Unialgal Cultures Research Articles

Plocamiomonas psychrophila gen. et sp. nov. (Pelagophyceae, Heterokontophyta), an Arctic marine nanoflagellate characterized by microscopy, pigments and molecular phylogeny

ABSTRACT During a field campaign in Baffin Bay (June, 1998), a sample dominated by small yellow brown biflagellates was collected from a small pocket of liquid water on the sea ice and established into a unialgal culture. Later it was given strain number CCMP2097 and deposited at the NCMA collection. Growth experiments over a range of temperatures and salinities indicated adaptation to variable Arctic conditions. This strain was studied here using light and transmission electron microscopy, HPLC for characterization of photosynthetic pigments and the relationship to related taxa was elucidated from molecular phylogeny. This integrative approach resulted in suggesting Plocamiomonas psychrophila gen. et sp. nov. the first systematically named Arctic species of the Pelagophyceae. Plocamiomonas had two unequally long flagella and the spherical cells measured 7.5 µm in diameter. Novel morphological characters included (1) a swelling on the mature flagellum, (2) a bi-layered theca, thin elongated arm-like structures emanating from the cell surface and (3) a thin filament above the flagellar transition region. This set of morphological characters represented a novel combination for the Pelagophyceae and together with evidence from the phylogenetic position of Plocamiomonas and eight uncultured cold-water pelagophytes supported the proposal of a new order Plocamiomonadales and a new family Plocamiomonadaceae.

Morphological and physiological responses of the cosmopolitan marine diatom Thalassiosira rotula to acidification

We investigated the effect of decreased pH on the morphology and nutrient physiology of the cosmopolitan marine diatom Thalassiosira rotula (CCMP3362) by acclimating unialgal cultures to two different CO2 gas concentrations under optimal light, temperature and nutrient conditions. At lower pH (higher CO2), T. rotula exhibited a reduction in cell diameter (7%), surface area (13%), and volume (20%), and an increase in surface area-to-volume ratio (7%). All measures of silicification in T. rotula, i.e., silica (SiO2) quota, rate of silicic acid (Si(OH)4) utilization, and elemental ratios of SiO2:C and SiO2:N, remained unchanged. Similarly, carbon (C) and nitrogen (N) quotas, ratios and utilization rates were mostly unaffected by pH. In contrast, the utilization rate of nitrate (ρNO3) was significantly lower at decreased pH when the rate was normalized by cell number instead of by cell volume. The changes in cell morphology found in this study under low pH were likely not large enough to significantly impact physiological processes and the role of this species in marine food webs and biogeochemical cycles. It is possible that the interactive effects of pH, temperature, light, and nutrient availability characteristic of different regions of the world ocean could result in stronger physiological and morphological responses in this widespread diatom. However, under constant optimal growth conditions, T. rotula was only mildly affected by changes in pH, and in particular the physiology and elemental stoichiometry of C, N and Si were not sensitive to acidification.

Ecophysiology of Kuwaiti macroalgae with special emphasis on temperature and salinity tolerance related to the conditions at desalination plant outfalls

Abstract Brine discharged from seawater desalination plants impacts marine life by exposure to increased salinity and, in some cases, temperature. However, the responses of individual species to such stress remains poorly known yet their understanding is essential for assessing and predicting the impacts of seawater desalination plants. In this study, unialgal cultures obtained by the germling emergence method of 34 taxa representative of the Rhodophyta, Chlorophyta and Phaeophyceae seaweeds in the Arabian Gulf, and isolated from the vicinity of two large desalination plants in Kuwait, were subjected to increased temperature and salinity under experimental conditions in the laboratory. The dataset is complemented by measurements of seawater temperature and salinity obtained at increasing distances from the outfalls of desalination plants and along the Kuwaiti coastline including from two pristine areas, Boubiyan and Fintas. Chlorophyta, especially Ulva spp., and Phaeophyceae displayed remarkable tolerance against hypersaline and thermal stress, suggesting that this group can cope better with adverse environmental conditions. Members of the Rhodophyta were considerably more sensitive to temperature increases.

Lipid degradation and photosynthetic traits after prolonged darkness in four Antarctic benthic diatoms, including the newly described species Planothidium wetzelii sp. nov.

In polar regions, the microphytobenthos has important ecological functions in shallow-water habitats, such as on top of coastal sediments. This community is dominated by benthic diatoms, which contribute significantly to primary production and biogeochemical cycling while also being an important component of polar food webs. Polar diatoms are able to cope with markedly changing light conditions and prolonged periods of darkness during the polar night in Antarctica. However, the underlying mechanisms are poorly understood. In this study, five strains of Antarctic benthic diatoms were isolated in the field, and the resulting unialgal cultures were identified as four distinct species, of which one is described as a new species, Planothidium wetzelii sp. nov. All four species were thoroughly examined using physiological, cell biological, and biochemical methods over a fully controlled dark period of 3 months. The results showed that the utilization of storage lipids is one of the key mechanisms in Antarctic benthic diatoms to survive the polar night, although different fatty acids were involved in the investigated taxa. In all tested species, the storage lipid content declined significantly, along with an ultrastructurally observable degradation of the chloroplasts. Surprisingly, photosynthetic performance did not change significantly despite chloroplasts decreasing in thylakoid membranes and an increased number of plastoglobules. Thus, a combination of biochemical and cell biological mechanisms allows Antarctic benthic diatoms to survive the polar night.

Comparative effects of temperature and salinity on growth of four harmful Chattonella spp. (Raphidophyceae) from tropical Asian waters

In Asia, four harmful raphidophyte species, Chattonella malayana, C. marina, C. subsalsa, and C. tenuiplastida, coexist in the tropical waters but only C. marina was detected in temperate waters. This occurrence pattern pointed to a potentially distinct ecophysiological niche occupancy and possible species dispersion. The growth physiology of these species isolated from tropical Southeast Asia was investigated using unialgal cultures in ten temperatures (13.0–35.5°C) and five salinities (15–35) to better understand the factors driving their distribution. The highest maximum specific growth rates were observed in C. subsalsa (0.65 ± 0.01 d-1), followed by C. malayana (0.47 ± 0.03 d-1), C. marina (0.45 ± 0.02 d-1), and C. tenuiplastida (0.39 ± 0.01 d-1). Their optimal temperatures were 28.0, 30.5, 25.5, and 30.5°C, respectively, of which C. marina preferred colder water. C. subsalsa exhibited a wider growth temperature range (20.5–35.5°C), followed by C. marina (20.5–30.5°C), C. tenuiplastida (23.0–33.0°C), and C. malayana (25.5–33.0°C). Optimal salinities were similar between C. subsalsa and C. malayana (30), and between C. marina and C. tenuiplastida (25), but C. subsalsa and C. marina exhibited a similar growth salinity range of 15–35, while C. malayana and C. tenuiplastida was 20–35. High values of Fv/Fm were observed in C. subsalsa and C. marina (> 0.5) in all tested conditions, but Fv/Fm of C. malayana and C. tenuiplastida were significantly lower at 20.5°C. All four species achieved a maximum cell density of > 104 cells mL-1 in their optimal conditions. Optimal temperatures in C. subsalsa and C. marina were identical to previous reports. The high adaptability of C. subsalsa in various temperatures and salinities suggests its high competitiveness and bloom potential. The high adaptability of C. marina in colder waters compared to other species likely contributes to its wide distribution in the temperate Asian waters. The narrow temperature window of C. malayana and C. tenuiplastida suggests their endemicity and limited distribution in the tropical waters. This study provides evidence about the occurrences and bloom potential of Chattonella spp. in Asia, but the endemicity versus dispersion issue remains unresolved.

Temperature as a factor controlling geographical distribution of greenNoctiluca scintillansin Southeast Asia

AbstractThe dinoflagellate Noctiluca scintillans exists in two forms, green and red Noctiluca, with and without the photosynthetic endosymbiont Pedinomonas noctilucae, respectively. In Southeast Asia, green Noctiluca has a northern boundary of distribution in the Luzon Strait and the Vietnamese coast, approximately corresponding to the 25°C isotherm. We examined whether this boundary arose from the growth dependence of Noctiluca on temperature and/or that of P. noctilucae. We analyzed the effect of temperature on growth by examining one and four unialgal cultures of red and green Noctiluca, respectively, and two monocultures of the endosymbiont. Growth dependence on temperature was markedly different between host cells of green Noctiluca and monocultures of the endosymbiont, although some variations in growing temperature were observed among strains of both Noctiluca and the endosymbiont. Green Noctiluca grew at a temperature range of 20–31°C, whereas P. noctilucae did not grow at low temperatures ≤25°C. Growth rates of green Noctiluca were considerably higher when fed compared to monocultures. Our findings demonstrate that the effect of temperature on P. noctilucae growth is a crucial factor controlling the geographical distribution of green Noctiluca in the South China Sea. An implication of this finding is discussed in the context of global warming.

The Western Lake Erie culture collection: A promising resource for evaluating the physiological and genetic diversity of Microcystis and its associated microbiome

Cyanobacterial harmful algal blooms (cyanoHABs) dominated by Microcystis spp. have significant public health and economic implications in freshwater bodies around the world. These blooms are capable of producing a variety of cyanotoxins, including microcystins, that affect fishing and tourism industries, human and environmental health, and access to drinking water. In this study, we isolated and sequenced the genomes of 21 primarily unialgal Microcystis cultures collected from western Lake Erie between 2017 and 2019. While some cultures isolated in different years have a high degree of genetic similarity (genomic Average Nucleotide Identity >99%), genomic data show that these cultures also represent much of the breadth of known Microcystis diversity in natural populations. Only five isolates contained all the genes required for microcystin biosynthesis while two isolates contained a previously described partial mcy operon. Microcystin production within cultures was also assessed using Enzyme-Linked Immunosorbent Assay (ELISA) and supported genomic results with high concentrations (up to 900 μg L⁻¹) in cultures with complete mcy operons and no or low toxin detected otherwise. These xenic cultures also contained a substantial diversity of bacteria associated with Microcystis, which has become increasingly recognized as an essential component of cyanoHAB community dynamics. These results highlight the genomic diversity among Microcystis strains and associated bacteria in Lake Erie, and their potential impacts on bloom development, toxin production, and toxin degradation. This culture collection significantly increases the availability of environmentally relevant Microcystis strains from temperate North America.

Multigene phylogeny reveals a cryptic diversity in the genus Dinobryon (Chrysophyceae) with integrative description of five new species.

The genus Dinobryon is one of the most recognizable chrysophyte genera, characterized by dendroid colonies with a biflagellate inside each cellulosic lorica. The representative forms of lorica are cylindrical, conical, vase, or funnel shaped, with undulation on the lorica wall. Traditionally, the morphological characteristics of the lorica and the colony organization have been used for the delimitation of Dinobryon species. To understand the taxonomy and phylogeny of colonial Dinobryon species, we performed molecular and morphological studies using 39 unialgal cultures and 46 single colony isolations from environmental specimens collected in Korea. We used a nuclear internal transcript spacer (ITS1-5.8S-ITS2) to find the genetic diversity of Dinobryon from environmental samples and a combined dataset from six gene sequences (nuclear SSU and LSU rRNA, plastid LSU rRNA, rbcL and psaA, and mitochondrial CO1 genes) for phylogenetic analysis. We found 15 different lineages based on the genetic diversity of the nuclear ITS sequences. The phylogenetic tree of the colonial species based on the combined multigene dataset were divided into 18 subclades, including five new species, each with unique molecular signatures for the E23-5 helix of the V4 region in the nuclear SSU rRNA and the E11-1 helix of D7b, and the E20-1 helix of D8 regions in the nuclear LSU rRNA. Morphological studies were focused on lorica dimension and shape, and stomatocyst morphology. The Dinobryon species showed similarities or differences in lorica morphologies between and within species, and also differences in lorica size between culture and environmental samples. Five Dinobryon species formed distinctive stomatocysts, their stomatocyst morphologies, including collar structure, surface ornamentation, and cyst shape, showed unique characteristics in each species and were useful for identification. Here, we propose five new species based on morphological and molecular evidences: D. cylindricollarium, D. exstoundulatum, D. inclinatum, D. similis, and D. spinum.

Domoic acid production by Pseudo-nitzschia australis: Re-evaluating the role of macronutrient limitation on toxigenicity

The toxigenic diatom Pseudo-nitzschia australis (Frenguelli), isolated from the California Current System (CCS), was examined in unialgal laboratory cultures to evaluate domoic acid (DA) production and cellular growth as a response to macronutrient limitation. Toxic blooms of P. australis are common in the coastal waters of eastern boundary upwelling systems (EBUS), including those of the CCS off the west coast of the United States where limitation by macronutrients, specifically silicon as silicic acid [Si(OH)4], or phosphorus as phosphate [PO43-], has been suggested to increase the production of DA by these diatoms. This study used batch cultures grown under conditions of macronutrient sufficiency and limitation, expected during and after a natural upwelling event, to determine whether PO43- or Si(OH)4 deficiency enhances the production of DA and the expected risk of DA toxicity in natural coastal ecosystems. These controlled lab studies demonstrate that despite increases in cell-specific DA concentrations found during the nutrient-limited stationary phase, DA production rates did not increase due to either PO43- or Si(OH)4 limitation, and total DA production rates were statistically greater during the nutrient-replete, exponential growth phase compared to the nutrient-limited, stationary phase. In addition, the relative contribution of particulate DA (pDA) and dissolved DA (dDA) varied markedly with growth phase, where the contribution of pDA to total DA (pDA + dDA) declined from an average of 70% under P- and Si-replete conditions to 49% under P-limited conditions and 39% under Si-limited conditions. These laboratory results demonstrate that macronutrient sufficiency does not regulate the biosynthetic production of DA by this strain of P. australis. This finding, together with a comparative analysis of the various equations employed to estimate DA production, suggests that the current paradigm of increased toxigenicity due to macronutrient limitation be carefully re-examined, particularly when attempting to forecast the toxic threat of DA to coastal ecosystems as a function of macronutrient availability.

The molecular mechanisms and environmental effects of modified clay control algal blooms in aquacultural water

Modified clay (MC) technology is an effective method for controlling harmful algal blooms (HABs). Based on field experience, a bloom does not continue after treatment with MC, even though the residual HAB biomass accounts for 20–30% of the initial biomass. Laboratory studies using unialgal cultures have found that MC could inhibit the growth of the residual algal cells to prevent HABs. Nevertheless, the phytoplankton in field waters is diverse. Therefore, unclassified complex mechanisms may exist. To illustrate the molecular mechanisms through which MC controls HABs in the field and verify the previous laboratory findings, a series of experiments and bioinformatics analyses were conducted using bloom waters from aquacultural ponds. The results showed that a 72.29% removal efficiency of algal biomass could effectively control blooms. The metatranscriptomic results revealed that the number of downregulated genes (131,546) was greater than that of upregulated genes (24,318) at 3 h after MC addition. Among these genes, several genes related to DNA replication were downregulated; however, genes involved in DNA repair were upregulated. Metabolism-related pathways were the most significantly upregulated (q < 0.05), including photosynthesis and oxidative phosphorylation. The results also showed that MC reduced most of the biomass of the dominant phytoplankton species, likely by removing apical dominance, which increased the diversity and stability of the phytoplankton community. In addition to reducing the pathogenic bacterial density, MC reduced the concentrations of PO43− (96.22%) and SiO32− (66.77%), thus improving the aquaculture water quality, altering the phytoplankton community structure (the proportion of Diatomea decreased, and that of Chlorophyta increased), and inhibiting phytoplankton growth. These effects hindered the rapid development of large phytoplankton biomasses and allowed the community structure to remain stable, reducing HAB threats. This study illustrates the molecular mechanisms through which MC controls HABs in the field and provides a scientific method for removing HABs in aquacultural waters.

Exploring diatom diversity through cultures - a case study from the Bow River, Canada

Diatom cultures can help answer taxonomic, biogeographic and ecological questions on a local and global scale. Unialgal cultures are derived from a single cell and provide abundant material for morphological and molecular analyses. The link between the historic morphological species concept and the molecular data is becoming increasingly important with the use of eDNA metabarcoding. Additionally, cultures provide insights into the life cycle of diatoms and thereby complement taxonomy and species ecology. In this study, we present an approach to extract benthic diatoms from an environmental sample to generate unialgal cultures. We explored diatom diversity in preserved assemblages and by culturing as many different taxa as possible from benthic freshwater samples taken on the same day from the Bow River in Calgary, Canada. With both methods we found a total of 221 different benthic diatom taxa, of which 182 were identified in the preserved diatom assemblages. Interestingly, an additional 39 taxa only appeared in the cultures. In total 129 strains were cultivated representing 71 different taxa. This study includes pictures of living cells demonstrating the additional merits of unialgal cultures, as they provide information on plastid details, auxospores and endosymbionts. Both, the identification of the diatom assemblages and the generation and identification of strains provide the foundation for additional water quality assessment tools, taxonomic insights and molecular references libraries.

Establishment and optimization of a new model organism to study early land plant evolution: Germination, cultivation and oospore variation of Chara braunii Gmelin, 1826.

For studying land plant evolution, the establishment and optimization of model organisms representing streptophytic algae, sister to land plants, is essential. Long-term cultivation experiments with Chara braunii S276 were performed over 8 years, since 4 years (Nov. 2018) under constant conditions. Additionally, short-term experiments for optimization of culture conditions were performed with three strains of C. braunii (S276, NIES-1604 and Lausiger Teiche, LaT-2708). Germination success after application of sterilization agents, addition of gibberellic acid and under different incubation conditions with respect to pre-treatment, irradiance regime and substrate was investigated in order to develop protocols for generative cultivation of at least unialgal cultures. The resulting cultivation protocols for C. braunii S276, allowing maintenance of vegetative as well as generative cultures are presented in detail, including protocols for germination induction and growth of sterilized and unsterilized oospores.

Перша знахідка представника роду Tenebriella (Cyanobacteria, Oscillatoriales) на мисі Казантип (Азовське море, Україна)

Studying terrestrial algae of the Kazantip Nature Reserve (Kerch Peninsula) and its environs, a morphologically distinctive homocytic filamentous cyanobacterium with dark coloured trichomes was found in the biological soil crust from the surface of the clay scree. Despite the peculiar coloration and rather large dimensions of the filaments, we failed to identify it even to the genus based on morphological features. Unfortunately, a molecular study of this material was impossible since an attempt to isolate it in the unialgal culture was unsuccessful. The recently described genus Tenebriella Hauerová, Hauer et Kaštovský separated from Oscillatoria sensu lato, has morphological features very close to our material. They include dark colored filaments, trichomes pale-greyish green to purple, slightly constricted at the cross walls, with short cells, gently tapering to the ends, and several last cells of the trichome often yellowish. Morphologically and ecologically Ukrainian record is very close to T. amphibia Hauerová, Hauer et Kaštovský, but is characterized by noticeably thinner trichomes. Morphological description of this species, photomicrographs, information on habitat in Ukraine and ecology, comparison with literature data are given.

How to Verify Non-Presence-The Challenge of Axenic Algae Cultivation.

Many phycological applications require the growth and maintenance of pure algae cultures. In some research areas, such as biochemistry and physiology, axenic growth is essential to avoid misinterpretations caused by contaminants. Nonetheless, axenicity—defined as the state of only a single strain being present, free of any other organism—needs to be verified. We compare the available methods to assess axenicity. We first purified unialgal Limnospira fusiformis cultures with an established series of axenicity treatments, and by including two additional treatment steps. The presumable axenic cultures were then tested for their axenic state by applying conventional tests on LB (lysogeny broth) agar-plates, 16S rRNA gene amplicon sequencing, flow-cytometry and epifluorescence microscopy. Only the plate tests indicated axenic conditions. We found a linear relationship between total cell counts of contaminants achieved by flow cytometry and epifluorescence microscopy, with flow cytometry counts being consistently higher. In addition, 16S rRNA gene amplicon sequencing demonstrated its superiority by not only being an efficient tool for axenicity testing, but also for identification of persistent contaminants. Although classic plate tests are still commonly used to verify axenicity, we found the LB-agar-plate technique to be inappropriate. Cultivation-independent methods are highly recommended to test for axenic conditions. A combination of flow-cytometry and 16S rRNA gene amplicon sequencing complement each other and will yield the most reliable result.

Pigment-based chemotaxonomy of the phytoplankton community from a marine mussel farming area

Microscopy technique is traditionally employed for assessing phytoplankton taxonomy. However, chemical approaches, such as the identification of biomarker pigments, allow to elucidate microalgae groups more quickly, in addition to detecting these valuable compounds. This study aimed to identify the pigment profile of microalgae species and to determine the seasonal pigment fluctuation in a marine mussel farm. Water samples were collected periodically between the years of 2014 and 2015. Phytoplanktonic organisms were isolated and cultivated in f/2 liquid medium under controlled temperature and light cycle. Cell content extracts from each unialgal culture and water sample were analyzed by High Performance Liquid Chromatography (HPLC). Pigment profiles of 13 marine microalgae strains were identified (12 Diatoms and 1 Dinoflagellate). Chlorophyll a and fucoxanthin were most present in Diatoms, while peridinin was the major carotenoid in Prorocentrum micans. Bacillariophyceae had higher density in the summer and fall. Dinophyceae, Cryptophyceae, Euglenophyceae, Prasinophyceae, Dictyophyceae and Ebriidea, had an increase in density during winter and spring. Following the trend of phytoplankton groups, pigments such as fucoxanthin, zeaxanthin, diadinoxanthin, chlorophyll c3 and c2, were identified throughout the year. Prasinoxanthin, peridinin, dinoxanthin and antheraxanthin were identified in samples collected during winter and spring.

Life cycle assessment of microalgae systems for wastewater treatment and bioproducts recovery: Natural pigments, biofertilizer and biogas

The aim of this study was to assess the potential environmental impacts associated with microalgae systems for wastewater treatment and bioproducts recovery. In this sense, a Life Cycle Assessment was carried out evaluating two systems treating i) urban wastewater and ii) industrial wastewater (from a food industry), with the recovery of bioproducts (i.e. natural pigments and biofertilizer) and bioenergy (i.e. biogas). Additionally, both alternatives were compared to iii) a conventional system using a standard growth medium for microalgae cultivation in order to show the potential benefits of using wastewater compared to typical cultivation approaches. The results indicated that the system treating industrial wastewater with unialgal culture had lower environmental impacts than the system treating urban wastewater with mixed cultures. Bioproducts recovery from microalgae wastewater treatment systems can reduce the environmental impacts up to 5 times compared to a conventional system using a standard growth medium. This was mainly due to the lower chemicals consumption for microalgae cultivation. Food-industry effluent showed to be the most promising scenario for bioproducts recovery from microalgae treating wastewater, because of its better quality compared to urban wastewater which also allows the cultivation of a single microalgae species. In conclusion, microalgae wastewater treatment systems are a promising solution not only for wastewater treatment but also to boost the circular bioeconomy in the water sector through microalgae-based product recovery.

The efficiency of microalgae-based remediation as a green process for industrial wastewater treatment

Wastewater treatment including microalgae is an economically promising and environmentally friendly alternative to conventional wastewater treatment due to the concurrent nutrient removal and production of high-value biomass. This work aimed to choose the most suitable type of wastewater and its appropriate dilution for sustaining microalgal growth by testing unialgal versus mixed microalgae cultures and raw versus autoclaved wastewater. Based on pilot experiments including microplate screening, unialgal cultures of Chlorella emersonii grown in 1:5 diluted, non-autoclaved wastewater were selected as the optimum scenario for further testing. In the main experiments, Chlorella emersonii was cultivated in Erlenmeyer flasks and bubble column reactors. Additionally, a photoautotrophic biofilm was grown in flumes. We studied three types of wastewater originating from a large municipal wastewater treatment plant, a brewery, and a dairy. Dairy wastewater performed best in all cultivation systems and resulted in a maximum biomass per irradiated area of around 145 μg cm−2 chlorophyll-a of Chlorella cultures in bubble columns, followed by 105 μg cm−2 in flumes run by a photoautotrophic biofilm. Furthermore, phosphorus reduction of 96 % for dairy, 40 % for the brewery, and 43 % for municipal wastewater were achieved in the flumes. Due to the easy harvest of algae biomass, low construction and operation costs, flumes represent a recommendable cultivation system for dairy wastewater treatment on a larger scale.

Strong and widespread cycloheximide resistance in Stichococcus-like eukaryotic algal taxa

This study was initiated following the serendipitous discovery of a unialgal culture of a Stichococcus-like green alga (Chlorophyta) newly isolated from soil collected on Signy Island (maritime Antarctica) in growth medium supplemented with 100 µg/mL cycloheximide (CHX, a widely used antibiotic active against most eukaryotes). In order to test the generality of CHX resistance in taxa originally identified as members of Stichococcus (the detailed taxonomic relationships within this group of algae have been updated since our study took place), six strains were studied: two strains isolated from recent substrate collections from Signy Island (maritime Antarctica) (“Antarctica” 1 and “Antarctica” 2), one isolated from this island about 50 years ago (“Antarctica” 3) and single Arctic (“Arctic”), temperate (“Temperate”) and tropical (“Tropical”) strains. The sensitivity of each strain towards CHX was compared by determining the minimum inhibitory concentration (MIC), and growth rate and lag time when exposed to different CHX concentrations. All strains except “Temperate” were highly resistant to CHX (MIC > 1000 µg/mL), while “Temperate” was resistant to 62.5 µg/mL (a concentration still considerably greater than any previously reported for algae). All highly resistant strains showed no significant differences in growth rate between control and treatment (1000 µg/mL CHX) conditions. Morphological examination suggested that four strains were consistent with the description of the species Stichococcus bacillaris while the remaining two conformed to S. mirabilis. However, based on sequence analyses and the recently available phylogeny, only one strain, “Temperate”, was confirmed to be S. bacillaris, while “Tropical” represents the newly erected genus Tetratostichococcus, “Antarctica 1” Tritostichococcus, and “Antarctica 2”, “Antarctica 3” and “Arctic” Deuterostichococcus. Both phylogenetic and CHX sensitivity analyses suggest that CHX resistance is potentially widespread within this group of algae.

Comparison of a Chilean strain of the ichthyotoxic phytoflagellate Heterosigma akashiwo (Raphidophyceae) with strains from France, Spain and New Zealand

ABSTRACT Heterosigma akashiwo is a bloom-forming planktonic flagellate that causes fish mortalities, which damages the aquaculture industry worldwide. In Chile, this species was reported for the first time in 1988 when it caused one of the most dramatic harmful algal blooms in the south of the country. At that time, an unialgal culture was set and used for a morphological characterization but, unfortunately, it was not maintained. In April 2013 a new outbreak of H. akashiwo was detected in the Maullín Estuary (41°37′S, 73°36′W), Southern Chile, causing fish mortalities in commercial salmon farms. An unialgal culture of this new Chilean population of H. akashiwo was obtained and has been maintained in the Culture Collection of Microalgae of the University of Concepción, Chile, under the code CCM-UdeC 225. The aim of this study was to compare the morphological and physiological attributes of this Chilean strain of H. akashiwo with those of strains from France, Spain and New Zealand, in order to identify eventual intraspecific variability among them. Cell size, growth parameters, pigments profiles, photosynthetic attributes and toxicity were determined for the strains, grown under identical culture conditions. The Chilean strain exhibited the highest growth and accumulated the highest proportion of the carotenoid fucoxanthin, while strains from Chile and Spain were the less light-tolerant and had similar toxicity. These results reinforce previous findings about the high level of intraspecific variability within H. akashiwo including, for the first time, a strain from South America.

Carotenoid profiling of Mytilus coruscus with unialgal cultures: Insights into biosynthetic pathways in tissues

The carotenoid profiling of Mytilus coruscus generally depends on the dietary microalgae, and the ingested carotenoids are modified and processed depending on the properties of different tissues. Although much research effort has focused on analyzing carotenoid profiling between microalgae and shellfish, the detailed metabolic pathways of dietary carotenoids in shellfish are poorly understood. In the present study, we carried out feeding experiments with different unialgal cultures (Chaetoceros mulleri and Dicrateria inornata) during the growth stages and examined the carotenoid profiles between the dietary microalgae and the corresponding tissues in order to study the resorption and metabolic transformations of dietary carotenoids in M. coruscus. The results showed that 1) carotenoids mainly occurred in the pre-spawning phase of M. coruscus; the contents of most carotenoids showed the same trends as in dietary microalgae. 2) An association analysis of the total content of carotenoids and relevant gene expression demonstrated that the main tissue for carotenoid synthesis in M. coruscus was the hepatopancreas. The synthetic reactions of the allenic to acetylenic carotenoids pathway and the 4-keto oxidative pathway in M. coruscus coexisted. 3) Quantitative analysis revealed that the carotenoids mytiloxanthin, alloxanthin, and pectenolone containing acetylene groups were selectively assimilated in gonad (69.3–74.1%), mantle (83.2–102%), and muscle (86.9–100%), forming a large percentage of the carotenoids present in those tissues. These results provide new insights into the biosynthetic and metabolic transformation pathways of carotenoids in M. coruscus.