Loggerhead Sea Turtles as Hosts of Diverse Bacterial and Fungal Communities.

Research on microbial communities associated with wild animals provides a valuable reservoir of knowledge that could be used for enhancing their rehabilitation and conservation. The loggerhead sea turtle (Caretta caretta) is a globally distributed species with its Mediterranean population categorized as least concern according to the IUCN Red List of Threatened Species as a result of robust conservation efforts. In our study, we aimed to further understand their biology in relation to their associated microorganisms. We investigated epi- and endozoic bacterial and endozoic fungal communities of cloaca, oral mucosa, carapace biofilm. Samples obtained from 18 juvenile, subadult, and adult turtles as well as 8 respective enclosures, over a 3-year period, were analysed by amplicon sequencing of 16S rRNA gene and ITS2 region of nuclear ribosomal gene. Our results reveal a trend of decreasing diversity of distal gut bacterial communities with the age of turtles. Notably, Tenacibaculum species show higher relative abundance in juveniles than in adults. Differential abundances of taxa identified as Tenacibaculum, Moraxellaceae, Cardiobacteriaceae, and Campylobacter were observed in both cloacal and oral samples in addition to having distinct microbial compositions with Halioglobus taxa present only in oral samples. Fungal communities in loggerheads' cloaca were diverse and varied significantly among individuals, differing from those of tank water. Our findings expand the known microbial diversity repertoire of loggerhead turtles, highlighting interesting taxa specific to individual body sites. This study provides a comprehensive view of the loggerhead sea turtle bacterial microbiota and marks the first report of distal gut fungal communities that contributes to establishing a baseline understanding of loggerhead sea turtle holobiont.

Phytoplankton diversity and chemotaxonomy in contrasting North Pacific ecosystems.

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.

More than just hitchhikers: a survey of bacterial communities associated with diatoms originating from sea turtles.

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.

Cultivating epizoic diatoms provides insights into the evolution and ecology of both epibionts and hosts.

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.

Characterization of oral and cloacal microbial communities of wild and rehabilitated loggerhead sea turtles (Caretta caretta).

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.

Diatom Genus Hyalosira (Rhabdonematales emend.) and Resolution of its Polyphyly in Grammatophoraceae and Rhabdonemataceae with a New Genus, Placosira, and Five New Hyalosira Species.

Hyalosira gene sequences are divided into two clades within different families. We examined authentic material of Hyalosira (isotype material of H. obtusangula, synonymous with H. delicatula) and voucher specimens of published sequences, and pooled our observations of Hyalosira-like taxa from benthic and epizoic habitats in several parts of the globe. The two molecular clades corresponded to two morphological groups, with Hyalosira obtusangula associated with Grammatophoraceae. We emend the description and provide lectotypification for Hyalosira and propose Placosira to encompass the taxa in the other clade, associated with Rhabdonemataceae. We propose that Hyalosira has uniseriate to triseriate striae, sometimes different on valve face and mantle. Copulae in most species had shallow septa, though in one they were moderately deep. All species had girdle bands bearing two rows of areolae separated by a midrib. We name five new species of Hyalosira. Morphologies of taxa in the Placosira clade were superficially similar to Hyalosira but differed in having areolae with ricae, a single row of areolae on the girdle bands, and tubular rimoportulae on the valve-face-mantle junction. Hyalosira hustedtiana Patrick should revert to its original position in Striatella until the appropriate genus can be determined. We emend Rhabdonematales to encompass Rhabdonemataceae, Grammatophoraceae and Tabellariaceae.

Detection of multi-drug resistance and AmpC ß-lactamase/extended-spectrum ß-lactamase genes in bacterial isolates of loggerhead sea turtles (Caretta caretta) from the Mediterranean Sea.

Sea turtles are useful sentinels to monitor the dissemination of antimicrobial resistance (AMR) in the marine coastal ecosystems. Forty Gram negative bacteria were isolated from wounds of 52 injured Caretta caretta, living in the Mediterranean Sea. Bacteria were identified using 16S rRNA gene sequencing and tested for susceptibility to 15 antibiotics. In addition, NGS amplicon sequencing was performed to detect the presence of AmpC ß-lactamase genes (blaAmpC) and extended-spectrum ß-lactamase (ESBL) genes (blaCTX-M,blaSHV,blaTEM). Seventy-five percent of the isolates (30/40 isolates) exhibited multidrug resistance (MDR) phenotypes and 32.5% (13/40 isolates) were confirmed to be positive for at least one gene. The variants of ESBLs genes were blaCTX-M-3,blaTEM-236 and blaSHV-12. Variants of the blaAmpCß-lactamase gene i.e., blaACT-24, blaACT-2, blaACT-17, blaDHA-4 and blaCMY-37, were also detected. In addition, 4 isolates were found simultaneously harboring CTX and AmpC genes while 2 strains harbored 3 genes (blaACT-2+TEM-236+SHV-12, and blaCTX-M-3+ACT-24+TEM-236).

A polyphasic approach to the study of the genus Nitzschia (Bacillariophyta): three new planktonic species from the Adriatic Sea.

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 and 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.

On Sea Turtle-associated Craspedostauros (Bacillariophyta), with Description of Three Novel Species.

The current study focuses on four species from the primarily marine diatom genus Craspedostauros that were observed growing attached to numerous sea turtles and sea turtle-associated barnacles from Croatia and South Africa. Three of the examined taxa, C. danayanus sp. nov., C. legouvelloanus sp. nov., and C. macewanii sp. nov., are described based on morphological and, whenever possible, molecular characteristics. The new taxa exhibit characters not previously observed in other members of the genus, such as the presence of more than two rows of cribrate areolae on the girdle bands, shallow perforated septa, and a complete reduction of the stauros. The fourth species, C. alatus, itself recently described from museum sea turtle specimens, is reported for the first time from loggerhead sea turtles rescued in Europe. A 3-gene phylogenetic analysis including DNA sequence data for three sea turtle-associated Craspedostauros species and other marine and epizoic diatom taxa indicated that Craspedostauros is monophyletic and sister to Achnanthes. This study, being based on a large number of samples and animal specimens analyzed and using different preservation and processing methods, provides new insights into the ecology and biogeography of the genus and sheds light on the level of intimacy and permanency in the host-epibiont interaction within the epizoic Craspedostauros species.

Assessment of holographic microscopy for quantifying marine particle size and concentration.

Holographic microscopy has emerged as a tool for in situ imaging of microscopic organisms and other particles in the marine environment: appealing because of the relatively larger sampling volume and simpler optical configuration compared to other imaging systems. However, its quantitative capabilities have so far remained uncertain, in part because hologram reconstruction and image recognition have required manual operation. Here, we assess the quantitative skill of our automated hologram processing pipeline (CCV Pipeline), to evaluate the size and concentration measurements of environmental and cultured assemblages of marine plankton particles, and microspheres. Over 1 million particles, ranging from 10 to 200 µm in equivalent spherical diameter, imaged by the 4-Deep HoloSea digital inline holographic microscope (DIHM) are analyzed. These measurements were collected in parallel with a FlowCam (FC), Imaging FlowCytobot (IFCB), and manual microscope identification. Once corrections for particle location and nonuniform illumination were developed and applied, the DIHM showed an underestimate in ESD of about 3% to 10%, but successfully reproduced the size spectral slope from environmental samples, and the size distribution of cultures (Dunaliella tertiolecta, Heterosigma akashiwo, and Prorocentrum micans) and microspheres. DIHM concentrations (order 1 to 1000 particles ml-1) showed a linear agreement (r 2 = 0.73) with the other instruments, but individual comparisons at times had large uncertainty. Overall, we found the DIHM and the CCV Pipeline required extensive manual correction, but once corrected, provided concentration and size estimates comparable to the other imaging systems assessed in this study. Holographic cameras are mechanically simple, autonomous, can operate at very high pressures, and provide a larger sampling volume than comparable lens-based tools. Thus, we anticipate that these characterization efforts will be rewarded with novel discovery in new oceanic environments.

Geographical variation in the diatom communities associated with loggerhead sea turtles (Caretta caretta).

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.

Winter picoplankton diversity in an oligotrophic marginal sea.

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.

Morphological diversity and phylogeny of the diatom genus Entomoneis (Bacillariophyta) in marine plankton: six new species from the Adriatic Sea.

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.

Distribution and diversity of marine picocyanobacteria community: Targeting of Prochlorococcus ecotypes in winter conditions (southern Adriatic Sea).

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 100m 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 novel type of colony formation in marine planktonic diatoms revealed by atomic force microscopy.

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.

Phytoplankton distribution across the southeast Adriatic continental and shelf slope to the west of Albania (spring aspect).

We present the first insight to the oceanography of the southeastern Adriatic Sea, where coastal water influenced by Albanian rivers comes into contact with the inflowing oligotrophic Eastern Adriatic Current (Ionian Surface Water and Levantine Intermediate Water). A distinct plankton distribution was observed on each side of the shelf break hydrographic boundary in May 2009, during gradual warming of the surface waters. The prochlorophytes accumulated along the nutricline above the shelf and continental slope. The phosphorus limited inshore waters were dominated by a small diatom Chaetoceros circinalis, dinoflagellates, cryptophytes, autotrophic picoplankton, and heterotrophic nanoplankton. The offshore surface layer was characterized by bigger nanoplankton (coccolithophorids, green flagellates). Low nutrient concentrations influence relatively low productivity not only above the Albanian shelf but also further to the north along the Montenegrine and Croatian coastal Adriatic Sea.