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Abstract 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.

Background Phytoplankton is the base of majority of ocean ecosystems. It is responsible for half of the global primary production, and different phytoplankton taxa have a unique role in global biogeochemical cycles. In addition, phytoplankton abundance and diversity are highly susceptible to climate induced changes, hence monitoring of phytoplankton and its diversity is important and necessary. Methods Water samples for phytoplankton and photosynthetic pigment analyses were collected in boreal winter 2017, along transect in the North Pacific Subtropical Gyre (NPSG) and the California Current System (CCS). Phytoplankton community was analyzed using light and scanning electron microscopy and photosynthetic pigments by high-performance liquid chromatography. To describe distinct ecosystems, monthly average satellite data of MODIS Aqua Sea Surface temperature and Chlorophyll a concentration, as well as Apparent Visible Wavelength were used. Results A total of 207 taxa have been determined, mostly comprised of coccolithophores (35.5%), diatoms (25.2%) and dinoflagellates (19.5%) while cryptophytes, phytoflagellates and silicoflagellates were included in the group “others” (19.8%). Phytoplankton spatial distribution was distinct, indicating variable planktonic dispersal rates and specific adaptation to ecosystems. Dinoflagellates, and nano-scale coccolithophores dominated NPSG, while micro-scale diatoms, and cryptophytes prevailed in CCS. A clear split between CCS and NPSG is evident in dendogram visualising LINKTREE constrained binary divisive clustering analysis done on phytoplankton counts and pigment concentrations. Of all pigments determined, alloxanthin, zeaxanthin, divinyl chlorophyll b and lutein have highest correlation to phytoplankton counts. Conclusion Combining chemotaxonomy and microscopy is an optimal method to determine phytoplankton diversity on a large-scale transect. Distinct communities between the two contrasting ecosystems of North Pacific reveal phytoplankton groups specific adaptations to trophic state, and support the hypothesis of shift from micro- to nano-scale taxa due to sea surface temperatures rising, favoring stratification and oligotrophic conditions.

Abstract Background Microbial communities of wild animals are being increasingly investigated to provide information about the hosts’ biology and promote conservation. Loggerhead sea turtles (Caretta caretta) are a keystone species in marine ecosystems and are considered vulnerable in the IUCN Red List, which led to growing efforts in sea turtle conservation by rescue centers around the world. Understanding the microbial communities of sea turtles in the wild and how affected they are by captivity, is one of the stepping stones in improving the conservation efforts. Describing oral and cloacal microbiota of wild animals could shed light on the previously unknown aspects of sea turtle holobiont biology, ecology, and contribute to best practices for husbandry conditions. Results We describe the oral and cloacal microbiota of Mediterranean loggerhead sea turtles by 16S rRNA gene sequencing to compare the microbial communities of wild versus turtles in, or after, rehabilitation at the Adriatic Sea rescue centers and clinics. Our results show that the oral microbiota is more sensitive to environmental shifts than the cloacal microbiota, and that it does retain a portion of microbial taxa regardless of the shift from the wild and into rehabilitation. Additionally, Proteobacteria and Bacteroidetes dominated oral and cloacal microbiota, while Kiritimatiellaeota were abundant in cloacal samples. Unclassified reads were abundant in the aforementioned groups, which indicates high incidence of yet undiscovered bacteria of the marine reptile microbial communities. Conclusions We provide the first insights into the oral microbial communities of wild and rehabilitated loggerhead sea turtles, and establish a framework for quick and non-invasive sampling of oral and cloacal microbial communities, useful for the expansion of the sample collection in wild loggerhead sea turtles. Finally, our investigation of effects of captivity on the gut-associated microbial community provides a baseline for studying the impact of husbandry conditions on turtles’ health and survival upon their return to the wild.

The loggerhead sea turtle is considered a keystone species with a major ecological role in Mediterranean marine environment. As is the case with other wild reptiles, their outer microbiome is rarely studied. Although there are several studies on sea turtle’s macro-epibionts and endo-microbiota, there has been little research on epibiotic microbiota associated with turtle skin and carapace. Therefore we aimed to provide the identification of combined epibiotic eukaryotic, bacterial and archaeal microbiota on Mediterranean loggerhead sea turtles. In this study, we sampled skins and carapaces of 26 loggerheads from the Mediterranean Sea during 2018 and 2019. To investigate the overall microbial diversity and composition, amplicon sequencing of 16S and 18S rRNA genes was performed. We found that the Mediterranean loggerhead sea turtle epibiotic microbiota is a reservoir of a vast variety of microbial species. Microbial communities mostly varied by different locations and seas, while within bacterial communities’ significant difference was observed between sampled body sites (carapace vs. skin). In terms of relative abundance, Proteobacteria and Bacteroidota were the most represented phyla within prokaryotes, while Alveolata and Stramenopiles thrived among eukaryotes. This study, besides providing a first survey of microbial eukaryotes on loggerheads via metabarcoding, identifies fine differences within both bacterial and eukaryotic microbial communities that seem to reflect the host anatomy and habitat. Multi-domain epi-microbiome surveys provide additional layers of information that are complementary with previous morphological studies and enable better understanding of the biology and ecology of these vulnerable marine reptiles.

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 Telaščica 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.

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.

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.

During the June 2010 survey of phytoplankton and physicochemical parameters in the Krka River estuary (eastern Adriatic Sea), a cryptophyte bloom was observed. High abundance of cryptophytes (maximum 7.9×106 cells l–1) and high concentrations of the class-specific biomarker pigment alloxanthine (maximum 2312 ng l–1) were detected in the surface layer and at the halocline in the lower reach of the estuary. Taxonomical analysis revealed that the blooming species was Plagioselmis cf. prolonga. Analysis of the environmental parameters in the estuary suggested that the bloom was supported by the slower river flow as well as the increased orthophosphate and ammonium concentrations. The first record of a cryptophyte bloom in the Krka River estuary may indicate that large-scale changes are taking place in the phytoplankton community. Such changes could have a major impact on the natural ecosystem dynamics and the mariculture production in the area.

Diatoms have evolved a variety of colonial life forms in which cells are connected by organic threads, mucilage pads or silicate structures. In this study, we provide the first description of a novel strategy of colony formation among marine planktonic diatoms. Bacteriastrum jadranum forms loose but regular chains with distinct heterovalvate terminal cells. The colonial cells and their siliceous projections, the setae, are not in direct contact; instead, they are enclosed within the optically transparent organic matrix. This cell jacket structure was detected by staining procedure with Alcian Blue, which showed that the polysaccharides are predominant matrix constituents and revealed that the jacket reaches the span of the setae. The scanning electron microscopy (SEM) observations showed distinguishable fibrillar network firmly associated with cells. Using atomic force microscopy (AFM), we were able to visualise and characterise the cell jacket structure at molecular resolution. At nanoscale resolution, the cell jacket appears as a cross-linked fibrillar network organised into a recognisable structure. The circular patches of self-repeating pattern (hexagonal pores with openings of 8-100 nm) are connected through thicker surrounding fibrils and reinforced by branching fibrils. The pore-forming fibrils within the patches are only 0.6-1.6 nm high, the surrounding fibrils connecting patches are 2.0-2.8 nm high, and the branching fibrils are considerably wider but not higher than 4.0 nm. The discovered polysaccharide fibrillar network is highly organised and delicately structured with a monomolecular fibril height of 0.6 nm. We conclude that the Bacteriastrum polysaccharide jacket represents an essential part of the cell, as the conjunction of the polymer network with the frustule appears to be extremely tight and such specific and unique patterns have never been found in self-assembled polysaccharide gel networks, which are usually encountered in the marine environment.

The species composition and size-structure of the phytoplanktoncommunity in the Boka Kotorska Bay (SE Adriatic Sea) were analysed withrespect to abundance and carbon biomass, together with the physico-chemicalparameters, with the aim of evaluating the predefined oligo-mesotrophicstatus of this transitional water ecosystem. Three stations locatedin the inner part of the Bay were sampled with seasonal frequencyin 2008/2009. Picophytoplankton cells were counted using flowcytometry; nanophytoplankton and microphytoplankton were identifiedand counted by light microscopy. The relative importance of thepicoplankton in the Bay, in terms of both abundance and biomass,during all the investigated seasons emphasized their significancein the phytoplankton community. Picocyanobacteria (Synechococcus)constituted a significant part of the summer assemblages withregard to both abundance (up to 3.38 × 108 cellsL-1)and carbon biomass (up to 73% of total phytoplankton carbon).The contribution of the nanophytoplankton was found to be generallylow (Skeletonema marinoi, dominated the microphytoplanktonfraction. S. marinoi was the most abundant in spring/winter(up to 2.86 imes 106 cells L-1) above the halocline(making a 96% contribution to the microphytoplankton). The potentiallytoxin-producing diatom Pseudo-nitzschia pseudodelicatissimawas recorded at abundances greater than 105 cells L-1, togetherwith Thalassionema frauenfeldii, as well as the dinoflagellatesProrocentrummicans and the potentially harmful P. minimum. The higher valuesof phytoplankton biomass and the dominance of phytoplankton speciesor groups with preferences for nutrient-enriched conditions appear tobe consistent with the oligo-mesotrophic status of this specific ecosystem.