Potentially toxic diatom genus Pseudo-nitzschia in the northern Adriatic Sea: ecological, taxonomic and molecular features

Uzorci fitoplanktona na području sjevernog Jadrana sakupljani su na dvije postaje, RV001 i SJ107, od 1972. do 2009. godine. Analiza ovog tridesetsedmogodišnjeg niza podataka omogućila je značajne spoznaje o dinamici fitoplanktona, višegodišnjim ciklusima, sukcesiji vrsta, te promjenama unutar fitoplanktonske zajednice sjevernog Jadrana. Ustanovljeno je da se abundancija ukupnog fitoplanktona, dijatomeja i nanofitoplanktona značajno smanjila nakon 2000 te godine, te je došlo do promjena u sastavu vrsta i razdobljima cvjetanja. Vrste roda Pseudo nitzschia dominirale su fitoplanktonskom zajednicom tijekom čitavog istraživanog razdoblja, a opažena je promjena u sastavu vrsta unutar roda. Krajem osamdesetih godina prošlog stoljeća fitoplanktonskom zajednicom sjevernog Jadrana dominirale su vrste iz grupe P. seriata, dok se grupa P. delicatissima po prvi put pojavila u 1978. i od tada joj učestalost pojavljivanja stalno raste. Grupa P. delicatissima u razdoblju od 1993.-2010. ima učestalost pojavljivanja od 76-97 %. Posljednjih godina praćena su ekološka, morfološka i toksična svojstva roda Pseudo nitzschia, a sustavno su uzimani i uzorci za kulture koje su taksonomski i molekularno okarakterizirane. Na osnovi detaljnijih morfoloških obilježja: broja strija i fibula, strukture areola, postojanju ili izostanku središnje pore za sada je potvrđeno postojanje sedam vrsta Pseudo nitzschia u sjevernom Jadranu. Određene su tri vrste iz P. seriata grupe i to su: P. fraudulenta, P. pungens i P. subfraudulenta, te četiri vrste iz grupe P. delicatissima i to su: P. manii, P. pseudodelicatissima, P. delicatissima i P. calliantha. Rod Pseudo nitzschia specifičan je i po tome što neke vrste imaju mogućnost sinteze domoične kiseline (DK), snažnog neurotoksina. Posebna pažnja posvećena je potencijalno toksičnoj vrsti P. calliantha koja je dominirala 2007. Većina pronađenih vrsta potvrđena je i molekularnim metodama sa različitim genetičkim markerima (18S, ITS1-5, 8S-ITS2, 28S). Napravljene su analize sličnosti, filogenetske rekonstrukcije i analize populacija vrsta Pseudo nitzschia. Regija ITS1 5, 8S ITS2 omogućila je analizu populacija, te je statističkom analizom parsimonije određeno i njihovo geografsko i evolucijsko podrijetlo. Najznačajniji rezultati pokazuju da su populacije iz sjevernog Jadrana autohtone i genetički odvojene od populacija iz Sredozemlja i ostalih regija diljem svijetaPhytoplankton samples were collected at two stations, RV001 and SJ107, in the northern Adriatic Sea from 1972 to 2009. The analysis of this 37 years long data set provided significant insights in the phytoplankton dynamics, species succession and changes in the phytoplankton community. It was found that the total abundance of phytoplankton, diatoms and nanophytoplankton significantly decreased after the year 2000, and a change in the species composition and blooming periods has been observed. Species from the genus Pseudo-nitzschia were among the most abundant and most frequent diatoms during the entire investigated period. Changes in the species composition within the genus have been observed. In the 1980ies the phytoplankton community was dominated by the P. seriata group. While the group P. delicatissima first appeared in the '80s and since then their frequency continuously increased. P. delicatissima has a frequency of appearance from 76-97% in the period from 1993-2010. Morphological, ecological and toxic properties of the genus Pseudo-nitzschia were studied in the recent years in the northern Adriatic. Monoclonal cultures were established for taxonomical and molecular identification. The existence of 7 different Pseudo-nitzschia species is confirmed in the northern Adriatic on the basis of detailed morphological characteristics. Three in the P. seriata group: P. fraudulenta, P. pungens and P. subfraudulenta, and four species in P. delicatissima group: P. mannii, P. pseudodelicatissima, P. delicatissima and P. calliantha. The genus Pseudo-nitzschia is associated with the production of domoic acid (DA), a strong neurotoxin. Special attention was given to a potentially toxic P. calliantha that dominated the bloom in 2007. Most of the identified species were confirmed by molecular methods with different genetic markers (18S, ITS1-5,8S-ITS2, 28S). Similarity representation after Neighbourjoining analysis, most parsimoneous phylogenetic reconstruction and analysis of populations confirmed that populations from the Northern Adriatic are separated and genetically different from the rest of the Mediterranean and the world (ITS1-5.8S-ITS2). It could be demonstrated that the investigated Pseudo-nitzschia populations of the northern Adriatic are indigenous and genetically separated from populations in the Mediterranean and other regions of the world

Colonization of diatoms and bacteria on artificial substrates in the northeastern coastal Adriatic Sea

Every surface that is immersed in seawater becomes rapidly covered with an unavoidable biofilm. Such biofilm formation, also known as fouling, is a complex multistage process and not yet thoroughly investigated. In this study, the succession of diatoms and bacteria was investigated during a one month exposure on an artificial substrate of plexiglass (polymer of methyl methacrylate) mounted above the seafloor at a depth of 5 m. For biofilm analyses, the fouling was investigated using selective agar plates, epifluorescence, light and electronic microscopy, as well as high performance liquid chromatography (HPLC) pigment analysis. During biofilm development, the abundance of all biofilm components increased and reached maximum values after a one month exposure. In the bacterial community, heterotrophic marine bacteria were dominant and reached 1.96 ± 0.79 × 104 colony forming units (CFU) cm–2. Despite the fact that faecal coliforms and intestinal enterococci were detected in the water column, faecal coliforms were not detected in the biofi lm and intestinal enterococci appeared after one month of exposure but in the negligible number of 60 ± 10 CFU cm–2. The phototrophic component of the biofilm was dominated by diatoms and reached a concentration of 6.10 × 105 cells cm–2, which was supported by pigment analysis with fucoxanthin as dominant pigment in a concentration up to 110 ng cm–2. The diatom community was dominated by Cylindrotheca closterium and other pennate benthic diatoms. A detailed taxonomic analysis by electronic microscopy revealed 30 different taxa of diatoms. The study confirmed that a plexiglass surface in a marine environment is susceptible to biofouling within 30 days of contact. Furthermore, the colonization process sequence firstly involved bacteria and cyanobacteria, and secondly diatoms, which together formed a primary biofilm in the sea

Development of periphytic diatoms on different artificial substrates in the Eastern Adriatic Sea

The settling of diatoms as fouling organisms on a certain substrate is greatly influenced by substrate characteristics and the preferences of a diatom community and diatom species. A distinction among substrates can be made by analysing the specific abundance and composition of diatoms on different substrates. In this study, 11 different artificial substrates were exposed to a marine environment for a period of 30 days. Abundance and taxonomic composition of periphytic diatoms was determined on each of the substrates and on shoots of the marine seagrass Posidonia oceanica. The aim was to compare diatom community structure on different newly colonized surfaces. On all surfaces examined, periphytic diatoms were the pioneering organisms with differences in quantitative and qualitative composition on the different substrates. Taxonomic analysis of diatom communities on the substrates examined revealed 41 diatom taxa, with the dominant genera Cylindrotheca, Amphora, Nitzschia, Cocconeis and Navicula. Given that all the examined artificial substrates were solid materials, differences in the abundance and species composition of diatoms found between the materials point to the substrates’ physical and chemical characteristics as a major influence on the final settling of diatoms. Knowledge from investigating the settlement of fouling organisms on anthropogenic substrates can have future use in management of waste materials that end up in the marine environment

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

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.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