Environmental Pollution 204 (2015) 271e279

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Combined thermal and herbicide stress in functionally diverse coral symbionts J.W. van Dam a, b, c, *, S. Uthicke c, V.H. Beltran c, J.F. Mueller d, A.P. Negri c a

Australian Institute of Marine Science, PO Box 41775, Casuarina, NT 0811, Australia The University of Queensland, School of Biological Sciences, St. Lucia, QLD 4072, Australia c Australian Institute of Marine Science, PMB 3, Townsville MC, Townsville, QLD 4810, Australia d The University of Queensland, National Research Centre for Environmental Toxicology, 39 Kessels Road, Coopers Plains, QLD 4108, Australia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 February 2015 Received in revised form 29 April 2015 Accepted 8 May 2015 Available online xxx

Most reef building corals rely on symbiotic microalgae (genus Symbiodinium) to supply a substantial proportion of their energy requirements. Functional diversity of different Symbiodinium genotypes, endorsing the host with physiological advantages, has been widely reported. Yet, the influence of genotypic specificity on the symbiont's susceptibility to contaminants or cumulative stressors is unknown. Cultured Symbiodinium of presumed thermal-tolerant clade D tested especially vulnerable to the widespread herbicide diuron, suggesting important free-living populations may be at risk in areas subjected to terrestrial runoff. Co-exposure experiments where cultured Symbiodinium were exposed to diuron over a thermal stress gradient demonstrated how fast-growing clade C1 better maintained photosynthetic capability than clade D. The mixture toxicity model of Independent Action, considering combined thermal stress and herbicide contamination, revealed response additivity for inhibition of photosynthetic yield in both tested cultures, emphasizing the need to account for cumulative stressor impacts in ecological risk assessment and resource management. © 2015 Published by Elsevier Ltd.

Keywords: Symbiodinium Diuron Thermal stress Combined stress Photoinhibition

1. Introduction Coral reefs are primarily formed by calcification processes of scleractinian corals and coralline algae, providing a structural basis for a host of reef-dwelling organisms. Most coral species rely on an obligate symbiotic association with intracellular dinoflagellates of the genus Symbiodinium for the majority of their energy acquisition (LaJeunesse and Thornhill, 2011; Muscatine, 1990). These microalgae convert sunlight into chemically bound energy through photosynthesis and translocate photosynthates to the coral host, allowing the coral to maintain growth and reproduction in an oligotrophic environment (Muscatine and Porter, 1977). However, coral reefs worldwide are in decline due to a range of anthropogenic pressures including climate change and reduced water quality (Hoegh-Guldberg et al., 2007; Wilkinson, 2008). These

* Corresponding author. Australian Institute of Marine Science, PO Box 41775, Casuarina, NT 0811, Australia. E-mail addresses: [email protected] (J.W. van Dam), [email protected] (S. Uthicke), [email protected] (V.H. Beltran), [email protected] (J.F. Mueller), [email protected] (A.P. Negri). http://dx.doi.org/10.1016/j.envpol.2015.05.013 0269-7491/© 2015 Published by Elsevier Ltd.

pressures are likely to increase in future years under projected climate scenarios (Collins et al., 2013), expanding industrialization, urbanization and agricultural activities, presenting a substantial risk to the biodiversity of tropical coral reefs and the services they provide (Moberg and Folke, 1999). The physical and biological properties of a coral's (micro)environment predominantly determines the specific genotypes of Symbiodinium the host partners with, whose qualities in turn are important regulators of the host's distribution (Baker, 2003; Cooper et al., 2011; Iglesias-Prieto and Trench, 1994; Rowan and Knowlton, 1995; van Oppen et al., 2001). Most species can host several taxa simultaneously, and may shift the dominant Symbiodinium type following an environmental cue to increase tolerance to prevailing conditions (Baker et al., 2004; Berkelmans and van Oppen, 2006). For example, some corals maintained under conditions of thermal stress displaced sensitive symbionts in favor of a more thermally tolerant type (Jones et al., 2008). While beneficial to overcome periods of adverse conditions, the increase in thermal tolerance has been described as a trade-off with major implications (Stat and Gates, 2011), as several empirical studies have shown how hosting a more thermal-tolerant strain of Symbiodinium is likely to come at the cost of reduced photophysiological output, calcification

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and growth (Cantin et al., 2009; Jones and Berkelmans, 2010; Little et al., 2004; Mieog et al., 2009). Reef-building corals are vulnerable to environmental change as they live close to their upper thermal limits (Berkelmans and Willis, 1999) and are sensitive to a range of contaminants (Fabricius, 2005). When sea surface temperatures (SSTs) rise above certain thresholds, high solar irradiance can overexcite the algal photosystems, overwhelming protective mechanisms and potentially damaging sensitive tissues in both symbiont and host. In corals and other symbiotic reef species, this may lead to the dissociation of the symbiotic relationship (bleaching) (Lesser, 2011). On Australia's Great Barrier Reef (GBR), highest SSTs occur in January and February (Lough, 2007), coinciding with monsoonal rainfall and river flooding events that deliver vast loads of fresh water, suspended sediments, dissolved nutrients and agrochemical residues into estuaries and coastal seas (Kroon et al., 2012). Thus nearshore reefs in the GBR lagoon are prone to suffer from combinations of cumulative stressors during episodes of thermal stress. Recent decades have seen increased agricultural applications of fertilizers and pesticides in coastal river catchments which has resulted in the ubiquitous presence of chemical residues in waterways and inshore areas of the GBR (Kennedy et al., 2012; Lewis et al., 2009; Shaw et al., 2010). Of particular ecological concern are the photosystem II (PSII) herbicides including diuron and atrazine as they are highly mobile and persistent (Brodie et al., 2012), while safety margins between chronic environmental concentrations and toxic concentrations are relatively small (van Dam et al., 2011). Although the highest concentrations of PSII herbicides on the GBR have been found in conjunction with major river floodings (Lewis et al., 2009), some highly potent ones such as diuron can be detected year round in the central region of the GBR (Kennedy et al., 2012). These compounds inhibit photochemical electron transport through PSII after binding to an electronacceptor protein, outcompeting the normal ligand for binding sites. In corals, this limits photosynthesis in the symbionts and energy flow to the host (Cantin et al., 2009). Chronic exposure can lead to coral bleaching (Cantin et al., 2007; Jones, 2004), as excitation energy cannot be transferred through the electron transport chain but is instead passed over to form damaging reactive oxygen. Despite the relatively low direct risk associated with herbicide pollution to corals as chronic environmental concentrations are generally low (Kennedy et al., 2012; Shaw et al., 2010), evidence is emerging that prolonged, low level herbicide exposure may increase the sensitivity of corals and other symbiotic phototrophs to further environmental stressors such as high SSTs (Negri et al., 2011; van Dam et al., 2012). Despite a wide body of research on the thermal stress physiology of corals and their diverse range of symbionts, very little is known regarding the sensitivity of different types of Symbiodinium to further aspects of water quality, e.g. their ability to tolerate contaminants or sedimentation, let alone how these parameters may influence their thermal sensitivity. Physical conditions such as temperature are likely to affect membrane properties, diffusion rates and biokinetic pathways of contaminants, thereby largely influencing a compound's toxicity (Holmstrup et al., 2010). In a similar fashion, contaminants may effectively lower threshold temperatures at which a stress response is initiated. Elevated SSTs in conjunction with PSII herbicides have previously been reported to elicit additive adverse effects in adult corals (Negri et al., 2011) and symbiotic foraminifera (van Dam et al., 2012), yet the physiological properties of the hosted symbionts were never considered in these mixed stressor studies. Here, we aim to further elucidate what role the genotype may play in a symbiont's response to combined physical and chemical stress, and how environmentally relevant concentrations of a PSII

herbicide may influence the thermal susceptibility of different strains of Symbiodinium. We apply a comparative approach to investigate the photochemical capacity of two distinct types of Symbiodinium dominant on the central section of the GBR: a generalist (fast-growing) strain of clade C1 against a specialist (thermal-tolerant) strain of clade D. First, PSII electron turnover in response to the commonly detected PSII herbicide diuron was assessed in long-term cultures (1e3 months; proxies for freeliving populations) and freshly isolated Symbiodinium (symbiotic cells outside the host's influence) of both strains. Next, cultures of both strains were exposed to increasing concentrations of diuron over a thermal stress gradient to examine any cumulative effects. With globally increasing SSTs potentially driving corals towards associations with more thermal-tolerant symbiont types (Stat and Gates, 2011; van Oppen et al., 2009), their capacity to respond to and manage additional environmental pressure may be reduced. 2. Methods 2.1. Preparation of Symbiodinium cultures Several healthy colonies (~30 cm diameter) of both Acropora tenuis and Acropora millepora were randomly sampled at ~6 m depth from an inshore, central GBR reef (19
10.2000 S, 146
51.1570 E). Colonies were maintained outdoors in partially shaded, 1000 L flow-through aquaria containing 5 mm filtered seawater (FSW) at 26e27
C. At high noon, maximum irradiance observed was 350 mmol quanta m2s1 PAR. The dominant Symbiodinium clades C1 (A. tenuis) and D (A. millepora) as hosted by the corals were confirmed by single strand conformation polymorphism (SSCP) following van Oppen et al. (2001). Symbiont types were selected due to their dominance on the central section of the GBR and their history as a laboratory test species. Part of each coral was stripped of its tissue using compressed air; tissue and algae were collected, filtered through a 20 mm mesh and centrifuged at 1600  g for 3 min. The supernatant was then gently decanted and the algal pellet resuspended in 0.2 mm FSW. This process was repeated 3 times before algal suspensions were pooled by type. Antibiotics (penicillin, neomycin, streptomycin and nystatin; at 100 mg mL1 each) and a diatom growth inhibitor (GeO2; 5 mg mL1) were added to the suspensions. After 24 h, the suspensions were washed 3 times before resuspension in sterile IMK medium (Wako Chemicals, USA) containing antibiotics (as above). Each parent coral hosted several strains of the dominant clades C1 and D Symbiodinium, resulting in polyspecific cultures for each clade. These will subsequently be referred to as the clade C1 and clade D cultures. Cultures were maintained in log growth phase by weekly transfer to fresh growth media for 1e3 months before use; dominant genotypes were reconfirmed prior to the start of each experiment. Cultures and test plates were maintained at 26
C, on a 14L:10D photoperiod under 50e60 mmol quanta m2s1 PAR irradiance, unless stated otherwise. 2.2. Chlorophyll fluorescence techniques Photochemical performance of the different symbiont types was estimated using the saturation pulse method (Schreiber, 2004) in a Maxi Imaging-PAM (I-PAM) (Walz GmbH) fluorometer. The effective quantum yield of PSII, DF/Fm0 , is directly proportional to photosynthetic energy conversion in PSII and the recommended parameter for use in ecotoxicology (Ralph et al., 2007). This is particularly important when dealing with PSII-inhibitors as these chemicals generally require light to elicit an effect (Ralph et al., 2007; Schreiber et al., 2007). Test procedures followed a standard

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I-PAM bioassay to assess phytotoxicity (Muller et al., 2008; Schreiber et al., 2007), implemented with slight modifications: i) cell densities for maximum test sensitivity and reproducibility were determined at 1.5  106 cells mL1; ii) actinic light settings were chosen so that test irradiance matched culture conditions (50e60 mmol quanta m2s1 PAR). 2.3. Diuron response of freshly isolated and long-term cultured Symbiodinium Diuron (N0 -[3,4-dichlorophenyl]-N,N-dimethylurea) stock solutions were prepared by dissolving analytical grade diuron (SigmaeAldrich) in 0.2 mm FSW (acetone carrier, final concentration in test media < 0.05% v/v). Two mL aliquots (1.5  106 cells mL1) of either long-term Symbiodinium culture were added to each well in duplicate 24-well cell culture plates and dosed with diuron stock to obtain final concentrations of 0.3, 1, 2, 3, and 10 mg L1 diuron, adding to a solvent control group (carrier only). Each treatment concentration was replicated 6 times. Following dosing, suspensions were well mixed and plates shelved under culture conditions. DF/Fm0 was assessed following a 24 h incubation period. Similar methods were applied to triplicate strains of Symbiodinium clade C1 and duplicate strains of clade D, freshly isolated from parent corals (isolation as above; washed 3 times in 0.2 mm FSW and resuspended in IMK growth media; no antibiotics were added). 2.4. Diuron response over a temperature gradient Five controlled-light incubators (14L:10D photoperiod, 50e60 mmol quanta m2s1 PAR irradiance) set at 26, 28, 30, 32 and 34
C (±0.2
C) were used for this experiment. Eight days prior to the start of the experiment, 10  100 mL aliquots of both C1 and D cultures were transferred to 250 mL flasks and placed in the 26
C incubator. After 24 h, duplicate flasks of each culture were heated over a 24 h period (maximum increase 0.33
C h1) by successively placing flasks in warmer incubators until each incubator held duplicate flasks of each culture. Seven days later and 2 h after the start of the photoperiod, 2 mL aliquots (1.5  106 cells mL1) of Symbiodinium suspensions from each flask were added to duplicate 24-well plates and dosed with diuron stock as described above to obtain final concentrations of 0.3, 1, 2, 3, 6, 10 and 30 mg L1 diuron, adding to a solvent control group (carrier only). Each treatment concentration was replicated 6 times. Following dosing, suspensions were well mixed and plates replaced in their respective incubators. DF/Fm0 was assessed after 24 h. 2.5. Data analysis General linear models were employed to test for differences between cultured and fresh symbionts and between symbiont types. Equal variance and normal distribution assumptions were evaluated via residual analysis. Yield data were arcsine transformed prior to analysis as these data represent fractions. Treatment effects were quantified by calculating the percentage inhibition of DF/Fm0 in treatment groups relative to the solvent control: % inhibition ¼ 100 x (1 (DF/Fm0 )sample/(DF/Fm0 )control). Equations modeling the response of PSII to diuron were obtained by fitting 4-parameter logistic curves to the inhibition data; from these curves diuron concentrations eliciting x% inhibition of DF/ Fm0 (ICx) were estimated. Evaluation of phytotoxicity through the combined effects of diuron and temperature was achieved in similar fashion. Here, ICx values and temperatures eliciting x% inhibition of DF/Fm0 (ITx) were estimated by evaluating the diuron response at the various temperatures and the temperature

Fig. 1. Relative inhibition (as % of control values) of DF/Fm0 in cultured (solid lines) and freshly isolated (dotted lines) Symbiodinium clade C1 and D strains exposed to 0e10 mg L1 diuron for 24 h n ¼ 6 wells per treatment. a) Symbiodinium clade C1 strains. b) Symbiodinium clade D strains. Data are means ± SE. Regression represents a 4-parameter logistic curve fitted to data means.

response at different diuron concentrations, respectively. Temperature effects were modeled by fitting 3rd degree polynomial curves to the inhibition data as the temperature response followed an optimum curve and the polynomial model provided the best fit. The reference mixture model of Independent Action (IA) was used as previously described (van Dam et al., 2012) to predict combined additive effects from the individual component effects. These predictors were then plotted against the observed data. Observed data means and confidence limits overlapping the zerointeraction line (derived through the model of IA) indicated response additivity; an observed response < predicted response indicated sub-additivity; and an observed response > predicted response indicated synergism. Contour plots modeling the inhibition of DF/Fm0 in response to combined thermal and herbicide exposure were generated through the mean inhibition of DF/Fm0 for each exposure combination, relative to maximum DF/Fm0 observed. Linear modeling was performed in NCSS 2007 (Number Crusher Statistical Software), concentration-response modeling and analysis in SigmaPlot 11.0 (Systat Software).

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Table 1 Parameters calculated from modeled DF/Fm0 inhibition curves and concentrationresponse experiments conducted at 26
C. IC10 and IC50 are the concentrations of diuron required to inhibit DF/Fm0 by 10 and 50%, respectively. DF/FM0 values in the absence of herbicide ranged from 0.34 to 0.55 for all cultures (50e60 mmol quanta m2s1 PAR irradiance). Clade

State

Curve fit (R2)

24 h IC10 [95% CI] (mg L1 diuron)

24 h IC50 [95% CI] (mg L1 diuron)

C1

Cultured Fresh isolates Cultured Fresh isolates

1.0 0.98 0.99 1.0

0.4 0.7 0.2 0.6

2.5 5.0 1.2 3.7

D

[0.3e0.5] [0.5e1.0] [0.1e0.3] [0.5e0.7]

[2.3e2.7] [4.1e6.0] [1.1e1.4] [3.4e3.9]

3. Results 3.1. Diuron response of freshly isolated and long-term cultured Symbiodinium The inhibition of DF/Fm0 in response to increasing diuron concentrations followed a classic logistic pattern in all tests (Fig. 1). Adverse effects were noted at very low concentrations: 10% inhibition of DF/Fm0 (IC10) was observed in all experiments at concentrations between 0.2 and 0.7 mg L1 diuron (Table 1). Both longterm cultures exhibited a significantly higher (p < 0.001) sensitivity to diuron (2e3-fold) than their freshly isolated counterparts (concentrations inhibiting DF/Fm0 by 50% (IC50) ¼ 1.2 mg L1 and 3.7 mg L1 for clade D and 2.5 mg L1 and 5.0 mg L1 for clade C1 respectively) after 24 h exposure (Table 1). Furthermore, the clade D culture tested twice as sensitive to diuron as the C1 culture (IC50 ¼ 1.2 mg L1 and 2.5 mg L1, respectively), whereas fresh isolates from the clade D corals were also more sensitive than the fresh C1 isolates (IC50 ¼ 3.7 mg L1 and 5.0 mg L1 diuron, respectively) yet the differences were less pronounced (Table 1). 3.2. Diuron response over a temperature gradient Both C1 and D cultures exhibited the highest effective quantum PSII yield (DF/Fm0 ) at 28
C (Table 2); no significant difference (p ¼ 0.444) was noted between cultures under these conditions. Yields at 28
C were subsequently taken as baseline values to derive concentration-response curves for diuron at different temperatures (Fig. 2) and to assess the response to combined thermal and herbicide stress (Fig. 3). In both cultures a rapid proportional reduction of DF/Fm0 was observed at temperatures  30
C. For the C1 culture

(Fig. 2a) the order of energy conversion in PSII in the absence of diuron was 28
C > 26
C > 30
C > 32
C (7%, 20% and 66% inhibition of DF/Fm0 relative to the 28
C reference, respectively). For the clade D culture (Fig. 2b) the order of energy conversion in PSII was 28
C > 30
C > 26
C > 32
C (9%, 15% and 55% inhibition DF/ Fm0 relative to the 28
C reference, respectively). Cultures of neither clade survived seven days at 34
C (data excluded from graphs and analyses). At 26
C, diuron response (IC50) for both cultures (Table 2) corresponded well with the response observed during the previous experiment (Table 1). With the exception of the 28
C controls, in all 26
C and 28
C treatments a significantly higher DF/Fm0 was noted for the clade C1e than for the clade D culture, irrespective of diuron concentration (p  0.012). An increased thermal tolerance was noted for the clade D culture in the 30
C and 32
C treatments in the absence of diuron, where significantly higher DF/Fm0 values were documented than for the C1 culture (p  0.020). However, once diuron was introduced, DF/Fm0 dropped more rapidly in the D than in the C1 cultures, such that once diuron reached  1 mg L1, DF/Fm0 in the C1 culture was always higher (p < 0.001), regardless of temperature (at this irradiance). As DF/Fm0 was equivalent for both cultures in the 28
C (no diuron) treatments and inhibition in each treatment was calculated proportionally to this value, ICx- and ITxvalues (Table 2) could be directly compared across the two cultures (where IT50 is the temperature that inhibited DF/Fm0 by 50%). No significant differences were noted between the cultures in their sensitivity to temperature (IT50) at different diuron concentrations (Table 2), however the clade D culture was found to be more sensitive to diuron than the C1 culture over the entire temperature range tested (IC50s 1.1e1.5 mg L1 and 2.0e2.1 mg L1 diuron, respectively; Table 2). Contour plots presenting inhibition of DF/Fm’ over the range of combined temperature and diuron exposures effectively demonstrated the differential characters of each culture's response to the cumulative stressors (Fig. 3). The more widely spaced contours noted for the C1 culture (Fig. 3a) in vertical direction as opposed to the D culture (Fig. 3b) suggest the C1 culture to be more tolerant to the DF/ Fm’-inhibiting effects of diuron, while the horizontally extending pattern of contours as found for the D culture is indicative of a slightly higher thermal stress tolerance. 3.3. Quantifying the combined effects of temperature and diuron on photosynthetic efficiency The mixture model of Independent Action (IA) was used to

Table 2 Effective diuron concentrations and temperatures where 50% inhibition of DF/Fm0 occurred (IC50 and IT50, respectively) [95% CI] in Symbiodinium clade C1 and D cultures exposed to 0e30 mg L1 diuron for 24 h at temperatures ranging from 26 to 32
C (Fig. 2). Values for each treatment were calculated relative to DF/Fm0 at 28
C in the absence of diuron. Diuron response for the different temperatures was modeled by fitting a 4-parameter logistic curve to the inhibition data; temperature response for the different diuron concentrations was modeled by fitting a 3rd degree polynomial curve to the inhibition data. Empty fields signify diuron concentrations or temperatures at which always  50% inhibition DF/Fm0 was observed. Temperature (
C)

Clade C1

Clade D

DF/Fm Solvent control [95% CI] 26 28 30 32 34

0.32 0.34 0.27 0.11 0

[0.31e0.32] [0.33e0.35] [0.25e0.29] [0.11e0.12]

Diuron (mg L1)

DF/Fm0 28
C [SE]

0 0.3 1 2 3 6 10

0.34 0.33 0.26 0.18 0.13 0.06 0

[0.33e0.35] [0.32e0.33] [0.26e0.26] [0.17e0.18] [0.12e0.13] [0.05e0.06]

Clade C1

Clade D 1

IC50 (mg L

0

0.30 0.35 0.32 0.16 0

[0.29e0.31] [0.33e0.36] [0.29e0.34] [0.14e0.17]

diuron) [95% CI]

2.1 [1.9e2.3] 2.1 [1.7e2.5] 2.0 [1.7e2.3] e e

1.1 [1.0e1.3] 1.5 [1.2e1.7] 1.3 [1.1e1.4] e e

IT50 (
C) [95% CI] 0.35 0.31 0.23 0.13 0.08 0 0

[0.33e0.36] [0.31e0.32] [0.22e0.24] [0.12e0.14] [0.08e0.09]

31.3 31.2 30.8 29.3 e e e

[30.8e31.7] [30.6e31.8] [29.3e32.0] [NC-30.9]

31.7 [30.7e32.9] 31.3 [30.4e32.3] 30.3 [28.1e31.4] e e e e

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Fig. 2. Relative inhibition (as % of control values) of DF/Fm0 in Symbiodinium clade C1 and D cultures exposed to 0e30 mg L1 diuron for 24 h at temperatures ranging from 26 to 32
C. n ¼ 6 wells per treatment. a) Symbiodinium clade C1 cultures. b) Symbiodinium clade D cultures. Data are means ± SE. Regression represents a 4-parameter logistic curve fitted to data means.

investigate any interactive effects of simultaneous temperature and diuron exposure. When presenting the predicted combined response (calculated through IA) plotted against the measured combined response (raw data), a very high level of agreement between the two parameters for both investigated cultures was revealed (Fig. 4). The great majority of datapoints overlapped the 1:1 line (measured inhibition of DF/Fm’ matching the predicted inhibition) or deviated only slightly (5%) from it, indicative of response additivity (individual diuron and thermal effects added up e no stressor interactions). For the C1 culture, a few datapoints in the 30
C and 32
C treatments were 5e20% below the 1:1 line, implying a slightly greater predicted than observed effect (subadditive interaction) at these temperatures (Fig. 4a). Conversely, some datapoints were observed up to 12% above the 1:1 line for the D culture, indicating slight synergy (observed effect greater than predicted) around 32
C (Fig. 4b). Most importantly, the greatest effects are observed when stressors are combined, which corresponds well with data presented in Figs. 2 and 3. 4. Discussion In corals, functional adaptation of the obligate symbionts is an important driver of host differentiation (Baker, 2003; Cooper et al., 2011; Rowan and Knowlton, 1995) and is often accompanied by physiological tradeoffs (Stat and Gates, 2011; van Oppen et al., 2009). We exposed two distinct strains of Symbiodinium to the widespread PSII herbicide diuron over a thermal gradient to investigate potential differences in response to diuron and the influence of the herbicide on thermal susceptibility, as well as the nature of a possible interaction of the two stressors. The two types of Symbiodinium considered here have been the subject of earlier comparative studies, wherein the clade C1 algae exhibited much faster growth (Little et al., 2004; Mieog et al., 2009), electron transport and carbon fixation rates (Cantin et al., 2009) than the clade D algae at unstressed conditions. However, once a thermal

stress threshold was exceeded, an increase in excitation pressure over PSII was noted in corals associated with clade C1 (but not in clade D) Symbiodinium (Mieog et al., 2009), a measure which is narrowly associated with chronic photoinhibition (Iglesias-Prieto et al., 2004) and a precursor to a bleaching response (Jones, 2004; Smith et al., 2005). We found the two investigated strains to vary considerably in their response to diuron. Effect concentrations inhibiting effective quantum PSII yield (DF/Fm0 ) (Tables 1 and 2) in the investigated Symbiodinium cultures were lower than reported for most other cultured microalgae (Magnusson et al., 2010), in the range of reported environmental concentrations (Kennedy et al., 2012; Lewis et al., 2009) and below current water quality guideline values for the GBR (GBRMPA, 2010), suggesting that populations of freeliving Symbiodinium may be at risk from herbicide exposure. The observed sensitivity of the clade D cultures to diuron could be especially consequential, as free-living Symbiodinium provide a pool for infection for larvae of many coral species not deriving symbionts through vertical transfer (parent to egg) (Coffroth et al., 2006). Photosystem II energy conversion in the clade D cultures was approximately three times as sensitive to diuron as in their freshly isolated counterparts (Table 1) and at least twice as sensitive as that of the C1 cultures or freshly isolated C1 symbionts considered here (Tables 1 and 2). Moreover, it tested twice as sensitive as DF/Fm0 in intracellular Symbiodinium (unknown genotypes) in a range of coral species (IC50 between 2.3 and 6 mg L1) (Jones and Kerswell, 2003; Jones et al., 2003; Negri et al., 2005), in a C2 strain hosted by A. millepora (IC50 of 2.9 mg L1) (Negri et al., 2011) and in freshly isolated Symbiodinium (unknown genotype) from Stylophora pistillata (IC50 of 5.5 mg L1) (Jones et al., 2003). Cultured Symbiodinium cells alternate between coccoid (nonmotile) and mastigote (motile) morphology (Fitt and Trench, 1983), where the mastigote stage is believed to allow for shortrange dispersal which, in the field, facilitates infection of aposymbiotic hosts. Symbiotic, intracellular algae occur

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Fig. 3. Contour plots for the relative inhibition of DF/Fm0 (as % of maximum DF/Fm0 observed) of Symbiodinium clade C1 and D cultures in response to combined herbicide and temperature stress. a) Symbiodinium clade C1 cultures. b) Symbiodinium clade D cultures.

predominantly in coccoid form, surrounded by a membrane of host origin (plasmalemma) beneath the cell wall (Colley and Trench, 1983). The plasmalemma is presumably still present shortly following isolation and provides an extra physical barrier that may protect the cell from xenobiotics and adverse conditions, possibly explaining the reduced sensitivity to diuron in fresh isolates as opposed to the longer-term cultures. In the only previous direct comparison of differential clade responses to diuron, Cantin et al. (2009) observed reductions in electron transport and carbon fixation rates in juvenile A. millepora corals hosting a C1 strain proportionally greater than corals hosting a clade D strain; however, IC50 was not quantified for DF/Fm0 to enable direct comparison. Nonetheless, both studies highlight the importance of accounting for symbiont genotype when reporting on the sensitivity of Symbiodinium to herbicides. The mechanism behind the apparent difference between the investigated strains in

Fig. 4. Comparison between observed and predicted (IA) combined effects of elevated temperature and diuron on DF/Fm0 in Symbiodinium clade C1 and D cultures. Datapoints overlapping the zero-interaction line indicate response additivity; datapoints underneath the additivity line indicate sub-additivity (observed effect < predicted effects); and datapoints above the additivity line indicate synergism (observed effect > predicted effects). n ¼ 6 wells per treatment. a) Symbiodinium clade C1 cultures. b) Symbiodinium clade D cultures. Data are means; error bars represent 95% CI.

their response to diuron remains unknown. Diuron is a plastoquinone analog that competes for binding places on the QB binding site of the D1 electron acceptor protein in PSII, effectively blocking linear electron transport, leading to a decrease in photochemical efficiency (reduced DF/Fm0 ). The binding of diuron-like inhibitors to the D1-protein is a reversible process, yet sustained exposure or high concentrations may result in chronic photoinhibition (Jones, 2005) when the rate of photodamage to the PSII reaction centers exceeds the rate of its repair (Aro et al., 1993). Different genetic variants of Symbiodinium have been reported to vary considerably in their capacity to repair damaged PSII reaction centers as a reaction to stress-induced photoinhibition (Takahashi et al., 2004,

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2009; Warner et al., 1999), and although this hypothesis remains untested, it is likely the differential response to diuron as observed here can be allocated to disparity in PSII protein turnover rates during photoinhibition. The photosystems of the investigated clade C1 and D Symbiodinium cultures performed optimally at 28
C, corresponding with the average summer maximum at the central GBR (Momigliano and Uthicke, 2013). At higher or lower temperatures, DF/Fm0 was impaired to some extent (Fig. 2). Temperatures  30
C inhibited DF/Fm0 to a greater extent in the C1 than D culture, consistent with the ‘thermal tolerance’ assumption that many known strains of clade D Symbiodinium demonstrate a relative resilience to elevated SSTs with regards to other clades, as often reported in the last decade (Baker et al., 2004; Berkelmans and van Oppen, 2006; Rowan, 2004). It has been argued that thermal stress limits the amount of energy translocated to the dark reactions of photosynthesis where CO2 is fixed (sink limitation) (Jones et al., 1998). Further evidence suggests that thermal damage to thylakoid membranes causes photoinhibition in cultured Symbiodinium (Tchernov et al., 2004). Photoinhibition is the process whereby excess absorbed light energy cannot be dissipated by the various quenching pathways and is instead passed over to oxygen, creating highly reactive oxygen radicals (Lesser, 2006). These damage photochemical and carbon fixation pathways in the symbionts, while DNA and tissue damage may occur in the host (Lesser, 2011). The breakdown in symbiosis (bleaching) is closely linked to photoinhibition and oxidative stress (Hill et al., 2004; Jones, 2004; Warner et al., 1999). In cnidarians, a number of photoprotective pigments and biochemical antioxidant agents exist that can limit the availability of reactive oxygen or reduce the amount of light energy converted (Lesser, 2006). Several experimental studies on corals hosting clade D Symbiodinium have demonstrated a high thermal and irradiance tolerance as opposed to other clades (Berkelmans and van Oppen, 2006; Iglesias-Prieto et al., 2004; Rowan, 2004) and although the exact means through which enhanced thermal tolerance is achieved remain unresolved, many clade D strains are likely to be better endorsed protectively than other clades. Moreover, as mentioned earlier, evidence exists that the rate of physical turnover of PSII reaction centers greatly influences a symbiont's sensitivity to photochemical stress (Takahashi et al., 2009; Warner et al., 1999). Most Symbiodinium clades embrace a rich diversity at a subcladal level (LaJeunesse and Thornhill, 2011) with varying functional optima (Baker, 2003; Rowan and Knowlton, 1995; van Oppen et al., 2001). Functional variation has even been observed in similar strains isolated from different regions (Howells et al., 2012). Therefore, contextualizing results obtained from laboratory experiments to ecologically relevant scenarios is challenging at best. Corals in their natural environment can simultaneously host a variety of Symbiodinium genotypes (Carlos et al., 2000; Rowan and Knowlton, 1995) and may regulate dominating fractions to complement prevailing environmental conditions (Baker et al., 2004; Berkelmans and van Oppen, 2006). Conversely, culturing under controlled conditions is a selective process that will favor specific strains to the detriment of others (Kinzie III et al., 2001); and Symbiodinium in culture are likely to optimize their physiology to culture conditions, further affecting stress response capacity. The results presented here were obtained exclusively with the use of chlorophyll a fluorescence techniques. These have been widely applied in algal and coral ecotoxicology in recent years and are especially suitable for early detection of inhibition of function within PSII (Ralph et al., 2005). However, despite its costeffectiveness, ease of use and accurate assessment of the photosynthetic apparatus, the use of PAM-fluorometry as a relevant

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toxicological method is debated, as photoinhibition is a reversible, sublethal stress response and would therefore be an inadequate indicator for ecosystem health (Ralph et al., 2007). However, several studies suggest that PSII inhibition as measured by PAMfluorometry correlates well with more established toxicity measures such as growth rates and biomass increase in macrophytes and microalgae (Küster and Altenburger, 2007; Magnusson et al., 2008); and bleaching in corals and symbiotic foraminifera (Negri et al., 2011; van Dam et al., 2012). Inhibition of DF/Fm0 in Symbiodinium by diuron and/or thermal stress may therefore imply detrimental effects on the growth and productivity of the algal cells. In a system where the response of a biological receptor to two stressors is estimated, the combined response is either a simple addition of each individual response, or the stressors can interact, resulting in a sub-additive or synergistic response (Berenbaum, 1989; Folt et al., 1999). The evaluation of the combined response is usually based upon a reference concept that predicts combined additive effects from the toxicity of the individual components (Berenbaum, 1985; Greco et al., 1995). The combined effect model of Independent Action (IA) was considered appropriate to explore the combined impacts of diuron and thermal stress as both affect energy conversion in PSII, yet have different molecular modes of action. The IA model revealed very consistent results for both symbiont types with only minor diversions from the response additive model (Fig. 4), indicative of how PSII electron flow in Symbiodinium is most impacted when elevated SSTs and diuron are combined and emphasizing the need to consider cumulative stressors in environmental risk management. As highest SSTs on the GBR are observed in austral summer (January and February (Lough, 2007)), when simultaneously monsoonal rainfall events deliver peak herbicide concentrations into nearshore areas of the lagoon (Kennedy et al., 2012), vulnerable reefs are likely to be exposed to combinations of these stressors. While thermal stress is most likely the main physiological driver behind stress-induced coral bleaching (Baker et al., 2008), recent evidence implies that water quality may strongly influence the vulnerability of corals to increased SSTs (Negri et al., 2011; Wooldridge, 2009), a notion supported by the research presented here. The common association of clade D Symbiodinium with corals exposed to relatively high levels of environmental stress (e.g. LaJeunesse et al., 2010; Oliver and Palumbi, 2009) has led to the hypothesis that this association is established primarily as a survival strategy (Stat and Gates, 2011). However, our results demonstrate how both freshly isolated and long-term cultured clade D Symbiodinium tested more sensitive to the herbicide than their clade C1 counterparts. Furthermore, while the clade D culture was more thermally tolerant than the clade C1 culture, the reverse was the case in the presence of diuron above 0.5 mg L1. This is a first indication that in the presence of low level herbicide contamination, stress-tolerance benefits for corals through association with clade D Symbiodinium may be moderated. Moreover, the high sensitivity of the cultured strains to diuron may have substantial implications for free-living populations in the environment, vital for infection of aposymbiotic coral larvae and facilitating restocking of bleached colonies. The ‘nugget of hope’ expressed by various authors on the adaptive strategy of corals associating with clade D Symbiodinium to survive projected increases in ocean temperature due to global warming (Berkelmans and van Oppen, 2006; Stat and Gates, 2011) may thus be impacted at sites with a potential for herbicide contamination. In view of this, our results lend further support to the hypothesis that management practices directed towards curbed inflow of contaminated waters can contribute to the longer-term protection of vulnerable coral reef ecosystems.

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