Altering the Sex Pheromone Cyclo(l-Pro-l-Pro) of the Diatom Seminavis robusta towards a Chemical Probe.

As a major group of algae, diatoms are responsible for a substantial part of the primary production on the planet. Pennate diatoms have a predominantly benthic lifestyle and are the most species-rich diatom group, with members of the raphid clades being motile and generally having heterothallic sexual reproduction. It was recently shown that the model species Seminavis robusta uses multiple sexual cues during mating, including cyclo(l-Pro-l-Pro) as an attraction pheromone. Elaboration of the pheromone-detection system is a key aspect in elucidating pennate diatom life-cycle regulation that could yield novel fundamental insights into diatom speciation. This study reports the synthesis and bio-evaluation of seven novel pheromone analogs containing small structural alterations to the cyclo(l-Pro-l-Pro) pheromone. Toxicity, attraction, and interference assays were applied to assess their potential activity as a pheromone. Most of our analogs show a moderate-to-good bioactivity and low-to-no phytotoxicity. The pheromone activity of azide- and diazirine-containing analogs was unaffected and induced a similar mating behavior as the natural pheromone. These results demonstrate that the introduction of confined structural modifications can be used to develop a chemical probe based on the diazirine- and/or azide-containing analogs to study the pheromone-detection system of S. robusta.

News from the Western Front: Guidelines for the excavation of casualties of the First and Second World War (Flanders, Belgium).

In cooperation with many other actors, the Flanders Heritage Agency (Flemish Government) set up a guideline with procedures how to act when human remains are found in the soil of Flanders (the northern part of Belgium). The basic principle is that human remains with an archaeological value always must be excavated by archaeologists and physical anthropologists, according to the Flemish legislation on archaeology. This also applies for casualties of the First and Second World War: excavating using archaeological techniques does not conflict at all with the principles of the Geneva Convention, on the contrary: this way of working guarantees a qualitative research that increases the chance of identification and delivers more details about the battle and war: a win-win.

Mating type specific transcriptomic response to sex inducing pheromone in the pennate diatom Seminavis robusta.

Sexual reproduction is a fundamental phase in the life cycle of most diatoms. Despite its role as a source of genetic variation, it is rarely reported in natural circumstances and its molecular foundations remain largely unknown. Here, we integrate independent transcriptomic datasets to prioritize genes responding to sex inducing pheromones (SIPs) in the pennate diatom Seminavis robusta. We observe marked gene expression changes associated with SIP treatment in both mating types, including an inhibition of S phase progression, chloroplast division, mitosis, and cell wall formation. Meanwhile, meiotic genes are upregulated in response to SIP, including a sexually induced diatom specific cyclin. Our data further suggest an important role for reactive oxygen species, energy metabolism, and cGMP signaling during the early stages of sexual reproduction. In addition, we identify several genes with a mating type specific response to SIP, and link their expression pattern with physiological specialization, such as the production of the attraction pheromone diproline in mating type - (MT-) and mate-searching behavior in mating type + (MT+). Combined, our results provide a model for early sexual reproduction in pennate diatoms and significantly expand the suite of target genes to detect sexual reproduction events in natural diatom populations.

Author Correction: The Seminavis robusta genome provides insights into the evolutionary adaptations of benthic diatoms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Seminavis robusta genome provides insights into the evolutionary adaptations of benthic diatoms.

Benthic diatoms are the main primary producers in shallow freshwater and coastal environments, fulfilling important ecological functions such as nutrient cycling and sediment stabilization. However, little is known about their evolutionary adaptations to these highly structured but heterogeneous environments. Here, we report a reference genome for the marine biofilm-forming diatom Seminavis robusta, showing that gene family expansions are responsible for a quarter of all 36,254 protein-coding genes. Tandem duplications play a key role in extending the repertoire of specific gene functions, including light and oxygen sensing, which are probably central for its adaptation to benthic habitats. Genes differentially expressed during interactions with bacteria are strongly conserved in other benthic diatoms while many species-specific genes are strongly upregulated during sexual reproduction. Combined with re-sequencing data from 48 strains, our results offer insights into the genetic diversity and gene functions in benthic diatoms.

Distinctive Growth and Transcriptional Changes of the Diatom Seminavis robusta in Response to Quorum Sensing Related Compounds.

In aquatic habitats, diatoms are frequently found in association with Proteobacteria, many members of which employ cell-to-cell communication via N-acyl homoserine lactones (AHLs). It has been suggested that diatoms could distinguish between beneficial and algicidal bacteria in their surroundings by sensing AHLs. Although some microalgae can interfere with AHL signaling, e.g., by releasing AHL mimics or degrading them, molecular responses to AHLs in microalgae are still unclear. Therefore, we tested the effects of short-chained AHLs, i.e., N-hexanoyl homoserine lactone (C6-HSL), N-3-hydroxyhexanoyl homoserine lactone (OH-C6-HSL), and N-3-oxohexanoyl homoserine lactone (oxo-C6-HSL) and long-chained AHLs, i.e., N-tetradecanoyl homoserine lactone (C14-HSL), N-3-hydroxytetradecanoyl homoserine lactone (OH-C14-HSL), and N-3-oxotetradecanoyl homoserine lactone (oxo-C14-HSL), on growth of the benthic diatom Seminavis robusta. All tested short-chained AHLs did not affect diatom growth, while long-chained AHLs promoted (C14-HSL) or inhibited (OH-C14-HSL and oxo-C14-HSL) growth. To investigate the physiological effects of these long-chained AHLs in more detail, an RNA-seq experiment was performed during which S. robusta was treated with the growth-promoting C14-HSL and the growth-inhibiting oxo-C14-HSL. One tetramic acid was also tested (TA14), a structural rearrangement product of oxo-C14-HSL, which also induced growth inhibition in S. robusta. After 3 days of treatment, analysis revealed that 3,410 genes were differentially expressed in response to at least one of the compounds. In the treatment with the growth-promoting C14-HSL many genes involved in intracellular signaling were upregulated. On the other hand, exposure to growth-inhibiting oxo-C14-HSL and TA14 triggered a switch in lipid metabolism towards increased fatty acid degradation. In addition, oxo-C14-HSL led to downregulation of cell cycle genes, which is in agreement with the stagnation of cell growth in this treatment. Combined, our results indicate that bacterial signaling molecules with high structural similarity induce contrasting physiological responses in S. robusta.

Associated Bacteria Affect Sexual Reproduction by Altering Gene Expression and Metabolic Processes in a Biofilm Inhabiting Diatom.

Diatoms are unicellular algae with a fundamental role in global biogeochemical cycles as major primary producers at the base of aquatic food webs. In recent years, chemical communication between diatoms and associated bacteria has emerged as a key factor in diatom ecology, spurred by conceptual and technological advancements to study the mechanisms underlying these interactions. Here, we use a combination of physiological, transcriptomic, and metabolomic approaches to study the influence of naturally co-existing bacteria, Maribacter sp. and Roseovarius sp., on the sexual reproduction of the biofilm inhabiting marine pennate diatom Seminavis robusta. While Maribacter sp. severely reduces the reproductive success of S. robusta cultures, Roseovarius sp. slightly enhances it. Contrary to our expectation, we demonstrate that the effect of the bacterial exudates is not caused by altered cell-cycle regulation prior to the switch to meiosis. Instead, Maribacter sp. exudates cause a reduced production of diproline, the sexual attraction pheromone of S. robusta. Transcriptomic analyses show that this is likely an indirect consequence of altered intracellular metabolic fluxes in the diatom, especially those related to amino acid biosynthesis, oxidative stress response, and biosynthesis of defense molecules. This study provides the first insights into the influence of bacteria on diatom sexual reproduction and adds a new dimension to the complexity of a still understudied phenomenon in natural diatom populations.

Author Correction: Attraction Pheromone of The Benthic Diatom Seminavis robusta: Studies on Structure-Activity Relationships.

The original version of this article unfortunately contained a mistake. The chemical structure of compound 6 in Fig. 1 was incorrect. The tested compound 6 in this study was (3S,8aS)-3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione as shown in the corrected version of Fig. 1 here.

Incomplete Reproductive Isolation Between Genetically Distinct Sympatric Clades of the Pennate Model Diatom Seminavis robusta.

Incomplete reproductive isolation between genetically distinct taxa provides an interesting opportunity for speciation and adaptation studies. This phenomenon is well-described in macro-organisms, but less experimental evidence is available for unicellular eukaryotes. Here, we document the sympatric occurrence of genetically differentiated populations of the pennate model diatom Seminavis robusta in coastal subtidal biofilm communities and show widespread potential for gene flow between them. Based on sequence variation in the plastid-encoded rbcL gene, three distinct clades were identified. Morphological variation between the clades reflected their phylogenetic relationships, with subtle differences in valve morphology in the most distant clade compared to the other two clades, which were indistinguishable. Using a large number of experimental crosses we showed that, although reproductive output was significantly lower compared to the majority of within-clade crosses, approximately 34.5% of the inter-clade crosses resulted in viable and fertile progeny. While the nature of the incomplete reproductive isolation remains unknown, its occurrence in natural diatom populations represents an additional mechanism contributing to population genetic structuring and adaptation and can spur further research into the mechanisms of species divergence and the maintenance of species identity in the presence of gene flow.

Attraction Pheromone of The Benthic Diatom Seminavis robusta: Studies on Structure-Activity Relationships.

Recently the first pheromone of a marine diatom was identified to be the diketopiperazine (S,S)-diproline. This compound facilitates attraction between mating partners in the benthic diatom Seminavis robusta. Interestingly, sexualized S. robusta cells are attracted to both the natural pheromone (S,S)-diproline as well as to its enantiomer (R,R)-diproline. Usually stereospecificity is a prerequisite for successful substrate-receptor interactions, and especially pheromone perception is often highly enantioselective. Here we introduce a structure-activity relationship study, to learn more about the principles of pheromone reception in diatoms. We analyzed the activity of nine different diketopiperazines in attraction and interference assays. The pheromone diproline itself, as well as a pipecolic acid derived diketopiperazine with two expanded aliphatic ring systems, showed the highest attractivity. Hydroxylatoin of the aliphatic rings abolished any bioactivity. Diketopiperazines derived from acyclic amino acids were not attrative as well. All stereoisomers of both the diproline and the pipecolic acid derived diketopiperazine were purified by enantioselective high-performance liquid chromatography, and application in bioactivity tests confirmed that attraction pheromone perception in this diatom is indeed not stereospecific. However, the lack of activity of diketopiperazines derived from acyclic amino acids suggests a specificity that prevents misguidance to sources of other naturally occurring diketopiperazines.

Expanding the toolbox for cryopreservation of marine and freshwater diatoms.

Diatoms constitute the most diverse group of microalgae and have long been recognised for their large biotechnological potential. In the wake of growing research interest in new model species and development of commercial applications, there is a pressing need for long-term preservation of diatom strains. While cryopreservation using dimethylsulfoxide (DMSO) as a cryoprotective agent is the preferred method for long-term strain preservation, many diatom species cannot be successfully cryopreserved using DMSO. Therefore, in this study, we studied cryopreservation success in six different diatom species, representing the major morphological and ecological diatom groups, using a range of DMSO concentrations and Plant Vitrification Solution 2 (PVS2) as an alternative cryoprotectant to DMSO. In addition, we tested whether suppressing bacterial growth by antibiotics accelerates the post-thaw recovery process. Our results show that the effects of cryoprotectant choice, its concentration and the addition of antibiotics are highly species specific. In addition, we showed that PVS2 and antibiotics are useful agents to optimize cryopreservation of algae that cannot survive the traditional cryopreservation protocol using DMSO. We conclude that a species-specific approach will remain necessary to develop protocols for diatom cryopreservation and to increase their representation in public culture collections.

A sex-inducing pheromone triggers cell cycle arrest and mate attraction in the diatom Seminavis robusta.

Although sexual reproduction is believed to play a major role in the high diversification rates and species richness of diatoms, a mechanistic understanding of diatom life cycle control is virtually lacking. Diatom sexual signalling is controlled by a complex, yet largely unknown, pheromone system. Here, a sex-inducing pheromone (SIP(+)) of the benthic pennate diatom Seminavis robusta was identified by comparative metabolomics, subsequently purified, and physicochemically characterized. Transcriptome analysis revealed that SIP(+) triggers the switch from mitosis-to-meiosis in the opposing mating type, coupled with the transcriptional induction of proline biosynthesis genes, and the release of the proline-derived attraction pheromone. The induction of cell cycle arrest by a pheromone, chemically distinct from the one used to attract the opposite mating type, highlights the existence of a sophisticated mechanism to increase chances of mate finding, while keeping the metabolic losses associated with the release of an attraction pheromone to a minimum.