Effects of Temperature and CO2-driven Seawater Acidification on the Physiology, Toxicity and Molecular Responses of Benthic Dinoflagellates Coolia spp.


Student thesis: Doctoral Thesis

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Award date9 Jul 2021


Benthic and epiphytic dinoflagellates are widely distributed around the world. Biologically active and toxic chemicals produced by dinoflagellates can potentially bio-accumulate and be bio-magnified in the marine food chain, which can lead to fish poisoning incidents, and thus have attracted scientific attention. One of the causative groups, Coolia Meunier (Dinophyceae), has been reported to produce algal toxins like cooliatoxin, yessotoxin, and 44-methylgambierone, and has shown toxicity effects on marine organisms. However, risk assessments of Coolia species on the health of coastal ecosystems are limited. The physiological responses and the underlying molecular mechanisms of Coolia species in response to climate changes have not been well studied.

Ocean temperature and CO2-driven ocean acidification have potentially far-reaching consequences for the physiology of many algal groups. Changes in physiological responses and the effects of toxic algae reflect their potential threats to marine ecosystems when algae are exposed to various environmental conditions. The responses include algal growth, carbon fixation, toxin production, and enzyme metabolism. Therefore, this study investigated the physiological performances, toxicological effects, and underlying mechanisms of Coolia species under the individual and combined effects of seawater temperature and pCO2 level.

In an initial experiment (Chapter 2), a temperate species of Coolia, C. canariensis, was selected to study the physiological responses of algae to different temperatures. Algae were exposed to seven temperatures ranging from 16 °C to 28 °C. The optimum growth temperature of C. canariensis was 24 °C, and the algal growth were significantly inhibited at higher temperatures of 26 °C and 28 °C. Moreover, C. canariensis showed high tolerance to temperature as low as 16 °C in regard to algal growth rate, compared with algae exposed to high temperatures. A significantly higher amount of chlorophyll a was, however, detected in C. canariensis after exposure to high temperatures of 26 °C (4.98 ± 0.52 µg·mg-1) and 28 °C (5.01 ± 0.45 µg·mg-1). The values of Fv/Fm (fluorescence-based maximum quantum yield for PSII) of C. canariensis were higher under high temperatures of 26 °C and 28 °C. The toxicity results showed that lipophilic algal extracts of C. canariensis from the seven temperatures showed low toxicity to Artemia larvae with 48-h mortality less than 14%; and hydrophilic algal extracts of C. canariensis from the seven temperatures showed no toxicity to Artemia larvae. To further investigate the underlying molecular mechanisms of the responses of C. canariensis to temperature, differentially expressed genes (DEGs) in C. canariensis under temperature treatments of 16 °C (low temperature), 24 °C (optimum temperature), and 28 °C (high temperature) were detected and discussed based on comparative transcriptomic analysis. Most of the DEGs were enriched in the plant-pathogen interaction pathway of C. canariensis under low temperature; the most enriched pathway of the algae under high temperature was RNA-transport.

In addition to less toxic Coolia species, the physiological responses of a tropical species of Coolia, C. tropicalis exposure to seven temperatures from 16 °C to 28 °C were studied (Chapter 3). The optimum growth temperature of C. tropicalis was 28 °C. Low temperature of 16 °C induced significantly adverse effects on algal growth and algal structural formations. C. tropicalis showed the lowest growth rate of 0.02 ± 0.01 div·d-1, and the highest deformation rate of 17.29 ± 2.42% under 16 °C. According to the correlations between cell dry weight and cell number of C. tropicalis under seven temperatures, the dry weight of a single algal cell significantly increased under low temperatures, i.e., 16 °C, 18 °C and 20 °C. Results showed high positive correlations between the amount of the pigment and temperature (Spearman r = 0.89), between the value of Fv/Fm and temperature (Spearman r = 0.96), and between the proportion of chlorophyll a and the value of Fv/Fm (Spearman r = 0.93).

Hydrophilic algal lysates of C. tropicalis showed toxic effects on Artemia larvae and marine medaka larvae after 48-h exposure in a concentration-response pattern. Algal lysates from cultures exposed to 28 °C showed significantly higher toxicity to Artemia larvae (EC50 = 0.5222 mg·mL-1, p < 0.05, Duncan test) compared with the toxicity of algal lysates at other lower temperatures. The 48-h LC50 values in marine medaka larvae showed a significantly negative correlation with temperature (Spearman r = -0.82, p = 0.0341). Hydrophilic algal extracts of C. tropicalis exposed to 24 °C showed the highest toxicity to marine medaka larvae (LC50 = 0.3611 mg·mL-1, p < 0.05, Duncan test). C. tropicalis exposed to 16 °C showed the lowest toxicity toward Artemia larvae and medaka larvae. The lipophilic algal extracts of C. tropicalis under all the seven temperatures showed no and low toxic effects on both marine medaka larvae and Artemia larvae, respectively. To investigate the molecular mechanisms involved in the changes of algae in response to temperature, DEGs in C. tropicalis under 16 °C, 24 °C and 28 °C were revealed using comparative transcriptomic analysis. Most of the DEGs were enriched in metabolic pathways of the algae under 24 °C and 28 °C when compared with those of the algae under 16 °C.

The toxic effects of hydrophilic algal lysates of C. tropicalis on marine medaka was further evaluated (Chapter 4). Various endpoints of toxicological responses were studied including the fish swimming activities, physiological performance, and stress-associated molecular responses. Two sub-lethal concentrations (0.025 and 0.035 mg·mL-1) of hydrophilic algal lysates were administrated to marine medaka larvae via water-borne exposure for 96 h. Treatment with C. tropicalis lysates inhibited swimming activity, activated spontaneous undirected locomotion, altered the nerve length ratio, and induced early development abnormalities, such as shorter eye diameter, body and axon length of medaka larvae. Consistent with these abnormalities, changes in the expression of genes associated with apoptosis (CASPASE-3 and BCL-2), inflammatory response (IL-1βand COX-2), oxidative stress (SOD), and energy metabolism (ACHE and VHA), were also observed.

In Chapter 5, the combined effects of temperature and CO2-driven seawater acidification on the physiological responses of three Coolia species, C. malayensis, C. canariensis, and C. tropicalis were studied. The three Coolia species were exposed to combined treatments of three pCO2 concentrations of a low level at 400 ppm (LC), a moderate high level at 1000 ppm (MC), and an extreme high level at 2000 ppm (HC), and three temperatures of a low temperature at 16 °C (LT), a moderate temperature at 24 °C (MT), and a high temperature at 28 °C (HT) for generations. The physiological performances of algae were examined by measuring algal growth rate, algal cell size, pigment content, photosynthetic ability, and enzyme activity. The growth rates of the three Coolia species significantly increased at higher temperatures of 24 °C and 28 °C. High pCO2 concentrations stimulated the algal growth of all the three Coolia species, especially under low temperature of 16 °C. The highest growth rates of C. malayensis were detected under HT × HC, followed by those of algae exposed to MT × MC, and HT × MC. C. canariensis and C. tropicalis showed the highest growth rates under MT × LC and HT × HC, respectively. The cell anterior-posterior (AP) length of Coolia was significantly affected by temperature and pCO2. Significantly longer cell AP length of C. malayensis and C. tropicalis was observed under low temperature of 16 °C. pCO2 enrichment showed species-specific effects on AP length of the algae. The photosynthetic activities of Coolia were significantly affected by temperature and pCO2. The Fv/Fm values of the three Coolia species were increased under 24 °C and 28 °C, while those values of C. malayensis and C. tropicalis were decreased under 16 °C. The activities of a carbon metabolism-related enzyme, sucrose synthase (SS), of the three Coolia species were significantly affected by temperature and pCO2, and species-specific responses of SS activity to different environmental factors were observed. Significantly higher SS activities were detected in C. canariensis and C. tropicalis under LT × LC.

The underlying mechanisms of the physiological responses of Coolia to pCO2 enrichment were further revealed using comparative transcriptomic analysis. Algal cultures of C. malayensis, C. canariensis, and C. tropicalis that were exposed to two high pCO2 levels of 1000 ppm and 2000 ppm, and a control level of 400 ppm under their optimum temperatures, were used for performing RNA sequencing in Chapter 5. In C. malayensis, most of the DEGs were enriched in the ribosome pathway of the algae under two pCO2 enrichment conditions compared with those algae in the control group. In C. tropicalis, most of the DEGs were enriched in the algal photosynthesis-antenna proteins pathway and citrate cycle pathway under 2000 ppm pCO2 compared with those in the control group. In C. canariensis, the DEGs were highly enriched in pathways of photosynthesis-antenna proteins, biosynthesis of amino acids, and photosynthesis under 2000 ppm compared with those algae under 400 ppm.

In summary, the effects of temperature and the interactions of temperature and pCO2 level under global climate change scenarios in marine benthic dinoflagellates C. malayensis, C. canariensis, and C. tropicalis were studied. Compared with low temperatures and moderate temperatures, high temperatures showed more obvious inhibition on the growth and photosynthetic activity of C. canariensis. Tropical member C. tropicalis could benefit from warmer seawater temperatures, and the algal toxicities were also significantly increased under high temperature exposure. Further toxicological studies demonstrated that exposure to the hydrophilic algal extracts for 96 h was associated with impaired swimming activity, abnormal spontaneous indirect locomotion, altered nerve length ratio, and early development defects of marine medaka larvae. This study advanced understanding of C. tropicalis-mediated toxic mechanisms in marine fish in early life stages and could contribute to future ecological risk assessment of toxic benthic dinoflagellates. In this study, temperature showed a stronger effect on physiological responses of the three Coolia species than CO2-driven seawater acidification did. C. tropicalis could benefit from combined effects of high temperature and high pCO2. All the three Coolia species showed very high sensitivity to pCO2 enrichment when they were exposed to low temperature. Further molecular validation should be conducted to fully determine the underlying mechanisms of physiological changes as a result of temperature and pCO2 changes. This study will help assess the ecological and human health risks of harmful dinoflagellates, as well as the changing risks under global climate change scenarios.