The SeaCoRe system for large scale kelp aquaculture: a plug-and-play, compatible, open-source system for the propagation and transport of clonal gametophyte cultures

The future of large-scale kelp aquaculture is standing at a crossroad, with the diverging paths being characterized by two fundamentally different cultivation methods that differ on how well gametophyte reproduction can be controlled. The cultivation method that does not directly control gametophyte reproduction is more widely utilized at the moment, but interest in better controlling gametophyte reproduction is growing steadily. Here, we validate a bioreactor system that overcomes a number of implementation challenges for this controlled reproductive method, expanding the possibility of clonal gametophyte cultivation outside of expensive laboratory settings. The main goals of this system include (i) the maintenance of clean gametophyte clonal cultures in non-sterile environments over prolonged periods of time, (ii) the production of large numbers of juvenile sporophytes, and (iii) effective transportation of gametophytes and sporophytes. The “SeaCoRe system” consists out of three parts that correspond to these three challenges: (1) clone-reactors, (2) a clone-inducer, and (3) a transporter. The validation of the system showed that delayed Saccharina latissima and Alaria esculenta gametophytes can grow reliably for 75 days in the clone-reactors. Initial gametophyte densities of 0.4 mg DW and 0.6 mg DW gametophtyes mL−1 were optimal for S. latissima and A. esculenta, resulting in reproductive successes of 604 and 422 sporophytes mL−1, respectively. Lastly, gametophyte transport was simulated, with high reproductive success still achieved within 19 days in ~ 20 °C environments. The SeaCoRe system helps unlock the full potential of large-scale kelp cultivation using multiannual delayed clonal.

Contribution to the current list of Nitzschia species (Bacillariophyceae) in Malaysia

Current occurrence of Nitzschia Hassall from marine and freshwater environment in Malaysia is compiled from current and previously published materials. Phytoplankton was collected using a 20 µm plankton net from waterfronts of Peninsular Malaysia and Borneo. Some Nitzschia cells were successfully isolated and established into clonal cultures. For morphology observation, organic matters of the net samples and cultures were oxidized and removed through acid treatment. Cleaned specimens were observed using light and electron microscopes. The morphology of the Nitzschia cells was described and micrographs are included. In addition to current field sampling, Nitzschia spp. records and distributions in Malaysia had also been compiled from secondary data. Sources of references whereby the Nitzschia species were listed in this article were cited accordingly. This article compiles a total of 11 Nitzschia spp. found in Malaysia including a few revisions and new records. This article could serve as an important reference for Nitzschia taxonomic and distributional study particularly in tropical region.Keywords: distribution, morphology, taxonomy, tropical

Tetraselmis jejuensis sp. nov. (Chlorodendrophyceae), a Euryhaline Microalga Found in Supralittoral Tide Pools at Jeju Island, Korea

We found the euryhaline microalga, Tetraselmis jejuensis sp. nov., which was adapted to supralittoral tide pools with salinities varying from 0.3–3.1%. Fifteen strains of T. jejuensis were isolated from Daejeong (DJ) and Yongduam (YO), and clonal cultures were established in the laboratory. Morphological characterization revealed that the cells have a compressed shape, four flagella emerging from a depression near the apex in two opposite pairs, a cup-shaped chloroplast containing one pyrenoid surrounded by starch, and eyespot regions not located near the flagellar base. T. jejuensis cells showed distinct characteristics compared to other Tetraselmis species. First, a regular subunit pattern with honeycomb-like structures was predominantly displayed on the surface in the middle of the cell body. Second, the pyrenoid was invaded by both cytoplasmic channels comprising electron-dense material separated from the cytoplasm, and two branches of small cytoplasmic channels (canaliculi) in various directions, which characterize the subgenus Tetrathele. Eyespot regions containing a large number of osmiophilic globules, packed closely together and arranged in subcircular close packing of diverse sizes, were dispersed throughout the chloroplast. In the phylogenetic analysis of small subunit (SSU) rDNA sequences, the 15 strains isolated from DJ and YO separated a newly branched clade in the Chlorodendrophyceae at the base of a clade comprising the T. carteriiformi/subcordiformis clade, T. chuii/suecica clade, and T. striata/convolutae clade. The strains in the diverging clade were considered to belong to the same species. The SSU rDNA sequences of the DJ and YO strains showed a maximum difference of 1.53% and 1.19% compared to Tetraselmis suecica (MK541745), the closest species of the family based on the phylogenetic analysis, respectively. Based on morphological, molecular, and physiological features, we suggest a new species in the genus Tetraselmis named Tetraselmis jejuensis, with the species name “jejuensis” referring to the collection site, Jeju Island, Korea.

The AcrAB-TolC Efflux Pump Impacts Persistence and Resistance Development in Stationary-Phase Escherichia coli Following Delafloxacin Treatment

Bacteria have a repertoire of strategies to overcome antibiotics in clinical use, complicating our ability to treat and cure infectious diseases. In addition to evolving resistance, bacteria within genetically clonal cultures can undergo transient phenotypic changes and tolerate high doses of antibiotics. These cells, termed persisters, exhibit heterogeneous phenotypes: the strategies that a bacterial population deploys to overcome one class of antibiotics can be distinct from those needed to survive treatment with drugs with another mode of action. It was previously reported that fluoroquinolones, which target DNA topoisomerases, retain the capacity to kill non-growing bacteria that tolerate other classes of antibiotics. Here, we show that in Escherichia coli stationary-phase cultures and colony biofilms, persisters that survive treatment with the anionic fluoroquinolone Delafloxacin depend on the AcrAB-TolC efflux pump. In contrast, we did not detect this dependence on AcrAB-TolC in E. coli persisters that survive treatment with three other fluroquinolone compounds. We found that the loss of AcrAB-TolC activity via genetic mutations or chemical inhibition not only reduces Delafloxacin persistence in non-growing E. coli MG1655 or EDL933 (an E. coli O157:H7 strain), it limits resistance development in progenies derived from Delafloxacin persisters that were given the opportunity to recover in nutritive media following antibiotic treatment. Our findings highlight the heterogeneity in defense mechanisms that persisters use to overcome different compounds within the same class of antibiotics. They further indicate that efflux pump inhibitors can potentiate the activity of Delafloxacin against stationary-phase E. coli and block resistance development in Delafloxacin persister progenies.

The effect of sodium thiosulfate in a culture medium on the sexual reproduction of three species of Bacillariophyta

It was studied the effect of sulfur in the form of sodium thiosulfate pentahydrate on the capability of clonal cultures of Pleurosigma sp., Toxarium undulatum, Pseudo-nitzschia calliantha to interbreed successfully and produce fertile offspring if they were cultivated in artificially prepared marine water (ESAW medium). Differences in the time of the beginning of the sexual process and the intensity of its passage for clones contained on ESAW with the addition of sodium thiasulfate and in the absence of it are shown.

Phosphate diester utilization by marine diazotrophs Trichodesmium erythraeum and Crocosphaera watsonii

In the phosphate-depleted oligotrophic ocean, microbes utilize various dissolved organic phosphorus (P) compounds as alternative P sources, using enzymes such as alkaline phosphatases. However, knowledge of such P acquisition mechanisms is limited, especially in association with the physiology of nitrogen-fixing organisms, which play a substantial role in marine biogeochemical cycling. We show that nonaxenic clonal cultures of 2 oceanic diazotrophs, Trichodesmium erythraeum and Crocosphaera watsonii, have the ability to utilize phosphate diester as their sole P source, using a model artificial compound—bis-p-nitrophenyl phosphate (bisNPP). Although both diazotroph cultures likely preferred phosphate monoester to diester, the expressed diesterase activity was theoretically sufficient to fulfill their P demands, and they showed significant growth in bisNPP-added media. Interestingly, a distinct difference in their growth trends was observed, with faster onset of growth by C. watsonii and delayed onset of growth by T. erythraeum. This indicates that the C. watsonii consortium can effectively and rapidly assimilate in situ diesters as alternative P sources in the field. Nonetheless, when considering the poor bisNPP utilization reported from other marine phytoplankton taxa, our results indicate that the utilization of particular diester compounds is a notable and advantageous strategy for both diazotroph consortia to alleviate P limitation in the oligotrophic ocean.

Human kidney clonal proliferation disclose lineage-restricted precursor characteristics

In-vivo single cell clonal analysis in the adult mouse kidney has previously shown lineage-restricted clonal proliferation within varying nephron segments as a mechanism responsible for cell replacement and local regeneration. To analyze ex-vivo clonal growth, we now preformed limiting dilution to generate genuine clonal cultures from one single human renal epithelial cell, which can give rise to up to 3.4 * 106 cells, and analyzed their characteristics using transcriptomics. A comparison between clonal cultures revealed restriction to either proximal or distal kidney sub-lineages with distinct cellular and molecular characteristics; rapidly amplifying de-differentiated clones and a stably proliferating cuboidal epithelial-appearing clones, respectively. Furthermore, each showed distinct molecular features including cell-cycle, epithelial-mesenchymal transition, oxidative phosphorylation, BMP signaling pathway and cell surface markers. In addition, analysis of clonal versus bulk cultures show early clones to be more quiescent, with elevated expression of renal developmental genes and overall reduction in renal identity markers, but with an overlapping expression of nephron segment identifiers and multiple identity. Thus, ex-vivo clonal growth mimics the in-vivo situation displaying lineage-restricted precursor characteristics of mature renal cells. These data suggest that for reconstruction of varying renal lineages with human adult kidney based organoid technology and kidney regeneration ex-vivo, use of multiple heterogeneous precursors is warranted.

Estimation of cell distribution heterogeneity at toxicological experiments with clonal cultures of benthic diatoms

An increase in anthropogenic pressure on coastal water areas requires regular monitoring of marine ecosystems. The appropriate bioindicators for indirect assessment of the quality of the near-shore environment are benthic diatom algae, which are a key element of coastal communities and are highly sensitive to environmental impact. Changes in the development of diatoms under the influence of various toxicants may be used as relevant tool for monitoring of marine environment quality. However, scientific and methodological approaches to application of benthic diatom algae as test objects remain unstudied. One of the important methodological problems is the assessment of the significance of the samples in experimental vessels when counting cells abundance at different stages of toxicological test. The study is focused on assessment of the statistical significance of the equality of the initial mean number of cells of clonal culture inoculum placed into each of the replicates, as well as the statistical uniformity of cell distribution over the entire bottom area of Petri dishes. We used clonal cultures of three benthic diatom species belonging to different classes of Bacillariophyta: Thalassiosira excentrica Cleve, 1903 (Coscinodiscophycea), Ardissonea crystallina (C. Agardh) Grunow, 1880 (Fragilariophyceae), and Pleurosigma aestuarii (Bréb. in Kütz.) W. Smith, 1853 (Bacillariophyceae). They significantly differ in valve morphology and life history (floating in water mass, attached to substrate, and motile). The results of statistical comparison of cell number variability in the experiment for all studied species confirmed the absence of significant differences between the mean values of the tested parameter at a standard significance level (0.05). It was shown that despite specific differences in cell growth rate during the experiment, the variability in cell number in the microscope viewing fields varies irregularly. The highest value of the variability coefficient was observed on the 5th day for the small-sized species T. excentrica (Cv = 42…55 %), and the lowest variability – for the large-cell species A. crystallina (Cv = 27…31 %). The absence of significant differences in cell number between three replicates (for each species) was established both during the initial placing of inoculum into the dishes and on the following days of the experiment. The conclusion is applicable for each of diatom species studied, which allows to consider all replicates as subsamples of the replicate sample and to average the results obtained at different stages of the toxicological experiment. The uniformity of cell distribution throughout experimental dishes bottom, which does not depend on species and absolute cell number, was statistically proven. The results obtained allow to statistically reliably estimate the changes in cell number at different stages of toxicological experiment according to replicate sampling, based on cell counting in a limited number of viewing fields.

Salinity optima for vegetative growth and sexual reproduction of the diatom Toxarium undulatum

Distribution of diatom algae is limited by their tolerance to environmental factors. Although a genus Toxarium has been evolving for more than 100 million years, it is represented by only two species. Toxarium undulatum is widely spread in tropical and subtropical seas, and it can be also found in the Black Sea, the salinity of which is twice lower than the oceanic one. Ecological and psychological characteristics research of this species is of great interest in terms of its relationship to salinity. T. undulatum clonal cultures were sampled in the Donuzlav Lake connected to the Black Sea (southwest of the Crimean Peninsula) and on Gran Canaria coast (Canary Islands archipelago). Experiments on the salinity tolerance limits showed, that the Black Sea clones were viable in a range of at least 30 ‰ (12 to 42 ‰). The same wide range of salinity tolerance with slightly higher values was observed among oceanic clones of this species. Optima of vegetative growth and sexual reproduction were determined. Optima of the Black Sea clones appeared to be 27.8 and 27.2 ‰, respectively, which was significantly higher than salinity observed in population habitat. Similar higher optima of vegetative growth and sexual reproduction, compared with those salinity values, at which natural population developed, were observed for a number of other Black Sea diatoms, which proved their oceanic (Mediterranean) origin. It was concluded that T. undulatum, along with other species, began to populate the Black Sea basin about seven thousand years ago after Mediterranean Sea water started to flow into the freshened Novoevksinsky Sea-Lake through the Bosporus Strait. However, the evolution rate did not allow bringing physiological and ecological characteristics of the species studied into full agreement with environmental conditions. Oceanic origin is evidently seen in its physiological reactions to salinity. Possibility of speciation due to settlement of the Black Sea with oceanic species is discussed.