Molecular cloning reveals co-occurring species behind red tide blooms of the harmful dinoflagellate Cochlodinium polykrikoides

Red tides caused by the marine dinoflagellate Cochlodinium polykrikoides Margalef pose significant environmental problems worldwide. Recently, the existence of severe blooms attributable to a single Cochlodinium Schütt species has been questioned by many researchers. Herein we investigated the dinoflagellate composition of harmful algal blooms (HABs) attributed to C. polykrikoides in Korean coastal waters at nine different stations (St.). The component species of Cochlodinium blooms were examined by using microscopic and gene-cloning methods. In the nine study areas, C. polykrikoides was the predominant species of HABs in St. 2, 4, 7, and St. 9. Based on the morphological identification, the bloom was initially thought to be caused only by C. polykrikoides; however, we detected additional bloom-forming dinoflagellates (Polykrikos schwartzii Bütschli and Polykrikos kofoidii Chatton), and diatoms (Pseudo-nitzschia americana (Hasle) Fryxell) along with C. polykrikoides. The parasitic dinoflagellates Amoebophrya Koeppen and Euduboscquella Coats, Bachvaroff & Delwiche were found to be co-located with Cochlodinium in our study, and for the first time, Cochlodinium fulvescens Iwataki, Kawami & Matsuoka was detected in Korea (west coast). These results suggest co-existence of multiple dinoflagellates in bloom populations of Cochlodinium and describe the composition of other dinoflagellate blooms (e.g., Polykrikos spp.) in Korean coastal regions. This co-occurrence may be considered during efforts to monitor and control HABs.     

Alteration of plankton communities and biogeochemical cycles by harmful Cochlodinium polykrikoides (Dinophyceae) blooms

Cochlodinium polykrikoides is a globally distributed, ichthyotoxic, bloom-forming dinoflagellate. Blooms of C. polykrikoides manifest themselves as large (many km²) and distinct patches with cell densities exceeding 10³ ml⁻¹ while water adjacent to these patches can have low cell densities (<100 cells ml⁻¹). While the effect of these blooms on fish and shellfish is well-known, their impacts on microbial communities and biogeochemical cycles are poorly understood. Here, we investigated plankton communities and the cycling of carbon, nitrogen, and B-vitamins within blooms of C. polykrikoides and compared them to areas in close proximity (<100 m) with low C. polykrikoides densities. Within blooms, C. polykrikoides represented more than 90% of microplankton (>20 μm) cells, and there were significantly more heterotrophic bacteria and picoeukaryotic phytoplankton but fewer Synechococcus. Terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA genes revealed significant differences in community composition between bloom and non-bloom samples. Inside the bloom patches, concentrations of vitamin B₁₂ were significantly lower while concentrations of dissolved oxygen were significantly higher. Carbon fixation and nitrogen uptake rates were up to ten times higher within C. polykrikoides bloom patches. Ammonium was a more important source of nitrogen, relative to nitrate and urea, for microplankton within bloom patches compared to non-bloom communities. While uptake rates of vitamin B₁ were similar in bloom and non-bloom samples, vitamin B₁₂ was taken up at rates five-fold higher (>100 pmol⁻¹ L⁻¹ d⁻¹) in bloom samples, resulting in turn-over times of hours during blooms. This high vitamin demand likely led to the vitamin B₁₂ limitation of C. polykrikoides observed during nutrient amendment experiments conducted with bloom water. Collectively, this study revealed that C. polykrikoides blooms fundamentally change microbial communities and accelerate the cycling of carbon, some nutrients, and vitamin B₁₂.     

Development of real-time RT-PCR for detecting viable Cochlodinium polykrikoides (Dinophyceae) cysts in sediment

Morphological observations have confirmed that cysts are produced by dinoflagellates. However, finding a seed bed or unknown cysts in field samples by microscopy is extremely time consuming. Real-time PCR has been used to facilitate the detection of dinoflagellate cysts in sediment. However, DNA from dead vegetative cells remaining on the surface sediment may persist for a long period of time, which can cause false positive DNA detection. In this study, a non-quantitative RNA targeted probe using real-time RT-PCR was developed for detection of viable cysts in sediment. Large-subunit rRNA was used to develop a species-specific RNA targeted probe for the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. The sediment samples were sieved and incubated at 30 °C for 3 h prior to RNA extraction to remove RNA from dead cells remaining in the sediment. Nested-PCR was conducted to maximize assay sensitivity. A field survey to determine the distribution of cysts at 155 sampling stations in the western and southern part of the Korean peninsula showed that C. polykrikoides cysts were detected at five sampling stations.     

Molecular phylogeny of the spiny-surfaced species of the dinoflagellate Prorocentrum with the description of P. thermophilum sp. nov. and P. criophilum sp. nov. (Prorocentrales,Dinophyceae)

Spiny-surfaced species of Prorocentrum form harmful algal blooms, and its taxonomic identity is obscure due to the size and shape variability. Molecular phylogenies reveal two major clades: one for P. cordatum with sequences mainly retrieved as P. minimum, and the other for P. shikokuense with sequences also retrieved as P. dentatum and P. donghaiense. Several closely related clades still need to be characterized. Here, we provide nuclear SSU and LSU rRNA genes, and nuclear ITS region (ITS1-5.8S gene-ITS2) sequences of the strain CCMP3122 isolated from Florida (initially named P. donghaiense) and strains Prorocentrum sp. RCC6871–2 from the Ross Sea, Antarctica. We describe Prorocentrum thermophilum sp. nov. based on the strain CCMP3122, a species also distributed in the open waters of the Gulf of Mexico, New Zealand, and the Arabian Gulf; and Prorocentrum criophilum sp. nov. based on the strain RCC6872, which is distributed in the Antarctic Ocean and Arctic Sea. Prorocentrum thermophilum is roundish (~14 μm long, ~12 μm wide), with an inconspicuous anterior spine-like prolongation under light microscopy, valves with tiny, short knobs (5–7 per μm²), and several (<7) large trichocyst pores (~0.3 μm) in the right valve, as well as smaller pores (~0.15 μm). Prorocentrum criophilum is round in valve view (~11 μm long, 10 μm wide) and asymmetrically roundish in lateral view, the periflagellar area was not discernible under light microscopy, valves with very tiny, short knobs (6–10 per μm²), and at least 12 large pores in the right valve. Other potentially undescribed species of spiny-surfaced Prorocentrum are discussed.     

Growth and epiphytic behavior of three Gambierdiscus species (Dinophyceae) associated with various macroalgal substrates

Species of the benthic dinoflagellate Gambierdiscus produce polyether neurotoxins that caused ciguatera fish/shellfish poisoning in human. The toxins enter marine food webs by foraging of herbivores on the biotic substrates like macroalgae that host the toxic dinoflagellates. Interaction of Gambierdiscus and their macroalgal substrate hosts is believed to shape the tendency of substrate preferences and habitat specialization. This was supported by studies that manifested epiphytic preferences and behaviors in Gambierdiscus species toward different macroalgal hosts. To further examine the supposition, a laboratory-based experimental study was conducted to examine the growth, epiphytic behaviors and host preferences of three Gambierdiscus species towards four macroalgal hosts over a culture period of 40 days. The dinoflagellates Gambierdiscus balechii, G. caribaeus, and a new ribotype, herein designated as Gambierdiscus type 7 were initially identified based on the thecal morphology and molecular characterization. Our results showed that Gambierdiscus species tested in this study exhibited higher growth rates in the presence of macroalgal hosts. Growth responses and attachment behaviors, however, differed among different species and strains of Gambierdiscus over different macroalgal substrate hosts. Cells of Gambierdiscus mostly attached to substrate hosts at the beginning of the experiments but detached at the later time. Localized Gambierdiscus-host interactions, as demonstrated in this study, could help to better inform efforts of sampling and monitoring of this benthic toxic dinoflagellate.     

Differences in the photoacclimation and photoprotection exhibited by two species of the ciguatera causing dinoflagellate genus,Gambierdiscus

In culture, Gambierdiscus spp. have been shown to prefer irradiances that are relatively low (≤250 μmol photons m⁻² s⁻¹) versus those to which they are frequently exposed to in their natural environment (>500 μmol photons m⁻² s⁻¹). Although several behavioral strategies for coping with such irradiances have been suggested, it is unclear as to how these dinoflagellates do so on a physiological level. More specifically, how do long term exposures (30 days) affect cell size and cellular chlorophyll content, and what is the photosynthetic response to short term, high irradiance exposures (up to 1464 μmol photons m⁻² s⁻¹)? The results of this study reveal that cell size and chlorophyll content exhibited by G. carolinianus increased with acclimation to increasing photon flux density. Additionally, both G. carolinianus and G. silvae exhibited reduced photosynthetic efficiency when acclimated to increased photon flux density. Photosynthetic yield exhibited by G. silvae was greater than that for G. carolinianus across all acclimation irradiances. Although such differences were evident, both G. carolinianus and G. silvae appear to have adequate biochemical mechanisms to withstand exposure to irradiances exceeding 250 μmol photons m⁻² s⁻¹ for at least short periods of time following acclimation to irradiances of up to 150 μmol photons m⁻² s⁻¹.     

Improved real-time PCR method for quantification of the abundance of all known ribotypes of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides by comparing 4 different preparation methods

Red tides by the ichthyotoxic dinoflagellate Cochlodinium polykrikoides have caused large scaled mortality of fish and great loss in aquaculture industry in many countries. Detecting and quantifying the abundance of this species are the most critical step in minimizing the loss. The conventional quantitative real-time PCR (qPCR) method has been used for quantifying the abundance of this species. However, when analyzing > 500 samples collected during huge C. polykrikoides red tides in South Sea of Korea in 2014, this conventional method and the previously developed specific primer and probe set for C. polykrikoides did not give reasonable abundances when compared with cell counting data. Thus improved qPCR methods and a new specific primer and probe set reflecting recent discovery of 2 new ribotypes have to be developed. A new species-specific primer and probe set for detecting all 3 ribotypes of C. polykrikoides was developed and provided in this study. Furthermore, because the standard curve between cell abundance and threshold cycle value (Ct) is critical, the efficiencies of 4 different preparation methods used to determine standard curves were comparatively evaluated. The standard curves were determined by using the following 4 different preparations: (1) extraction of DNA from a dense culture of C. polykrikoides followed by serial dilution of the extracted DNA (CDD method), (2) extraction of DNA from each of the serially diluted cultures with different concentrations of C. polykrikoides cultures (CCD method), (3) extraction of DNA from a dense field sample of C. polykrikoides collected from natural seawater and then dilution of the extracted DNA in serial (FDD method), and (4) extraction of DNA from each of the serially diluted field samples having different concentrations of C. polykrikoides (FCD method). These 4 methods yielded different results. The abundances of C. polykrikoides in the samples collected from the coastal waters of South Sea, Korea, in 2014–2015, obtained using the standard curves determined by the CCD and the FCD methods, were the most similar (0.93–1.03 times) and the second closest (1.16–1.33 times) to the actual cell abundances obtained by enumeration of cells. Thus, our results suggest that the CCD method is a more effective tool to quantify the abundance of C. polykrikoides than the conventional method, CDD, and the FDD and FCD methods.     

Development of compound microsatellite markers in the harmful dinoflagellate Cochlodinium polykrikoides (Dinophyceae)

We isolated 15 polymorphic microsatellites from Cochlodinium polykrikoides. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from two to 15, and the estimate of gene diversity was from 0.083 to 0.880 across the 15 microsatellites. We consider that these loci have a potential to reveal the genetic structure and gene flow among C. polykrikoides populations.     

Identification of the resting cyst of Cochlodinium polykrikoides Margalef (Dinophyceae,Gymnodiniales) in Korean coastal sediments

This study provides the first morphological features of resting cysts of Cochlodinium polykrikoides collected from Korean coastal sediments. Evidence for the existence of resting cysts of C. polykrikoides is based on the morphological and molecular phylogenetic data of the germinated cells and a resting cyst. The morphology of the resting cysts differed from that reported previously in sediments and culture experiments. The distinct feature is that the cyst body was covered by the reticulate ornaments and spines.     

Dynamics of bacterial community structure during blooms of Cochlodinium polykrikoides (Gymnodiniales,Dinophyceae) in Korean coastal waters

Recent studies of dinoflagellates have reported that blooms can be closely related to the characteristics of the associated bacteria, but studies of the correlation between the toxic dinoflagellate, Cochlodinium polykrikoides and their associated bacterial community composition has not been explored. To understand this correlation, changes in bacterial community structure through the evolution of a C. polykrikoides bloom in Korean coastal waters via clone library analysis were investigated. Although there were no apparent changes in physio-chemical factors during the onset of the C. polykrikoides bloom, the abundance of bacteria bourgeoned in parallel with C. polykrikoides densities. Alpha-, gamma-proteobacteria and Flavobacteria were found to be dominant phyletic groups during C. polykrikoides blooms. The proportion of gamma-proteobacteria was lower (11.8%) during peak of the bloom period compared to the post-bloom period (26.2%). In contrast, alpha-proteobacteria increased in dominance during blooms. Among the alpha-proteobacteria, members of Rhodobacterales abruptly increased from 38% of the alpha-proteobacteria before the bloom to 74% and 56% during the early bloom and peak bloom stages, respectively. Moreover, multiple sites concurrently hosting C. polykrikoides blooms also contained high portions of Rhodobacterales and principal component analysis (PCA) demonstrated that Rhodobacterales had a positive, significant correlation with C. polykrikoides abundances (p ≤ 0.01, Pearson correlation coefficients). Collectively, this study reveals the specific clades of bacteria that increase (Rhodobacterales) and decrease (gamma-proteobacteria) in abundance C. polykrikoides during blooms.     

Mixotrophy in the phototrophic harmful alga Cochlodinium polykrikoides (Dinophycean): prey species, the effects of prey concentration, and grazing impact

We first reported here that the harmful alga Cochlodinium polykrikoides, which had been previously known as an autotrophic dinoflagellate, was a mixotrophic species. We investigated the kinds of prey species and the effects of the prey concentration on the growth and ingestion rates of C. polykrikoides when feeding on an unidentified cryptophyte species (Equivalent Spherical Diameter, ESD = 5.6 microm). We also calculated grazing coefficients by combining field data on abundances of C. polykrikoides and co-occurring cryptophytes with laboratory data on ingestion rates obtained in the present study. Cocholdinium polykrikoides fed on prey cells by engulfing the prey through the sulcus. Among the phytoplankton prey offered, C. polykrikoides ingested small phytoplankton species that had ESD's < or = 11 microm (e.g. the prymnesiophyte Isochrysis galbana, an unidentified cryptophyte, the cryptophyte Rhodomonas salina, the raphidophyte Heterosigma akashiwo, and the dinoflagellate Amphidinium carterae). It did not feed on larger phytoplankton species that had ESD's > or = 12 microm (e.g. the dinoflagellates Heterocapsa triquetra, Prorocentrum minimum, Scrippsiella sp., Alexandrium tamarense, Prorocentrum micans, Gymnodinium catenatum, Akashiwo sanguinea, and Lingulodinium polyedrum). Specific growth rates of C. polykrikoides on a cryptophyte increased with increasing mean prey concentration, with saturation at a mean prey concentration of approximately 270 ng C ml(-1) (i.e. 15,900 cells ml(-1)). The maximum specific growth rate (mixotrophic growth) of C. polykrikoides on a cryptophyte was 0.324 d(-1), under a 14:10 h light-dark cycle of 50 microE m(-2) s(-1), while its growth rate (phototrophic growth) under the same light conditions without added prey was 0.166 d(-1). Maximum ingestion and clearance rates of C. polykrikoides on a cryptophyte were 0.16 ng C grazer(-1)d(-1) (9.4 cells grazer(-1)d(-1)) and 0.33 microl grazer(-1)h(-1), respectively. Calculated grazing coefficients by C. polykrikoides on cryptophytes were 0.001-0.745 h(-1) (i.e. 0.1-53% of cryptophyte populations were removed by a C. polykrikoides population in 1 h). The results of the present study suggest that C. polykrikoides sometimes has a considerable grazing impact on populations of cryptophytes.     

Removal of harmful algal cells (Karenia brevis) and toxins from seawater culture by clay flocculation

Harmful algal blooms (HABs) occur worldwide causing serious threat to marine life, and to public health through seafood-borne illnesses and exposure to toxin-containing marine aerosol. This study was undertaken to assess the ability of phosphatic clay to remove the toxic dinoflagellate, Karenia brevis, and the potent neurotoxins (brevetoxins) produced by this species. Results showed that the addition of an aqueous slurry of 0.75 g (dry weight) clay to 3 l of K. brevis culture, containing 5×10⁶ and 10×10⁶ cells/l, removed 97±4% of brevetoxins from the water column within 4 h after the addition of clay. Clay flocculation of extra-cellular brevetoxins, released from cells ruptured (lyzed) by ultrasonication, removed 70±10% of the toxins. Addition of the chemical flocculant, polyaluminum chloride (PAC), removed all of the extra-cellular toxins. A 14 day study was undertaken to observe the fate of brevetoxins associated with clay flocculation of viable K. brevis cells. At 24 h following the clay addition, 90±18% of the toxins were removed from the water column, along with 85±4% of the cells. The toxin content of clay diminished from 208±13 μg at Day 1, to 121±21 μg at Day 14, indicating that the phosphatic clay retained about 58% of the toxins throughout the 14-day period. These studies showed the utility of natural clay as a means of reducing adverse effects from HABs, including removal of dissolved toxins, in the water column, although considerable work clearly remains before this approach can be used on natural blooms in open waters.     

Phylogenetic relationships of Cochlodinium polykrikoides Margalef (Gymnodiniales,Dinophyceae) from the Mediterranean Sea and the implications of its global biogeography

Although the diversity of dinoflagellates has been intensively studied in several locations in the Mediterranean Sea since the 1950s, it is only during the last two decades that the morphotype of the toxic unarmoured dinoflagellate Cochlodinium polykrikoides Margalef has been detected, coinciding with its apparent worldwide expansion in marine coastal waters. In this study, vegetative cells of C. polykrikoides morphotype from the Catalan coast (NW Mediterranean Sea) were detected and isolated, and the DNA from collected cells was sequenced. While in the Mediterranean Sea, detections are scarce and C. polykrikoides is consistently present at low concentrations, we reported exceptional blooms of this species, in which the maximum abundance reached 2 × 10⁴ cells L⁻¹. Partial LSU rDNA region sequences showed that most C. polykrikoides populations from the Catalan coast formed a new differentiated ribotype, but others were included within the ‘Philippines’ ribotype, demonstrating their coexistence in the Mediterranean Sea. Thus, the current biogeographic nomenclature of the ribotypes is likely to be invalid with respect to the available information from populations comprising the ‘Philippines’ ribotype. The phylogeny suggests the existence of cryptic species that should be evaluated for species-level status. Accordingly, the ribotype determination must be carefully evaluated for all detections and bloom events, since accurate characterization of the morphology, ecophysiology and distribution of the ribotypes are not well resolved.     

Harmful dinoflagellate Cochlodinium polykrikoides impairs the feeding behavior of larval sheepshead minnows (Cyprinodon variegatus)

Research evaluating the toxicity of the harmful dinoflagellate Cochlodinium (a.k.a. Margalefidinium) polykrikoides has been dominated by acute bioassays while the sublethal effects remain less well understood. This study examined the sublethal effects of C. polykrikoides exposure on the feeding behavior of larval estuarine fish. Sheepshead minnow (Cyprinodon variegatus) larvae were used in feeding experiments which assessed the total consumption of zooplankton prey (i.e., Artemia nauplii) over defined time periods. Larvae exposed to intermediate concentrations (i.e., 10² cells ml⁻¹) of clonal cultures of C. polykrikoides saw statistically significant reductions (range = 10%–81%) in the Artemia consumed compared to controls (i.e., filtered seawater, culture media or nontoxin producing dinoflagellate). These reductions were found independent of whether the larvae were fed or starved prior to experimentation. As these concentrations are similar to those typically found during mild blooms or at the periphery of dense blooms, these findings have significant implications for the feeding behavior of ichthyoplankton.     

Morphology,ultrastructure, and molecular phylogeny of Wangodinium sinense gen. et sp. nov. (Gymnodiniales,Dinophyceae) and revisiting of Gymnodinium dorsalisulcum and Gymnodinium impudicum

The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium‐like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop‐shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W. sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G. dorsalisulcum and G. impudicum, e.g., a short sulcal intrusion in G. dorsalisulcum; nuclear chambers in G. impudicum and G. dorsalisulcum; and a chloroplast enveloped by two membranes in G. impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G. dorsalisulcum, G. impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.     

Temporal changes in the cyst densities of Pyrodinium bahamense var. compressum and other dinoflagellates in Manila Bay, Philippines

Temporal variation in the type and abundance of dinoflagellate cysts in Manila Bay, Philippines, is established using ²¹⁰Pb-dated sediment cores. At least 17 dinoflagellate cyst species, including those of the toxic species, Pyrodinium bahamense var. compressum, were identified. P. bahamense may have been present in the area since at least the 1920s. Total cyst density has increased beginning about 1988 to 1998 coinciding with records of P. bahamense blooms in the area. Heterotrophs have always dominated the cysts assemblage. These changes in the dinoflagellate record and the P. bahamense blooms in recent years may have been induced by the interplay of warmer temperatures, high rainfall leading to higher river discharge and less turbulent waters due to passage of few tropical cyclones.     

Morphology,phylogeny, dynamics,and ichthyotoxicity of Pheopolykrikos hartmannii (Dinophyceae) isolates and blooms from New York,USA

We report on morphological observations, phylogenetic analyses, bloom dynamics, and ichthyotoxicity of the common but poorly characterized dinoflagellate Pheopolykrikos hartmannii (Zimmermann) Matsuoka et Fukuyo. From 2008 to 2010 in the Forge River Estuary, NY, USA, P. hartmannii bloomed during summer and early fall, achieving densities exceeding 8,000 cells · mL^?1 and often dominating microphytoplankton communities. Large subunit (LSU) and small subunit (SSU) rDNA sequences demonstrated that NY isolates of P. hartmannii sequences were 99%–100% identical to P. hartmannii isolates from eastern US and Korea. In both the LSU and SSU rDNA phylogenies, the clades containing P. hartmannii sequences were distinct sister clades to those composed of Polykrikos schwartzii and P. kofoidii. In the LSU rDNA phylogeny, however, the clade composed of P. hartmannii and a sequence of the photosynthetic Polykrikos lebourae was well separated from the clade composed of 10 entries of Polykrikos schwartzii and P. kofoidii. In addition, a gap of ~180 bases was observed when the LSU rDNA sequences of P. hartmannii were aligned with P. schwartzii and P. kofoidii but was not observed in the alignment between P. hartmannii and P. lebourae. Using scanning electron microscopy, several morphological features previously not reported for P. hartmannii were observed: a ventral groove located in the sulcus, a deep arc‐like apical concavity within the area of apical groove, scale‐like vesicles, and a shallow, completely enclosed, loop‐like apical groove. Resting cysts with arrow‐like surface spines were produced heterothallically by crossing clonal isolates and germinated single gymnoid cells. Finally, filtered and unfiltered bloom water from the Forge River and clonal cultures of P. hartmannii exhibited acute ichthyotoxicity to juvenile sheepshead minnows (Cyprinodon variegates) and aeration did not mitigate this effect, suggesting P. hartmannii is an ichthyotoxic, harmful alga.