Shining Light on Photosynthesis in the Harmful Dinoflagellate Karenia mikimotoi–Responses to Short-Term Changes in Temperature, Nitrogen Form, and Availability

Karenia mikimotoi is a toxic bloom-forming dinoflagellate that sometimes co-blooms with Karenia brevis in the Gulf of Mexico, especially on the West Florida Shelf where strong vertical temperature gradients and rapid changes in nitrogen (N) can be found. Here, the short-term interactions of temperature, N form, and availability on photosynthesis–irradiance responses were examined using rapid light curves and PAM fluorometry in order to understand their interactions, and how they may affect photosynthetic yields. Cultures of K. mikimotoi were enriched with either nitrate (NO3−), ammonium (NH4+), or urea with varying amounts (1, 5, 10, 20, 50 µM-N) and then incubated at temperatures of 15, 20, 25, 30 °C for 1 h. At 15–25 °C, fluorescence parameters (Fv/Fm, rETR) when averaged for all N treatments were comparable. Within a given light intensity, increasing all forms of N concentrations generally led to higher photosynthetic yields. Cells appeared to dynamically balance the “push” due to photon flux pressure and reductant generation, with consumption in overall metabolism (“pull” due to demand). However, at 30 °C, all fluorescence parameters declined precipitously, but differential responses were observed depending on N form. Cells enriched with urea at 30 °C showed a smaller decline in fluorescence parameters than cells treated with NO3− or NH4+, implying that urea might induce a photoprotective mechanism by increasing metabolic “pull”.

Biochemical and Physiological Responses of Harmful Karenia mikimotoi to Algicidal Bacterium Paracoccus homiensis O-4

Harmful algal blooms caused by Karenia mikimotoi frequently occur worldwide and severely threaten the marine environment. In this study, the biochemical and physiological responses of K. mikimotoi to the algicidal bacterium Paracoccus homiensis O-4 were investigated, and the effects on the levels of reactive oxygen species (ROS), malondialdehyde content, multiple antioxidant systems and metabolites, photosynthetic pigments, and photosynthetic index were examined. The cell-free supernatant in strain O-4 significantly inhibited K. mikimotoi cell growth. The bacterium caused the K. mikimotoi cells to activate their antioxidant defenses to mitigate ROS, and this effect was accompanied by the upregulation of intracellular antioxidant enzymes and non-enzyme systems. However, the overproduction of ROS induced lipid peroxidation and oxidative damage within K. mikimotoi cells, ultimately leading to algal death. In addition, the photosynthetic efficiency of the algal cells was significantly inhibited by O-4 and was accompanied by a reduction in photosynthetic pigments. This study indicates that O-4 inhibits K. mikimotoi through excessive oxidative stress and impaired photosynthesis. This research into the biochemical and physiological responses of K. mikimotoi to algicidal bacteria provides insights into the prophylaxis and control of harmful algal blooms via interactions between harmful algae and algicidal bacteria.

Proteome Analysis of Whole-Body Responses in Medaka Experimentally Exposed to Fish-Killing Dinoflagellate Karenia mikimotoi

Karenia mikimotoi is a well-known harmful algal bloom species. Blooms of this dinoflagellate have become a serious threat to marine life, including fish, shellfish, and zooplanktons and are usually associated with massive fish death. Despite the discovery of several toxins such as gymnocins and gymnodimines in K. mikimotoi, the mechanisms underlying the ichthyotoxicity of this species remain unclear, and molecular studies on this topic have never been reported. The present study investigates the fish-killing mechanisms of K. mikimotoi through comparative proteomic analysis. Marine medaka, a model fish organism, was exposed to K. mikimotoi for a three-part time period (LT25, LT50 and LT90). Proteins extracted from the whole fish were separated by using two-dimensional gel electrophoresis, and differentially expressed proteins were identified with reference to an untreated control. The change in fish proteomes over the time-course of exposure were analyzed. A total of 35 differential protein spots covering 19 different proteins were identified, of which most began to show significant change in expression levels at the earliest stage of intoxication. Among the 19 identified proteins, some are closely related to the oxidative stress responses, energy metabolism, and muscle contraction. We propose that oxidative stress-mediated muscle damage might explain the symptoms developed during the ichthyotoxicity test, such as gasping for breath, loss of balance, and body twitching. Our findings lay the foundations for more in-depth studies of the mechanisms of K. mikimotoi’s ichthyotoxicity.

Nutrient Alteration Drives the Impacts of Seawater Acidification on the Bloom-Forming Dinoflagellate Karenia mikimotoi

Seawater acidification and nutrient alteration are two dominant environmental factors in coastal environments that influence the dynamics and succession of marine microalgae. However, the impacts of their combination have seldom been recorded. A simulated experimental system was set up to mimic the effects of elevated acidification on a bloom-forming dinoflagellate, Karenia mikimotoi, exposed to different nutrient conditions, and the possible mechanism was discussed. The results showed that acidification at different pH levels of 7.6 or 7.4 significantly influenced microalgal growth (p<0.05) compared with the control at pH 8.0. Mitochondria, the key sites of aerobic respiration and energy production, were impaired in a pH-dependent manner, and a simultaneous alteration of reactive oxygen species (ROS) production occurred. Cytochrome c oxidase (COX) and citrate synthase (CS), two mitochondrial metabolism-related enzymes, were actively induced with acidification exposure, suggesting the involvement of the mitochondrial pathway in coping with acidification. Moreover, different nutrient statuses indicated by various N:P ratios of 7:1 (N limitation) and 52:1 (P limitation) dramatically altered the impacts of acidification compared with those exposed to an N:P ratio of 17:1 (control), microalgal growth at pH 7.4 was obviously accelerated with the elevation of the nutrient ratio compared to that at pH 8.1 (p<0.05), and nutrient limitations seemed beneficial for growth in acidifying conditions. The production of alkaline phosphatase (AP) and acid phosphatase (AcP), an effective index indicating the microalgal growth status, significantly increased at the same time (p<0.05), which further supported this speculation. However, nitrate reductase (NR) was slightly inhibited. Hemolytic toxin production showed an obvious increase as the N:P ratio increased when exposed to acidification. Taken together, mitochondrial metabolism was suspected to be involved in the process of coping with acidification, and nutrient alterations, especially P limitation, could effectively alleviate the negative impacts induced by acidification. The obtained results might be a possible explanation for the competitive fitness of K. mikimotoi during bloom development.

Bioavailability of Organic Phosphorus Compounds to the Harmful Dinoflagellate Karenia mikimotoi

Karenia mikimotoi is one of the most well-known harmful bloom species in temperate coastal waters. The present study investigated the characteristics of alkaline phosphatase (APase) and phosphodiesterase (PDEase) activities in hydrolysis of two phosphomonoesters (adenosine triphosphate (ATP) and ribulose 5-phosphate (R5P)) and a phosphodiester (cyclic adenosine monophosphate (cAMP)) in K. mikimotoi and compared its growth and physiological responses to the different forms of phosphorus substrates. K. mikimotoi produced comparable quantities of APase and PDEase to hydrolyze the organic phosphorus substrates, although hydrolysis of the phosphomonoesters was much faster than that of the phosphodiester. The growth of K. mikimotoi on organic phosphorus substrates was comparable to or better than that on inorganic phosphate. The difference in particulate organic nutrients (carbon, nitrogen, and phosphorus) and hemolytic activity supported different rates of hydrolysis-assimilation of the various organic phosphorus substrates by K. mikimotoi. The hemolytic activities of K. mikimotoi in the presence of organic phosphorus substrates were several times those in the presence of inorganic phosphate during the exponential phase. This suggested the potential important role of organic phosphorus in K. mikimotoi blooms.

The Bloom-Forming Dinoflagellate Karenia mikimotoi Adopts Different Growth Modes When Exposed to Short or Long Period of Seawater Acidification

Impacts of ocean acidification (OA) on noncalcifying organisms and the possibly responsible mechanism have aroused great research interests with the intensification of global warming. The present study focused on a noxious, noncalcifying, bloom-forming dinoflagellate, Karenia mikimotoi (K. mikimotoi), and its variation of growth patterns exposed to different periods of seawater acidification with stressing gradients was discussed. The dinoflagellates under short-time acidifying stress (2d) with different levels of CO2 presented significant growth inhibition (p < 0.05). The cell cycle was obviously inhibited at S phase, and the photosynthetic carbon fixation was also greatly suppressed (p < 0.05). Apoptosis was observed and the apoptotic rate increased with the increment of pCO2. Similar tendencies were observed in the key components of mitochondrial apoptotic pathway (the mitochondrial membrane potential (MMP), Caspase-3 and -9, and Bax/Bcl-2 ratio). However, under prolonged stressing time (8 d and 15 d), the growth of dinoflagellates was recovered or even stimulated, the photosynthetic carbon fixation was significantly increased (p < 0.05), the cell cycle of division presented little difference with those in the control, and no apoptosis was observed (p > 0.05). Besides, acidification adjusted by HCl addition and CO2 enrichment resulted in different growth performances, while the latter had a more negative impact. The results of present study indicated that (1) the short-time exposure to acidified seawater led to reduced growth performance via inducing apoptosis, blocking of cell cycle, and the alteration in photosynthetic carbon fixation. (2) K. mikimotoi had undergone adaptive changes under long-term exposure to CO2 induced seawater acidification. This further demonstrated that K. mikimotoi has strong adaptability in the face of seawater acidification, and this may be one of the reasons for the frequent outbreak of red tide. (3) Ions that dissociated by the dissolved CO2, instead of H+ itself, were more important for the impacts induced by the acidification. This work thus provides a new perspective and a possible explanation for the dominance of K. mikimotoi during the occurrence of HABs.