Identification of the heterotrophic nanoflagellate Bilabrum latius in the southern Adriatic (Mediterranean Sea).

Heterotrophic flagellates (HF) represent an important protist group in marine ecosystem functioning. Characterised by high taxonomic diversity, identification and classification of HF is often difficult using classical methods of light microscopy (LM). Complementing LM with molecular methods, such as barcoding, enables reliable taxonomic identification of even small size nanoflagellates that share similar or unnoticeable morphological features. The order Bicosoecida is a group of heterotrophic nanoflagellates that are important part of protist plankton and benthic communities of the world oceans. Recently, on the basis of high-resolution light microscopy and barcoding, a new bicosoecid genus, Bilabrum, was described with B. latius sp. as a type species. Our study reports on identification of B. latius from co-culture with the diatom species Chaetoceros affinis isolated from fresh plankton samples collected in the southern Adriatic. This detection of the Adriatic B.latius represents first record of this species outside its up to now known and described habitat (deep-sea sediment of the South - East Atlantic Ocean) and in diatom co-culture.

A metabarcode based (species) inventory of the northern Adriatic phytoplankton.

Background: The northern Adriatic is characterised as the coldest and most productive marine area of the Mediterranean, which is due to high nutrient levels introduced by river discharges, the largest of which is the Italian Po River (at the same time also the largest freshwater input into the Mediterranean). The northern Adriatic is a very shallow marine ecosystem with ocean current patterns that result in long retention times of plankton in the area. The northern Adriatic phytoplankton biodiversity and abundance are well-studied, through many scientific and long-term monitoring reports. These datasets were based on phytoplankton morphological traits traditionally obtained with light microscopy. The most recent comprehensive eastern Adriatic phytoplankton checklist was published more than 20 years ago and is still valuable today. Since phytoplankton taxonomy and systematics are constantly being reviewed (partly also due to new molecular methods of species identification that complement classical methodologies), checklists need to be updated and complemented. Today, metabarcoding of molecular markers gains more and more importance in biodiversity research and monitoring. Here, we report the use of high throughput sequencing methods to re-examine taxonomic richness and provide updated knowledge of phytoplankton diversity in the eastern northern Adriatic to complement the standardised light microscopy method. New information: This study aimed to report an up-to-date list of the phytoplankton taxonomic richness and phylogenetic relationships in the eastern northern Adriatic, based on sequence variability of barcoding genes resolved with advanced molecular tools, namely metabarcoding. Here, metabarcoding is used to complement standardised light microscopy to advance conventional monitoring and research of phytoplankton communities for the purpose of assessing biodiversity and the status of the marine environments. Monthly two-year net sampling targeted six phytoplankton groups including Bacillariophyceae (diatoms) and Chrysophyceae (golden algae) belonging to Ochrophyta, Dinophyceae (dinoflagellates), Cryptophyceae (cryptophytes), Haptophyta (mostly coccolithophorids) and Chlorophyta with Prasinophyceae (prasinophytes) and Chlorophyceae (protist green algae). Generated sequence data were taxonomically assigned and redistributed in two kingdoms, five classes, 32 orders, 49 families and 67 genera. The most diverse group were dinoflagellates, comprising of 34 found genera (48.3%), following by diatoms with 23 (35.4%) and coccolithophorids with three genera (4.0%). In terms of genetic diversity, results were a bit different: a great majority of sequences with one nucleotide tolerance (ASVs, Amplicon sequence variants) assigned to species or genus level were dinoflagellates (83.8%), 13.7% diatoms and 1.6% Chlorophyta, respectively. Although many taxa have not been detected that have been considered as common in this area, metabarcoding revealed five diatoms and 20 dinoflagellate genera that were not reported in previous checklists, along with a few species from other targeted groups that have been reported previously. We here describe the first comprehensive 18S metabarcode inventory for the northern Adriatic Sea.

Characterisation and toxicological activity of three different Pseudo-nitzschia species from the northern Adriatic Sea (Croatia).

Diatoms of the genus Pseudo-nitzschia are cosmopolitans spread in seas and oceans worldwide, with more than 50 described species, dozens of which have been confirmed to produce domoic acid (DA). Here, we characterized and investigated the toxicological activity of secondary metabolites excreted into the growth media of different Pseudo-nitzschia species sampled at various locations in the northern Adriatic Sea (Croatia) using human blood cells under in vitro conditions. The results revealed that three investigated species of the genus Pseudo-nitzschia were capable of producing DA indicating their toxic potential. Moreover, toxicological data suggested all three Pseudo-nitzschia species can excrete toxic secondary metabolites into the surrounding media in addition to the intracellular pools of DA, raising concerns regarding their toxicity and environmental impact. In addition, all three Pseudo-nitzchia species triggered oxidative stress, one of the mechanisms of action likely responsible for the DNA damage observed in human blood cells. In line with the above stated, our results are of great interest to environmental toxicologists, the public and policy makers, especially in light of today's climate change, which favours harmful algal blooms and the growth of DA producers with a presumed negative impact on the public health of coastal residents.

Morpho-physiological adaptations of Leptocylindrus aporus and L. hargravesii to phosphate limitation in the northern Adriatic.

The northern Adriatic is highly productive and shallow area characterized by numerous spatio-temporal gradients (e.g. nutrients, salinity, temperature). It is strongly influenced by numerous freshwater inputs, mainly from Po river. Its current systems as well as Po river, generates gradients of phosphate availability with an expressed N/P imbalance and phosphate limitation. A number of recent studies characterized these gradients as major factors affecting abundance and composition of microphytoplankton communities. Focus of this study is on two Leptocylindrus species, Leptocylindrus aporus (F.W. French & Hargraves) D. Nanjappa & A. Zingone 2013 and Leptocylindrus hargravesii D. Nanjappa & A. Zingone 2013. Species belonging to Leptocylindrus genus are frequently observed and have high abundances and also high contributions to the microphytoplankton community in this area. We focused on their morphological and physiological responses to phosphate limitation in situ and also performed in vitro experiments. In this study we report data on species specific growth rates under phosphorus (P) deplete and P rich conditions, localization and characteristics of alkaline phosphate activity, phosphate uptake rates as well as their morphological differences in P deplete versus P rich conditions. Our in vitro experiments showed that both Leptocylindrus species morphologically reacted similarly to phosphorus depletion and showed significantly elongated pervalvar axis in P depleted conditions if compared to P rich conditions. Also average chain lengths increased when in P depleted conditions. Two previously mentioned adaptations indicate their tendency to increase cellular surface areas available for alkaline phosphatase. Chlorophyll fluorescence of both species significantly decreased in P depleted medium. Although both species morphologically reacted similarly, our experiment demonstrated significant differences in physiological reactions to P depleted conditions.

Insights into the life strategy of the common marine diatom Chaetoceros peruvianus Brightwell.

Chaetoceros peruvianus is a marine diatom species with circumglobal distribution. While frequently observed, it appears never to dominate the marine phytoplankton community hence it can be characterized as a rather opportunistic, generalistic species. Here we present ecological interpretations from a long-term data set on marine microphytoplankton in the northern Adriatic Sea, where the abundancies and relative contributions of C. peruvianus were observed along a set of steep ecological gradients. Limited supply of dissolved inorganic phosphate was identified as the driving ecological factor for this ecosystem. In parallel C. peruvianus was cultivated in monoclonal cultures and its morphological and physiological reaction to replete and phosphorus depleted medium was analysed. C. peruvianus reacted to phosphorus depletion by an increase in cell height and length as well as thickness and length of setae. This morphological reaction included an increase in cellular volume and calculated carbon content. Additionally, it represents the transition between two described morphological varieties, C. peruvianus and C. peruvianus var. robusta. C. peruvianus showed a significant induction of extracellular alkaline phosphatase activity if grown in phosphate depleted medium. Microscopical analysis demonstrated this activity to be located exclusively on the setae of the cells.

The Ecology of One Cosmopolitan, One Newly Introduced and One Occasionally Advected Species from the Genus Skeletonema in a Highly Structured Ecosystem, the Northern Adriatic.

The diatom genus Skeletonema is globally distributed and often an important constituent of the phytoplankton community. In the marine phytoplankton of the northern Adriatic Sea, we found three species of the genus Skeletonema: Skeletonema menzelii, Skeletonema marinoi and Skeletonema grevillei. Making use of the steep ecological gradients that characterise the northern Adriatic, along which we could observe those species, we report here on the ecological circumstances under which those species thrive and how their respective populations are globally connected. This is the first detailed ecological study for the species S. grevillei. This study is also the first report for S. grevillei for the Adriatic Sea and Mediterranean together with additional electron microscopic details on fresh in situ samples for this species. S. marinoi appears to clearly prefer strong freshwater influence and high nutrient concentrations delivered by low salinity waters. It can outcompete other diatom species and dominate microphytoplankton blooms. S. grevillei on the other hand appears to thrive in high nutrient concentrations triggered by water column mixing. It also appears to prefer higher salinity waters and coastal embayments. Genetic analysis of S. grevillei demonstrated a peculiar dissimilarity with isolates from coastal waters off Yemen, India, Oman and China. However, a closely related sequence was isolated from coastal waters off Japan. These results indicate that S. grevillei is an introduced species, possibly transported by ballast waters. S. menzelii is a sporadic visitor in the northern Adriatic, advected from rather oligotrophic middle Adriatic waters and never dominates the phytoplankton community in the northern Adriatic.