Can the release of dopamine by Ulvaria obscura into intertidal waters affect the development of Fucus distichus, a co-occurring alga?
Our experimental goal was to measure the effects of dopamine on a co-occurring macro alga called Fucus distichus.
A unique macroalga called Ulvaria obscura occurs from the mid intertidal to the shallow subtidal zones of the North Pacific and North Atlantic shores (Fig. 1). This algae releases dopamine (Fig. 2) into the surrounding waters. The dopamine oxidizes to form dopamine quinone (Fig. 3) in concentrations high enough to affect co-occurring marine biota, possibly disrupting entire ecosystems. We hypothesized that dopamine exudates would influence growth patterns of co-existing marine algae. To test our hypothesis we exposed fertilized embryos of Fucus distichus (Fig. 4), a co-occurring macroalga to three concentrations of dopamine (Fig. 5) at three time intervals to see what effect, if any, it might have on embryonic germination rates and development.
Algal blooms have been known to occur in marine as well as fresh water environments. Blooms arise as a result of an increase in nutrients, often termed eutrophication. These conditions have adverse effects on surrounding species (Peterson et al.,1994; Fletcher, 1996; Valiela et al., 1997; Raffaelli et al., 1998; Cummins et al., 2004). Harmful algal blooms (HAB’s) can negatively affect co-occurring biota. The bloom species can cover and shade other algal species, interfere with the amount of oxygen in the water leading to anoxic conditions and deplete nutrients needed by surrounding organisms. Ulvaria Obscura is one of the dominant components of algal blooms that occur from the mid intertidal to the shallow subtidal zones of North Pacific and North Atlantic shores. U. Obscura is different from other species of algae because it produces dopamine. It is not fully understood why U. obscura releases dopamine, but it has been hypothesized that it may be a feeding deterrent (Van Alstyne et al., 2006). Dopamine is a compound with an amine group linked to a catechol (an aromatic ring with two hydroxyl groups) center (Figure 1; 3,4-dihydroxyphenylethylamine). Dopamine is a neurotransmitter that allows communication between a neuron and a cell. Dopamine has an important function in the physiology of animals. However, in marine algae such as the Ulvaria species, its function is not fully understood.
Materials and Methods
An experiment was conducted in January, 2012 to determine if dopamine released from U. obscura affects other algae that co-occur in the same habitat. We collected Fucus distichus specimens from the shore of the Shannon Point Marine Center, Anacortes, WA, USA. The alga grows extensions from the stipe that lead to the filamentous branches called vesicles where the gametes develop. The vesicles were cleaned with a toothbrush to remove dirt and debris. The vesicles were rinsed 3 times using filtered sea water (FSW). Afterwards, the vesicles were placed in 9 inch diameter finger bowls with 50ml filtered sea water and placed in an incubator (12⁰C) and monitored daily. After two days, we collected the fertilized eggs released from the vesicles. The vesicles were removed and discarded. The eggs were collected in 0.2 micron filters and re-suspended in 100mL of filtered sea water. The experiment was conducted by preparing five replicate samples for the three concentrations at the different exposure times. Dopamine hydrochloride (Sigma 12K1175) was added to yield concentrations of 100µM, 250µM and 500µM. The fertilized eggs were then placed into the finger bowls in their respective filters for 1, 3, or 48 hours at each concentration. Sample blanks were treated similarly with the exception of the addition of dopamine. All of the bowls were then placed in the incubator for the designated time intervals. At the end of the exposure time, the embryos were fixed with a hydrochloric acid/ iodine solution. A compound microscope was used to count the number of embryos that had germinated and measure the rhizoid length. The 2-way ANOVA using concentration and time as factors was used to evaluate results.
Our results show that both germination success and rhizoid length of F.distichus embryos were affected by dopamine. The 2 way ANOVA (Table 1) showed asignificant time by concentration interaction.
Survival: After 1 hour and 3 hours there were no significant differences in the number of germinated embryos (Figure 1). After 48 hours of exposure, however, the control group showed a higher percent germination than the 100µM treatment. The middle concentration of 250µM and the high concentration of 500µM had much lower percent germination.
Length: 2 way ANOVA (Table 2) showed a significant interaction between time and concentration with respect to rhizoid length. After one hour of exposure to the three dopamine concentrations, there was no significant difference in the length of rhizoids (Figure 2). However, after a 3 hour exposure, all three treatments showed a decrease in rhizoid growth with the most significant drop in length for 500µM concentration. At the end of 48 hours, all sample treatments had smaller rhizoid lengths. The biggest effect was in the two higher concentrations of dopamine: 250µM and 500µM.
Based on our experimental results, dopamine affected the germination of Fucus distichus embryos. The percent germination droped if the exposure time was long enough. The higher concentrations of dopamine drastically reduced rhizoid length within the first three hours of exposure. Our work did not test the possibility that the effects were the result of the oxidation and formation of ROS species from dopamine. It has been determined experimentally that dopamine is released into surrounding medium when U obscura is stressed (Van Alstyne et al., submitted). Part of the ambiguity rests with the chemistry of dopamine, which, once released, oxidizes to yield reactive oxygen species (ROS) such as hydrogen peroxide, superoxide and hydroxyl radicals (Stokes et al., 1999) which can damage protein, lipid and amino acids (Fridovich, 1978; Asada and Takahashi, 1987; Halliwell and Gutteridge, 1989; Dummermuth et al.,2003). This auto oxidation also produces dopamine quinone, characterized by reddening of the water (Stokes et al., 1999; Whitehehad et al., 2001) surrounding the algae and progresses to darker shades of red as time passes, eventually becoming a dark black non-soluble tar. The dopamine quinone covalently binds to DNA and cysteine residues thus inactivating proteins (Stokes et al,. 1999). Overall harmful algal blooms of which Ulvaria obscura is a dominant component can interfere with ocean sediment, disrupt water flow and damage co-occurring species due to hypoxic conditions. Harmful algal blooms occur worldwide. A better understanding of such occurrences may lead to more efficient control and the protection of susceptible habitats.