Effects of reduced dissolved oxygen concentrations on physiology and fluorescence of hermatypic corals and benthic algae

Introduction

Over the last several decades coral cover has been declining around the globe (Gardner et al., 2003; Bruno & Selig, 2007). These losses in live coral cover are often associated with increases in the abundance of fleshy algae (Hughes, 1994; McCook, 1999). Multiple causes for these so called “phase shifts” have been identified including warming and subsequent coral bleaching and mortality or local anthropogenic influences such as pollution and overfishing (Bellwood et al., 2004; McManus & Polsenberg, 2004; Hughes et al., 2010). The underlying competitive mechanisms of coral-algae interactions still remain poorly understood (McCook, 2001; Jompa & McCook, 2003; Smith et al., 2006). However, the outcomes of these competitive interaction processes are critical for determining the resulting relative abundance of these organisms in coral reef habitats (McCook, 2001).

Both corals and algae use various physical mechanisms (e.g., sweeper tentacles, messentarial filaments, abrasion, shading) to compete with one another for the limited substratum available on the benthos (Nugues & Roberts, 2003; Van Veghel, Cleary & Bak, 1996; Coyer et al., 1993; Tanner, 1995). In addition, such competition may also involve direct chemical mechanisms, such as allelopathy (Bak & Borsboom, 1984), or the enhancement of disease transfer (Nugues et al., 2004) to other sessile organisms (McCook, Jompa & Diaz-Pulido, 2001). Furthermore, an indirect mechanism has also been suggested where organic carbon released by algae promotes microbial activity, which in turn can affect coral physiology and causes damage to the holobiont. The main driver of coral mortality in this proposed scenario is hypoxia resulting from intensified microbial activity and respiration at the coral/algal interface (Smith et al., 2006). There is increasing evidence that algal exudates can cause microbe-mediated coral mortality but more information is needed about the specific mechanisms involved (Barott et al., 2009).

It is known that oxygen concentrations well below saturation level can occur on coral reefs (Nilsson & Östlund-Nilsson, 2004; Wild et al., 2010). In such complex environments, in situ oxygen levels can fluctuate widely on a diurnal cycle and can be very low at night when organismal respiration dominates the landscape (Haas et al., 2010a; Wild et al., 2010). The extent of this diurnal fluctuation is thereby largely influenced by the biological nature of the local benthic community (Niggl, Haas & Wild, 2010). For example, dissolved oxygen (DO) concentrations in water surrounding algae-dominated areas can be lower than in adjacent coral-dominated reef areas, reaching DO concentrations as low as 4 mg L⁻¹ (Haas et al., 2010a).

Benthic algae are known to release a significant portion of their daily fixed carbon as photosynthetates, primarily carbohydrates (Haas & Wild, 2010), thus enriching the immediate surrounding water with dissolved organic carbon (DOC) (Khailov & Burlakova, 1969; Brylinsky, 1977; Haas et al., 2010a; Haas et al., 2010b). It is also known that water column microbial communities consume some of this DOC. As a result of this carbon uptake (Nelson et al., 2011; Haas et al., 2011) increased microbial metabolism and concomitant consumption of oxygen via respiration (Wild et al., 2010; Haas et al., 2010a), can result in locally confined (from the mm to cm spatial scale) but sometimes severe hypoxic conditions (Barott et al., 2009). These hypoxic conditions are likely most severe at night when all taxa are consuming oxygen via respiration but recent studies have shown that increased biological oxygen demand may even outweigh photosynthetic oxygen release during daylight (Haas et al., 2013). Clearly the characteristics of coral algal interaction zones will depend upon the taxa present (their species and relative abundance) and their respective metabolic rates. Despite this variability, controlled laboratory experiments have shown that these hypoxic conditions can be reversed with the addition of antibiotics suggesting that bacteria play a key role in these interactions (Smith et al., 2006).

There is thus strong evidence that hypoxia plays an important role in coral-algae competition and therefore on processes structuring coral reef communities (McCook, 2001). However, much less is known about how corals and algae individually respond to reduced oxygen conditions. Assuming that algal exudates can fuel microbial community metabolism, resulting in hypoxic conditions at the coral — algal interface, algae need to be more tolerant to these hypoxic conditions than corals to be the competitive superior. The goals of the present study were to investigate the differential tolerance and responses of a common Indo-Pacific coral and a green macroalga to reduced DO conditions in independent incubation experiments.

Material and Methods

This study was conducted in controlled laboratory conditions at the Scripps Institution of Oceanography (SIO) in San Diego, California from July 17 to 27, 2011. The hermatypic coral Acropora yongei initially provided by the Birch Aquarium at Scripps in 2010 has been maintained in culture under optimal growing conditions at Marine Biology Research Division Experimental Aquarium Facility at SIO (Roth et al., 2010). The Birch Aquarium at Scripps also supplied specimens of the green alga Bryopsis pennata specifically for this study. All samples were grown in controlled environments, allowing for better comparison amongst treatments, which is somewhat simplified and possibly not thoroughly reflecting variation and adaptation likely found in-situ. Organisms were kept in identical flow-through tanks with filtered (nominal pore size 50 µm) and temperature controlled seawater at least 10 days prior to the experiment (from now on referred to as “seawater”). Seawater temperature was monitored at 5 min intervals with data loggers (Onset HOBO^® Pendant UA-002-64) and was 26.3 ± 0.4°C for the duration of the experiment.

Adequate irradiance was provided to the corals and algae by artificial illumination (2 × 54 W 6000 K Aquablue⁺, 1 × 54 W 6000 K Midday, and 1 × 54 W Actinic⁺, Geismann, Germany) placed 80 cm above the surface water level with a daylight cycle of 12–12 h. The resulting photosynthetically active radiation (PAR) was 120 µmol quanta m⁻² s⁻¹, as measured with a LICOR LI-193 Spherical Quantum Sensor.

Results

Overall, the different oxygen treatments significantly affected various metrics of coral health while no significant impacts were found on algal health and/or physiology. This was clear in particular for the fluorescence analyses that were, however, specific to each organism (see specific sections below). When considering the analysis common to both the coral and the algae, the effects of oxygen depletion treatment were greater for the maximum quantum yield, while the effective quantum yield and the photosynthetic oxygen production were less affected (Table S1). Maximum quantum yield appeared affected significantly by the oxygen treatment and species, as well as their interaction; thus the oxygen depletion had effects that were different between coral and algae with regards to the maximum quantum yield (species*oxygen treatment of Max. QY; Table S1). As for the effective quantum yield, it showed significant difference between organisms, but in both species this parameter showed no significant variation with the oxygen treatment (whether oxygen treatment or species*oxygen treatment; Table S1). As for the photosynthetic oxygen production, it was not significantly different between organisms, yet was a parameter sensitive to oxygen treatment especially when considering it within species (species*oxygen treatment; Table S1).

Photosynthetic oxygen production measurements were not different over the course of the experiment for all algae treatments and for corals in the ambient (6–8 mg L⁻¹) and decreased (4–6 mg L⁻¹) oxygen treatment. Corals in the low oxygen treatment (2–4 mg L⁻¹) showed a significant decline in oxygen production rates (52.7 ± 12.7% of the initial values) over the experimental period (Fig. 1.1, Table 2). All algae displayed an initial tendency to decrease on the first experimental day but recovered from day one onward, and reached their initial levels again by the end of the experimental period (Fig. 1.1A).

PAM measurements. Over the course of the 10-day experiment dark-adapted QY values recovered for all specimens to pre-experimental values, except for corals subjected to the low oxygen treatment. In this treatment the dark-adapted QY values gradually declined until they reached ∼70% of their initial values on day three and stayed at this level thereafter (Figs. 1.2B–1.2E). Decreases in dark-adapted QY values of corals subjected to low oxygen treatment were significantly greater at the end of the experiment than of any other coral treatment (Tukey p < 0.05) (Fig. 1.2E, Table 2).

Daylight adapted QY measurements followed the same pattern as the dark-adapted QY (data not shown). After a slight initial decrease, daylight adapted QY recovered in all treatments to pre-experimental conditions, except in the low oxygen coral treatment, which stayed at ∼80% of the initial value. This treatment showed significantly greater decreases than all other treatments.

Daylight and dark-adapted QY values were always in the same range and, with one exception, dark-adapted QY values were always higher for the same specimen on the same day as daylight adapted QY. Only on day 10 in the 2–4 mg L⁻¹ oxygen treatment all coral specimens showed significantly higher daylight adapted QY than the dark-adapted QY values (paired sample T-test; p = 0.038; Fig. S1).

Fluorescence analysis of algae showed significantly higher fraction of red pixels at the end of the experiment for all treatments (F = 27.46, p < 0.001), yet with no detectable difference in the rate of increase between the respective oxygen treatments (Fig. 1.3). Although there was an initial drop in the fraction of red pixels, which was similar with all parameters measured for algae (Figs. 1.3A–1.3C), a strong increase in the fraction of red pixels in algae samples from day 3 onward suggests a constant growth or increase in chlorophyll concentration for these specimens (Figs. 1.3A, 1.3D and 1.3E)

Coral fluorescence analysis revealed significant decreases in green intensity between the 2–4 and 4–6 mg L⁻¹ treatments compared to the 6–8 mg L⁻¹ control treatment at the end of the experiment (F_(2,14) = 4.60, p = 0.029) (Fig. 1.4E, Table 2). Green fluorescence intensity values for the 6–8 mg L⁻¹ control treatment stayed in the same range throughout the experiment (Fig. 1.4A). The fluorescence intensities in the 4–6 mg L⁻¹ treatment gradually declined over the course of the experiment, while in the 2–4 mg L⁻¹ treatment they decreased rapidly to the point of not being detectable after day 3 (Figs. 1.4A–1.4E). Because intensity measurements were calculated for each individual pixel on the green channel, we also determined whether the number of pixels in the green channel changed, representing the area of fluorescence in the samples. While the area of green fluorescence remained similar for corals in the 6–8 mg L⁻¹ control treatment over the duration of the experiment, there was a gradual decrease in the 4–6 mg L⁻¹ treatment; the area of green fluorescence was about 3× smaller than the control by the end of the experiment (Figs. 1.5A, 1.5C–1.5E). The fraction of green pixels in corals showed significantly greater decreases in the 2–4 mg L⁻¹ oxygen treatment than in the 4–6 and 6–8 mg L⁻¹ treatments by the end of the experiment (F_(2,15) = 63.21, p < 0.001), being about 10× smaller than in the controls (Figs. 1.5A–1.5E). The fraction of green fluorescent pixels rapidly declined within the first three experimental days (Fig. 1.5A) and was barely detectable by the end of the experiment.

The intensity of fluorescence and the amount of pixels producing green fluorescence are inherently correlated and they were thus multiplied in order to obtain one single combined “green fluorescence parameter” (Fig. S2). This parameter was significantly different amongst corals from all three oxygen treatments at the end of the experiment (Table 2).

In contrast, the increase in the signal of coral red fluorescence in the 2–4 mg L⁻¹ (Figs. 1.6A–1.6C) was significantly higher than in the 6–8 mg L⁻¹ control treatment at the end of the experiment (Table 2). The 4–6 mg L⁻¹ treatment showed slight increases in the intensity of red pixels over the experimental period, but values were not significantly different from those of either of the other treatments at the end of the experiment (Figs. 1.6D and 1.6E; Table 2).

Visual census indicated that all corals subjected to low (2–4 mg L⁻¹) oxygen treatments had lesions and partial tissue loss within the first 3 days and all specimens were dead or dying, with no coral tissue covering the calcareous coral skeleton, after day 3 (Fig. 2). These specimens rapidly became discolored (in bright field) instead of staying white, likely the result of a microbial film and/or endolithic or micro-algae growing in/on the skeleton. No other experimental specimens, under any of the other treatments showed visually detectable signs of stress or tissue loss during the course of the experiment.

Repeated-measures-ANOVA showed that algae and corals had distinct responses to the various oxygen treatments, but also that these responses were primarily driven by different factors (Table S2). The algae showed little response to the oxygen treatments (responsible for 0.6% of changes in red fluorescence, and up to 12.5% for O₂ production). The majority of effects observed in algae were driven by the Time factor (ranging from 14.4% for the Effective QY to 47.5% for the Maximum QY) and the Residual factor (ranging from 31.1% for the maximum QY to 50.5% for the Effective QY) (Table S2).

The influence of these factors was differently distributed for corals. The Treatment factor contributed from 14.1% (for daylight oxygen production rates) to 41.0% (for green fluorescence fraction) of the observed variability and was always (except for O₂ production) a statistically significant factor (Maximum QY: F = 9.998, p = 0.0017; Effective QY: F = 8.386, p = 0.0036; Green fluorescence intensity: F = 8.699, p = 0.0035; Green fluorescence fraction: F = 33.453, p < 0.0001; Green fluorescence intensity * fraction: F = 21.752, p < 0.0001; Red fluorescence intensity: F = 17.223, p = 0.0001). The Time factor contributed less to the variability but was still important, with contribution ranging from 9.2% (for green fluorescence fraction) to 37.6% (for daylight oxygen production rates). These results indicate that for the corals, daytime oxygen production rates were the least affected parameter after repeated exposure to nighttime hypoxia, while the fraction of green fluorescence was the most affected. Repetition factor effects ranged from 1.5% for O₂ production to 15.6% for the Maximum QY, and from 15.6% for the red fluorescence fraction to 39.3% for the Green fluorescence fraction. Such a large effect was also observed for the red fluorescence of algae (37.7%) thus suggesting that measuring fluorescence from complex-shaped samples (such as organisms) as a strong inherent variability due to repetition that needs to be taken into account. This variability also evolved with time since the Repetition * Time factor was often significant, ranging from 10.7% for the Green Fluorescence intensity to 22.4% for the Green Fluorescence fraction, while ranging from 9.3% for oxygen production to 23.3% for Effective QY. As for the Residual factor, it was usually lower in corals than for algae, ranging from 4.6% for the Green Fluorescence fraction to 37.5% for the O₂ production (Table S2).

Discussion

Here we examined the response of a common species of coral and reef algae to experimentally manipulated oxygen conditions reported to occur at night in coral reef environments, where DO can decrease dramatically in certain areas, due to organismal respiration and microbial metabolism. Hypoxia, defined as dissolved oxygen concentrations below 2 mg L⁻¹ (Stevenson & Wyman, 1991) has been repeatedly described in coral reef ecosystems, especially within interaction zones or reef interstices (Shashar, Cohen & Loya, 1993; Barott et al., 2009). Hypoxia has also been hypothesized to play a role in coral mortality at the location of coral algal interaction zones (Smith et al., 2006; Barott et al., 2009). This study demonstrates for the first time that low oxygen concentrations can have deleterious effects on the health and physiology of a hermatypic coral, Acropora yongei, while causing little to no effect on the common green alga Bryopsis pennata.

Conclusion

Although extremely low oxygen concentrations (2–4 mg L⁻¹) had severe impacts on A. yongei specimens over a short period of time (Fig. 1; Table S1), we also show that corals were able to tolerate reduced oxygen concentrations reasonably well (within 4–6 mg L⁻¹). Nighttime oxygen concentrations between 4 and 6 mg L⁻¹, which are commonly found during the early morning hours in various reef locations (Kraines et al., 1996; Niggl, Haas & Wild, 2010; Wild et al., 2010; Haas et al., 2010a), showed some effects on fluorescent proteins, but not on the physiological performance of the respective corals over the experimental period of 10 days. A surplus of oxygen consumption to naturally occurring low nighttime DO concentrations, as may be facilitated by proximate algae in combination with algal exudate induced increases in microbial oxygen consumption, may however exceed the tolerance (< 4 mg L⁻¹) and lead to rapid tissue loss and colonization of the calcareous coral structure by algae (Fig. 2). Additional evidence on the importance of this interaction mechanism is given by studies conducted by Barott et al. (2009), who showed that oxygen concentrations on the interfaces of coral algae interaction zones were on average 3.2 ± 0.5 and 2.9 ± 0.4 mg L⁻¹ when algae were the superior competitors (Pocillopora verrucosa vs. mixed red turf algae and Montipora sp. vs. mixed red turf algae, respectively); an oxygen concentration which is just below the coral tolerance threshold suggested by the present study, but not harmful to the algae we studied. The study further revealed that for coral algae interaction zones, where both organisms were in a stable state or corals were the superior competitor (Favia sp., Montipora sp. and Pocillopora sp. vs. crustose coralline algae), oxygen concentrations on the interfaces were on average 7.9 ± 0.7 mg L⁻¹, i.e., well above the suggested threshold of 4 mg L⁻¹.

Finally, our study presents two main findings: (1) the alga Bryopsis pennata was significantly more tolerant than the hermatypic coral Acropora yongei to extremely low oxygen concentrations (<4 mg L⁻¹), and (2) the coral could tolerate decreased oxygen concentrations up to a given point. The threshold (∼4 mg L^{- 1}) is below reported diurnal oscillations facilitated by the coral reef community metabolism, but lies within oxygen concentrations reported from the interface along coral algae interaction zones. Beyond this threshold the corals used in this study experienced rapid loss of tissue and death of the whole organism. This study therefore suggests that hypoxia may be a factor influencing competitive interactions between the reef-building corals such as A. yongei and common benthic algae such as B. pennata. Further research will investigate whether such mechanisms could be generalized to other species of corals and algae.

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