The effect of iron on Chilean Alexandrium catenella growth and paralytic shellfish toxin production as related to algal blooms.

The dinoflagellate Alexandrium catenella is a well-known paralytic shellfish toxin producer that forms harmful algal blooms (HABs) worldwide. Blooms of this species have repeatedly brought severe ecological and economic impacts to Chile, especially in the southern region, where the shellfish and salmon industries are world-famous. The mechanisms of such HABs have been intensively studied but are still unclear. Nutrient overloading is one of the often-discussed drivers for HABs. The present study used the A. catenella strain isolated from southern Chile to investigate how iron conditions could affect their growth and toxin production as related to HAB. Our results showed that an optimum concentration of iron was pivotal for proper A. catenella growth. Thus, while excess iron exerted a toxic effect, low iron media led to iron insufficiency and growth inhibition. In addition, the study shows that the degree of paralytic shellfish toxin production by A. catenella varied depending on the iron concentration in the culture media. The A. catenella strain from southern Chile produced GTX1-4 exclusively in the fmol cell-1 scale. Based on these findings, we suggest that including iron and paralytic shellfish toxin measurements in the fields can improve the current HAB monitoring and contribute to an understanding of A. catenella bloom dynamics in Chile.

Alexandriicola marinus gen. nov., sp. nov., a new member of the family Rhodobacteraceae isolated from marine phycosphere.

Two yellow-pigmented bacterial strains, LZ-14 T and ABI-LZ29, were isolated from the cultivable phycosphere microbiota of the highly toxic marine dinoflagellate Alexandrium catenella LZT09 and demonstrated obvious microalgae growth-promoting potentials toward the algal host. To elucidate the taxonomic status of the two bioactive bacterial strains, they were subjected to a polyphasic taxonomic characterization. Both strains were found to be Gram-negative, aerobic, rod-shaped and motile; to contain Q-10 as the predominant ubiquinone; summed feature 8, C16:0, C18:1 ω7c 11-methyl and summed feature 3 as the major fatty acids; and diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and two unidentified phospholipids as the predominant polar lipids. Based on the phylogenetic analysis, phylogenomic inferences and phenotypic characteristics, the strains could be clearly distinguished from phylogenetically closely related species and formed a distinct monophyletic lineage in the family Rhodobacteraceae. The size of the draft genome of strain LZ-14 T is 4.615 Mb, with a DNA G + C content of 63.3 mol%. It contains ten predicted secondary metabolite biosynthetic gene clusters and core genes for bacterial exopolysaccharide biosynthesis. Therefore, strain LZ-14 T (= CCTCC AB 2017230 T = KCTC 62342 T) represents a novel species of a new genus, for which the name Alexandriicola marinus gen. nov., sp. nov., is proposed.

Taxonomic and Bioactivity Characterizations of Mameliella alba Strain LZ-28 Isolated from Highly Toxic Marine Dinoflagellate Alexandrium catenella LZT09.

Microalgae host varied microbial consortium harboring cross-kingdom interactions with fundamental ecological significance in aquatic ecosystems. Revealing the complex biofunctions of the cultivable bacteria of phycosphere microbiota is one vital basis for deeply understanding the mechanisms governing these dynamic associations. In this study, a new light-yellow pigmented bacterial strain LZ-28 was isolated from the highly-toxic and harmful algal bloom-forming dinoflagellate Alexandrium catenella LZT09. Collective phenotypic and genotypic profiles were obtained to confidently identify this strain as a new Mameliellaalba member. Comparative genomic analysis showed that strain LZ-28 shared highly similar functional features with other four marine algae-derived M. alba strains in spite of their distinctive isolation sources. Based on the bioactivity assaying, the mutual growth-promoting effects between bacterial strain LZ-28 and algal strain LZT09 were observed. After the culture conditions were optimized, strain LZ-28 demonstrated an extraordinary production ability for its bioflocculanting exopolysaccharides (EPS). Moreover, the portions of two monosaccharides glucose and fucose of the EPS were found to positively contribute to the bioflocculanting capacity. Therefore, the present study sheds light on the similar genomic features among the selected M. alba strains, and it also reveals the potential pharmaceutical, environmental and biotechnological implications of active EPS produced by this new Mameliella alba strain LZ-28 recovered from toxic bloom-forming marine dinoflagellate.

Maricaulis alexandrii sp. nov., a novel active bioflocculants-bearing and dimorphic prosthecate bacterium isolated from marine phycosphere.

An aerobic, Gram-stain-negative, straight or curved rods and dimorphic prosthecate bacterium designated as strain LZ-16-1T was isolated from phycosphere microbiota of routinely laboratory-cultured and highly-toxic marine dinoflagellate Alexandrium catenella LZT09. Strain LZ-16-1T produces active bioflocculanting exopolysaccharides (EPS). Cells were dimorphic with non-motile prostheca, or non-stalked and motile by a single polar flagellum. Growth occurred at 10-40 °C, pH 5-9 and 1-8% (w/v) NaCl, with optimum growth at 25 °C, pH 7-8 in the presence of 2-4% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain LZ-16-1T was affiliated to the genus Maricaulis, and closely related to M. parjimensis MCS 25T (99.5%) and M. virginensis VC-5T (99.0%). However, based on genome sequencing and phylogenomic calculations, the average nucleotide identity (ANI) and digtal DNA-DNA genome hybridization (dDDH) values between strains LZ-16-1T and its closest relative, M. parjimensis MCS 25T were only 85.0 and 20.9%, respectively. The dominant fatty acids of strain LZ-16-1T were summed feature 8, C16:0, C17:0, C18:0, C18:1 ω9c and summed feature 9. Major polar lipids were sulfoquinovosyl diacylglycerol, six glycolipids, one unidentified phospholipid and one unidentified polar lipid. The predominant isoprenoid quinone was Q-10. The DNA G + C content calculated from the genome was 63.6 mol%. Physiological and chemotaxonomic characterizations further confirmed the distinctiveness of strain LZ-16-1T from other Maricaulis members. Thus, strain LZ-16-1T represents a novel species of the genus Maricaulis, for which the name Maricaulis alexandrii sp. nov. (type strain LZ-16-1T = KCTC 72194T = CCTCC AB 2019006T) is proposed.

Low Temperature and Cold Stress Significantly Increase Saxitoxins (STXs) and Expression of STX Biosynthesis Genes sxtA4 and sxtG in the Dinoflagellate Alexandrium catenella.

Toxic dinoflagellate Alexandrium spp. produce saxitoxins (STXs), whose biosynthesis pathway is affected by temperature. However, the link between the regulation of the relevant genes and STXs' accumulation and temperature is insufficiently understood. In the present study, we evaluated the effects of temperature on cellular STXs and the expression of two core STX biosynthesis genes (sxtA4 and sxtG) in the toxic dinoflagellate Alexandrium catenella Alex03 isolated from Korean waters. We analyzed the growth rate, toxin profiles, and gene responses in cells exposed to different temperatures, including long-term adaptation (12, 16, and 20 °C) and cold and heat stresses. Temperature significantly affected the growth of A. catenella, with optimal growth (0.49 division/day) at 16 °C and the largest cell size (30.5 µm) at 12 °C. High concentration of STXs eq were detected in cells cultured at 16 °C (86.3 fmol/cell) and exposed to cold stress at 20→12 °C (96.6 fmol/cell) compared to those at 20 °C and exposed to heat stress. Quantitative real-time PCR (qRT-PCR) revealed significant gene expression changes of sxtA4 in cells cultured at 16 °C (1.8-fold) and cold shock at 20→16 °C (9.9-fold). In addition, sxtG was significantly induced in cells exposed to cold shocks (20→16 °C; 19.5-fold) and heat stress (12→20 °C; 25.6-fold). Principal component analysis (PCA) revealed that low temperature (12 and 16 °C) and cold stress were positively related with STXs' production and gene expression levels. These results suggest that temperature may affect the toxicity and regulation of STX biosynthesis genes in dinoflagellates.

Saccharospirillum alexandrii sp. nov., isolated from the toxigenic marine dinoflagellate Alexandrium catenella LZT09.

A Gram-stain-negative, strictly aerobic, non-motile and non-pigmented spirillum, designated strain LZ-5T, was isolated from cultures of the paralytic shellfish poisoning (PSP) toxin-producing marine dinoflagellate Alexandrium catenella LZT09 collected from the Zhoushan sea area in the East China Sea during an algal bloom. The isolate grew at 4-40 °C (optimum, 25-33 °C) and pH 5.0-9.0 (optimum, 7.5) in the presence of 0.5-10 % (w/v) NaCl (optimum, 4.0 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain LZ-5T clearly belonged to the genus Saccharospirillum of the family Saccharospirillaceae. Strain LZ-5T shared highest 16S rRNA gene sequence similarity with Saccharospirillum impatiens EL-105T (98.9 %), Saccharospirillum mangrovi HK-33T (97.2 %), Saccharospirillum correiae CPA1T (96.8 %), Saccharospirillum salsuginis YIM-Y25T (96.8 %) and Saccharospirillum aestuarii IMCC 4453T (95.1 %). The average nucleotide identity and in silico DNA-DNA hybridization between strain LZ-5T and the two most closely related Saccharospirillum strains, S. impatiens EL-105T and S. mangrovi HK-33T, were 82.2 and 19.3 %, and 72.2 and 13.2 %, respectively. The predominant respiratory quinone of strain LZ-5T was Q-8, and the major fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The polar lipids of strain LZ-5T were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), glycolipid (GL), two unidentified glycophospholipids (GPLs), three unidentified aminophospholipids (APLs) and two unidentified lipids. The genomic DNA G+C content was 57.2 mol%. On the basis of this polyphasic characterization, strain LZ-5T represents a novel species of the genus Saccharospirillum, for which the name Saccharospirillum alexandrii sp. nov. is proposed. The type strain is LZ-5T (=KCTC 62460T=CCTCC AB2017232T).

Haliea alexandrii sp. nov., isolated from phycosphere microbiota of the toxin-producing dinoflagellate Alexandrium catenella.

A Gram-negative, aerobic, non-motile, non-spore-forming and rod-shaped bacterium, named strain LZ-16-2T, was isolated from the phycosphere microbiota of the paralytic shellfish poisoning toxin-producing marine dinoflagellate Alexandrium catenella LZT09. Strain LZ-16-2T grew optimally at 28 °C at pH 6.5 and with 3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain LZ-16-2T fell within the genus Haliea and was most closely related to Haliea salexigens DSM 19537T, with which the new isolate exhibited 98.5 % 16S rRNA gene sequence similarity. The major respiratory quinone was Q-8. The predominant cellular fatty acids were C17 : 1 ω8c, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C17 : 1 ω6c, C11 : 0 3-OH and C17 : 0. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The average nucleotide identity and in silico DNA-DNA genome hybridization relatedness values between strain LZ-16-2T and its closest relative, H. salexigens DSM 19537T, were 92.8 and 55.1 %, respectively. The DNA G+C content was 61.3 mol%. Differential phenotypic properties and phylogenetic distinctiveness distinguished strain LZ-16-2T from all other members of the genus Haliea. On the basis of the polyphasic characterization, strain LZ-16-2T represents a novel species of the genus Haliea, for which the name Haliea alexandrii sp. nov. is proposed. The type strain is LZ-16-2T (=KCTC 62344T=CCTCC AB2017229T).

Limnobacter alexandrii sp. nov., a thiosulfate-oxidizing, heterotrophic and EPS-bearing Burkholderiaceae isolated from cultivable phycosphere microbiota of toxic Alexandrium catenella LZT09.

A novel Gram-negative, aerobic, motile and short rod-shaped bacterium with exopolysaccharides production, designated as LZ-4T, was isolated from cultivable phycosphere microbiota of harmful algal blooms-causing marine dinoflagellate Alexandrium catenella LZT09 which produces paralytic shellfish poisoning toxins. Strain LZ-4T was able to use thiosulfate (optimum concentration 10 mM) as energy source for bacterial growth. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain LZ-4T belonged to the genus Limnobacter, showing high 16S rRNA gene sequences similarities with L. thiooxidans DSM 13612T (99.4%), L. humi NBRC 11650T (98.2%) and L. litoralis NBRC 105857T (97.2%), respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between LZ-4T and L. thiooxidans DSM 13612T were 78.9 and 21.9%, respectively. Both values were far lower than the thresholds (95-96% for ANI and 70% for dDDH) generally accepted for new species delineation. The respiratory quinone of strain LZ-4T was Q-8. The dominant cellular fatty acids were determined as summed feature 3 (C16:1 ω6c/ω7c), summed feature 8 (C18:1 ω6c/ω7c) and C16:0. Polar lipids profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and three unidentified polar lipids. The genomic DNA G+C content of strain LZ-4T was 52.5 mol%. Based on polyphasic characterization, strain LZ-4T represents a novel species of the genus Limnobacter, for which the name Limnobacter alexandrii sp. nov. is proposed. The type strain is LZ-4T (=CCTCC AB 2019004T =KCTC 72281T).

Experimental exposure of the mussel Mytilus platensis (d'Orbigny, 1842) to the dinoflagellate Alexandrium catenella from Argentine Patagonia.

Individuals of Mytilus platensis were exposed to Alexandrium catenella to evaluate the accumulation and metabolization of paralytic shellfish toxins (PST) over a period of 25 days. Mussels were collected from the intertidal zone of Cerro Avanzado, Argentine Patagonia. After 16 days, the toxins in the tissues of mussels were detected by the methods of mouse bioassay and high performance liquid chromatography with fluorometric detection (HPLC-FDL). The accumulation kinetics of PST toxins in M. platensis fed with A. catenella fitted to a linear function, in which the accumulation rate was 31.2 µg STX eq kg-1 day-1. After 16 days, the PST toxin level in tissues of mussels reached 1178 µg STX eq kg-1 exceeding the safety limit for human consumption (800 µg STX eq kg-1 tissue), whereas the highest PST toxin level was reached at the end of the experimentation (1613 µg STX eq kg-1) at 25 days. Differences in the toxin profile of the dinoflagellates and the tissues of the mussels confirmed biotransformation of PST in the mussel digestive system. The toxin profile of M. platensis was dominated by the gonyautoxins GTX1 and GTX4, while the toxin profile of A. catenella was dominated by the N-sulfocarbamoyl toxin C2. To our knowledge, this is the first experimentation on a laboratory scale of PST toxins accumulation in M. platensis with a native strain of A. catenella of Argentine Patagonia.

iTRAQ-Based Quantitative Proteomic Analysis of a Toxigenic Dinoflagellate Alexandrium catenella at Different Stages of Toxin Biosynthesis during the Cell Cycle.

Paralytic shellfish toxins (PSTs) are a group of potent neurotoxic alkaloids that are produced mainly by marine dinoflagellates. PST biosynthesis in dinoflagellates is a discontinuous process that is coupled to the cell cycle. However, little is known about the molecular mechanism underlying this association. Here, we compared global protein expression profiles of a toxigenic dinoflagellate, Alexandrium catenella, collected at four different stages of toxin biosynthesis during the cell cycle, using an isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic approach. The results showed that toxin biosynthesis occurred mainly in the G1 phase, especially the late G1 phase. In total, 7232 proteins were confidently identified, and 210 proteins exhibited differential expression among the four stages. Proteins involved in protein translation and photosynthetic pigment biosynthesis were significantly upregulated during toxin biosynthesis, indicating close associations among the three processes. Nine toxin-related proteins were detected, and two core toxin biosynthesis proteins, namely, sxtA and sxtI, were identified for the first time in dinoflagellates. Among these proteins, sxtI and ompR were significantly downregulated when toxin biosynthesis stopped, indicating that they played important roles in the regulation of PST biosynthesis. Our study provides new insights into toxin biosynthesis in marine dinoflagellates: nitrogen balance among different biological processes regulates toxin biosynthesis, and that glutamate might play a key modulatory role.

qPCR assays for Alexandrium fundyense and A. ostenfeldii (Dinophyceae) identified from Alaskan waters and a review of species-specific Alexandrium molecular assays.

Paralytic shellfish poisoning (PSP) poses a serious health threat in Alaska and prevents effective utilization of shellfish resources by subsistence and recreational harvesters. Substantial economic losses also affect shellfish growers during PSP events. The toxins responsible for PSP are produced by dinoflagellates in the genus Alexandrium. Despite the persistent threat posed by PSP and the long history of shellfish toxicity research, there is still confusion concerning the Alexandrium species that cause PSP in Alaska. The primary objective of this study was to identify the toxic Alexandrium species present in Alaska and to develop polymerase chain reaction (PCR) assays for use in screening phytoplankton and sediment samples. Before developing the PCR assays for this study, we evaluated published assays and many were not adequate because of primer dimer formation or because of cross-reactivity. Rather than continue to grapple with the uncertainty and inadequacy of published assays, we developed new assays for the Alexandrium species most likely to be present in Alaska. Only Alexandrium fundyense Group I and A. ostenfeldii were identified from four sampling regions from southeast Alaska to Kodiak Island, indicating that these two species are widely distributed. PCR assays for these two species were converted to quantitative (q)PCR format for use in monitoring programs. During the course of this study, we realized that a systematic evaluation of all published (~150) Alexandrium species-specific assays would be of benefit. Toward this objective, we collated published Alexandrium PCR, qPCR, and in situ hybridization assay primers and probes that targeted the small-subunit (SSU), internal transcribed spacer (ITS/5.8S), or D1-D3 large-subunit (LSU) (SSU/ITS/LSU) ribosomal DNA genes. Each individual primer or probe was screened against the GenBank database and Alexandrium gene sequence alignments constructed as part of this study. These data were used to identify a suite of species-specific Alexandrium assays that can be recommended for evaluation by the global harmful algal bloom community.

iTRAQ-based quantitative proteomic analysis of a toxigenic dinoflagellate Alexandrium catenella and its non-toxic mutant.

Paralytic shellfish toxins (PSTs) are a group of potent neurotoxic alkaloids produced by cyanobacteria and dinoflagellates. The PST biosynthesis gene cluster and several toxin-related proteins have been unveiled in cyanobacteria, yet little is known about dinoflagellates. Here, we compared the protein profiles of a toxin-producing dinoflagellate Alexandrium catenella (ACHK-T) and its non-toxic mutant (ACHK-NT), and characterized differentially displayed proteins using a combination of the iTRAQ-based proteomic approach and the transcriptomic database. Totally 3488 proteins were identified from A. catenella, and proteins involved in carbohydrate, amino acid and energy metabolism were the most abundant. Among them, 185 proteins were differentially displayed: proteins involved in amino acid biosynthesis, protein and carbohydrate metabolism and bioluminescence were more abundant in ACHK-T, while proteins participating in photosynthesis, fatty acid biosynthesis, and the processes occurring in peroxisome displayed higher abundances in ACHK-NT. Seven toxin-related proteins were identified but they varied insignificantly between the two strains. Different carbon and energy utilization strategies were potentially related to the toxin producing ability, and the regulation mechanism of PST biosynthesis was more complex in dinoflagellates. Our study provides the first comprehensive dataset on the dinoflagellate proteome and lays the groundwork for future proteomic study.

Multispecies mass mortality in the Beagle Channel associated with paralytic shellfish toxins.

The Beagle Channel is a Subantarctic semi-estuarine environment at the southern tip of South America, where intoxication events associated with harmful algal blooms have been reported since 1886, including a world record in toxicity due to Alexandrium catenella in 1992. Toxic algae affect public health and ecosystem services, particularly mussel aquaculture and fisheries management. During the austral summer of 2022, an intense bloom of A. catenella (5 × 104 cells L-1) occurred in the Beagle Channel, leading to the second most toxic event in the area, with mussel toxicity reaching 197,266 µg STXeq kg-1. This event was synchronous with the mortality of marine organisms from different trophic levels and terrestrial fauna, i.e., two Fuegian red foxes and a southern caracara. Stomach content and liver samples from dead kelp gulls (Larus dominicanus), Magellanic penguins (Spheniscus magellanicus), papua penguins (Pygoscelis papua), and imperial cormorants (Leucocarbo atriceps), presented variable paralytic shellfish toxins (PST) levels (up to 3427 µg STXeq kg-1) as measured by high performance liquid chromatography (HPLC), suggesting that deaths were associated with high PST toxicity level. The different toxin profiles found in phytoplankton, zooplankton, squat lobsters (Grimothea gregaria), Fuegian sprat (Sprattus fuegensis), and seabirds evidenced possible toxin transformation along the food web and the possible transfer vectors. The unexpected detection of PST in terrestrial fauna (up to 2707 µg STXeq kg-1) suggested intoxication by scavenging on squat lobsters, which had high toxicity (26,663 µg STXeq kg-1). PST trace levels were also detected in a liver sample of a dead false killer whale (Pseudorca crassidens), an oceanic odontocete stranded on the coast during the bloom. Overall, our results denote the exceptional nature of the toxic, multispecies mortality event and that toxins may propagate to several levels of the food web in this Subantarctic environment.

Ingestion of paralytic shellfish toxins in a carnivorous gastropod (Chorus giganteus): effects on their elemental composition and reproductive traits.

The producer of paralytic shellfish toxin (PST), Alexandrium catenella, is one of the main generators of HABs in the coasts of Chile. Its presence produces ecological and economic damage, directly affecting filter-feeding organisms, and indirectly to other organism through the trophic chain. The objective of this research was to identify the effect of a toxic diet on the energetic and reproductive parameters of the carnivorous snail Chorus giganteus. Two groups of snails were used, one fed with toxic prey (bivalves fed with A. catenella), and the other fed with non-toxic prey. Both treatments were maintained under these conditions for 63 days, then, elemental composition (C, N) and energy content were estimated, and fecundity parameters were analyzed. The results indicate that snails fed with toxic prey had a lower percentage of C and C/N ratio. The energy content was significantly lower in intoxicated snails. Regarding fecundity parameters, a higher number of egg-masses were produced by toxic snails, however, only 62% of these showed embryonic development, with 57% hatching success. A negative relationship was identified between the mean PST concentration, quantified in snails, and the number of egg-masses produced per aquarium. In the aquarium where the snails had highest average PST concentration (1200 ± 820 µg STX.2HCL eq. Kg-1) there was no oviposition, while egg-masses were only produced by snails in aquaria where the average concentration did not exceed 360 ± 160 µg STX.2HCL eq. Kg-1. It is likely that, with low levels of accumulated PST, C. giganteus activates its oviposition process as a response to toxin-induced stress, generating a higher energy expenditure supported by a redirection of its reserves. However, when the intoxication presents higher levels, the reproductive process could be inhibited, similar to what has been identified in other molluscs.

Quantification of Alexandrium catenella (Group I) using sxtA4-based digital PCR for screening of paralytic shellfish toxins in Jinhae-Masan Bay, Korea.

We employed a detection method to quantify Alexandrium catenella (Group I), one of the causative species for paralytic shellfish poisoning (PSP) in Jinhae-Masan Bay, Korea, targets sxtA4, via chip-based digital PCR. Additionally, we explored the dynamics of Alexandrium during the spring of 2022 using an rDNA-based quantitative PCR (qPCR) assay to enhance the performance of the dPCR assay. In matching dPCR results with PSP monitoring reports, we optimized a cell regulatory threshold of 102 cells L-1, the maximum cell density when shellfish harvesting was permitted, for the dPCR assay. This threshold functioned similar to the PST threshold used in mouse bioassays (MBAs). Furthermore, we validated a total concordance rate of 83.8 % between the two assays for 2020-2022, reaching a maximum of 96.2 % in 2020. Thus, the result of dPCR could complement MBAs, facilitating the early detection of PSP outbreaks.

Allelochemicals determine competition and grazing control in Alexandrium catenella.

The production of allelochemicals by the toxigenic dinoflagellate Alexandrium catenella is one of the suggested mechanisms to facilitate its bloom formation and persistence by outcompeting other phototrophic protists and reducing grazing pressure. In Southern California, toxic events caused by A. catenella and paralytic shellfish toxins (PSTs) regularly impact coastal ecosystems; however, the trophic interactions and mechanisms promoting this species in a food web context are still not fully understood. In the present study, we combined a dynamical mathematical model with laboratory experiments to investigate potential toxic and allelochemical effects of an A. catenella strain isolated off the coast of Los Angeles, Southern California, on competitors and a common zooplankton consumer. Experiments were conducted using three toxigenic strains of A. catenella, comparing the new Californian isolate (Alex Cal) to two strains previously described from the North Sea, a lytic (Alex2) and non-lytic (Alex5) strain, testing for donor density-dependent effects on two phytoplankton species (Rhodomonas salina, Tetraselmis sp.) and on the rotifer Brachionus plicatilis. Bioassays revealed a steep decline in competitor and consumer populations with increasing Alex Cal concentrations, indicating an intermediate lytic activity compared to the North Sea strains (lytic Alex2 and non-lytic Alex5). The rotifer fed and grew well on the PST- toxic, but non-lytic Alex5 strain, while its survival significantly decreased with increasing concentrations of the two lytic strains Alex Cal and Alex 2, indicating that negative effects on the rotifer were mediated by allelochemicals rather than PST-toxins. Mixed culture experiments including both competitors and consumers demonstrated that the intensity of allelochemical effects not only depended on the A. catenella density but also on the target density. Negative effects on grazers were alleviated by co-occurring competitors with a lower sensitivity to allelochemicals, thus reducing harmful compounds and allowing grazing control on the dinoflagellate to come into effect again. Results from mixed culture experiments were supported by the mathematical approach used in this study which was calibrated with data from simple monoculture growth, pairwise competition and predator-prey experiments, demonstrating the applicability of this model approach to predict the outcome of more complex food web dynamics at the community level.

Proteomic and toxicological analysis of the response of dinoflagellate Alexandrium catenella to changes in NaNO3 concentration.

Dinoflagellates of the genus Alexandrium cause Harmful Algal Blooms (HABs) in coastal waters worldwide, damaging marine environments, aquaculture, and human health. They synthesize potent neurotoxic alkaloids known as PSTs (i.e., Paralytic Shellfish Toxins), the etiological agents of PSP (i.e., Paralytic Shellfish Poisoning). In recent decades, the eutrophication of coastal waters with inorganic nitrogen (e.g., nitrate, nitrite, and ammonia) has increased the frequency and scale of HABs. PSTs concentrations within Alexandrium cells can increase by up to 76% after a nitrogen enrichment event; however, the mechanisms that underlie their biosynthesis in dinoflagellates remains unclear. This study combines mass spectrometry, bioinformatics, and toxicology and investigates the expression profiles of PSTs in Alexandrium catenella grown in 0.4, 0.9 and 1.3 mM NaNO3. Pathway analysis of protein expression revealed that tRNA amino acylation, glycolysis, TCA cycle and pigment biosynthesis were upregulated in 0.4 mM and downregulated in 1.3 mM NaNO3 compared to those grown in 0.9 mM NaNO3. Conversely, ATP synthesis, photosynthesis and arginine biosynthesis were downregulated in 0.4 mM and upregulated in 1.3 mM NaNO3. Additionally, the expression of proteins involved in PST biosynthesis (sxtA, sxtG, sxtV, sxtW and sxtZ) and overall PST production like STX, NEO, C1, C2, GTX1-6 and dcGTX2 was higher at lower nitrate concentrations. Therefore, increased nitrogen concentrations increase protein synthesis, photosynthesis, and energy metabolism and decrease enzyme expression in PST biosynthesis and production. This research provides new clues about how the changes in the nitrate concentration can modulate different metabolic pathways and the expression of PST biosynthesis in toxigenic dinoflagellates.

Nitrogen liberated via allelopathy can promote harmful algal blooms.

Allelopathy is a biological mechanism that can promote harmful algal blooms (HAB) via the inhibition of sympatric phytoplankton. While nutrient loading can also promote HABs, the ability of allelopathy to stimulate HABs via the regeneration of nutrients has yet to be explored. To examine the impacts of allelopathically liberated N on HAB species, a series of experiments were performed using multiple allelopathic HAB species including the dinoflagellates Alexandrium catenella and Margalefidinium polykrikoides, and the pelagophyte, Aureoumbra lagunensis. These HAB species were paired with the cosmopolitan dinoflagellate, Akashiwo sanguinea, that was labeled with 15NO3- or 15NH4+, allowing the release and transfer of N to be traced as a time course during allelopathic interactions. During all experiments, the allelopathic inhibition of Akashiwo was accompanied by increases in cell densities, growth rates, and the δ15N content of the HAB species, evidencing the transfer of N liberated from Akashiwo. The cellular transfer of 15N and release of dissolved N was higher when Akashiwo was grown with 15NO3- compared to 15NH4+ suggesting a differential subcellular-compartmentalization of N sources. Regardless of the type of N, HAB species obtained 60 - 100% of their cellular N from lysed Akashiwo cells and there was an enrichment of the δ15N content of the dissolved NH4+ pool post-lysis of Akashiwo. Collectively, the results demonstrate that beyond facilitating species succession, allelopathy can supply HABs with N and, therefore, is likely important for promoting and sustaining HABs. Given that allelopathy is known to be a dose-dependent process, allelopathy may induce a positive feedback loop, whereby competitors are lysed, N is liberated, HABs are intensified and, in turn, become more strongly allelopathic.

Small and patchy is enough: An example about how toxic HAB events can spread through low resting cyst loads.

The frequency of harmful algal blooms (HABs) has increased over the last two decades, a phenomenon enhanced by global climate change. However, the effects of climate change will not be distributed equally, and Chile has emerged as one important, vulnerable area. The Chilean Patagonian region (41‒56°S) hosts two marine ecoregions that support robust blue economies via wild fisheries, aquaculture, and tourism. However, the harmful algal bloom-forming dinoflagellate Alexandrium catenella, a causative agent of paralytic shellfish poisoning outbreaks, threatens the viability of blue industries in this region and others worldwide. Despite the proliferation of A. catenella blooms over the last few decades, the role of sedimentary resting cysts in the recurrence of harmful algal blooms and the species' northward expansion across Chilean Patagonia is not well understood. As a resting cyst-producing species, the sediment-cyst dynamics of A. catenella likely contribute to the geographical expansion and bloom recurrence of this species. For this purpose, we analyzed a decade of A. catenella surface sediment cyst records across the two ecoregions of the Chilean Patagonian System that were further stratified into five subregions based on water temperature, salinity, dissolved oxygen, and nutrient characteristics. We also analyzed spatio-temporal cyst dynamics in a pre-, during-, and post-bloom scenario of the Chiloense ecoregion (more northern) of the Magellanic province. Our results indicated highly variable A. catenella resting cyst abundances, with a maximum of 221 cysts cm-3 recorded in 2002 after an intense bloom. Generalized linear mixed models and linear mixed models found that sampling season, subregion, and Total Organic Matter (%) explained resting cyst presence and density. The results also demonstrated the presence of A. catenella cysts in northern subregions, evidencing the northward geographical expansion observed during the last few decades. The risks of A. catenella bloom recurrence from small, patchy resting cyst distributions across broad geographical areas and under changing environmental conditions are discussed.

Grazers modify the dinoflagellate relationship between toxin production and cell growth.

Although the typical framework for studies and models of bloom dynamics in toxigenic phytoplankton is predominantly based on abiotic determinants, there is mounting evidence of grazer control of toxin production. We tested for the effect of grazer control of toxin production and cell growth rate during a laboratory-simulated bloom of the dinoflagellate Alexandrium catenella. We measured cellular toxin content and net growth rate when cells were exposed to copepod grazers (direct exposure), copepod cues (indirect exposure), and no copepods (control) throughout the exponential, stationary, and declining phases of the bloom. During the simulated bloom, cellular toxin content plateaued after the stationary phase and there was a significantly positive relationship between growth rate and toxin production, predominantly in the exponential phase. Grazer-induced toxin production was evident throughout the bloom, but highest during the exponential phase. Induction was greater when cells were directly exposed to grazers rather than their cues alone. In the presence of grazers toxin production and cell growth rate were negatively related, indicating a defense-growth trade-off. Further, a fitness reduction associated with toxin production was more evident in the presence than the absence of grazers. Consequently, the relationship between toxin production and cell growth is fundamentally different between constitutive and inducible defense. This suggests that understanding and predicting bloom dynamics requires considering both constitutive and grazer-induced toxin production.