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Natural Variation in Responses to Acute Heat and Cold Stress in a Sea Anemone Model System for Coral Bleaching.


In the absence of a significant adaptive response, most of the world's coral reef cover is projected to vanish within the century (Frieler et al., 2012; Logan et al., 2014). Yet the complex nature of coral stress responses challenges our ability to accurately predict evolutionary potential. In addition to abiotic factors, coral stress responses depend on interactions among multiple organisms including the coral animal, its endosymbiotic microalgae, and numerous other mucus-or skeleton-associated microbes, altogether termed the coral holobiont (Vega Thurber et al., 2009). In particular, the health of coral reef ecosystems hinges on mutualistic symbioses between corals and dinoflagellate microalgae of the genus Symbiodinium. Symbiodinium, a hyperdiverse taxon comprising nine subgeneric clades (A-I), also forms nutritional symbioses with giant clams, foraminiferans, and other cnidarians (e.g., sea anemones and jellyfish) (Pochon et al., 2006; Pochon and Gates, 2010). Living inside cnidarian gastrodermal cells, Symbiodinium accesses inorganic nutrients in return for compounds produced through photosynthesis. Exogenous stressors induce expulsion of Symbiodinium cells or photosynthetic pigments from host tissues, or "bleaching," causing coral mortality in severe cases (Venn et al., 2006).

The primary trigger of coral bleaching in natural environments is elevated temperature, though exceedingly low temperatures, high solar radiation, nutrients, disease, and pollutants can also induce or exacerbate bleaching (Weis, 2008). Severe bleaching and mortality associated with cold temperatures have been described for tropical coral populations from the Great Barrier Reef (GBR), the Caribbean, and the Arabian Gulf (Coles and Fadlallah, 1991; Hoegh-Guldberg et al., 2005; Lirman et al., 2011). These natural bleaching events were associated with exposure to temperatures less than 16 [degrees]C for up to 140 h in the Florida Reef Tract, surface temperatures down to 9 [degrees]C during predawn low tides for 2 d for the GBR, and 4 d where temperatures dropped below 12 [degrees]C in the Arabian Gulf (Coles and Fadlallah, 1991; Hoegh-Guldberg et al., 2005; Lirman et al., 2011). Far fewer studies have investigated tolerance to natural stressors other than high temperature, but an experimental study of the coral Acropora yongei exposed to 26 [+ or -] 5 [degrees]C suggests that short-term exposure to cold stress (<5 d) may be more detrimental to photosynthesis and growth rate compared to heat stress, though the reverse may be true under long-term stress (Roth et al., 2012). As ocean temperatures become both warmer and more variable and reef ecosystems are exposed to combined local and global stressors, improved understanding of coral resilience in the context of multiple stressors is essential (Pendleton et al., 2016).

Evidence exists supporting several mechanisms of temperature stress tolerance. For example, association with different Symbiodinium species or genotypes has a substantial influence on holobiont thermal tolerance (Rowan et al., 1997; Rowan, 2004; Howells et al., 2012). Intraspecific genetic diversity in hosts, a major determinant of adaptive potential, also contributes to the considerable variation in bleaching observed in natural coral populations. Comparisons of reciprocally transplanted corals in American Samoa suggest that 38% of the difference in heat tolerance between populations of Acropora hyacinthus is attributable to fixed effects, including holobiont genotype, whereas 62% can be attributed to acclimatization achievable over 2 y (Palumbi et al., 2014). Additional studies highlight the importance of host genotype in shaping heat tolerance of Porites astreoides and altering photochemistry of Symbiodinium in Acropora palmata subjected to cold stress (Kenkel et al., 2013; Parkinson et al., 2015). These studies have gone far toward characterizing genetic variation in responses to single stressors in corals. However, the extent to which bleaching resistance may vary in the same population under different or combined stressors remains unclear.

A major obstacle to investigating functional variation with genotype-level resolution is the difficulty of performing laboratory experiments with corals. Slow-growing corals are challenging to maintain, making long-term study by different investigators on the same genetic background very difficult. For this reason and others, the tropical sea anemone Aiptasia sp. has been used extensively as a laboratory model for studies of cnidarian-dinoflagellate symbiosis (Weis et al., 2008). Aiptasia grows rapidly and reproduces asexually through pedal laceration, enabling clonal strains to be maintained in the laboratory over long periods of time. Furthermore, extensive genomic resources are available, including a reference genome for Aiptasia strain CC7 and transcriptomes for both Aiptasia and its Symbiodinium (Lehnert et al., 2014; Baumgarten et al., 2015; Xiang et al., 2015). Like many corals, Aiptasia associates with a diversity of Symbiodinium genotypes across its geographic range: global populations most often harbor Symbiodinium minutum (ITS2 subclade type B1), though populations in the western Atlantic have been observed in association with other Symbiodinium species corresponding to types B2, A4, and, rarely, C1 (Thornhill et al., 2013; Grajales et al., 2016). In addition to genomic resources, numerous cellular and laboratory protocols for Aiptasia are also available, including several established methods for Symbiodinium quantification. Destructive sampling methods include cell counts via hemocytometer, flow cytometry, and quantitative polymerase chain reaction (qPCR) (Mayfield et al., 2009; Krediet et al., 2015). Nondestructive methods based on anemone color and chlorophyll concentration have also been developed (Berneref al., 1993; Johnson and Goulet, 2007). Nondestructive measurements of chlorophyll concentration often utilize in vivo quantification of chlorophyll auto-fluorescence intensity as a measure of chlorophyll a concentration and a proxy of Symbiodinium density (Berner et al., 1993; Detournay et al., 2012; Hawkins et al., 2016b).

In this study, we applied the Aiptasia model system to investigate natural variation in bleaching response over time using a nondestructive symbiont quantification method. We evaluated the hypothesis ([H.sub.1]) that Aiptasia exhibits genetic variation in bleaching response by exposing six genetically distinct anemone strains to two different temperature stress regimes. On discovery of significant variation in bleaching, we further hypothesized that ([H.sub.1A]) some holobiont genotypes are most resistant to breakdown, regardless of stressor type; ([H.sub.1B]) trade-offs are associated with resistance to dissimilar stressors, such that holobionts most resistant to one bleaching stress are least resistant to another; and ([H.sub.1C]) bleaching responses to different stressors are independent. In addition to bleaching, we tracked mortality and changes in anemone behavior over time to investigate alternative responses to thermal stress.

Materials and Methods

Anemone strains

We analyzed bleaching responses of six genetically distinct laboratory strains of Aiptasia sea anemones in response to two different stressors (Table 1). Aiptasia strains were initially provided by the laboratories of J. Pringle (CC7 and H2; Stanford University, Stanford, CA), D. Kemp (DD3; University of Georgia, Athens), V. Weis (MMB and GM10; Oregon State University, Corvallis), and E. Meyer (EM5; Oregon State University). To confirm that each strain represented a unique genotype, we sequenced two Symbiodinium-specific loci and six Arprasia-specific loci from three clonal representatives of each strain. DNA was extracted from whole frozen anemones using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). Symbiodinium types associated with each sample were genotyped as clade A or B based on sequencing of chloroplast photosystem II protein D1 (psbA) using PCR primers 5'-GGATGGGTAGAGAATGGGAATTCAG-3' and 5'-CGAGAGTTATTGAAGGAAGCATATTG-3' modified from Barbrook et al. (2006). Further designation as type B1 or B2 was based on chloroplast 23S ribosomal DNA (cp23s) sequencing (Lajeunesse et al., 2012). For Aiptasia genotyping, primers amplified DNA from six genes chosen from a random subset of Aiptasia gene models (Table A1; Baumgarten et al., 2015). All PCR reactions were performed with EconoTaq DNA Polymerase (Lucigen, Middleton, WI) according to the manufacturer's instructions, with an annealing temperature of 56 [degrees]C. Amplicons were sequenced using BigDye Terminators (Applied Biosystems, Waltham, MA). Phylogenetic networks for Aiptasia were constructed in Splits-Tree4 version 4.13.1 (Huson and Bryant, 2006), based on sequences from 6 genes concatenated into a single 3.4-kb alignment with uncorrected pairwise distances and averaging across heterozygous sites. Sequences are available in GenBank under accession numbers KU847812-KU847847.

Laboratory bleaching experiment

Bleaching experiments were conducted in May-June 2015, using anemones maintained and propagated in laboratory aquaria for a minimum of 1 y. Seven weeks before temperature stress, 30 small polyps per strain with average oral disk diameter (ODD) 2.24 [+ or -] 0.65 (SD) mm were transferred to individual 60 x 15-mm polystyrene petri dishes and assigned to 2 experimental blocks (Fig. 1). Anemones of each strain were placed in stacks of three, matched based on ODD to control for effect of size on bleaching response (Muscatine et al., 1991). Anemones were maintained at 25 [degrees]C in an I36LL incubator (Percival Scientific, Perry, IA) under a blue fluorescent light (Marine-GLO T8 15-W aquarium bulb, Hagen, Mansfield, MA) providing a maximum 15 [micro]mol photon [m.sup.-2] [s.sup.-1] at the top of stacks on a light/dark cycle of 12h : 12h. Stacks in a block and dishes within a stack were rearranged daily to randomize effects of light variation. Once weekly, anemones were fed freshly hatched Artemia salina (Carolina Biological Supply, Burlington, NC), and water changes were performed within 24 h of feeding.

After seven weeks of acclimation to incubator conditions, anemones were exposed to one of two distinct bleaching treatments. One anemone from each stack was randomly assigned to a heat-shock, cold-shock, or control treatment (Fig. 1). Treatments were designed not to be environmentally relevant but to facilitate comparisons with published studies that frequently use cold-shock temperatures of 4 [degrees]C and heat-stress temperatures of 34 [degrees]C to elicit a marked bleaching response in Aiptasia (Steen and Muscatine, 1987; Muscatine et al., 1991; Dunn et al., 2004, 2007; Goulet et al., 2005; Tolleter et al., 2013; Bieri et al., 2016). For heat treatment, anemones were moved to an identical Percival incubator as for controls and subjected to an experimental stress regime similar to that of Tolleter et al. (2013): temperature was increased to 34 [degrees]C over 10 h and maintained at 34 [degrees]C for 48 h before returning anemones to the control incubator. For cold treatments, longer exposure times proved lethal, so anemones were exposed to 2 consecutive pulses of temperature stress: a 5-h exposure to 4 [degrees]C, with 48 h of recovery in the control incubator, and a second 2.5-h exposure to 4 [degrees]C, with cold shocks initiated at the beginning of the dark phase of the light/dark cycle. Stress treatments for the second experimental block were initiated four days after stress treatments for the first block.

Measurement of bleaching response

We quantified anemone bleaching across four time points using an in vivo chlorophyll autofluorescence method developed by N. Kirk and colleagues at Oregon State University, who previously verified concordance of chlorophyll autofluorescence with Symbiodinium density via qPCR, cell counts, and chlorophyll a concentration (N. L. Kirk, K. R. Corey, T. Tivey, M. A. Coffroth, E. Meyer, and V. M. Weis, Oregon State University, unpubl. data). Briefly, fluorescence measurements were obtained by collecting images of each anemone using a Zeiss Stemi 2000-C stereomicroscope (Thornwood, NY) equipped with a Canon EOS Rebel 100D digital camera (Melville, NY) and red filter to attenuate wavelengths <500 nm. All photos were taken at the end of the dark phase of the light/dark cycle. Anemones were illuminated immediately before photos were taken with 470 nm blue light from a CooLED pE-100 light source (Andover, Hampshire, UK) under 100% intensity (-2500 [micro]mol photon [m.sup.-2] [S.sup.-1] ). The light source was turned off immediately after a 1.3-s image exposure time. Images of all anemones in a block were obtained in the morning at 4 time points: 10 d before temperature stress (day 0), the day of temperature stress initiation (day 10), 3 d after initiation of heat shock (day 13), and 5 d after exposure (day 18). Images were randomly assigned a numerical identifier before analysis with ImageJ, version 1.48 (Schneider et al., 2012). The mean gray value of 20 measurements taken with the ellipse tool over the brightest areas of each image was used to calculate average fluorescence. Region-specific measurements were also taken for the tentacles (base), oral disk (central and peripheral), and body column, based on the average of five separate measurements per region per image. Region-specific measurements were not taken if the region was not visible in the image as a result of the orientation of the anemone in the petri dish.

Initial analyses of the fluorescence data served three purposes: (1) to confirm the relationship between fluorescence and bleaching status, (2) to assess stability in fluorescence values over time in our experiment, and (3) to examine natural variation in fluorescence of different regions across anemone strains. To test whether raw fluorescence values were a significant predictor of visual bleaching score, anemone color at the end of the experiment was scored through comparison to a color reference card (CoralWatch Coral Health Chart, St. Lucia, Queensland, Australia). Color scores were based on a 6-point brightness and saturation scale (1 = lightest; 6 = darkest) within 4 color hues (Siebeck et al., 2006). Anemone color was analyzed using ordered logistic regression, with color score as the dependent variable and fluorescence at day 18 as the predictor, using the "polr" function of the MASS package, version 7.3-45 (Venables and Ripley, 2002).

Stability of measurements in controls was compared across four time points. Fluorescence values were negatively skewed even after data transformation, and so statistical analyses were performed using generalized linear mixed models (GLMMs). To minimize missing data, this analysis was restricted to measurements taken for tentacles and over the brightest areas of anemones (n = 10 per strain). Analyses were implemented with the "glmer" function of the lme4 package, version 1.1.12, specifying a gamma distribution and the identity link function (Bates et al., 2015). Model selection was performed through stepwise regression, with the likelihood ratio test as the search criterion and a significance level of [alpha] = 0.01. Strain, anatomical region measured, day, size, block, and interaction terms between previously included predictors were investigated as possible explanatory variables for inclusion and removal at each step of the model selection process, with individual anemone as a random effect to account for repeated measures over time. Post hoc tests were conducted with the "glht" function of the multcomp package, version 1.4-6 (Hothorn et al., 2008).

We also investigated natural variation in fluorescence values across different anemone strains and anatomical regions before temperature stress (n = 30 per strain). The difference between measurements on the same individual and region from days 0 and 10 was not significantly different from 0, and so fluorescence values from the 2 time points were averaged before further analysis (t = -1.76, df = 583, P = 0.08, pairwise t-test). Average fluorescence was analyzed using generalized linear models (GLMs), implemented with the "glm" function of the stats package, version 3.3.0, and specifying a gamma distribution with the identity link function (R Core Team, 2014). Model selection was performed as described for GLMMs, except region, strain, assigned treatment group, experimental block, and size were the possible explanatory variables of interest.

Bleaching response ([DELTA]F) was calculated as the difference in fluorescence of a treated anemone compared to the size-matched control anemone assigned to the same stack. Statistical analyses of [DELTA]F were performed as described for GLMs except that results for each day and region were analyzed separately. Strain, experimental block, and anemone size were the possible explanatory variables of interest for model selection. All statistical analyses were implemented in R, version 3.3.0 (R Core Team, 2014). R code and raw data files to re-create the analyses are available online (Bellis, 2016).

Behavior and mortality

For each time point, we also investigated the effects of temperature stress on behavior and mortality. Based on photos taken for each time point, anemones were scored as "expanded" (tentacles and oral disk fully visible), "retracted" (tentacles and oral disk withdrawn), "detached" (not attached to a petri dish, with healthy tissue appearance), or "not alive" (tissue heavily degraded such that anatomical features were not distinguishable). In addition, several expanded anemones appeared to be missing significant proportions of tentacles on day 13. Correspondingly, we scored images from all time points for short tentacles, characterized as having all tentacles less than 1/4 of the ODD. Measurements of tentacle length and ODD were performed in ImageJ (Schneider et al., 2012).


We measured bleaching of six Aiptasia strains over time in response to two thermal stress regimes (Fig. 2; Table 1). The six strains included members of two major genetic networks previously described for Aiptasia: the "global" network (GM10 and H2) and the U.S. South Atlantic network (CC7, DD3, and MMB) (Thornhill et al., 2013; Grawunder et al., 2015). Anemone strains were genetically distinct, but a strong phylogenetic signal was not observed based on 3.4 kb of aligned DNA sequence including 85 polymorphic sites (Fig. 2; Table A1). Anemones hosted Symbiodinium types B1, B2, or A4. Only a single Symbiodinium genotype was detected within each anemone strain. Though our approach lacked resolution to detect genotypes present at low frequency, only a single Symbiodinium genotype was observed in three strains (CC7, GM10, and DD3) that were included in a previous whole genome sequencing-based analysis (Bellis et al., 2016).

Fluorescence as a measure of bleaching status

Fluorescence was a highly significant predictor of visual bleaching status when measured in tentacles (t = 9.01, P < 0.001, ordered logistic regression) and over the brightest image areas (t = 9.46, P < 0.001, ordered logistic regression). The effect size was the same for both regions: for every unit increase in measured fluorescence value, anemones were 1.2 times as likely to be assigned a darker color score (Fig. 3). Correspondingly, we used the change in chlorophyll auto-fluorescence ([DELTA]F) between size-matched pairs of control and treated anemones to measure bleaching response.

Consistency of measurements over time in our experiment was investigated by comparing fluorescence in controls across four time points. Day was a significant predictor of raw fluorescence in controls (P < 0.001, likelihood-ratio test [LRT]). The effect of day was driven by day 18, and measurements taken on day 18 were 7.5 [+ or -] 0.6 (SE) fluorescence units lower on average than measurements taken at time points during the first 2 wk of the experiment. It is likely that this difference resulted from changes in microscope hardware associated with a maintenance visit that occurred between day 13 and day 18, of which we were unaware until after the experiment. Because the lower fluorescence detected on day 18 likely pertained to all anemones, it was not expected to influence our analyses of bleaching response, [DELTA]F, measured as the difference in fluorescence values between size-matched pairs of control and treated anemones on the same day. In addition, variation among strains was similar on day 18 compared to the previous days (Table 2). However, for severely bleached anemones with measured fluorescence values close to the lower detection limit, it is possible that our estimates of [DELTA]F on day 18 may underestimate the true bleaching response. After effects of day, region, and strain were taken into account, block and size were not significant predictors of fluorescence in controls (P = 0.55 and P = 0.17, respectively, LRT).

Chlorophyll autofluorescence was also compared across anatomical regions before temperature stress (n = 30 per strain). Chlorophyll autofluorescence at 25 [degrees]C varied significantly across strains and regions measured (Fig. 4; Table 3). In general, fluorescence values measured over the brightest areas of each image were similar to measurements from the periphery of the oral disk but higher than measurements taken at the base of tentacles, the center of the oral disk, or the body column (Table 3; Fig. 4). The effect of anatomical region on chlorophyll autofluorescence also depended on strain, with the most notable strain x region interaction effects for tentacles (Table 3). Strains DD3, EM5, and H2 had lower fluorescence than CC7 and GM10 when measured at the base of tentacles, while GM10 had significantly higher fluorescence in tentacles than all other strains (all P < 0.001, Tukey honest significant difference [HSD]). Sample sizes for measurements of body column and oral disk were sometimes less than 30 due to the orientation of anemones in an image (e.g., the side vs. the bottom of the petri dish), which may have reduced power to detect significant differences among strains for regions other than tentacles (Fig. 4). There was also some evidence for a relationship between anemone size and chlorophyll autofluorescence (P = 0.05, LRT for comparison of full and reduced GLM). However, the predicted increase of 0.59 fluorescence units for every millimeter increase in ODD was minimal compared to the range of ODD sizes investigated in the study. No significant differences between treatment groups or experimental blocks were observed prior to temperature stress after accounting for strain and region measured.

Natural variation in bleaching response

We measured bleaching responses of anemone strains the morning after completion of temperature treatments (day 13) and five days after recovery (day 18). Bleaching responses were analyzed as the magnitude of the difference in chlorophyll autofluorescence between a treated anemone and a size-matched control anemone of the same strain ([DELTA]F). In the corresponding analyses (Tables 4-6), intercepts not significantly different from zero suggest similar Symbiodinium density in control and treated anemones. Larger, more positive values indicate a stronger bleaching response.

All strains bleached moderately in response to cold stress, but little evidence was observed to support significant strain-specific differences (Fig. 5). Raw fluorescence values in the brightest areas were typically higher than for tentacles, with less resolution to distinguish darker anemones (Fig. 3). This difference could contribute to the greater [DELTA]F and larger range of values observed for tentacles on day 13 compared to the brightest areas (Table 4). However, similar results for model selection were obtained for both regions, with strain as the only significant predictor of [DELTA]F at day 13 and the interceptonly model exhibiting the best fit at day 18 (Table 4). Post hoc tests did not reveal significant differences in pairwise comparisons between strains based on brightest image areas on either day (P > 0.01, Tukey HSD). Conversely, post hoc comparisons revealed decreased [DELTA]F in tentacles of DD3 compared to EM5 or GM10 on day 13 (P = 0.006 and P = 0.005, respectively, Tukey HSD).

In contrast to the overall lack of strain-specific bleaching responses to cold shock, significant differences were identified among strains in response to heat stress (Figs. 6, 7). Two strains originally collected from Hawaii, both associated with Symbiodinium type B1, bleached severely in response to heat stress, whereas other strains exhibited resistance to heat-induced bleaching. Results were generally similar for measurements from the brightest areas of images compared to tentacles (Tables 5, 6). For measurements from the brightest areas of images, significant effects of strain, experimental block, and anemone size were detected for both days (Table 5). On day 13, significant differences between strains were observed only in block B, with strains H2 and GM10 exhibiting significantly greater bleaching compared to CC7, DD3, and MMB (GM10 only) (P < 0.01, Tukey HSD). Block effects were less prominent for day 18, on which both GM10 and H2 had significantly greater bleaching responses compared to all other strains in both blocks (P < 0.01, Tukey HSD) (Table 5). No recorded differences could be found to explain the origin of these block effects; and block effects differed across strains, with more severe bleaching of three strains from block A compared to B, more severe bleaching of two strains from block B compared to A, and the effect of block for one strain differing across days (Table 5). After accounting for strain and block, larger anemones responded more severely to heat stress, with an average decrease of 5.96 [+ or -] 2.01 (SE) (day 13) or 3.43 [+ or -] 1.27 (SE) (day 18) fluorescence units for every 1-mm increase in ODD (P = 0.005, day 13; P = 0.010, day 18) (Table 5). A similar effect of anemone size on bleaching responses to cold shock has been previously demonstrated (Muscatine et al., 1991).

Change in chlorophyll autofluorescence in response to heat stress was also measured for tentacles (Fig. 6). Block effects were detected on day 13, on which bleaching in block B was less severe than responses for block A. However, block effects were less apparent for tentacle measurements than for the brightest areas, as evidenced by absence of strain x block interaction effects on day 13 or a main effect of block on day 18 (Table 6). H2 was significantly more bleached than CC7, EM5, or MMB on day 13 and more bleached than DD3 or MMB on day 18 (P < 0.01, Tukey HSD). GM10 was excluded from comparisons of tentacle fluorescence on day 13, because of the large number of retracted individuals on this day, but exhibited greater changes in fluorescence in response to heat stress than all other strains besides H2 on day 18 (P < 0.01, Tukey HSD). Smaller [DELTA]F values for DD3 and MMB compared to EM5 and CC7 suggested that Florida strains might exhibit higher resistance to heat-induced bleaching when measured with respect to tentacles (0.005 [less than or equal to] P [less than or equal to] 0.009, Tukey HSD) (Figs. 6, 7).

Behavior and mortality

In addition to bleaching, we tracked behavior and mortality in response to temperature stress. Behavioral responses included detachment from petri dishes and retraction of the oral disk and tentacles. Behavioral responses were most apparent on day 13 for 3 strains in particular: the heat-susceptible anemone strain GM10 and 2 heat-resistant strains that hosted Symbiodinium type A4 (CC7 and MMB). On day 13, 4 anemones were detached from their petri dish, and 11 anemones were retracted; whereas on days prior to temperature stress, no unattached anemones and up to 3 retracted anemones were observed on either day. All 11 retracted anemones from day 13 had been subjected to heat stress and comprised 4 MMB individuals, 1 DD3 individual, 1 CC7 individual (which later died), and 5 GM10 individuals. The four unattached anemones included two anemones exposed to heat stress (one CC7 individual and a GM10 individual that later died) and two GM10 individuals exposed to cold shock. One cold-shocked GM10 individual was still unattached at day 18, along with 1 MMB individual that had been exposed to heat stress.

Throughout the experiment, anemones were also observed to shorten their tentacles, characterized by having all tentacles shorter than ~1/4 of the ODD. Detached tentacles were not observed during the experiment or in acquired images; shorter, stubby tentacles may therefore have resulted from either substantial degradation or contraction of the tentacle tissue. Short tentacles were most apparent at day 13; no individuals were recorded with short tentacles prior to day 13, and only 3 individuals were recorded on day 18, suggesting that tentacles had either significantly expanded or regenerated by this time. Twenty individuals exhibited short tentacles on day 13, including 4 cold-stressed individuals and 16 heat-stressed individuals (5 CC7 anemones, 5 GM10 anemones, and 6 MMB anemones). Mortality was minimal during the experiment, involving one GM10 individual exposed to heat shock and three CC7 individuals (one from each of the three treatments).


In this study, we identified strain-specific variation in bleaching in response to acute heat stress but not in response to cold shock in the sea anemone model system Aiptasia. This finding has important implications for understanding cellular mechanisms of the bleaching response and thermal tolerance in cnidarian-dinoflagellate symbiosis.

Consistent with the findings of N. Kirk and colleagues (unpubl. data), chlorophyll autofluorescence generally proved to be a reliable indicator of bleaching response across diverse Aiptasia genotypes (Fig. 3), but several factors warrant further discussion. First, reductions in chlorophyll fluorescence can also occur as a result of photochemical and nonphotochemical quenching (NPQ), and NPQ increases under conditions associated with bleaching stress (Miiller et al., 2001; Hill et al., 2005). However, relaxation of most NPQ processes occurs on timescales of seconds to hours and thus would be expected to have a greater effect during and immediately after bleaching stress rather than after five days of recovery under control conditions (Maxwell and Johnson, 2000). Furthermore, microscopic observations of the 2 severely bleached strains revealed large numbers of Symbiodinium cells being expelled from the coelenteron after anemones were returned to room temperature; only small numbers of Symbiodinium cells remained in host tissues at day 18, suggesting that low fluorescence values in this study were due to loss of Symbiodinium rather than reductions in chlorophyll fluorescence associated with quenching or change in chlorophyll concentration per symbiont cell (Jones, 1997). However, for moderately bleached anemones, potential changes in chlorophyll autofluorescence during recovery of algal symbionts that were damaged but not expelled cannot be excluded.

A second factor that may influence results reported here is vertical stratification of Symbiodinium within host tissues. Correspondingly, measurements of tentacles may be more robust than measurements of other regions, since variation associated with differential layering of body column and oral disk tissues due to anemone position in images is avoided. In keeping with this assertion, tentacle fluorescence was found to be more sensitive for distinguishing among darker anemones than measurements over the brightest areas of images (Fig. 6), which were generally higher than tentacle measurements and became saturated at lower color scores (Fig. 3). However, future studies should exercise caution when basing interpretations on raw tentacle fluorescence, since our study also indicated significant variation in tentacle chlorophyll fluorescence among strains under control conditions (Fig. 4). Importantly, if the shorter tentacles for some strains on day 13 resulted from tissue contraction, higher fluorescence measurements in contracted tentacles could also cause underestimation of bleaching response.

Our results revealed significant genetic variation associated with response to acute heat stress in Aiptasia holobionts, with severe bleaching of two strains from Hawaii but minimal bleaching of strains from the U.S. South Atlantic (Fig. 6). Because strains were acclimated to laboratory culture conditions for more than 1 y and no differences were observed between strains collected from the same location more than 30 y apart (GM10 vs. H2), we attribute the majority of the observed variation in bleaching to genetic differences among holobionts. Importantly, both susceptible strains harbored Symbiodinium type B1, and so it is unclear how much of the variation between susceptible and tolerant strains may be attributable to host genotype, symbiont genotype, or genotype-genotype interaction. With evidence for impaired photosynthetic performance at elevated temperatures in Aiptasia anemones hosting clade B compared to clade A Symbiodinium, it is not surprising that in our study anemones naturally harboring Symbiodinium type B1 bleached more severely under heat stress than anemones hosting Symbiodinium clade A (Perez et al., 2001; Goulet et al., 2005). However, to our knowledge, this is the first study to demonstrate resistance to heat-induced bleaching in Aiptasia lineages naturally harboring Symbiodinium psygmophilum (type B2). This may be particularly interesting given that S. psygmophilum is considered a cold-tolerant lineage and rapidly recovers photosynthetic efficiency after weeks of exposure to 10 [degrees]C, whereas Symbiodinium minutum (type B1) does not (Thornhill et al., 2008; Lajeunesse et al., 2012).

Importantly, it is likely that the observed variability in holobiont heat-stress responses results from distinctive photobiological characteristics of A4, B1, and B2 lineages in combination with host behavioral and/or genetic factors (Goulet et al., 2005; Suggett et al., 2015). In support of this argument, we observed some indication that elevated temperature differentially influenced the behavior or physiology of CC7 and MMB, two heat-resistant strains that hosted type A4 Symbiodinium, and GM10, a heat-susceptible strain that harbored type B1 Symbiodinium. Responses included shortened tentacles in all 3 strains and retraction of MMB and GM10 anemones following heat stress on day 13. In temperate anemone species, retraction of the tentacles and oral disk by sea anemones is a behavior that may serve to minimize oxidative stress by reducing rates of photosynthesis via shading of the Symbiodinium (Shick and Dykens, 1984). Though the irradiance used in our experiment was much lower than irradiances previously demonstrated to cause photoinhibition in Aiptasia under short-term heat exposure (~6 h at 34 [degrees]C), longer exposure times in our experiment (2 d at 34 [degrees]C) or nonphotosynthetically derived sources of reactive oxygen species could contribute to these responses (Nii and Muscatine, 1997; Goulet et al., 2005; Dunn et al., 2012; Tolleter et al., 2013).

In response to a pulsed cold shock, we observed moderate reductions in chlorophyll autofluorescence across all strains but negligible strain-specific differences, even for strains hosting Symbiodinium lineages that exhibit cold tolerance in culture (Thornhill et al., 2008; Lajeunesse et al., 2012). In genetically distinct colonies of the coral Acropora palmata associated with a single Symbiodinium "fitti" strain, Parkinson et al (2015) observed significant variation in photosynthetic performance under extreme cold stress correlated with differences in host gene expression. It is possible that significant variation in other traits related to symbiosis physiology may exist in Aiptasia under extreme cold stress but that negligible variation in outward bleaching response masks dynamic molecular interactions between host and symbiont.

With no evidence to suggest heat-susceptible strains were either more or less tolerant to cold shock (Fig. 7), our findings support [H.sub.1c], the hypothesis of independence in bleaching responses between the two thermal stress regimes tested. A similar pattern was observed at the species level for Florida corals, whose resistance to warm-water anomalies was a poor predictor of responses to a severe cold-water event in 2010 (Lirman et al., 2011). Though inclusion of additional strains or use of different bleaching treatments may alter our conclusions, our results could suggest different cellular mechanisms of bleaching under disparate thermal stress regimes (i.e., in situ degradation, exocytosis, host cell detachment, host cell apoptosis, and/or host cell necrosis) (Weis, 2008). Support for each of these mechanisms has been observed in Aiptasia subjected to various hypothermic and/or hyperthermic stress regimes, but much remains unknown regarding the relative importance of and potential interactions among these mechanisms under different environmental contexts (Steen and Muscatine, 1987; Gates et al., 1992; Dunn et al., 2004, 2007; Weis, 2008). A recent study utilizing Aiptasia strain CC7 suggested that expulsion of intact Symbiodinium was the predominant bleaching mechanism under short-term heat and light stress, whereas in situ degradation and host cell detachment occurred at a high frequency only during acute cold shock (Bieri et al., 2016). However, important roles of host cell detachment and apoptosis under hyperthermic stress have been supported in other investigations utilizing the Aiptasia model system (Gates et al., 1992; Dunn et al., 2004, 2007; Paxton et al., 2013). One distinction between Bieri et al. and these latter studies is that the latter focused on Hawaiian Aiptasia, found in our study to be relatively heat susceptible compared to CC7 when harboring their endogenous Symbiodinium.

Rather than the nature of the stressor, variation in bleaching mechanisms under different stressors may be driven by the severity of stress ultimately perceived by the holobiont. One interpretation of our results is that severe photoinhibition of Symbiodinium minutum under acute heat stress markedly increased production in harmful reactive oxygen species (ROS) compared to anemones hosting Symbiodinium linucheae and Symbiodinium psygmophilum (Goulet et al., 2005), whereas photoinhibition in all three species was similar under pulsed cold shock. Only recently have we begun to understand the cellular signaling cascades that eventually lead to loss of Symbiodinium (Weis, 2008; Weston et al., 2015). However, ROS concentrations that overwhelm antioxidant responses of the holobiont are thought to play a central role in triggering early stages of the bleaching response (Lesser, 2006; Weis, 2008). In the most widely accepted model, temperature and/or light stress generate ROS in Symbiodinium chloroplasts through backup of excitation energy at photosystem II; this may occur through damage to the water-splitting D1 protein of photosystem II, reduced photosystem repair, or inhibition of the dark reactions of photosynthesis (Douglas, 2003; Takahashi et al., 2004; Weis, 2008). Heat-induced bleaching in the absence of light has also been demonstrated in Aiptasia, suggesting that nonphotosynthetically derived ROS or other molecular signals such as nitric oxide could contribute to variation in bleaching responses (Nii and Muscatine, 1997; Dunn et al., 2012; Hawkins et al., 2013; Tolleter et al., 2013). Indeed, ROS production is typically higher in Aiptasia lacking symbionts compared to symbiotic anemones, concomitant with downregulation of host ROS-scavenging pathways in the symbiotic state (Nii and Muscatine, 1997; Oakley et al., 2016). Distinct Symbiodinium genotypes may further contribute to holobiont bleaching variation through differences in ROS-mediating activities such as production of antioxidants or [O.sub.2] consumption (Rodriguez-Lanetty et al., 2006; Hawkins et al., 2016a). Future studies comparing diverse Aiptasia strains and their associated Symbiodinium under different stress regimes could hold the key for linking holobiont production of ROS, antioxidants, and other molecules to diverse cellular pathways mediating breakdown of the symbiosis.


We wish to thank N. Kirk, K. Corey, E. Meyer, and V. Weis for sharing details of the algal quantification method ahead of publication. We thank R. Edlund for assistance with maintenance of anemone cultures and Symbiodinium genotyping, E. Meyer for use of the stereomicroscope, J. Nixon at the Center for Genome Research and Biocomputing at Oregon State University for sequencing assistance, and V. Weis and three reviewers for their instructive comments on early versions of the manuscript. C. Crowder, S. Guermond, E. Hambleton, D. Kemp, J. Pringle, and V. Weis provided anemone strains. This material is based upon work supported by a National Science Foundation Graduate Research Fellowship (0946928 to ESB).

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Department of Integrative Biology, Oregon State University, Corvallis, Oregon 97331

Received 5 January 2017; Accepted 18 August 2017; Published online 8 December 2017.

(*) To whom correspondence should be addressed. E-mail:

Abbreviations: GBR, Great Barrier Reef; GLM, generalized linear model; GLMM, generalized linear mixed model; LRT, likelihood ratio test; NPQ, nonphotochemical quenching; ODD, oral disk diameter; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; ROS, reactive oxygen species.

Table A1
Genotyping of Aiptasia host strains based on six nuclear genes

Gene          Annotation

AIPGENE25508  Cadherin, EGF LAG
              7-pass G-type receptor
              3 (CELSR3)
AIPGENE19740  Adrenergic receptor,
              beta 1 (ADRB1)
AIPGENE9245   Patched domain-containing
              2 (PTCHD2)
AIPGENE18961  Calcium-sensing
AIPGENE19577  Atrophin-1-interacting protein 1 (AIP1)
AIPGENE26625  A disintegrin and
              metalloproteinase with
              thrombospondin motifs
              17 (ADAMTS17)

Gene          Primer sequences            Amplicon (bp)  Alignment (bp)

AIPGENE25508  5'-CCAACGCTACAGGATATACG-3'       529            466

AIPGENE19740  5'-AGTCAGAGCGCTTTTAATCC-3'       513            348
AIPGENE9245   5'-TCACTCATGTCAGCGAAATG-3'       858            760

AIPGENE18961  5'-CATTGGACGCATTAACAACG-3'       843            746
AIPGENE19577  5'-ATTTCCGTGTTTCTGGTCAG-3'       690            545
AIPGENE26625  5'-TAAGCGTTAGAAAATCCGGC-3'       625            567

Gene          No. of SNPs  Heterozygosity (a)

AIPGENE25508       5              0.20

AIPGENE19740      10              0.37

AIPGENE9245       20              0.22

AIPGENE18961      13              0.35

AIPGENE19577      11              0.32
AIPGENE26625      26              0.21

(a) Heterozygosity is reported as the average fraction of heterozygous
genotypes per polymorphic site. SNPs, single nucleotide polymorphisms.

Table 1
Aiptasia strains studied

Strain                           Symbiodinium   Alternative
name    Origin                    ITS2 type    strain names

CC7     Wilmington, North             A4
DD3     Key Largo, Florida,           B2            FL1
EM5     Aquarium, likely Texas        B2
        Gulf, 2013
GM10    Coconut Island, Hawaii,       B1       HI1; Aiptasia
        1979                                   pulchella "A"
H2      Coconut Island, Hawaii        B1
MMB     St. Augustine, Florida,       A4

ITS, internal transcribed spacer.

Table 2
Analysis of raw fluorescence measurements in controls

                Estimate  Standard error  t-value  P-value

Intercept       43.21          1.55        27.78   <0.00I
 10              0.44          0.66         0.66    0.51
 13              0.76          0.67         1.15    0.25
 18             -7.53          0.60       -12.60   <0.001
 Tentacle       -6.19          1.13        -5.47   <0.001
 DD3             5.23          2.18         2.42    0.02
 EM5             3.06          2.18         1.40    0.16
 GM10            3.69          2.12         1.73    0.08
 H2              3.62          2.16         1.67    0.09
 MMB            -1.46          2.16        -0.68    0.50
 Tentacle:DD3   -9.45          1.61        -5.88   <0.001
 Tentacle:EM5   -9.04          1.58        -5.78   <0.001
 Tentacle:GM10   5.36          1.74         3.08    0.002
 Tentacle:H2    -5.81          1.61        -3.61   <0.001
 Tentacle:MMB   -5.25          1.50        -3.49   <0.001

P-values lower than 0.01 are shown in bold and italics.

Table 3
Fluorescence across strains and anatomical regions at 25 [degrees]C

                       Estimate  Standard error  t-value  P-value

Intercept               45.66         0.98       46.73    <0.001
 Tentacle               -7.58         1.27       -5.96    <0.001
 Disc_Central           -5.51         1.40       -3.93    <0.001
 Disc_Peripheral         0.58         1.67        0.35     0.729
 Column                 -4.75         1.49       -3.18     0.002
 DD3                     3.67         1.44        2.55     0.011
 EM5                     2.31         1.42        1.63     0.104
 GM10                    3.53         1.44        2.46     0.014
 H2                      3.60         1.44        2.50     0.013
 MMB                    -3.92         1.32       -2.96     0.003
 Tentacle:DD3          -11.50         1.78       -6.48    <0.001
 Disc_Central:DD3       -2.84         1.96       -1.45     0.147
 Disc_Peripheral:DD3    -1.46         2.24       -0.65     0.513
 Column:DD3             -2.16         3.40       -0.64     0.526
 Tentacle:EM5           -7.65         1.78       -4.30    <0.001
 Disc_Central:EM5       -2.54         1.96       -1.29     0.197
 Disc_Peripheral:EM5    -5.02         2.24       -2.24     0.026
 Column:EM5              4.06         2.12        1.91     0.056
 Tentacle:GM10           5.44         1.93        2.82     0.005
 Disc_Central:GM10      -3.59         1.98       -1.81     0.070
 Disc_Peripheral:GM10   -0.56         2.33       -0.24     0.811
 Column:GM10             0.42         2.21        0.19     0.850
 Tentacle:H2            -9.84         1.79       -5.50    <0.001
 Disc_Central:H2        -5.88         1.94       -3.03     0.003
 Disc_Peripheral: H2    -1.24         2.26       -0.55     0.583
 Column:H2               4.42         2.42        1.82     0.069
 Tentacle:MMB           -3.01         1.69       -1.78     0.076
 Disc_Central:MMB       -2.64         1.84       -1.43     0.152
 Disc_Peripheral:MMB     0.24         2.21        0.11     0.915
 Column:MMB              1.61         1.98        0.82     0.415

Shown are results from a generalized linear model with CC7 measured
over the brightest areas of each image as the reference category.
Measurements for days 0 and 10 from the same individual and region
were averaged. P-values lower than 0.01 are shown in bold and italics.

Table 4
Parameter estimates for generalized linear models (GLMs) of bleaching
response magnitude to pulsed cold shock immediately following
treatment (day 13) and 5 d after recovery (day 18)

                                Estimate   error    t-value  P-value

Cold shock (brightest, day 13)
 Intercept                       8.77       2.17    11.40    <0.001
  DD3                            9.98       3.74     2.67     0.010
  EM5                           -1.30       2.99    -0.43     0.667
  GM10                          -1.37       2.99    -0.46     0.649
  H2                             5.62       3.44     1.63     0.108
  MMB                            5.20       3.41     1.53     0.133
Cold shock (brightest, day 18)
 Intercept                      14.28       1.05    28.77    <0.001
Cold shock (tentacles, day 13)
 Intercept                      18.19       3.11    12.93    <0.001
  DD3                            5.88       4.73     1.24     0.219
  EM5                           -9.24       3.92    -2.36     0.022
  GM10                          -9.42       3.92    -2.41     0.020
  H2                             2.02       4.64     0.44     0.666
  MMB                           -3.78       4.20    -0.90     0.372
Cold shock (tentacles, day 18)
 Intercept                      11.74       1.08    31.30    <0.001

P-values lower than the significance threshold of 0.01 are shown in
bold and italics.

Table 5
Parameter estimates for generalized linear models of bleaching
response to acute heat stress immediately following treatment (day 13)
and 5 d after recovery (day 18)

                                Estimate   error    t-value  P-value

Heat shock (brightest, day 13)
 Intercept                        3.84      5.15     3.85    <0.001
  DD3                            -2.40      5.10    -0.47     0.640
  EM5                            -0.98      5.43    -0.18     0.857
  GM10                            6.47      5.84     1.11     0.273
  H2                              9.23      6.02     1.53     0.132
  MMB                            -2.72      5.30    -0.51     0.610
  B                             -17.31      4.02    -4.31    <0.001
 Size                             5.96      2.01     2.97     0.005
  DD3:B                          11.92      6.19     1.92     0.060
  EM5:B                          12.98      6.60     1.97     0.055
  GM10:B                         28.63      8.34     3.43     0.001
  H2:B                           21.00      8.21     2.56     0.014
  MMB:B                           7.19      6.05     1.19     0.241
Heat shock (brightest, day 18)
 Intercept                        2.53      4.21     4.41    <0.001
  DD3                            -5.81      3.95    -1.47     0.149
  EM5                           -12.21      3.80    -3.21     0.002
  GM10                           15.98      5.12     3.12     0.003
  H2                             13.16      4.91     2.68     0.010
  MMB                            -5.23      4.07    -1.29     0.205
  B                              -4.73      3.98    -1.19     0.242
 Size                             3.43      1.27     2.71     0.010
  DD3:B                          -2.16      4.54    -0.48     0.636
  EM5:B                          14.28      4.73     3.02     0.004
  GM10:B                          7.81      7.00     1.12     0.271
  H2:B                           15.61      6.89     2.27     0.029
  MMB:B                           2.38      4.77     0.50     0.620

P-values lower than 0.01 are shown in bold and italics.

Table 6
Parameter estimates for generalized linear models (GLMs) of bleaching
response to acute heat stress measured in tentacles

                        Estimate  Standard error  t-value  P-value

Heat shock (tentacles,
day 13)
 Intercept               3.69          3.17        8.11    <0.001
 Strain (a)
  DD3                    5.24          3.68        1.43     0.163
  EM5                    2.66          3.40        0.78     0.439
  H2                    19.95          4.92        4.05    <0.001
  MMB                   -7.53          3.76       -2.01     0.053
  B                     -7.93          2.78       -2.86     0.007
Heat shock (tentacles,
day 18)
 Intercept               5.22          3.08        8.84    <0.001
  DD3                   -9.73          3.50       -2.78     0.008
  EM5                    1.48          4.11        0.36     0.720
  GM10                  22.75          6.44        3.53     0.001
  H2                    18.88          5.77        3.27     0.002
  MMB                   -9.62          3.50       -2.75     0.009

(a) Tentacle measurements were not obtained for GM10 at day 13 since 4
of 5 anemones were retracted and tentacles were missing from the
remaining anemone in block A. P-values lower than the significance
threshold of 0.01 are shown in bold and italics.
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Author:Bellis, Emily S.; Denver, Dee R.
Publication:The Biological Bulletin
Date:Oct 1, 2017
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