Three Novel Bacteria Associated with Two Centric Diatom Species from the Mediterranean Sea, Thalassiosira rotula and Skeletonema marinoi

Diatoms are a successful group of microalgae at the base of the marine food web. For hundreds of millions of years, they have shared common habitats with bacteria, which favored the onset of interactions at different levels, potentially driving the synthesis of biologically active molecules. To unveil their presence, we sequenced the genomes of bacteria associated with the centric diatom Thalassiosira rotula from the Gulf of Naples. Annotation of the metagenome and its analysis allowed the reconstruction of three bacterial genomes that belong to currently undescribed species. Their investigation showed the existence of novel gene clusters coding for new polyketide molecules, antibiotics, antibiotic-resistance genes and an ectoine production pathway. Real-time PCR was used to investigate the association of these bacteria with three different diatom clones and revealed their preference for T. rotula FE80 and Skeletonema marinoi FE7, but not S. marinoi FE60 from the North Adriatic Sea. Additionally, we demonstrate that although all three bacteria could be detected in the culture supernatant (free-living), their number is up to 45 times higher in the cell associated fraction, suggesting a close association between these bacteria and their host. We demonstrate that axenic cultures of T. rotula are unable to grow in medium with low salinity (<28 ppt NaCl) whereas xenic cultures can tolerate up to 40 ppt NaCl with concomitant ectoine production, likely by the associated bacteria.

Integrated RNA-seq and Proteomic Studies Reveal Resource Reallocation towards Energy Metabolism and Defense in Skeletonema marinoi in Response to CO2 Increase

Rising atmospheric CO2 concentrations are causing ocean acidification (OA) with significant consequences for marine organisms. Because CO2 is essential for photosynthesis, the effect of elevated CO2 on phytoplankton is more complex and the mechanism is poorly understood. Here we applied RNA-seq and iTRAQ proteomics to investigate the impacts of CO2 increase (from ∼400 to 1000 ppm) on the temperate coastal marine diatom Skeletonema marinoi. We identified 32,389 differentially expressed genes (DEGs) and 1,826 differentially expressed proteins (DEPs) from elevated CO2 conditions, accounting for 48.5% of total genes and 25.9% of total proteins we detected, respectively. Elevated pCO2 significantly inhibited the growth of S. marinoi, and the ‘omic’ data suggested that this might be due to compromised photosynthesis in the chloroplast and raised mitochondrial energy metabolism. Furthermore, many genes/proteins associated with nitrogen metabolism, transcriptional regulation, and translational regulation were markedly up-regulated, suggesting enhanced protein synthesis. In addition, S. marinoi exhibited higher capacity of ROS production and resistance to oxidative stress. Overall, elevated pCO2 seems to repress photosynthesis and growth of S. marinoi, and through massive gene expression reconfiguration induce cells to increase investment in protein synthesis, energy metabolism and antioxidative stress defense, likely to maintain pH homeostasis and population survival. This survival strategy may deprive this usually dominant diatom in temperate coastal waters of its competitive advantages in acidified environments.Importance Rising atmospheric CO2 concentrations are causing ocean acidification with significant consequences for marine organisms. Chain-forming centric diatoms of Skeletonema is one of the most successful groups of eukaryotic primary producers with widespread geographic distribution. Among the recognized 28 species, S. marinoi can be a useful model for investigating the ecological, genetic, physiological, and biochemical characteristics of diatoms in temperate coastal regions. In this study, we found that the elevated pCO2 seems to repress photosynthesis and growth of S. marinoi, and through massive gene expression reconfiguration induce cells to increase investment in protein synthesis, energy metabolism and antioxidative stress defense, likely to maintain pH homeostasis and population survival. This survival strategy may deprive this usually dominant diatom in temperate coastal waters of its competitive advantages in acidified environments.

Insights into the Light Response of Skeletonema marinoi: Involvement of Ovothiol

Diatoms are one of the most widespread groups of microalgae on Earth. They possess extraordinary metabolic capabilities, including a great ability to adapt to different light conditions. Recently, we have discovered that the diatom Skeletonema marinoi produces the natural antioxidant ovothiol B, until then identified only in clams. In this study, we investigated the light-dependent modulation of ovothiol biosynthesis in S. marinoi. Diatoms were exposed to different light conditions, ranging from prolonged darkness to low or high light, also differing in the velocity of intensity increase (sinusoidal versus square-wave distribution). The expression of the gene encoding the key ovothiol biosynthetic enzyme, ovoA, was upregulated by high sinusoidal light mimicking natural conditions. Under this situation higher levels of reactive oxygen species and nitric oxide as well as ovothiol and glutathione increase were detected. No ovoA modulation was observed under prolonged darkness nor low sinusoidal light. Unnatural conditions such as continuous square-wave light induced a very high oxidative stress leading to a drop in cell growth, without enhancing ovoA gene expression. Only one of the inducible forms of nitric oxide synthase, nos2, was upregulated by light with consequent production of NO under sinusoidal light and darkness conditions. Our data suggest that ovothiol biosynthesis is triggered by a combined light stress caused by natural distribution and increased photon flux density, with no influence from the daily light dose. These results open new perspectives for the biotechnological production of ovothiols, which are receiving a great interest for their biological activities in human model systems.

De Novo Transcriptome Assembly and Gene Expression Profiling of the Copepod Calanus helgolandicus Feeding on the PUA-Producing Diatom Skeletonema marinoi

Diatoms are the dominant component of the marine phytoplankton. Several diatoms produce secondary metabolites, namely oxylipins, with teratogenic effects on their main predators, crustacean copepods. Our study reports the de novo assembled transcriptome of the calanoid copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi. Differential expression analysis was also performed between copepod females exposed to the diatom and the control flagellate Prorocentrum minimum, which does not produce oxylipins. Our results showed that transcripts involved in carbohydrate, amino acid, folate and methionine metabolism, embryogenesis, and response to stimulus were differentially expressed in the two conditions. Expression of 27 selected genes belonging to these functional categories was also analyzed by RT-qPCR in C. helgolandicus females exposed to a mixed solution of the oxylipins heptadienal and octadienal at the concentration of 10 µM, 15 µM, and 20 µM. The results confirmed differential expression analysis, with up-regulation of genes involved in stress response and down-regulation of genes associated with folate and methionine metabolism, embryogenesis, and signaling. Overall, we offer new insights on the mechanism of action of oxylipins on maternally-induced embryo abnormality. Our results may also help identify biomarker genes associated with diatom-related reproductive failure in the natural copepod population at sea.

Arctic marine ice nucleating aerosol: a laboratory study of microlayer samples and algal cultures

In recent years, sea spray and the biological material it contains has received increased attention as a source of ice nucleating particles (INPs). Such INPs may play a role in remote marine regions, where other sources of INPs are scarce or absent. Marine aerosol is of diverse nature, so identifying sources of INPs is challenging. One fraction of marine bioaerosol, phytoplankton and their exudates, has been a particular focus of marine INP research. In our study we attempt to address three main questions. Firstly, we compare the ice nucleating ability of two common phytoplankton species with Arctic seawater microlayer samples using the same instrumentation to see if these phytoplankton species produce ice nucleating material with sufficient activity to account for the ice nucleation observed in Arctic microlayer samples. We present first measurements of the ice nucleating ability of two predominant phytoplankton species, Melosira arctica, a common Arctic diatom species and Skeletonema marinoi, a ubiquitous diatom species across oceans worldwide. To determine the potential effect of nutrient conditions and characteristics of the algal culture, such as the amount of organic carbon associated with algal cells, on the ice nucleation activity, the Skeletonema marinoi was grown under different nutrient regimes. From comparison of the ice nucleation data of the algal cultures to those obtained from a range of sea surface microlayer (SML) samples obtained during three different field expeditions to the Arctic (ACCACIA, NETCARE, ASCOS) we found that although these diatoms do produce ice nucleating material, they were not as ice active as the investigated microlayer samples. Secondly, to improve our understanding of local Arctic marine sources as atmospheric INP we applied several aerosolisation techniques to analyse the ice nucleating ability of aerosolised microlayer and algae samples. The aerosols were generated either by direct nebulisation of the undiluted bulk solutions, or by the addition of the samples to a sea spray simulation chamber filled with artificial seawater. The latter method generates aerosol particles using a plunging jet to mimic the process of oceanic wave-breaking. We observed that the aerosols produced using this approach can be ice active indicating that the ice nucleating material in seawater can indeed transfer to the aerosol phase. Thirdly, we attempted to measure ice nucleation activity across the entire temperature range relevant for mixed-phase clouds using a suite of ice nucleation measurement techniques- an expansion cloud chamber, a continuous flow diffusion chamber, and a cold stage. In order to compare the measurements made using the different instruments, we have normalised the data in relation to the mass of salt present in the nascent sea spray aerosol. At temperatures above 248 K some of the SML samples were very effective at nucleating ice, but there was substantial variability between the different samples. In contrast, there was much less variability between samples below 248 K.