Cell cycle arrest and biochemical changes accompanying cell death in harmful dinoflagellates following exposure to bacterial algicide IRI-160AA

Abstract

Bacteria may play a role in regulating harmful algal blooms, but little is known about the biochemical and physiological changes associated with cell death induced by algicidal bacteria. Previous work characterized an algicidal exudate (IRI-160AA) produced by Shewanella sp. IRI-160 that is effective against dinoflagellates, while having little to no effect on other phytoplankton species in laboratory culture experiments. The objective of this study was to evaluate biochemical changes associated with cell death and impacts on the cell cycle in three dinoflagellate species (Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum) after exposure to IRI-160AA. In this study, IRI-160AA induced cell cycle arrest in all dinoflagellates examined. Several indicators for programmed cell death (PCD) that are often observed in phytoplankton in response to a variety of stressors were also evaluated. Cell death was accompanied by significant increases in DNA degradation, intra- and extracellular ROS concentrations and DEVDase (caspase-3 like) protease activity, which have been associated with PCD in other phytoplankton species. Overall, results of this investigation provide strong evidence that treatment with the bacterial algicide, IRI-160AA results in cell cycle arrest and induces biochemical changes consistent with stress-related cell death responses observed in other phytoplankton.

Figure 1. Cell density in dinoflagellate cultures after exposure to IRI-160AA.

(A) P. minimum, (B) K. veneficum and (C) G. instriatum after the addition of 10% (v/v) f/2 (gray squares) or IRI-160AA (black triangles). Points represent the average of triplicates measured every 2 hours for 14 hours (G. instriatum) or 22 hours (P. minimum and K. veneficum). Error bars are one standard deviation of the mean for each point (n = 3). The light gray rectangle identifies the light/dark transition.

Figure 2. Cell cycle analyses for all three dinoflagellates.

The percentage of cells in each phase at each time point for P. minimum (A,B), K. veneficum (C,D) and G. instriatum (E,F) after a 10% (v/v) f/2 medium (A,C,E) or IRI-160AA (B,D,F) treatment. Phases were distinguished using the Watson Model and partitioned into G₁ (gray), S (dark gray), and G₂/M (light gray). Bars represent concatenated triplicates at 2 hour time intervals. Rectangles under each panel indicate the light/dark transition for reference.

Figure 3

The proportion of P. minimum (A), K. veneficum (B) and G. instriatum (C) cells containing sub G₁ DNA, which were excluded from the cell cycle analysis. Data points represent concatenated triplicates at 2 hour time intervals of controls (gray squares) and IRI-160AA treatments (black triangles) after a 10% (v/v) addition to algal cultures. The large gray rectangle indicates the light/dark transition for reference. Panel C, Inset: Representative histograms for concatenated triplicates of G. instriatum control (left) and treatment (right) were included to show the proportion of cells with sub G₁ DNA at 12 hours after algicide addition (in black).

Figure 4. Endogenous ROS production.

ROS production after addition of algicide IRI-160AA to P. minimum, K. veneficum and G. instriatum. Bars represent the mean (n = 3) fluorescence intensity per cell by volume (bio-volume) for controls (dark gray) and algicide treatments (light gray). Error bars represent +/−1 standard deviation. Significant differences between controls and algicide treatments for each species are indicated by asterisks: *p < 0.05; **p < 0.01; ***p < 0.001. The horizontal line represents an axis break. The inset figure represents algicidal activity associated with the endogenous ROS experiment at 3 hours incubation with IRI-160AA.

Figure 5. Extracellular production of H₂O₂.

H₂O₂ production after addition of algicide IRI-160AA to P. minimum (dashed line), K. veneficum (dotted line) and G. instriatum (solid line). Points represent the net [average concentration in treatment – average concentration in control; (n = 2)] change in H₂O₂ production (μM) per cell, normalized to cell bio-volume. The break in data for P. minimum was due to electrical interference. The inset figure represents algicidal activity associated with the H₂O₂ experiment where a third replicate was run alongside the H₂O₂ sensors. For inset: error bars represent one standard deviation of the mean (n = 3).

Figure 6. DEVDase enzyme activity: time series.

DEVDase activity after addition of algicide IRI-160AA to G. instriatum after 18, 24 and 42 hours exposure. Relative activity was normalized to protein content. Bars represent triplicate means of the DEVDase activity expressed as relative fluorescence units per hour per μg of total protein in the control (dark gray) and algicide treatments (light gray). Error bars represent one standard deviation of the mean (n = 3). Significant differences between and treatments are indicated by asterisks: *p < 0.05; **p < 0.01; ***p < 0.001. An X on controls bars indicates little to no DEVDase activity. The inset figure represents algicidal activity accompanying the DEVDase experiment. For inset: error bars show one standard deviation of the mean (n = 3).

Figure 7. DEVDase enzyme activity.

DEVDase activity after addition of algicide IRI-160AA expressed as relative fluorescence units per hour per μg of protein. Activity was measured 18 (P. minimum) or 24 (K. veneficum and G. instriatum) hours after addition of the algicide and normalized to total protein content. Bars represent triplicate means of the DEVDase activity in the presence (light gray) and absence (dark gray) of the Ac-DEVD-CHO inhibitor. Error bars represent one standard deviation of the mean (n = 3). An X indicates little to no DEVDase activity in controls. Significant differences between controls and treatments for each species are next to the species name in the margins while significant differences between + and − inhibitor are above the light gray bars (+inhibitor). In both cases significance is indicated by asterisks: *p < 0.05; **p < 0.01; ***p < 0.001. The inset figure represents algicidal activity accompanying the DEVDase experiment. For inset: error bars represent one standard deviation of the mean (n = 3).