Cytotoxicity of Prymnesium parvum extracts and prymnesin analogs on epithelial fish gill cells RTgill-W1 and the human colon cell line HCEC-1CT.

Harmful algal blooms kill fish populations worldwide, as exemplified by the haptophyte microalga Prymnesium parvum. The suspected causative agents are prymnesins, categorized as A-, B-, and C-types based on backbone carbon atoms. Impacts of P. parvum extracts and purified prymnesins were tested on the epithelial rainbow trout fish gill cell line RTgill-W1 and on the human colon epithelial cells HCEC-1CT. Cytotoxic potencies ranked A > C > B-type with concentrations spanning from low (A- and C-type) to middle (B-type) nM ranges. Although RTgill-W1 cells were about twofold more sensitive than HCEC-1CT, the cytotoxicity of prymnesins is not limited to fish gills. Both cell lines responded rapidly to prymnesins; with EC50 values for B-types in RTgill-W1 cells of 110 ± 11 nM and 41.5 ± 0.6 nM after incubations times of 3 and 24 h. Results of fluorescence imaging and measured lytic effects suggest plasma membrane interactions. Postulating an osmotic imbalance as mechanisms of toxicity, incubations with prymnesins in media lacking either Cl-, Na+, or Ca2+ were performed. Cl- removal reduced morphometric rearrangements observed in RTgill-W1 and cytotoxicity in HCEC-1CT cells. Ca2+-free medium in RTgill-W1 cells exacerbated effects on the cell nuclei. Prymnesin composition of different P. parvum strains showed that analog composition within one type scarcely influenced the cytotoxic potential, while analog type potentially dictate potency. Overall, A-type prymnesins were the most potent ones in both cell lines followed by the C-types, and lastly B-types. Disturbance of Ca2+ and Cl- ionoregulation may be integral to prymnesin toxicity.

The combined effect of pH and dissolved inorganic carbon concentrations on the physiology of plastidic ciliate Mesodinium rubrum and its cryptophyte prey.

Ocean acidification is caused by rising atmospheric partial pressure of CO2 (pCO2) and involves a lowering of pH combined with increased concentrations of CO2 and dissolved in organic carbon in ocean waters. Many studies investigated the consequences of these combined changes on marine phytoplankton, yet only few attempted to separate the effects of decreased pH and increased pCO2. Moreover, studies typically target photoautotrophic phytoplankton, while little is known of plastidic protists that depend on the ingestion of plastids from their prey. Therefore, we studied the separate and interactive effects of pH and DIC levels on the plastidic ciliate Mesodinium rubrum, which is known to form red tides in coastal waters worldwide. Also, we tested the effects on their prey, which typically are cryptophytes belonging to the Teleaulax/Plagioslemis/Geminigera species complex. These cryptophytes not only serve as food for the ciliate, but also as a supplier of chloroplasts and prey nuclei. We exposed M. rubrum and the two cryptophyte species, T. acuta, T. amphioxeia to different pH (6.8 - 8) and DIC levels (∼ 6.5 - 26 mg C L-1) and assessed their growth and photosynthetic rates, and cellular chlorophyll a and elemental contents. Our findings did not show consistent significant effects across the ranges in pH and/or DIC, except for M. rubrum, for which growth was negatively affected only by the lowest pH of 6.8 combined with lower DIC concentrations. It thus seems that M. rubrum is largely resilient to changes in pH and DIC, and its blooms may not be strongly impacted by the changes in ocean carbonate chemistry projected for the end of the 21st century.

Local adaptation through countergradient selection in northern populations of Skeletonema marinoi.

Marine microorganisms have the potential to disperse widely with few obvious barriers to gene flow. However, among microalgae, several studies have demonstrated that species can be highly genetically structured with limited gene flow among populations, despite hydrographic connectivity. Ecological differentiation and local adaptation have been suggested as drivers of such population structure. Here we tested whether multiple strains from two genetically distinct Baltic Sea populations of the diatom Skeletonema marinoi showed evidence of local adaptation to their local environments: the estuarine Bothnian Sea and the marine Kattegat Sea. We performed reciprocal transplants of multiple strains between culture media based on water from the respective environments, and we also allowed competition between strains of estuarine and marine origin in both salinities. When grown alone, both marine and estuarine strains performed best in the high-salinity environment, and estuarine strains always grew faster than marine strains. This result suggests local adaptation through countergradient selection, that is, genetic effects counteract environmental effects. However, the higher growth rate of the estuarine strains appears to have a cost in the marine environment and when strains were allowed to compete, marine strains performed better than estuarine strains in the marine environment. Thus, other traits are likely to also affect fitness. We provide evidence that tolerance to pH could be involved and that estuarine strains that are adapted to a more fluctuating pH continue growing at higher pH than marine strains.

Effects of Glacial Flour on Marine Micro-plankton: Evidences from Natural Communities of Greenlandic Fjords and Experimental Studies.

Meltwater runoff from glaciers carries particles, so-called glacial flour that may affect planktonic organisms and the functioning of marine ecosystems. Protist microplankton is at the base of marine food webs and thus plays an important role in sustaining important ecosystem services. To assess the effect of glacial flour on photoautotrophic, heterotrophic and mixotrophic microplankton, the spatial distribution of these trophic groups was studied in four Greenlandic fjords during summer. The results suggest that the abundance of the autotrophic microplankton was affected by the glacier meltwater due to reduced light penetration and nutrient availability. The abundance of heterotrophic and mixotrophic microplankton were not apparently affected by the glacier meltwater. Incubation experiments were conducted on the natural population and in laboratory cultures of two mixoplanktonic ciliate species. The experiments on the natural population revealed that none of the trophic groups were affected by the suspended material at concentrations up to 50 mg L-1. The experiments on cultures gave no indication that glacial flour was ingested by the mixoplanktonic ciliates. Growth rates of cultured ciliates were not affected by the glacial flour addition. These results suggest that heterotrophic and mixotrophic microplankton are not affected by glacial flour as much as autotrophic microplankton.

Impacts of inorganic nutrients on the physiology of a mixoplanktonic ciliate and its cryptophyte prey.

Many marine planktonic ciliates retain functional chloroplasts from their photosynthetic prey and use them to incorporate inorganic carbon via photosynthesis. While this strategy provides the ciliates with carbon, little is known about their ability to incorporate major dissolved inorganic nutrients, such as nitrogen and phosphorus. Here, we studied how ciliates respond to different concentrations of dissolved inorganic nitrogen and phosphorus. Specifically, we tested the direct and indirect effects of nutrient availability on the ciliate Strombidium cf. basimorphum fed the cryptophyte prey Teleaulax amphioxeia. We assessed responses in the rates of growth, ingestion, photosynthesis, inorganic nutrient uptake, and excretion. Our results show that the prey changed its carbon content depending on the nutrient concentrations. Low inorganic nutrient concentrations increased S. cf. basimorphum growth and prey ingestion. The higher carbon content of the prey under these low nutrient conditions likely supported the growth of the ciliate, while the higher carbon:nutrient stoichiometry of the prey led to the higher ingestion rates. The low carbon content of the prey at high nutrient concentrations resulted in reduced growth of S. cf. basimorphum, which indicates that carbon acquired via photosynthesis in the ciliate cannot compensate for the ingestion of prey with low carbon content. In conclusion, our findings show S. cf. basimorphum is not able to utilize dissolved inorganic nitrogen and phosphorus for growth, and this species seems to be well adapted to exploit its prey when grown at low nutrient conditions.

The coupling between irradiance, growth, photosynthesis and prymnesin cell quota and production in two strains of the bloom-forming haptophyte, Prymnesium parvum.

Prymnesium parvum causes harmful algal blooms worldwide that are often associated with massive fish-kills and subsequent economic losses. Most of our knowledge of the toxicity of P. parvum derives from bioassays since methods for the identification and quantification of their toxins have been lacking. Recently, a quantitation method was developed for the causative lytic toxins, the prymnesins. Here, we for the first time present data on the influence of irradiance on cellular content and production of prymnesins under nutrient replete conditions in two P. parvum strains, which both produce B-type prymnesins. Large differences were observed between the two strains with regard to the influence of irradiance on prymnesin cell quota and production rates. At the highest irradiance level (550 µmol photons m-2 s-1), the cellular prymnesin quota was thirty times higher in strain K-0081 strain than in K-0374. The cellular prymnesin quota and production rates were closely linked to rates of growth and photosynthesis in strain K-0081, while this was not the case for K-0374. Yet, growth rate did explain the differences in prymnesin quota in the two strains. Consequently, the maximum prymnesin production rate (414 attomol cell-1 d-1) was only about three times higher in strain K-0081 than in K-0374, and revealed an optimum at the same irradiance of 200 µmol photons m-2 s-1 in both strains. At low irradiance levels, the difference in production rates between both strains became smaller, with 41 and 49 attomol cell-1 d-1 for K-0081 and K-0374, respectively. The carbon content of prymnesins made up for ∼3% and <1% of the total cellular carbon content in strains K-0081 and K-0374, respectively. The fraction of extracellular dissolved prymnesins was measured for strain K-0081, where it accounted for 14-30% of total prymnesin concentration in the cultures, irrespective of irradiance. The concentrations of prymnesins released to the water by the K-0081 strain were not significantly influenced by irradiance. Overall, we observed comparable responses in growth and photosynthesis of both tested strains toward changes in irradiance. However, the effects of irradiance on prymnesin quota and production rates were remarkably different between the two strains.

The strengths and weaknesses of Live Fluorescently Labelled Algae (LFLA) to estimate herbivory in protozooplankton and mixoplankton.

The Live Fluorescently Labelled Algae (LFLA) technique has been used numerous times to estimate microzooplankton herbivory. Yet, it is unknown how mixoplankton (i.e., single-cell organisms that can combine phototrophy and phagotrophy) affect the outcome of this technique. Hence, we conducted a broad-spectrum assessment of the strengths and weaknesses of the LFLA technique, using several mixoplanktonic and protozooplanktonic grazers. Species from different taxonomic groups and different feeding mechanisms were tested in short-term experiments (ca. 5 h) in the laboratory, at different prey concentrations and during light and dark periods of the day. Overall, our findings suggest that the LFLA technique, due to its short-term nature, is an effective tracker of diel ingestion and digestion rates, and can detect new mixoplanktonic predators. We recommend that, irrespective of the prey concentration, incubations to measure grazing rates with this technique should generally be concluded within 1 h (adaptable to the environmental temperature). Nevertheless, our results also call for caution whenever using LFLA in the field: feeding mechanisms other than direct engulfment (like peduncle feeding) may provide severely biased ingestion rates. Furthermore, size and species selectivity are very hard to circumvent. To reduce the effects of selectivity, we propose the combined use of two distinctly coloured fluorochromes (i.e., distinct emission spectra). With this modification, one could either label different size ranges of prey or account for species-specific interactions in the food web.

Mixoplankton interferences in dilution grazing experiments.

It remains unclear as to how mixoplankton (coupled phototrophy and phagotrophy in one cell) affects the estimation of grazing rates obtained from the widely used dilution grazing technique. To address this issue, we prepared laboratory-controlled dilution experiments with known mixtures of phyto-, protozoo-, and mixoplankton, operated under different light regimes and species combinations. Our results evidenced that chlorophyll is an inadequate proxy for phytoplankton when mixoplankton are present. Conversely, species-specific cellular counts could assist (although not fully solve) in the integration of mixoplanktonic activity in a dilution experiment. Moreover, cell counts can expose prey selectivity patterns and intraguild interactions among grazers. Our results also demonstrated that whole community approaches mimic reality better than single-species laboratory experiments. We also confirmed that light is required for protozoo- and mixoplankton to correctly express their feeding activity, and that overall diurnal grazing is higher than nocturnal. Thus, we recommend that a detailed examination of initial and final plankton communities should become routine in dilution experiments, and that incubations should preferably be started at the beginning of both day and night periods. Finally, we hypothesize that in silico approaches may help disentangle the contribution of mixoplankton to the community grazing of a given system.

Gill Transcriptomic Responses to Toxin-producing Alga Prymnesium parvum in Rainbow Trout.

The gill of teleost fish is a multifunctional organ involved in many physiological processes, including protection of the mucosal gill surface against pathogens and other environmental antigens by the gill-associated lymphoid tissue (GIALT). Climate change associated phenomena, such as increasing frequency and magnitude of harmful algal blooms (HABs) put extra strain on gill function, contributing to enhanced fish mortality and fish kills. However, the molecular basis of the HAB-induced gill injury remains largely unknown due to the lack of high-throughput transcriptomic studies performed on teleost fish in laboratory conditions. We used juvenile rainbow trout (Oncorhynchus mykiss) to investigate the transcriptomic responses of the gill tissue to two (high and low) sublethal densities of the toxin-producing alga Prymnesium parvum, in relation to non-exposed control fish. The exposure time to P. parvum (4-5 h) was sufficient to identify three different phenotypic responses among the exposed fish, enabling us to focus on the common gill transcriptomic responses to P. parvum that were independent of dose and phenotype. The inspection of common differentially expressed genes (DEGs), canonical pathways, upstream regulators and downstream effects pointed towards P. parvum-induced inflammatory response and gill inflammation driven by alterations of Acute Phase Response Signalling, IL-6 Signalling, IL-10 Signalling, Role of PKR in Interferon Induction and Antiviral Response, IL-8 Signalling and IL-17 Signalling pathways. While we could not determine if the inferred gill inflammation was progressing or resolving, our study clearly suggests that P. parvum blooms may contribute to the serious gill disorders in fish. By providing insights into the gill transcriptomic responses to toxin-producing P. parvum in teleost fish, our research opens new avenues for investigating how to monitor and mitigate toxicity of HABs before they become lethal.

Retention of Prey Genetic Material by the Kleptoplastidic Ciliate Strombidium cf. basimorphum.

Many marine ciliate species retain functional chloroplasts from their photosynthetic prey. In some species, the functionality of the acquired plastids is connected to the simultaneous retention of prey nuclei. To date, this has never been documented in plastidic Strombidium species. The functionality of the sequestered chloroplasts in Strombidium species is thought to be independent from any nuclear control and only maintained via frequent replacement of chloroplasts from newly ingested prey. Chloroplasts sequestered from the cryptophyte prey Teleaulax amphioxeia have been shown to keep their functionality for several days in the ciliate Strombidium cf. basimorphum. To investigate the potential retention of prey genetic material in this ciliate, we applied a molecular marker specific for this cryptophyte prey. Here, we demonstrate that the genetic material from prey nuclei, nucleomorphs, and ribosomes is detectable inside the ciliate for at least 5 days after prey ingestion. Moreover, single-cell transcriptomics revealed the presence of transcripts of prey nuclear origin in the ciliate after 4 days of prey starvation. These new findings might lead to the reconsideration of the mechanisms regulating chloroplasts retention in Strombidium ciliates. The development and application of molecular tools appear promising to improve our understanding on chloroplasts retention in planktonic protists.

Metabolic Reliance on Photosynthesis Depends on Both Irradiance and Prey Availability in the Mixotrophic Ciliate, Strombidium cf. basimorphum.

Many species of the ciliate genus Strombidium can acquire functional chloroplasts from a wide range of algal prey and are thus classified as generalist non-constitutive mixotrophs. Little, however, is known about the influence of irradiance and prey availability on their ability to exploit the photosynthetic potential of the chloroplasts, and how this may explain their spatial and temporal distribution in nature. In this study, inorganic carbon uptake, growth, and ingestion rates were measured for S. cf. basimorphum under three different irradiances (10, 40, and 120 µmol photons m-2 s-1) when acclimated to three different prey densities (5 × 103, 1 × 104, and 4 × 104 cells mL-1), as well as when allowed to deplete the prey. After prey depletion, cultures survived without prey longest (∼6 days) at the medium irradiance treatment (40 µmol photons m-2 s-1), while ciliate density, inorganic carbon uptake rates, and cellular chl-a content declined fastest at the highest irradiance treatment. This indicates that the ciliates may be unable to maintain the chloroplasts functionally without replacement at high irradiances. Ingestion rates were not shown to be significantly influenced by irradiance. The maximum gross growth efficiency (GGE) in this study (1.1) was measured in cultures exposed to the medium test irradiance and lowest prey density treatment (5 × 103 cells mL-1). The relative contribution of inorganic carbon uptake to the ciliate carbon budget was also highest in this treatment (42%). A secondary GGE peak (0.99) occurred when cultures were exposed to the highest test irradiance and the medium prey density. These and other results suggest that S. cf. basimorphum, and other generalist non-constitutive mixotrophs, can flexibly exploit many different environmental conditions across the globe.

Polyketide synthase genes and molecular trade-offs in the ichthyotoxic species Prymnesium parvum.

Prymnesium parvum is a bloom forming haptophyte that has been responsible for numerous fish kill events across the world. The toxicity of P. parvum has been attributed to the production of large polyketide compounds, collectively called prymnesins, which based on their structure can be divided into A-, B- and C-type. The polyketide chemical nature of prymnesins indicates the potential involvement of polyketide synthases (PKSs) in their biosynthesis. However, little is known about the presence of PKSs in P. parvum as well as the potential molecular trade-offs of toxin biosynthesis. In the current study, we generated and analyzed the transcriptomes of nine P. parvum strains that produce different toxin types and have various cellular toxin contents. Numerous type I PKSs, ranging from 37 to 109, were found among the strains. Larger modular type I PKSs were mainly retrieved from strains with high cellular toxin levels and eight consensus transcripts were present in all nine strains. Gene expression variance analysis revealed potential molecular trade-offs associated with cellular toxin quantity, showing that basic metabolic processes seem to correlate negatively with cellular toxin content. These findings point towards the presence of metabolic costs for maintaining high cellular toxin quantity. The detailed analysis of PKSs in P. parvum is the first step towards better understanding the molecular basis of the biosynthesis of prymnesins and contributes to the development of molecular tools for efficient monitoring of future blooms.

An under-ice bloom of mixotrophic haptophytes in low nutrient and freshwater-influenced Arctic waters.

The pelagic spring bloom is essential for Arctic marine food webs, and a crucial driver of carbon transport to the ocean depths. A critical challenge is understanding its timing and magnitude, to predict its changes in coming decades. Spring bloom onset is typically light-limited, beginning when irradiance increases or during ice breakup. Here we report an acute 9-day under-ice algal bloom in nutrient-poor, freshwater-influenced water under 1-m thick sea ice. It was dominated by mixotrophic brackish water haptophytes (Chrysochromulina/ Prymnesium) that produced 5.7 g C m-2 new production. This estimate represents about half the annual pelagic production, occurring below sea ice with a large contribution from the mixotrophic algae bloom. The freshwater-influenced, nutrient-dilute and low light environment combined with mixotrophic community dominance implies that phagotrophy played a critical role in the under-ice bloom. We argue that such blooms dominated by potentially toxic mixotrophic algae might become more common and widespread in the future Arctic Ocean.

Limits to the cellular control of sequestered cryptophyte prey in the marine ciliate Mesodinium rubrum.

The marine ciliate Mesodinium rubrum is famous for its ability to acquire and exploit chloroplasts and other cell organelles from some cryptophyte algal species. We sequenced genomes and transcriptomes of free-swimming Teleaulax amphioxeia, as well as well-fed and starved M. rubrum in order to understand cellular processes upon sequestration under different prey and light conditions. From its prey, the ciliate acquires the ability to photosynthesize as well as the potential to metabolize several essential compounds including lysine, glycan, and vitamins that elucidate its specific prey dependency. M. rubrum does not express photosynthesis-related genes itself, but elicits considerable transcriptional control of the acquired cryptophyte organelles. This control is limited as light-dependent transcriptional changes found in free-swimming T. amphioxeia got lost after sequestration. We found strong transcriptional rewiring of the cryptophyte nucleus upon sequestration, where 35% of the T. amphioxeia genes were significantly differentially expressed within well-fed M. rubrum. Qualitatively, 68% of all genes expressed within well-fed M. rubrum originated from T. amphioxeia. Quantitatively, these genes contributed up to 48% to the global transcriptome in well-fed M. rubrum and down to 11% in starved M. rubrum. This tertiary endosymbiosis system functions for several weeks, when deprived of prey. After this point in time, the ciliate dies if not supplied with fresh prey cells. M. rubrum represents one evolutionary way of acquiring photosystems from its algal prey, and might represent a step on the evolutionary way towards a permanent tertiary endosymbiosis.

Karmitoxin production by Karlodinium armiger and the effects of K. armiger and karmitoxin towards fish.

The dinoflagellate Karlodinium armiger has a huge impact on wild and caged fish during blooms in coastal waters. Recently, a new toxin, karmitoxin, was chemically characterized from K. armiger and a quantification method was established, thereby allowing investigations of the fish killing mechanism. K. armiger is not able to grow in standard growth media that are based on nitrate as a nitrogen source, and successful cultures of this species have only been achieved in mixotrophic cultures after addition of a prey source. Here we show that addition of ammonium (up to 50 µM) to the growth media is a good alternative, as K. armiger batch cultures achieve growth rates, which are comparable to growth rates reached in mixotrophic cultures. Karmitoxin production (1.9 and 2.9 pg cell-1 d-1) and cellular karmitoxin content (8.72 ± 0.25 pg cell-1 and 7.14 ± 0.29 pg cell-1) were in the same range, though significantly different, in prey-fed cultures and monocultures supplied with ammonium, respectively. Net production of karmitoxin stopped when the K. armiger cultures reached stationary growth phase, indicating no accumulation of karmitoxin in cells or growth media. Toxicity tests towards sheepshead minnow fish larvae indicated rapid death of the fish larvae when exposed to high K. armiger cell concentrations (LT50 of 2.06 h at 44.9 × 103 cells mL-1 cultivated with ammonium). Purified toxins caused the same physical damage to fish larvae as living K. armiger cultures. An exposure of purified karmitoxin to fish larvae and rainbow trout gill cells indicated that the fish larvae were about three times less sensitive than gill cells. When comparing the effect of purified toxins with the effect of whole K. armiger cultures, twice the toxin concentration of the purified toxins was needed to cause the same effect. Although a loss of karmitoxin of twenty percent was observed during the incubation, this could not explain the apparent discrepancy. Other factors, like a direct effect of the K. armiger cells on the fish larvae or other, yet unknown toxins may influence the effect of whole cell cultures. To study the effects of released karmitoxin, fish larvae were exposed to a K. armiger culture that was treated with HP-20 resin, which adsorbs extracellular karmitoxin. The 24 h HP-20 treatment resulted in a K. armiger culture that had 37% less total karmitoxin, without a reduction in cell concentration, and a reduced toxic effect was observed in the HP-20 treated culture, as compared to non-treated controls. Fish larvae that were exposed to HP-20 treated culture were immobilized, but survived during the 12 h exposure, whereas the exposure to non-treated culture led to high mortality of the fish larvae. Direct observations under the microscope revealed no evidence of micropredation of K. armiger on the fish larvae during any of the exposures. Thus, the results presented here, indicate that released karmitoxin is the main cause for fish kills by K. armiger. Finally, we found that juvenile rainbow trout were six times more sensitive than fish larvae towards K. armiger, indicating that juvenile fish are more sensitive to K. armiger in bloom situations than early larval stages.

Effects of the DSP-toxic dinoflagellate Dinophysis acuta on clearance and respiration rate of the blue mussel, Mytilus edulis.

Diarrheic Shellfish Poisoning toxins (DST) are a severe health risk to shellfish consumers and can be a major problem for the shellfish industry. Bivalve molluscs can accumulate DST via ingestion of toxic dinoflagellates like Dinophysis spp., which are the most prominent producers of DST. The effects of DST-containing dinoflagellate Dinophysis acuta on bivalve clearance and respiration rate were investigated in the blue mussel (Mytilus edulis) exposed to different algal densities in a controlled laboratory study. Results showed that M. edulis exposed to D. acuta displayed a reduced clearance rate compared to M. edulis exposed to equivalent bio-volumes of the non-toxic cryptophyte Rhodomonas salina. Furthermore, M. edulis ceased to feed on D. acuta after 1 to 4 h, depending on D. acuta densities. The quickest response was observed at the highest densities of D. acuta. The estimated total amount of DST accumulated in the M. edulis exceeded the regulatory limit for human consumption and furthermore, intoxication of the M. edulis seemed to occur faster at high cell toxicity rather than at high cell density. However, respiration rates were, similar, irrespective of whether M. edulis were fed single diets of R. salina, D. acuta or a mixed diet of both algal species. In conclusion, the DST-containing D. acuta had a severe negative effect on the clearance of M. edulis, which can affect the conditions of the M. edulis negatively. Hence, DST may cause low quality M. edulis, due to reduced feeding when exposed to DST-containing D. acuta.

Dynamic CO2 and pH levels in coastal, estuarine, and inland waters: Theoretical and observed effects on harmful algal blooms.

Rising concentrations of atmospheric CO2 results in higher equilibrium concentrations of dissolved CO2 in natural waters, with corresponding increases in hydrogen ion and bicarbonate concentrations and decreases in hydroxyl ion and carbonate concentrations. Superimposed on these climate change effects is the dynamic nature of carbon cycling in coastal zones, which can lead to seasonal and diel changes in pH and CO2 concentrations that can exceed changes expected for open ocean ecosystems by the end of the century. Among harmful algae, i.e. some species and/or strains of Cyanobacteria, Dinophyceae, Prymnesiophyceae, Bacillariophyceae, and Ulvophyceae, the occurrence of a CO2 concentrating mechanisms (CCMs) is the most frequent mechanism of inorganic carbon acquisition in natural waters in equilibrium with the present atmosphere (400 µmol CO2 mol-1 total gas), with varying phenotypic modification of the CCM. No data on CCMs are available for Raphidophyceae or the brown tide Pelagophyceae. Several HAB species and/or strains respond to increased CO2 concentrations with increases in growth rate and/or cellular toxin content, however, others are unaffected. Beyond the effects of altered C concentrations and speciation on HABs, changes in pH in natural waters are likely to have profound effects on algal physiology. This review outlines the implications of changes in inorganic cycling for HABs in coastal zones, and reviews the knowns and unknowns with regard to how HABs can be expected to ocean acidification. We further point to the large regions of uncertainty with regard to this evolving field.