Introduction

Coastal or inshore coral populations, which comprise approximately one-third of the GBR (Hopely et al. 2007), are predicted to be the most affected by local environmental and anthropogenic stressors (Wooldridge 2009; Brodie et al. 2011). Generally, inshore corals are exposed to acute disturbances, including wet season river plumes with associated decreased salinities, increased suspended solid loads and enhanced nutrient enrichment (e.g. De’ath and Fabricius 2010; Fabricius et al. 2013b), as well as chronic pressures associated with coastal development and deteriorated water quality [e.g. Singapore, Browne et al. (2015); Florida Keys, Kenkel et al. (2015)]. In combination with increasing global stress from climate change, these local stressors have a high potential to affect the health and survival of corals on inshore reefs.

Identifying how stressful an environment is for coral requires an understanding of coral responses under a range of environmental conditions. For example, moderate levels of nutrients and shading can provide benefits for some corals through higher photosynthetic efficiency (Browne et al. 2015) and improved nutritional status (Anthony et al. 2007), but poor water quality (i.e. increased suspended particulates and nutrients) can smother corals with particulates (e.g. Weber et al. 2012), decrease photosynthesis, calcification and energy stores (e.g. Cunning and Baker 2013; Fabricius et al. 2013a), and disrupt coral-Symbiodinium symbioses (e.g. Flores et al. 2012). Some commonly measured indicators of overall coral health are coral cover, rates of growth, diversity and survival (e.g. Connell et al. 1997; Bruno and Selig 2007; Sweatman et al. 2011; De’ath et al. 2012; Anderson et al. 2015). However, biochemical and physiological markers of coral health can respond more rapidly, provide early warning of deterioration in overall condition, and provide enhanced understanding of the fitness of coral populations (e.g. Grottoli et al. 2006; Mydlarz et al. 2010; Levas et al. 2016; Rocker et al. 2017).

Corals have high trophic plasticity, obtaining nutrition both through autotrophy from endosymbiotic dinoflagellates (Muscatine and Porter 1977; Teece et al. 2011; Baumann et al. 2014) and through heterotrophy from plankton, particulate matter and dissolved organic and inorganic nutrients (Sebens et al. 1996; Anthony and Fabricius 2000; Ferrier-Pagès et al. 2003; Houlbrèque and Ferrier-Pagès 2009; Teece et al. 2011; Hinrichs et al. 2013; Hughes and Grottoli 2013; Baumann et al. 2014). Environmental conditions such as turbidity (characterised by light and particulate levels) and nutrient concentrations can represent either a nutritional resource or a source of stress, dependent on the species of coral (Anthony and Fabricius 2000). Furthermore, heterotrophic feeding efforts can be influenced by morphology of coral species (i.e. large versus small polyps and mounding versus branching), surface area to volume ratio, type of tentacles and nematocysts and even Symbiodinium abundance (e.g. Houlbrèque and Ferrier-Pagès 2009; Teece et al. 2011). Assessing the sources of autotrophic nutrition may provide an indication of the dependence of coral colonies on Symbiodinum populations, as well as potentially healthy or detrimental population sizes, while assessing sources of heterotrophic nutrition may provide insights into both positive and negative effects of sedimentation and nutrients within the surrounding environment of the coral. Understanding sources of nutrition and energy is therefore fundamental to elucidating the fitness of the coral holobiont.

Scleractinian corals have a high lipid content ranging between 10 and 46% of total organic dry weight (Harland et al. 1992). Lipids provide a nearly 2-fold greater source of energy (39.5 kJ kg−1) compared to both proteins (23.6 kJ kg−1) and carbohydrates (17.2 kJ kg−1; Bureau et al. 2002), thus knowledge of deviations from baseline levels of lipids is an important indicator of the nutritional status of a coral. In general, half of coral lipids are committed to long-term energy storage (i.e. wax esters and triacylglycerols), while the other half are structural (i.e. phospholipids and sterols) and involved in maintaining cell structure and integrity (Joseph 1979; Edmunds and Davies 1986; Latyshev et al. 1991; Harland et al. 1993; Saunders et al. 2005; Imbs et al. 2010). Fatty acids (FA) are the building blocks of lipids, and the types of individual FA and FA classes and their relative abundances can be unique among taxa (Volkman 1999). FA are used in the majority of physiological processes in animals (e.g. respiration, growth, reproduction; studies on corals include: Stimson 1987; Ward 1995; Yamashiro et al. 1999), and play a vital role in coral metabolism and stress resistance (Hulbert 2003; Imbs et al. 2015). Saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) are both efficiently metabolised, thus they are typically used for rapid energy production (Tocher 2003). In comparison, polyunsaturated fatty acids (PUFA) are more structurally complex and are used to produce signalling molecules and compounds involved in cellular activity and immune function (Tocher 2003). PUFA are necessary for a wide range of physiological actions, including immune responses, inflammatory responses, neural functioning, reproduction, and the maintenance of cell membrane fluidity (Funk 2001; Tocher 2003). Individual FA and FA classes may aid in distinguishing between autotrophic and heterotrophic sources of nutrition of corals (Al-Moghrabi et al. 1995; Treignier et al. 2008) and thus, monitoring changes in the FA composition of coral tissues may be a valuable tool for assessing coral health. This suggests that studies of FA composition and key FA ratios can provide useful insights into coral health and condition.

The quantity of lipid reserves combined with FA composition of marine organisms reflect the biochemical and ecological conditions of their environment (Bergé and Barnathan 2005). Environmental factors that can influence lipid reserves and FA composition include light (Harland et al. 1992; Al-Moghrabi et al. 1995; Oku et al. 2003), depth (Harland et al. 1992), temperature (Ward 1995; Ben-David-Zaslow and Benayahu 1999; Oku et al. 2003) and feeding strategy (Meyers et al. 1978; Meyers 1979; Szmant-Froelich and Pilson 1980; Teece et al. 2011). Lipid reserves decrease in bleached corals (Porter et al. 1989; Fitt et al. 1993; Yamashiro et al. 2005; Rodrigues and Grottoli 2007; Levas et al. 2018), yet increase when exposed to additional particulate nutritional sources (Meyers et al. 1978; Meyers 1979; Szmant-Froelich and Pilson 1980; Conlan et al. 2017). However, some studies have found environmental factors, such as depth, temperature and pCO2, do not necessarily affect lipid reserves (Harland et al. 1991, 1992; Grottoli et al. 2004; Strahl et al. 2016). As many of these environmental factors are correlated and interact (Crain et al. 2008), determining responses to seasonal and environmental variation will provide further indication of how coral health and condition vary with environmental change.

An appropriate balance of long-chain polyunsaturated fatty acids (LC PUFA) is necessary to promote proper biochemical and physiological functioning, maintain structural integrity of cellular membranes, and retain energy reserves (Russo 2009; Richier et al. 2010). n-3 LC PUFA are essential precursors to compounds involved in anti-inflammatory regulation, cellular fluidity and immunity (Tocher 2003; Russo 2009). These FA include eicosapentaenoic acid (20:5n-3, EPA), which functions in anti-inflammatory processes and docosahexaenoic acid (22:6n-3, DHA), which functions in fluidity of cell membranes (e.g. Dalsgaard et al. 2003; Guil-Guerrero 2007; Russo 2009). Conversely, bioactive arachidonic acid (20:4n-6, ARA), an n-6 LC PUFA, is required for the synthesis of pro-inflammation health hormones, responsible for water transport across membranes and an important component of the immune response (e.g. Tocher 2003; Russo 2009). Concentrations of n-3 and n-6 PUFA, as well as relative proportions and ratios of these PUFA (i.e. the balance of these), can provide insights into overall health and functioning through nutritional and oxidative processes (i.e. fatty acid oxidation; Clarke and Jump, 1993), as well as indicators of potential disease (e.g. Simopoulos 2008; Nomura et al. 2013). Therefore, the following FA ratios have been suggested as putative FA health indicators: n-3:n-6, n-3 LC:n-6 LC, EPA:ARA and EPA:DHA.

Syntheses of FA occur in both the coral host animal and its Symbiodinium (Oku et al. 2003; Dunn et al. 2012; Kabeya et al. 2018; Fig. 1), and represent a principal biochemical link between these two symbiotic partners (Kellogg and Patton 1983; Patton and Burris 1983). Despite misconceptions based on generalisations, lower animals (including corals) are able to synthesise PUFA (Dunn et al. 2012; Monroig et al. 2013); furthermore, the direct de novo biosynthetic mechanisms of n-3 PUFA in invertebrates (including corals) have been established (Kabeya et al. 2018). Although vertebrates do not possess Δ12 and Δ15 desaturases (Dewick 1997), which metabolise FA by removing hydrogen atoms and create carbon–carbon double bonds, the presence and function of these desaturases has been found in many invertebrates, including Acropora millepora (Kabeya et al. 2018). Generally in photosynthesising invertebrates, 16:0 (palmitic acid; PA), the most common FA found in animals and other organisms, is converted into 18:3n-3 and 18:2n-6 (n-3 and n-6 FA, respectively), which are precursors to LC PUFA (Fig. 1). Specifically, 16:0 is elongated into 18:0 (stearic acid, SA) and metabolised by Δ9 desaturase into 18:1n-9 (oleic acid, OA). 18:1n-9 is further metabolised by Δ12 desaturase into 18:2n-6 (linoleic acid; LA) followed by Δ15 desaturase into 18:3n-3 (α-linolenic acid; ALA; Kellogg and Patton 1983; Dalsgaard et al. 2003). It has been suggested that 18:3n-3 and 18:2n-6 are produced in Symbiodinium cells and then translocated to tissues of the coral host (Kellogg and Patton 1983; Patton and Burris 1983; Harland et al. 1993; Dalsgaard et al. 2003; Papina et al. 2003; Bachok et al. 2006; Teece et al. 2011); however, de novo synthesis of these n-3 PUFA is also possible by the coral host itself (Kabeya et al. 2018). Furthermore, heterotrophic feeding is an additional source of n-3, n-6 and n-9 FA (18:1n-9, 18:2n-6 and 18:3n-3; Al-Moghrabi et al. 1993; Hinrichs et al. 2013; Meyers 1979; Seemann et al. 2013) and hence might be elevated under enhanced particulate and nutrient conditions. Determining the source of these FA is not as clear and simple as previously thought (i.e. host de novo synthesis, symbiont de novo synthesis, heterotrophy), but could provide further information on the health status of the coral holobiont, as 18:2n-6 and 18:3n-6 are essential for proper physiological functioning, metabolism and stress resilience.

Fig. 1
figure 1

Schematic diagram of fatty acid (FA) biosynthetic pathways potentially occurring in the coral holobiont (including the coral host tissues and the Symbiodinium). FA classes are indicated across the top and include saturated FA (SFA), monounsaturated FA (MUFA), and polyunsaturated FA (PUFA). Bold denotes essential FA; arachidonic acid (20:4n-6, ARA), docosahexaenoic acid (22:6n-3, DHA), and eicosapentaenoic acid (20:5n-3, EPA). (Modified from Dalsgaard et al. 2003 and Monroig et al. 2013). Blue arrows represent desaturation by specified desaturases (ΔX), red arrows represent elongation by specified elongases (EloX), and black arrows represent peroxisomal β-oxidation (β), which cleaves the FA chain

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Many marine studies that use FA as health indicators often assume stable or constant spatio-temporal patterns (Dethier et al. 2013), therefore it is vital to determine if FA profiles of corals vary across time and space. Assessment of seasonal (Oku et al. 2003) and spatial variation (Teece et al. 2011; Seemann et al. 2013) in FA composition of corals has been limited, restricting our capacity to evaluate the potential of FA ratios as health indicators. To evaluate the viability of FA composition as a health indicator of corals, spatial and temporal changes in FA composition of the coral Acropora tenuis were examined in situ along water quality gradients in two inshore regions of the Great Barrier Reef. The aim was to determine if variation in FA composition was correlated with variation in biochemical attributes of coral health and condition, as assessed and defined in Rocker et al. (2017). This study highlights the seasonal nature of FA composition in corals, the potential for FA ratios as coral health indicators and the relationships between FA composition and coral health attributes, including growth and survival.

Materials and methods

Study site and sampling design

This study was conducted in conjunction with the spatial and temporal study of biochemical and physiological health attributes described in Rocker et al. (2017). Briefly, corals were sampled along two water quality gradients in two regions in the central Great Barrier Reef, Australia, with three sites per region at increasing distance from rivers and with declining long-term mean concentrations of suspended sediments and nutrients (Fig. 2). All sites are part of the established Inshore Marine Monitoring Program (MMP) run by the Australian Institute of Marine Science (AIMS; Thompson et al. 2014). The Burdekin region included the following three sites: Geoffrey Bay (B1; Magnetic Island), Pandora Reef (B2), and Pelorus Island (B3; Table 1). The Whitsunday region included Pine Island (W1), Day Dream Island (W2), and Double Cone Island (W3; Table 1). In both regions, variation in water quality between sites has been attributed to variation in their exposure to river runoff (Fabricius et al. 2013b, 2016). Water quality is herein defined by principal component values (PC1) from Rocker et al. (2017) (Suppl. Figure 1). Specifically, three individual and three composite variables define PC1. Temperature, salinity, and dissolved organic carbon are the individual variables. Composite variables are particulates (i.e. chlorophyll, turbidity, total suspended solids, particulate organic carbon, particulate nitrogen, and particulate phosphorus), dissolved organic nutrients (i.e. dissolved organic nitrogen and dissolved organic phosphorus), and dissolved inorganic nutrients (i.e. silicate, dissolved inorganic nitrogen and dissolved inorganic phosphorus; Suppl. Figure 1; Rocker et al. 2017). The differences in water quality between sites within the Burdekin region are more distinct compared to differences between sites within the Whitsunday region (Table 1; Suppl. Figure 1, 2, and 3; Rocker et al. 2017). For example, concentrations of total suspended solids are three times higher at B1 than B3 with less variation (1.77 ± 0.26 mg l−1 vs. 0.61 ± 0.37 mg l−1), compared to W1 and W3 with higher variation (3.77 ± 0.76 mg l−1 and 1.49 ± 0.20 mg l−1, respectively; (Fabricius et al. 2013b; Thompson et al. 2014; Rocker et al. 2017).

Fig. 2
figure 2

Map of the central Great Barrier Reef showing the location of inshore sites: three sites within the Burdekin region (orange) and three sites within the Whitsunday region (green). Each region contains sites along gradients of increasing distances from the source of river runoff selected for assessment of fatty acid composition. The main rivers, the Burdekin River in the Burdekin region and the Proserpine, O‘Connell and Pioneer Rivers in the Whitsunday region, are marked as black lines

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Table 1 Site-specific information regarding the six inshore sites within the Burdekin and the Whitsunday regions
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Twelve fragmented coral colonies of A. tenuis (approximately 15 × 15 cm2) were collected from depths of 1–3 m below datum near the MMP transects and placed on stainless steel racks at 2 m depth a few metres away from the MMP transects (Table 1; Rocker et al. 2017). Small pre-reproductive colonies were used to minimise potential changes associated with coral reproduction and spawning events (Figueiredo et al. 2012). These colonies were sampled four times to document variation in FA composition in the wet season (19–22 February 2013, 10–22 February 2014), the early dry season (15–18 June 2013), and the late dry season (4–9 October 2013). For sites B1, B2, B3 and W1, racks were established in October 2012, but for logistical reasons sites W2 and W3 were not established until February 2013 and June 2013, respectively. At each site and during each sampling point, two branches, 2 to 6 cm long, were collected from the middle of each of the 12 coral colonies. These 12 colonies (only 8 colonies were available at B3) were used to ensure repeated colony comparison (Table 1). Samples were immediately snap-frozen in liquid nitrogen for further analyses.

Fatty acid composition

Total lipid concentration and FA composition of coral colonies were determined following Conlan et al. (2017). Crushed, ground and freeze-dried samples were sub-sampled and weighed for total lipid and FA (~ 90% of the sample) and ash-free dry weight analyses (~ 10%). Data of total lipid concentration and ash-free dry weight are presented in Rocker et al. (2017).

Total lipid extracts from the coral holobiont samples were reconstituted in 1.0 ml of dichloromethane:methanol (MeOH) (2:1) for saponification. Two ml of 5% potassium hydroxide (KOH) in 80/20 MeOH/H2O water was added and samples were heated to 60 °C for 3 h. After cooling, 1.0 ml MilliQ water was added. Samples were purified three times with the addition of 1.8 ml 4:1 hexane:CHCl3 (DCM), a 10 s vortex, centrifugation for 3 min at 1000 rpm, and collection of the aqueous MeOH/H2O extract.

The aqueous MeOH/H2O extracts were methylated at 100 °C for 1 h with 2.0 ml acetyl chloride:methanol (1:10) after addition of 100 ul internal standard C23:0 (0.75 mg ml−1). Two ml of potassium carbonate (1 M) and 1.7 ml hexane were added and samples were vortexed for 10 s then centrifuged at 1000 rpm for 3 min at room temperature. FA were analysed with gas chromatography—FID (Agilent Technologies 7890A, USA) following Conlan et al. (2017). Areas of resulting individual FA peaks were corrected by theoretical relative response factors (Ackman 2002) and identified and quantified against known external standards (mixed and individual standards from Sigma-Aldrich, Inc., St. Louis, USA and NuChek Prep Inc., Elysian, USA) using GC ChemStation software (Agilent Technologies, USA).

FA concentration was standardised to weight of total lipid concentration and expressed as mg FA g lipid−1 for quantitative comparisons. Individual FA concentrations, sums of FA classes, ratios of putative FA health indicators (i.e. n-3:n-6, n-3 LC:n-6 LC, EPA:ARA and EPA:DHA), and percentage composition of FA were calculated to further explore how investment in FA varies among reefs exposed to differing, seasonal water qualities among seasons.

Statistical analyses

Analyses were performed using R 2.15.3 (R Core Team 2014). Spatio-temporal trends in FA percentage composition were characterised with principal component analyses (PCA) using the package ‘vegan’ (Oksanen et al. 2015). Generalised linear models (GLMs) were performed on principal components considered important (standard deviations greater than 1.0 establish a principal component that explains more of the total variance than would be expected by chance; Al-Kandari and Jolliffe 2005), with the three explanatory factors in the model—date, region and site (nested within region)—all treated as fixed factors.

Differences in FA (total concentration, concentrations of different classes and of individual FA (mg FA g lipid−1), and putative FA health indicator ratios), among the three potential explanatory factors (date, region and site nested within region) were explored with generalised linear mixed models (GLMMs) using the packages ‘lme4’ (Bates et al. 2015) and ‘afex’ (Singmann et al. 2016). A Gamma error distribution and log-link function were used to meet model assumptions of the former three comparisons; FA health indicator ratios were run with a Gaussian error distribution. Colony identification was included as a random factor to account for repeated sampling and maintain assumptions of independence. Pairwise t-tests were used to reveal post hoc homogenous dates and sites.

Spearman’s rank correlation analyses were performed to test for associations between FA concentrations and a number of coral health measures including growth, symbiont density, skeletal density and ash-free dry weight (from Rocker et al. 2017). To control for type 1 error in statistical tests of FA composition, the Benjamini-Yukutieli (BY) multiple correction was used (false discovery rate corrected α = 0.009; Benjamini and Yekutieli 2001).

Results

Fatty acid composition of the coral holobiont Acropora tenuis

Seventeen FA were used in analyses of the coral colonies from the Burdekin and Whitsunday regions (split by region for ease of visualisation; Fig. 3). The relative contributions of individual FA to overall FA percentage composition of A. tenuis colonies varied among sites and between seasons. Corals from the Burdekin sites were separated along PC1, which explained 47.6% of the variation in FA composition (Fig. 3a; Suppl. Table 1). Corals at site B1 (closest to river mouth) had the highest PC1 scores and corals at site B3 (furthest from river) had the lowest PC1 scores. The FA composition of B1 corals was characterised by higher percentages of short chain, less unsaturated FA (14:0, 16:0, 16:1n-7, 18:1n-9, 18:2n-6, 18:3n-6 and 18:4n-3 with the exception of 20:3n-6; Fig. 3a). In contrast, B3 corals were characterised by FA that included both n-3 and n-6 LC PUFA (20:5n-3, 22:5n-3, 20:4n-6 and 22:4n-6; Fig. 3a). Less differentiation was detected among sites within the Whitsunday region along PC1 (Fig. 3b).

Fig. 3
figure 3

Biplot of principal component analysis of the fatty acid percentage compositions of Acropora tenuis from the two regions: a Burdekin region and b Whitsunday region. The percentage composition of fatty acids within lipid extractions were from coral holobionts sampled in February 2013 (open square), June 2013 (open circle), October 2013 (open triangle) and February 2014 (open daimond). Grey boxes indicate wet season

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Seasonal variation in FA composition for both inshore regions separated the different sampling occasions along PC2, which explained 14.2% of the overall variation. Generally, corals sampled in the dry season (July 2013 and October 2013) had positive scores, whereas corals sampled in the wet seasons (February 2013 and February 2014) had negative scores. FA associated with the dry season were 21:0, 16:1n-7, 18:1n-9, 20:1n-9, 20:5n-3, 22:5n-3 and 22:6n-3 (Fig. 3). Those associated with the wet season were 14:0, 16:0, 17:0, 18:0, 18:4n-3, 18:2n-6, 20:3n-6, 20:4n-6 and 22:4n-6. With the exception of 18:4n-3, a strong seasonal split of PUFA was detected, with n-3 FA associating with the dry season (20:5n-3, 22:5n-3 and 22:6n-3) and n-6 FA associating with the wet season (18:2n-6, 18:3n-6, 20:3n-6, 20:4n-6 and 22:4n-6).

Overall, concentrations of FA classes and individual FA (mg FA g lipid−1; Suppl. Table 2a, 2b and 3, Suppl. Figure 4, 5a, and 5b) of A. tenuis within the two GBR inshore regions varied among sampling times, and were significantly higher in dry seasons (June and October 2013) compared to wet seasons (February 2013 and February 2014).

Putative fatty acid health indicator ratios

All four PUFA ratios in the tissues of A. tenuis were significantly affected by sampling date, region and site (within region; Fig. 4, Table 2). Overall, the ratio of n-3:n-6 FA was 0.864 ± 0.011 (mean ± SE). The overall ratio of n-3 LC:n-6 LC was 1.046 ± 0.016, whereas the overall EPA:ARA ratio was slightly lower (0.805 ± 0.015). The overall EPA:DHA ratio (which examines the relative proportions of two n-3 LC PUFA) was 2.073 ± 0.458.

Fig. 4
figure 4

Comparisons of fatty acid ratios investigated as coral health indicators for the coral holobiont Acropora tenuis: (a, b, c) n-3:n-6; (d, e, f) n-3 LC:n-6 LC; (g, h) EPA:ARA (significant interaction of date and region); and (i, j, k) EPA:DHA. Mean FA ratios (± SE) are compared among sampling dates [February 2013 (open square), June 2013 (open circle), October 2013 (open triangle) and February 2014 (open diamond)]; between regions [Burdekin (orange) and Whitsunday (green)]; and among sites within regions (B1, B2, B3, W1, W2 and W3). Letters indicate homogenous groups identified by pairwise post hoc analyses. Grey panels indicate wet season. Fatty acid classes and individual fatty acids are shown in Suppl. Figure 4, 5a, and 5b

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Table 2 Effects of date, region and site (within region) on fatty acid health ratios of Burdekin and Whitsunday corals
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Ratios for two of the FA health indicators (n-3:n-6 and n-3 LC:n-6 LC) differed significantly among dates and sites (Fig. 4a–f; Table 2). Ratios were significantly higher in the early dry season (June 2013) compared to the wet seasons (February 2013 and February 2014; Fig. 4a, d) and significantly higher in the Whitsunday region compared to the Burdekin region (Fig. 4b, e). Sites with relatively good water quality in both the Burdekin and Whitsunday regions (B3 and W3) had higher ratios, where as mid-gradient sites (B2 and W2) generally had lowest ratios (Fig. 4c, f, h).

The EPA:DHA ratio also differed among sampling dates; significantly higher ratios were detected in February 2014 compared to all other sampling dates (Fig. 4i; Table 2). Corals in the Whitsunday region had higher EPA:DHA ratios compared to those in the Burdekin region (Fig. 4j) with the exception of B3 being similar to W3. In both regions, corals at sites with better water quality had the highest ratios (Fig. 4k). In the Whitsunday region, the EPA:DHA ratio was approximately 1.5-fold higher at W3 compared to W1 and W2 (Fig. 4k). In the Burdekin region, the EPA:DHA ratio for corals at B3 was approximately 2-fold higher than for corals at B1 and B2 (Fig. 4k).

Fatty acid correlations with coral health attributes

Positive and negative correlations were detected between FA concentrations (individual, classes and ratios) and coral health attributes (i.e. symbiont density, skeletal density and ash-free dry weight) of A. tenuis colonies among reefs along water quality gradients in the Burdekin and Whitsunday regions (Table 3). While total FA concentration did not correlate with any of the health attributes, symbiont density was positively correlated with eight individual FA (FDR corrected-α = 0.009; FA: 14:0, 16:0, 16:1n-7, 18:1n-9, 18:2n-6, 18:3n-6, 18:4n-3 and 22:6n-3), two FA classes (SFA and MUFA) and one health indicator ratio (n-3 LC:n-6 LC). Symbiont density was also negatively correlated with three individual FA (17:0, 20:5n-3 (EPA) and 20:4n-6 (ARA)) and EPA:DHA ratio, whereas skeletal density was positively correlated with EPA:DHA ratio. Growth of coral colonies was not correlated with any components of FA composition.

Table 3 Summary of significant correlations between fatty acid classes, individual fatty acids, and health indicator ratios in Acropora tenuis from the Burdekin and Whitsunday regions, and other coral health attributes: (a) symbiont density, (b) skeletal density, and (c) ash-free dry weight
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Discussion

Fatty acid variation across water quality gradients

This study documents temporal and spatial variation in FA composition of the coral A. tenuis along water quality gradients. Spatial variation in FA percentage composition was evident among sites in the Burdekin region where sites spanned a strong water quality gradient, but not in the Whitsunday region where environmental variation is weaker (e.g. Hinrichs et al. 2013; Rocker et al. 2017; Strahl et al. unpublished) and geographical distances among sites are shorter (up to 30 km, compared to 65 km for the Burdekin sites). This pattern was also detected in a range of other coral health attributes, including skeletal density, ash-free dry weight and total lipid concentration (Rocker et al. 2017; Strahl et al. unpublished). Although we cannot unequivocally demonstrate that differences in FA composition are due to water quality, measurable differences in environmental variation in conjunction with differences in FA acquisition (as well as more general biochemical attributes and maintenance), provide support for the use of FA as coral holobiont health indicators in A. tenuis (this study and Rocker et al. 2017).

SFA, MUFA and n-6 LC PUFA were in higher proportions at the reduced water quality site closest to the river mouth (B1), within the Burdekin region. High levels of n-6 PUFA, specifically ARA, have been found throughout the food chain within the Queensland region (Johns et al. 1979). Higher proportions of ARA, 22:4n-6, EPA and 22:5n-3 were found in corals further from the river (B2 and B3). Corals at these sites (B2 and B3) have less access to the both autotrophic (smaller Symbiodinium populations with less total chlorophyll content; Rocker et al. 2017; Strahl et al. unpublished) and heterotrophic sources (suspended particulates and nutrients; Rocker et al. 2017) suggesting acquisition and maintenance of LC PUFA, particularly ARA and EPA and their derivatives, is essential. Furthermore, photosynthesis rates of corals (and total chlorophyll content) within the Burdekin region decreased as both distance from river mouth increased and suspended particulates decreased (Strahl et al. unpublished). Selective retention and deposition of these LC PUFA are vital in maintaining proper physiological functioning of corals through eicosanoid hormone synthesis, tissue biosynthesis and chemical messaging (Glencross 2009).

Seasonal variation of n-3 and n-6 PUFA

The contributions of n-3 PUFA and n-6 PUFA to overall FA composition of A. tenuis varied strongly between seasons. Higher percentages of n-3 PUFA were found in the dry season (June and October 2013), when light, turbidity, particulates and temperatures are generally lower than in the wet season (see Rocker et al. 2017). This could indicate that corals are consuming prey containing higher quantities of the n-3 LC PUFA, or alternatively, that these FA are preferentially retained at the expense of other FA. The ~ 75% increase in n-6 LC PUFA concentrations, compared to the ~ 60% increase in n-3 LC PUFA, between wet and dry seasons is consistent with this latter interpretation. The maintenance of higher proportions of n-3 LC PUFA could offer a physiological advantage to corals by improving cell membrane fluidity, which permits diffusion of solutes and electrolytes across membranes (Hazel and Williams 1990). High n-3 LC PUFA concentrations also enhance electron flow in chloroplasts when light levels are reduced (Mock and Kroon 2002). It is therefore possible that the higher concentrations of this FA class were associated with optimal functioning of Symbiodinium symbionts under lower light conditions in dry seasons.

The higher concentrations of n-6 PUFA in both wet seasons influenced overall FA composition. The Austral wet season is associated with high summer temperatures, and commonly high levels of river runoff and consequently higher levels of particulates and dissolved nutrients (Thompson et al. 2014; Rocker et al. 2017). The higher levels of 18:2n-6, 20:3n-6, 20:4n-6 (ARA), and 22:4n-6 could reflect heterotrophic nutritional sources, as n-6 LC PUFA, particularly ARA, are dominant in the food chain in these regions of the GBR, from marine algae up to fish (e.g. Armstrong et al. 1994). Differentiation between Symbiodinium-produced (autotrophically sourced), coral host-produced, and heterotrophically sourced FA was not possible in this study, as corals with the greatest Symbiodinium density were also exposed to the highest particulate and nutrient loads masking the individual effects of either autotrophy or heterotrophy.

The higher proportions of n-6 PUFA could also be related to higher summer temperatures as they are involved in membrane fluidity and inflammation (e.g. Nettleton 1995). Additionally, unsaturation of FA is reduced (leading to an increase in SFA) with extreme heat stress in algae (e.g. Kneeland et al. 2013). Therefore, PUFA can provide a physiological advantage at the cellular level enabling corals to maintain the structure and integrity of cellular membranes at higher temperatures.

Putative fatty acid health indicator ratios

Similar patterns of variation in the n-3:n-6 ratios were found across water quality sites in the Burdekin and Whitsunday regions. Healthy cell membrane composition is highly dependent on PUFA acquisition and maintenance. Elevated levels of n-6 LC PUFA, particularly ARA, increase the rigidity and inflammation of cellular membranes (Simopoulos 2008). Conversely, intrinsic properties of n-3 LC PUFA, particularly EPA, include anti-inflammation and increased fluidity of cell membranes (e.g. Russo 2009). Increased n-3 LC PUFA composition is linked to improved growth, survival and stress resistance (Bachok et al. 2006). Acquisition, synthesis and maintenance of n-3 LC PUFA are essential to balance n-6 LC PUFA inflammation and low n-3 LC:n-6 LC ratios are usually due to excess intake of n-6 LC PUFA (Simopoulos 2008). This suggests that healthier FA compositions of corals should be indicated by higher relative n-3 PUFA compared to n-6 PUFA. As such, these ratios may be maintained through a higher relative contribution of heterotrophic feeding to the corals’ energetic needs at the sites with lower water quality (i.e. B1), and a higher relative contribution of photosynthesis in relatively good water quality (i.e. B3).

The ratio of n-3:n-6 in A. tenuis varied dependent on the season and supports findings from other marine invertebrates among seasons, with higher n-3:n-6 ratios in the spring proposed to be linked to food availability and spawning cycles. For example, the prawn, Crangon crangon, had an n-3:n-6 ratio of 15.16 ± 0.76 in spring, compared to 0.63 ± 0.003 in autumn (Mika et al. 2014) hypothesised to be a results of the collection of food post-winter and the autumn decrease associated with reproduction. The clam, Ensis siliqua, had a ratio of 5.21 ± 0.25 in spring compared to 3.66 ± 0.15 during the remaining seasons (Baptista et al. 2014), potentially related with gametogenic maturation and spawning. Higher n-3:n-6 ratios, found in this study, associated with the spring, or the dry season, further supports the hypothesis of selective n-3 PUFA retention as a mechanism for coping for reduced food availability or cooler temperatures, as well as storage for reproductive investments.

The overall (holobiont) n-3:n-6 ratio of 0.87 ± 0.03 for A. tenuis obtained here is comparable to ratios found for other invertebrates with photosymbionts. A. tenuis recruits on a variety of feeding regimes had n-3:n-6 ratios range from 0.7 ± 0.16 to 1.02 ± 0.19, while A. tenuis larvae (aposymbiotic) had an n-3:n-6 ratio of 0.19 ± 0.01 (Conlan et al. 2017). Several studies have compared these ratios between host tissues/cell layers and photosymbionts to determine the respective FA contributions of biologically distinct components. When the coral holobiont was split into animal host tissues (Turbinaria reniformis) and photosynthetic Symbiodinium cells, the respective n-3:n-6 ratios were 0.25 and 1.50; in comparison, their heterotrophic zooplankton food source had a mean ratio of 4.80 (Treignier et al. 2008). Similarly, when the symbiotic sea anemone Anemonia viridis was split into epidermal tissue, gastrodermal tissue and Symbiodinium cells, the n-3:n-6 ratios were 2.3, 3.8 and 4.0, respectively (Revel et al. 2016). However, these ratios may not accurately depict the health of the holobiont, as seasonal variation found in these ratios within this study may occur due to total lipid and FA reserves of the symbiotic host, Symbiodinium density and potentially Symbiodinium type; furthermore, this provides a strong indication that the presence of n-6 PUFA could be dictated by Symbiodinium populations (Imbs et al. 2014).

As FA indicator ratios are highly dependent on nutritional sources and environmental conditions (e.g. Dalsgaard et al. 2003), these ratios can be used in energy budgets to assess the trophic level of a variety of taxa (e.g. Seemann et al. 2013), including the coral host, its endosymbionts, and the combined coral holobiont (e.g. Tolosa et al. 2011). The EPA:DHA ratio can indicate the degree of carnivory and relative reliance on autotrophic versus heterotrophic nutritional sources given DHA is highly conserved through the food web (i.e. high EPA:DHA indicates a low trophic level and higher reliance on autotrophy; Dalsgaard et al. 2003). Therefore, corals at site B3 were at a lower trophic level and more reliant on autotrophy compared to corals at B1 and B2. The relative proportions of FA contributed by the animal host compared to the Symbiodinium, as well as different Symbiodinium clades could also influence the trophic levels. Symbiodinium associations within the Burdekin region differed significantly among sites; only clade C1-associations were found at B1, whereas a combination of C1/C2 mixed- and C2-associations were found at B3 (Rocker et al. 2017). Furthermore, the FA compositions of Symbiodinium clades C1 and D change differently under thermal stress (> 28 °C; Kneeland et al. 2013), although differences in FA composition were not detected prior to thermal stress.

Correlations of fatty acids with coral health attributes

Individual FA are not indicators of species-specific nutritional sources (i.e. Symbiodinium versus particulate food sources), but the presence and combination of FA can be indicative of taxonomic classes of marine primary producers (e.g. Bergé and Barnathan 2005). SFA and MUFA, including 14:0, 16:0, 16:1n-7 and 18:1n-9, were positively correlated with Symbiodinium density. These FA can be biomarkers of symbionts, as they can be produced by Symbiodinium (e.g. Figueiredo et al. 2012). However, it must be emphasised that these individual FA can also be obtained through heterotrophic food sources and manufactured de novo by the coral host. Negative correlations of the essential FA (EPA, DHA and ARA) with Symbiodinium density could suggest less healthy FA profiles could be related to high Symbiodinium densities (i.e. the holobiont FA profile could be dominated by FA contributed by the Symbiodinium and not the coral host). These FA are essential for general physiological functioning of the holobiont, including maintenance of symbiont populations, metabolism, immune response and respiration (e.g. Yamashiro et al. 1999; Hulbert 2003).

This study presented a comprehensive assessment of the FA composition of A. tenuis colonies along inshore water quality gradients of the GBR, identifying variation in n-3 LC and n-6 LC PUFA percentage composition associated with variation in both water quality and seasonal factors. FA composition was also spatially distinct over geographical distances at the regional scale (i.e. Burdekin versus Whitsunday regions). Ratios of LC PUFA (n-3:n-6 ratios) respond to fluctuations in both water quality and seasonal factors associated with wet versus dry seasons, suggesting that they are potential candidates for indicators of coral holobiont health; however, strong seasonal variation may negate these derived ratios as water quality indicators. Although there are other coral health indicators that may be easier and cheaper to measure, FA play a vital role in coral metabolism and stress resistance and are necessary for physiological processes, such as immune responses and cellular integrity. FA profiles provide additional depth and insights into the functioning of underlying mechanisms. Therefore, FA may not be a practical tool for large-scale monitoring but can provide deep insight into coral holobiont health and its interaction with the environment in more focused studies. Future research should aim to further discern the influence of autotrophic versus heterotrophic nutritional sources on FA profiles, as well as investigate how FA composition changes in the face of novel and/or stressful environmental conditions.