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Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH 4 F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell ("single nuclei") genomics, transcriptomics and proteomics in different diatom species.

Diatoms are the largest group of heterokont algae with more than 100,000 species. As one of the single-celled photosynthetic organisms that inhabit marine, aquatic and terrestrial ecosystems, diatoms contribute ~ 45% of global primary production. Despite their ubiquity and environmental significance, very few diatom plastid genomes (plastomes) have been sequenced and studied. This study explored patterns of nucleotide substitution rates of diatom plastids across the entire suite of plastome protein-coding genes for 40 taxa representing the major clades. The highest substitution rate was lineage-specific within the araphid 2 taxon Astrosyne radiata and radial 2 taxon Proboscia sp. Rate heterogeneity was also evident in different functional classes and individual genes. Similar to land plants, proteins genes involved in photosynthetic metabolism have lower synonymous and nonsynonymous substitutions rates than those involved in transcription and translation. Significant positive correlations were identified between substitution rates and measures of genomic rearrangements, including indels and inversions, which is a similar result to what was found in legume plants. This work advances the understanding of the molecular evolution of diatom plastomes and provides a foundation for future studies.

The secreted mucilage trails of the diatom Navicula sp. in the process of motility were studied by scanning electron microscopy (SEM), transmission electron microscope (TEM), atomic force microscopy (AFM) and Raman spectra etc. Contrary to previous studies, force measurement was taken directly on the mucilage trails of live cells using the method of in situ force mapping by AFM. The retraction force curve presented an increased tip-substrate peak and a small saw-tooth pattern tip-mucilage peak. Especially, same measurements on various substrates with different surface energy revealed that the mucilage trails actually functioned as a medium increasing the adhesive force between the diatom and substrates, which is crucial to diatom's adhesion and locomotion. In addition, the mechanical properties of mucilage trails were quite different from mucilage strands in the maximum adhesive force and the maximum polymer extension length. Raman spectra indicated the difference in compositions that both of the two kinds of mucilages had proteins and polysaccharide, but the mucilage strands contained some other components with C=O, -CH 2 - and -CH 3 asymmetric and symmetric stretches. This research hammers out more precise information about mucilage trails which would be useful in terms of diatom motility and biofouling prevention.

Diatoms are photosynthetic microalgae, a group with a major environmental role on the planet due to the biogeochemical cycling of silica and global fixation of carbon. However, they can evolve into harmful blooms through a resourceful communication mechanism, not yet fully understood. Here, we demonstrate that a population of diatoms under darkness show quasi-periodic electrical oscillations, or intercellular waves. The origin is paracrine signaling, which is a feedback, or survival, mechanism that counteracts changes in the physicochemical environment. The intracellular messenger is related to Ca 2+ ions since spatiotemporal changes in their concentration match the characteristics of the intercellular waves. Our conclusion is supported by using a Ca 2+ channel inhibitor. The transport of Ca 2+ ions through the membrane to the extracellular medium is blocked and the intercellular waves disappear. The translation of microalgae cooperative signaling paves the way for early detection and prevention of harmful blooms and an extensive range of stress-induced alterations in the aquatic ecosystem.

The concentration of CO 2 in many aquatic systems is variable, often lower than the K M of the primary carboxylating enzyme Rubisco, and in order to photosynthesize efficiently, many algae operate a facultative CO 2 concentrating mechanism (CCM). Here we measured the responses of a marine diatom, Thalassiosira pseudonana, to high and low concentrations of CO 2 at the level of transcripts, proteins and enzyme activity. Low CO 2 caused many metabolic pathways to be remodeled. Carbon acquisition enzymes, primarily carbonic anhydrase, stress, degradation and signaling proteins were more abundant while proteins associated with nitrogen metabolism, energy production and chaperones were less abundant. A protein with similarities to the Ca 2+ / calmodulin dependent protein kinase II_association domain, having a chloroplast targeting sequence, was only present at low CO 2 . This protein might be a specific response to CO 2 limitation since a previous study showed that other stresses caused its reduction. The protein sequence was found in other marine diatoms and may play an important role in their response to low CO 2 concentration., Competing Interests: The authors declare no competing financial interests.

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.