Temperature ranges for survival and growth of juvenile Saccharina sculpera (Laminariales, Phaeophyta) and applications for field cultivation

Saccharina sculpera is highly valued for human consumption and value-added products. However, natural resources of this kelp have decreased sharply and it is in danger of extinction. Resources recovery through cultivation is being trialed to enable the sustainable use of this species. In this study, the temperature range for survival and optimal growth of juvenile S. sculpera was identified and applied to field cultivation. This study investigated the survival and growth of juvenile S. sculpera under six temperatures (i.e., 5, 10, 15, 16, 18, and 20°C) and two light intensities (i.e., 20 and 40 μmol photons m-2 s-1) in an indoor culture experiment. In these experiments, the blade length decreased at 16°C under the both light intensities. The thalli died at 20°C and 20 μmol photons m-2 s-1, and at 18‒20°C and 40 μmol photons m-2 s-1. During the field cultivation, early growth of S. sculpera was highest at the 5 m depth and growth decreased as the water depth increased. When the initial rearing depth was maintained without adjustment throughout the cultivation period (from December to October), all the cultivated S. sculpera plants died during August and September. However, S. sculpera plants lowered from 5 to 15 m and grew to 90.8 ± 13.1 cm in July. The seawater temperature at 15 m depth was similar to the upper level of thermal tolerance demonstrated by juvenile S. sculpera in the indoor culture experiments (16°C or lower). The plants were subsequently lowered to 25 m depth in August, which eventually led to their maturation in October. The present study confirmed that improved growth rates and a delay in biomass loss can be achieved by adjusting the depth at which the seaweeds are grown during the cultivation period. These results will contribute to the establishment of sustainable cultivation systems for S. sculpera.

Ecophysiology of the kleptoplastidic dinoflagellate Shimiella gracilenta: I. spatiotemporal distribution in Korean coastal waters and growth and ingestion rates

To explore the ecophysiological characteristics of the kleptoplastidic dinoflagellate Shimiella gracilenta, we determined its spatiotemporal distribution in Korean coastal waters and growth and ingestion rates as a function of prey concentration. The abundance of S. gracilenta at 28 stations from 2015 to 2018 was measured using quantitative realtime polymerase chain reaction. Cells of S. gracilenta were detected at least once at all the stations and in each season, when temperature and salinity were 1.7–26.4°C and 9.9–35.6, respectively. Moreover, among the 28 potential prey species tested, S. gracilenta SGJH1904 fed on diverse prey taxa. However, the highest abundance of S. gracilenta was only 3 cells mL-1 during the study period. The threshold Teleaulax amphioxeia concentration for S. gracilenta growth was 5,618 cells mL-1, which was much higher than the highest abundance of T. amphioxeia (667 cells mL-1). Thus, T. amphioxeia was not likely to support the growth of S. gracilenta in the field during the study period. However, the maximum specific growth and ingestion rates of S. gracilenta on T. amphioxeia, the optimal prey species, were 1.36 d-1 and 0.04 ng C predator- 1 d-1, respectively. Thus, if the abundance of T. amphioxeia was much higher than 5,618 cells mL-1, the abundance of S. gracilenta could be much higher than the highest abundance observed in this study. Eurythermal and euryhaline characteristics of S. gracilenta and its ability to feed on diverse prey species and conduct kleptoplastidy are likely to be responsible for its common spatiotemporal distribution.

Consensus channelome of dinoflagellates revealed by transcriptomic analysis sheds light on their physiology

Ion channels are membrane protein complexes mediating passive ion flux across the cell membranes. Every organism has a certain set of ion channels that define its physiology. Dinoflagellates are ecologically important microorganisms characterized by effective physiological adaptability, which backs up their massive proliferations that often result in harmful blooms (red tides). In this study, we used a bioinformatics approach to identify homologs of known ion channels that belong to 36 ion channel families. We demonstrated that the versatility of the dinoflagellate physiology is underpinned by a high diversity of ion channels including homologs of animal and plant proteins, as well as channels unique to protists. The analysis of 27 transcriptomes allowed reconstructing a consensus ion channel repertoire (channelome) of dinoflagellates including the members of 31 ion channel families: inwardly-rectifying potassium channels, two-pore domain potassium channels, voltage-gated potassium channels (Kv), tandem Kv, cyclic nucleotide-binding domain-containing channels (CNBD), tandem CNBD, eukaryotic ionotropic glutamate receptors, large-conductance calcium-activated potassium channels, intermediate/small-conductance calcium-activated potassium channels, eukaryotic single-domain voltage-gated cation channels, transient receptor potential channels, two-pore domain calcium channels, four-domain voltage-gated cation channels, cation and anion Cys-loop receptors, small-conductivity mechanosensitive channels, large-conductivity mechanosensitive channels, voltage-gated proton channels, inositole-1,4,5- trisphosphate receptors, slow anion channels, aluminum-activated malate transporters and quick anion channels, mitochondrial calcium uniporters, voltage-dependent anion channels, vesicular chloride channels, ionotropic purinergic receptors, animal volage-insensitive cation channels, channelrhodopsins, bestrophins, voltage-gated chloride channels H+/Cl- exchangers, plant calcium-permeable mechanosensitive channels, and trimeric intracellular cation channels. Overall, dinoflagellates represent cells able to respond to physical and chemical stimuli utilizing a wide range of Gprotein coupled receptors- and Ca2+-dependent signaling pathways. The applied approach not only shed light on the ion channel set in dinoflagellates, but also provided the information on possible molecular mechanisms underlying vital cellular processes dependent on the ion transport.

A guide to phylotranscriptomic analysis for phycologists

Phylotranscriptomics is the study of phylogenetic relationships among taxa based on their DNA sequences derived from transcriptomes. Because of the relatively low cost of transcriptome sequencing compared with genome sequencing and the fact that phylotranscriptomics is almost as reliable as phylogenomics, the phylotranscriptomic analysis has recently emerged as the preferred method for studying evolutionary biology. However, it is challenging to perform transcriptomic and phylogenetic analyses together without programming expertise. This study presents a protocol for phylotranscriptomic analysis to aid marine biologists unfamiliar with UNIX command-line interface and bioinformatics tools. Here, we used transcriptomes to reconstruct a molecular phylogeny of dinoflagellate protists, a diverse and globally abundant group of marine plankton organisms whose large and complex genomic sequences have impeded conventional phylogenic analysis based on genomic data. We hope that our proposed protocol may serve as practical and helpful information for the training and education of novice phycologists.

Bioluminescence capability and intensity in the dinoflagellate Alexandrium species

Some species in the dinoflagellate genus Alexandrium are bioluminescent. Of the 33 formally described Alexandrium species, the bioluminescence capability of only nine species have been tested, and eight have been reported to be bioluminescent. The present study investigated the bioluminescence capability of seven Alexandrium species that had not been tested. Alexandrium mediterraneum, A. pohangense, and A. tamutum were bioluminescent, but A. andersonii, A. hiranoi, A. insuetum, and A. pseudogonyaulax were not. We also measured the bioluminescent intensity of A. affine, A. fraterculus, A. mediterraneum, A. ostenfeldii, A. pacificum, A. pohangense, A. tamarense, and A. tamutum. The mean 200-second-integrated bioluminescence intensity per cell ranged from 0.02 to 32.2 × 104 relative luminescence unit per cell (RLU cell-1), and the mean maximum bioluminescence intensity per cell per second (BLMax) ranged from 0.01 to 10.3 × 104 RLU cell-1 s-1-1. BLMax was significantly correlated with the maximum growth rates of Alexandrium species, except for A. tamarense. A phylogenetic tree based on large subunit ribosomal DNA (LSU rDNA) showed that the bioluminescent species A. affine, A. catenella, A. fraterculus, A. mediterraneum, A. pacificum, and A. tamarense formed a large clade. However, the toxicity or mixotrophic capability of these species was split. Thus, their bioluminescence capability in this clade was more consistent than their toxicity or mixotrophic capability. Phylogenetic trees based on LSU rDNA and the luciferase gene of Alexandrium were consistent except for A. pohangense. The results of the present study can provide a basis for understanding the interspecific diversity in bioluminescence of Alexandrium.

Amphidinium stirisquamtum sp. nov. (Dinophyceae), a new marine sand-dwelling dinoflagellate with a novel type of body scale

Amphidinium species are amongst the most abundant benthic dinoflagellates in marine intertidal sandy ecosystems. Some of them produce a variety of bioactive compounds that have both harmful effects and pharmaceutical potential. In this study, Amphidinium cells were isolated from intertidal sand collected from the East China Sea. The two strains established were subjected to detailed examination by light, and scanning and transmission electron microscopy. The vegetative cells had a minute, irregular, and triangular-shaped epicone deflected to the left, thus fitting the description of Amphidinium sensu stricto. These strains are distinguished from other Amphidinium species by combination characteristics: (1) longitudinal flagellum inserted in the lower third of the cell; (2) icicle-shaped scales, 276 ± 17 nm in length, on the cell body surface; (3) asymmetrical hypocone with the left side longer than the right; and (4) presence of immotile cells. Therefore, they are described here as Amphidinium stirisquamtum sp. nov. The molecular tree inferred from small subunit rRNA, large subunit rRNA, and internal transcribed spacer-5.8S sequences revealed that A. stirisquamtum is grouped together with the type species of Amphidinium, A. operculatum, in a fully supported clade, but is distantly related to other Amphidinium species bearing body scale. Live A. stirisquamtum cells greatly affected the survival of rotifers and brine shrimp, their primary grazers, making them more susceptible to predation by the higher tropic level consumers in the food web. This will increase the risk of introducing toxicity, and consequently, the bioaccumulation of toxins through marine food webs.

Thermal plasticity of growth and chain formation of the dinoflagellates Alexandrium affine and Alexandrium pacificum with respect to ocean acidification

The amount of CO2 absorbed by the oceans continues to rise, resulting in further acidification, altering some functional traits of phytoplankton. To understand the effect of elevated partial pressures of CO2 (pCO2) on functional traits of dinoflagellates Alexandrium affine and A. pacificum, the cardinal temperatures and chain formation extent were examined under two pCO2 (400 and 1,000 μatm) over the range of temperature expected to be associated with growth. The growth rate and chain formation extent of A. affine increased with higher pCO2, showing significant changes in cardinal temperatures and a substantial increase in middle chain-length (4‒8 cells) fractionation under elevated pCO2 condition. By contrast, there were no significant differences in specific growth rate and any chain-length fractionation of A. pacificum between ambient and elevated pCO2 conditions. The observed interspecies variation in the functional traits may reflect differences in ability of species to respond to environmental change with plasticity. Moreover, it allows us to understand the shifting biogeography of marine phytoplankton and predict their phenology in the Korea Strait.

A simple method to produce fragment seedstock for aquaculture of Pterocladiella capillacea (Gelidiales, Rhodophyta)

Raw material of gelidioid red algae yielding high-quality agar has been in short supply due to overharvesting, but in situ farming of gelidioids has not been practical due to their slow growth. To produce vegetative seedstock of a cosmopolitan species, Pterocladiella capillacea, we investigated the number and length of regenerated branches arising from sectioned fragments during 3 weeks of laboratory culture at 10, 15, 20, and 25°C. All sectioned fragments formed axis-like branches mostly from the upper cut edge and stolon-like branches mostly from the lower cut edge, showing a high capacity of regeneration and intrinsic bipolarity. At 20°C, the number of regenerated branches increased to 2.74 ± 1.29 on the upper cut edge and 4.26 ± 2.66 on the lower cut edge. Our study reveals that the use of fragments bearing regenerated branches as seedstock can be a simple method to initiate fast propagation for mass cultivation in the sea or outdoor tank.

Characterization of Coolia spp. (Gonyaucales, Dinophyceae) from Southern Tunisia: first record of Coolia malayensis in the Mediterranean Sea

This study provides the first report of the presence of Coolia malayensis in the Mediterranean Sea, co-occurring with C. monotis. Isolated strains from the Gulf of Gabès, Tunisia (South-eastern Mediterranean) were identified by morphological characterization and phylogenetic analysis. Examination by light and scanning electron microscopy revealed no significant morphological differences between the Tunisian isolates and other geographically distant strains of C. monotis and C. malayensis. Phylogenetic trees based on ITS1-5.8S-ITS2 and D1‒D3/28S rDNA sequences showed that C. monotis strains clustered with others from the Mediterranean and Atlantic whereas the C. malayensis isolate branched with isolates from the Pacific and the Atlantic, therefore revealing no geographical trend among C. monotis and C. malayensis populations. Ultrastructural analyses by transmission electron microscopy revealed the presence of numerous vesicles containing spirally coiled fibers in both C. malayensis and C. monotis cells, which we speculate to be involved in mucus production.

Downregulation of PyHRG1, encoding a novel secretory protein in the red alga Pyropia yezoensis, enhances heat tolerance

An increase in seawater temperature owing to global warming is expected to substantially limit the growth of marine algae, including Pyropia yezoensis, a commercially valuable red alga. To improve our knowledge of the genes involved in the acquisition of heat tolerance in P. yezoensis, transcriptomes sequences were obtained from both the wild-type SG104 P. yezoensis and heat-tolerant mutant Gy500. We selected 1,251 differentially expressed genes that were up- or downregulated in response to the heat stress condition and in the heat-tolerant mutant Gy500, based on fragment per million reads expression values. Among them, PyHRG1 was downregulated under heat stress in SG104 and expressed at a low level in Gy500. PyHRG1 encodes a secretory protein of 26.5 kDa. PyHRG1 shows no significant sequence homology with any known genes deposited in public databases to date. However, PyHRG1 homologs were found in other red algae, including other Pyropia species. When PyHRG1 was introduced into the single-cell green alga Chlamydomonas reinhardtii, transformed cells overexpressing PyHRG1 showed severely retarded growth. These results demonstrate that PyHRG1 encodes a novel red algae-specific protein and plays a role in heat tolerance in algae. The transcriptome sequences obtained in this study, which include PyHRG1, will facilitate future studies to understand the molecular mechanisms involved in heat tolerance in red algae.