Martin Pfannkuchen

AFM imaging of extracellular polymer release by marine diatom Cylindrotheca closterium (Ehrenberg) Reiman & J.C. Lewin.

Extracellular polysaccharide production by marine diatoms is a significant route by which photosynthetically produced organic carbon enters the trophic web and may influence the physical environment in the sea. This study highlights the capacity of atomic force microscopy (AFM) for investigating diatom extracellular polysaccharides with a subnanometer resolution. Here we address a ubiquitous marine diatom Cylindrotheca closterium, isolated from the northern Adriatic Sea, and its extracellular polymeric substance (EPS) at a single cell level. We applied a simple procedure for AFM imaging of diatom cells on mica under ambient conditions (in air) to achieve visualization of their EPS with molecular resolution. The EPS represents a web of polysaccharide fibrils with two types of cross‐linking: fibrils association forming junction zones and fibril–globule interconnections with globules connecting two or more fibrils. The fibril heights were 0.4–2.6 nm while globules height was in the range of 3–12 nm. Polymer networks of native gel samples from the Northern Adriatic and the network formed by polysaccharides extracted from the C. closterium culture share the same features regarding the fibril heights, pore openings and the mode of fibril association, proving that the macroscopic gel phase in the Northern Adriatic can be formed directly by the self‐assembly of diatom released polysaccharide fibrils. Copyright

Candidatus Paraholospora nucleivisitans, an intracellular bacterium in Paramecium sexaurelia shuttles between the cytoplasm and the nucleus of its host.

An intracellular bacterium was discovered in two isolates of Paramecium sexaurelia from an aquarium with tropical fish in Münster (Germany) and from a pond in the Wilhelma zoological-botanical garden, Stuttgart (Germany). The bacteria were regularly observed in the cytoplasm of the host, but on some occasions they were found in the macronucleus of the host cell. In these cases, only a few, if any, bacteria were observed remaining in the cytoplasm. The bacterium was not infectious to P. sexaurelia or other species of Paramecium and appeared to be an obligate intracellular bacterium, while bacteria-free host cells were completely viable. The fluorescence in situ hybridisation (FISH) and comparative 16SrDNA sequence analyses showed that the bacterium belonged to a new genus, and was most closely, yet quite distantly, related to Holospora obtusa. In spite of this relationship, the new bacteria differed from Holospora by at least two biological features. Whereas all Holospora species reside exclusively in the nuclei of various species of Paramecium and show a life cycle with a morphologically distinct infectious form, for the new bacterium no infectious form and no life cycle have been observed. For the new bacterium, the name Candidatus Paraholospora nucleivisitans is suggested. The host P. sexaurelia is usually known from tropical and subtropical areas and is not a species typically found in Germany and central Europe. Possibly, it had been taken to Germany with fish or plants from tropical or subtropical waters. Candidatus Paraholospora nucleivisitans may therefore be regarded as an intracellular neobacterium for Germany.

Depth-Related Alkaloid Variation in Mediterranean Aplysina Sponges

Total amounts and patterns of bromoisoxazoline alkaloids of Aplysina sponges from Croatia (Mediterranean Sea) were analyzed along an underwater slope ranging from 1.8 to 38.5 m. Total amounts of alkaloids varied from sample to sample and showed no correlation with depth. In contrast, striking differences of alkaloid patterns were found between sponges from shallow sites (1.8 - 11.8 m) and those collected from deeper sites (11.8 - 38.5 m). Sponges from shallow depths consistently exhibited alkaloid patterns typical for Aplysina aerophoba with aerophobin-2 (2) and isofistularin-3 (3) as main constituents. Sponges from deeper sites (below 11.8 m) resembled Aplysina cavernicola with aerothionin (4) and aplysinamisin- 1 (1) as major compounds. The typical A. cavernicola pigment 3,4-dihydroxyquinoline- 2-carboxylic acid (6), however, could not be detected in A. aerophoba sponges but was replaced by the A. aerophoba pigment uranidine (5) which appeared to be present in all sponge samples analyzed. During transplantation experiments sponges from sites below 30 m featuring the A. cavernicola chemotype of bromoisoxazoline alkaloids were translocated to shallower habitats (10 m). The alkaloid patterns in transplanted sponges were found to be stable over a period of 12 months and unaffected by this change in depth. In a further experiment, clones of Aplysina sponges from shallow depths of 5 - 6 m resembling the A. aerophoba chemotype were either kept in situ under natural light conditions or artificially shaded by excluding photosynthetically active radiation (PAR). Neither 4 nor 1 were detected in artificially shaded sponges over an observation period of 12 months. In summary, two chemically distinct types of Aplysina sponges were discovered in this study that proved to be remarkably stable with regard to the bromoisoxazoline patterns and unaffected either by changing the light conditions or depth. It is not clear presently whether the Aplysina sponges collected from depths < 11.8 m represent a new chemotype of A. cavernicola lacking the pigment 6 or whether we have incidentally come across a so far undescribed species of the genus Aplysina.

Diversity, occurrence, and habitats of the diatom genus Bacteriastrum (Bacillariophyta) in the northern Adriatic Sea, with the description of B. jadranum sp. nov.

Abstract Ongoing phytoplankton monitoring programs over the last 12 years in the northern Adriatic Sea showed that the diatom genus Bacteriastrum is an important component of the pelagic diatom assemblage. The main goals of this study were to identify which Bacteriastrum species occur in the northern Adriatic Sea and describe their distributions. Bacteriastrum comosum and B. hyalinum f. princeps were rare, while B. delicatulum, B. hyalinum, and B. mediterraneum were frequently present, together with the newly proposed species B. jadranum, described herein. This new species is weakly silicified and is mainly characterized by a large aperture between each cell of the filament. Cells are connected to each other by 8–12 setae, which cross at a distance equivalent to the diameters of 2–3 cells. There is no difference in the orientation of the setae on the two terminal cells, hence the species is placed in the section Isomorpha. The new species was recorded throughout the study area, in both coastal and offshore waters, with higher cell abundances in the vicinity of the Po River during the September–October period. The maximum cell abundance of B. jadranum was 244.2×103cells l-1in the surface waters at 44.75666667ºN, 12.75ºE in September 2006, sometimes representing up to 46% of the entire phytoplankton assemblage, as recorded in the surface waters at 45.06833333ºN, 13.515ºE in October 2008.

Integrative taxonomy of four Clathrina species of the Adriatic Sea, with the first formal description of Clathrina rubra Sarà, 1958

The taxonomy of the class Calcarea has long relied on histological and morphological characters, with a few molecular studies done. In character-poor genera, such as Clathrina, an integrative taxonomy, associating morphological and molecular tools, greatly aids in the species identification. In this study, we describe four Clathrina species from the northern Adriatic Sea using morphology and DNA sequences together with observations on their ecology. Clathrina clathrus and Clathrina blanca have been previously reported for the Adriatic Sea; however, it is the first time that a DNA sequence of C. blanca has been obtained. Clathrina cf. hondurensis, a Caribbean species, is being reported in the Adriatic Sea for the first time, and we present the first description and DNA sequence of Clathrina rubra, a red species originally mentioned from Naples.

Heterologous expression of DsRed2 in young sponges (Porifera).

Sponges (Porifera) are currently considered to be the first branch off the Urmetazoa, common ancestors of all multicellular animals or metazoa. Research in the field of the developmental biology of sponges was restricted to morphological observations. Nowadays, research is mainly concentrated on larval development, primarily dealing with tissue formation. Already since 1907, methods for developing functional sponges from stem cells have been at hand. Functional freshwater sponges can be grown from stem cell populations originating from gemmulae. A number of poriferan sequences with high similarity to regulative genes in higher metazoa have already been found. We have now succeeded in heterologously expressing the red fluorescent protein DsRedN1 under the control of the cytomegalovirus promoter in young specimens of the freshwater sponge Spongilla lacustris. The protein folded correctly, polymerized and subsequently was detected by fluorescence microscopy. Reporting this expression system, we now consider this appealing system for early meatazoan development to be ready for molecular developmental biology and functional genetics research.

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

Abstract 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 biofilm 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 co lonization process sequence firstly involved bacteria and cyanobacteria, and secondly diatoms, which together formed a primary biofilm in the sea.

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.

Characterizing an Anoxic Habitat: Sulfur Bacteria in a Meromictic Alpine Lake

Although a relatively high diversity of eukaryotic organisms exists in anoxic habitats, these extreme environments are mainly characterized by their prokaryotic communities. The meromictic alpine Lake Alat in Bavaria, Germany exhibits a stable chemocline throughout the year. In contrast to many other freshwater habitats, the purple sulfur bacteria bloom is a thick, well-defined layer. The different bacterial assemblages involved in the sulfur cycle create a stable and permanent environment. Chemical and thermal stratification make this lake a very unique lentic habitat. The thermo- and the chemocline were described by measurements of physical parameters, such as temperature, oxygen, and conductivity, as well as the chemobiological measurements of particulate organic carbon and nitrogen concentrations. With the analyses of environmental DNA samples, we studied the bacterial diversity within this thermocline. Universal bacterial 16S rDNA as well as specific Chromatiaceae 16S rDNA PCR were cloned. Analyses showed that almost all randomly chosen clones were among γ-proteobacteria more specifically; sequences are most similar to the family Chromatiaceae, but few δ-proteobacteria occur.

Multigene phylogeny and morphology of newly isolated strain of Pseudo-nitzschia mannii Amato & Montresor (Adriatic Sea)

An increasing number of cryptic and pseudo-cryptic species have been found within many newly described diatom species. To resolve the phylogenetic relationships of the genus Pseudo-nitzschia, molecular markers are being widely used in combination (or separately) with different morphological characters. Sequence analysis of ribosomal DNA markers (18S, ITS and 28S) and morphological analyses of Pseudo-nitzschia mannii strain (CIM_D-4), isolated from the Telašćica Bay (Adriatic Sea), differentiate it from all other currently reported strains of this species.