝ in shellfish (mostly oysters) affecting them in both positive and negative ways. In this study, historical and recent review regarding blue diatoms and physiological and behavioural effect of marennine as well as challenges in shellfish farming from diatom perspective is addressed. This study is also a first record of blue Haslea diatom in the South Adriatic Sea during BIOTA (Bio-tracing Adriatic Water Masses) cruise in March 2016. Investigated blue diatom was cultured in laboratory and morphologically analysed with light microscopy. Diatom investigations are very important for better understanding of the ecology of specific marine area, but also for the economy, aquaculture and tourism. The emergence of green coloured flesh of shellfish in the Adriatic Sea has not been recorded yet, but this finding of the blue diatom from genus Haslea does not rule out this possibility in the future.

In recent years, biofilm-forming diatoms have received increased attention as sea turtle epibionts. However, most of the research has focused on carapace-associated taxa and communities, while less is known about diatoms growing on sea turtle skin. The current study investigated diatom diversity on the skin of loggerhead sea turtle heads detached from the carcasses found along the Adriatic coast between 1995 and 2004 and stored frozen for a prolonged period of time. By using both light and scanning electron microscopy we have found diatom frustules in 7 out of 14 analysed sea turtle samples. Altogether, 113 diatom taxa were recorded, with a minimum of seven and a maximum of 35 taxa per sample. Eight taxa, Achnanthes elongata, Berkeleya cf. fennica, Chelonicola sp., Licmophora hyalina, Nagumoea sp., Navicula sp., Nitzschia cf. lanceolata, and Poulinea lepidochelicola exceeded 5% of relative abundance in any one sample. The presumably obligately epizoic diatom taxa, A. elongata, Chelonicola sp., and P. lepidochelicola, dominated in six loggerhead samples, contributing up to 97.1% of the total diatom abundance. These observations suggest that on the sea turtle skin highly specialised taxa gain even greater ecological advantage and dominance over the co-occurring benthic forms than in the carapace biofilms. The suitability of frozen sea turtle skin specimens for diatom analysis and limitations of this approach are discussed.

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.

The diatom genus Proschkinia is a common element of biofilms covering diverse substrata in saline inland or shallow coastal environments. It can be distinguished from other naviculoid taxa by its lanceolate valves with a fistula located within the central area and numerous open girdle bands with a U-shaped cross-section and a single row of perforations on the internal side of the fold. Despite this distinct morphology, frustules of Proschkinia are typically weakly silicified and often overlooked when cleaned diatom material is analysed. The current paper describes six new species of Proschkinia: P. browderiana sp. nov., P. lacrimula sp. nov., P. maluszekiana sp. nov., P. sulcata sp. nov., P. torquata sp. nov. and P. vergostriata sp. nov., found in numerous samples of marine organisms, such as sea turtles (including sea turtle museum specimens), sea turtle-associated barnacles and seagrass from across the three oceans. Some of the newly described taxa were found on multiple individuals belonging to different sea turtle species, whereas others were in samples collected from different continents. Molecular phylogenetic analysis indicated that examined Proschkinia strains formed a monophyletic clade, sister to Fistulifera.

Summary Unusual occurrence of planktonic diatom species, Chaetoceros bacteriastroides and Chaetoceros pseudosymmetricus , was noticed in three different marine ecosystems of Adriatic Sea: the Krka Estuary and Telascica Bay in the Central Adriatic, and in southern Adriatic offshore. From 2010 to 2015, these two Chaetoceros species were recorded in heterogeneous environmental conditions and in a very low abundances. Both species are regarded as very rare in world oceans, and consequently knowledge of their distribution and ecology is rather poor. Primarily described from tropical waters and showing Indo-Pacific distribution, C . bacteriastroides and C . pseudosymmetricus findings in Adriatic represent the northernmost records in world's oceans and seas. For C. pseudosymmetricus this is also the first occurrence in European seas. Areal expansion and introduction of new phytoplankton species in the Adriatic Sea might be related to different circulation regimes in the Ionian Sea and the concurrent rise in sea temperature in the Mediterranean in the last decade. Recent investigations have shown that entering currents, of either Atlantic/Western Mediterranean or Eastern Mediterranean origin, modify the composition of the plankton community in the Adriatic by bringing different newcomers.

Adriatic, the northernmost part of the Mediterranean Sea, due its oligotrophy, topography, and hydrology dynamics, and complex circulation patterns, was suggested as an important study site for rapid climatology impacts. Its southern part is mainly oligotrophic and dominated by picophytoplankton, with cyanobacteria as main representatives. Diversity and distribution patterns of different Prochlorococcus ecotypes were investigated by molecular tools and flow cytometry during the winter convection event in the southern Adriatic (BIOTA winter cruise; February/March 2015). Phylogenetic diversity based on clone libraries of the 16S–23S ribosomal DNA ITS region, as well as flow cytometry (histograms of red fluorescence), indicated presence of 2 different Prochlorococcus in the Adriatic. HLI, as a typical clade for Mediterranean Sea, was likewise found to be dominant Prochlorococcus in the Adriatic, followed by less abundant LLI clade. In addition, Prochlorococcus were found to co-occur with diverse Synechococcus population (53% and 47% of obtained ITS sequences, respectively). Different Prochlorococcus ecotypes had similar patterns of vertical distribution, predominantly occupying upper 100 m depth layer, but their distribution was clearly affected by the heterogeneity of hydrological conditions, nitrogen concentration and temperature along vertical and horizontal sampling points. Different studies pointed out that, as a consequence of climate changes, serious alteration of biological and ecological patterns are already taking place Therefore, understanding of the distribution and abundance of picophytoplankton in Adriatic, being still limited, is much needed baseline for predicting possible biogeochemical impact of future environmental changes.

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.

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 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.