Regulation of Apoptotic Pathways by Stylophora pistillata (Anthozoa, Pocilloporidae) to Survive Thermal Stress and Bleaching

Elevated seawater temperatures are associated with coral bleaching events and related mortality. Nevertheless, some coral species are able to survive bleaching and recover. The apoptotic responses associated to this ability were studied over 3 years in the coral Stylophora pistillata from the Gulf of Eilat subjected to long term thermal stress. These include caspase activity and the expression profiles of the S. pistillata caspase and Bcl-2 genes (StyCasp and StyBcl-2-like) cloned in this study. In corals exposed to thermal stress (32 or 34°C), caspase activity and the expression levels of the StyBcl-2-like gene increased over time (6–48 h) and declined to basal levels within 72 h of thermal stress. Distinct transcript levels were obtained for the StyCasp gene, with stimulated expression from 6 to 48 h of 34°C thermal stress, coinciding with the onset of bleaching. Increased cell death was detected in situ only between 6 to 48 h of stress and was limited to the gastroderm. The bleached corals survived up to one month at 32°C, and recovered back symbionts when placed at 24°C. These results point to a two-stage response in corals that withstand thermal stress: (i) the onset of apoptosis, accompanied by rapid activation of anti-oxidant/anti-apoptotic mediators that block the progression of apoptosis to other cells and (ii) acclimatization of the coral to the chronic thermal stress alongside the completion of symbiosis breakdown. Accordingly, the coral's ability to rapidly curb apoptosis appears to be the most important trait affecting the coral's thermotolerance and survival

Traditional and phylogenetic approaches in the diagnosis and identification of pathogens in mariculture.

Traditionally, the most common approach to diagnosis of microbial fish pathogens has relied on in vitro isolation of the microorganism and the information provided by its phenotypic features. However, viruses are generally highly species-specific and established cell lines do not neces- sarily show cytopathic effect, many species of bacteria are difficult or impossible to culture in vitro, while parasitic microorganisms often have a complex life cycle that requires propagation in live hosts. An increasing number of microbial pathogens are identified today by molecular meth- ods, without the need for isolation. A PCR direct method for detection and identification of Mycobacterium marinum based on the 16S rRNA gene sequence was successfully developed already 13 years ago at NCM. Comparison of the 16S rRNA sequence of Streptococcus iniae isolates revealed that, despite phenotypic, biochemical and pathogenetic similarities, marine and freshwater isolates were different strains. With time, however, it has become clear that 16S rRNA gene sequences alone are often insufficient to detect variation within bacterial species, and today other specific loci are also being employed. More recently, on the basis of hsp65 gene in addition to 16S rRNA gene, Israeli M. marinum isolates in marine and freshwater fish were found to belong to two distinct strains, and both were different from Israeli M. marinum clinical (human) isolates. Specific 18S rDNA probes for detection of elusive life stages of two myx- osporean parasites, Kudoa iwatai and Enteromyxum leei, in sea bream and sea bass are being employed in studies conducted over the last few years at NCM. By using whole-genome struc- tures rather than single gene sequences, two fingerprinting techniques - Amplified Fragment Length Polymorphism (AFLP) and Randomly Amplified Polymorphic DNA (RAPD) - have pro- vided a generally higher level of precision in genotyping. However, while the AFLP method revealed broad polymorphism among S. iniae isolates, the RAPD method did not provide addi- tional information. These examples show that not all regions of the DNA are equally useful in diagnosis and genotyping and therefore there is no single “best” molecular method. Molecular strategies have provided a phylogenetic approach to determining identification and taxonomic position by grouping closely related organisms that share a relatively recent ancestry into clus- ters. Although the question remains of how much genetic diversity is permissible in a discrete cluster for its members to be regarded as a single taxon, the ability to place a microorganism in a given taxon on the basis of its evolutionary development is of importance: if known members of the same family do not have a “clean bill” concerning their pathogenicity, any related organ- ism may be justifiably regarded as a potential offender. Traditional methods and molecular meth- ods provide different levels of information: only their combination offers a comprehensive insight into the microorganism’s nature

Lineage-specific symbionts mediate differential coral responses to thermal stress

Abstract Background Ocean warming is a leading cause of increasing episodes of coral bleaching, the dissociation between coral hosts and their dinoflagellate algal symbionts in the family Symbiodiniaceae. While the diversity and flexibility of Symbiodiniaceae is presumably responsible for variations in coral response to physical stressors such as elevated temperature, there is little data directly comparing physiological performance that accounts for symbiont identity associated with the same coral host species. Here, using Pocillopora damicornis harboring genotypically distinct Symbiodiniaceae strains, we examined the physiological responses of the coral holobiont and the dynamics of symbiont community change under thermal stress in a laboratory-controlled experiment. Results We found that P. damicornis dominated with symbionts of metahaplotype D1-D4-D6 in the genus Durusdinium (i.e., PdD holobiont) was more robust to thermal stress than its counterpart with symbionts of metahaplotype C42-C1-C1b-C1c in the genus Cladocopium (i.e., PdC holobiont). Under ambient temperature, however, the thermally sensitive Cladocopium spp. exhibited higher photosynthetic efficiency and translocated more fixed carbon to the host, likely facilitating faster coral growth and calcification. Moreover, we observed a thermally induced increase in Durusdinium proportion in the PdC holobiont; however, this “symbiont shuffling” in the background was overwhelmed by the overall Cladocopium dominance, which coincided with faster coral bleaching and reduced calcification. Conclusions These findings support that lineage-specific symbiont dominance is a driver of distinct coral responses to thermal stress. In addition, we found that “symbiont shuffling” may begin with stress-forced, subtle changes in the rare biosphere to eventually trade off growth for increased resilience. Furthermore, the flexibility in corals’ association with thermally tolerant symbiont lineages to adapt or acclimatize to future warming oceans should be viewed with conservative optimism as the current rate of environmental changes may outpace the evolutionary capabilities of corals. Video Abstrac

Additional file 1 of Lineage-specific symbionts mediate differential coral responses to thermal stress

Additional file 1: Fig. S1. Sampling information and dominant symbionts in P. damicornis. Fig. S2. Morphology of P. damicornis. Fig. S3. Neighbor-joining phylogenetic tree reconstructed based on the full-length ITS (ITS1-5.8S-ITS2) sequences amplified in coral nrDNA of P. damicornis colonies collected at the LHT and HH fringing reefs. Fig. S4. Maximum-parsimony (MP) phylogenetic tree and haplotype network reconstructions of ITS2 sequences amplified from Symbiodiniaceae nrDNA in the selected coral samples collected in HH. Fig. S5. Maximum-parsimony (MP) phylogenetic tree and haplotype network reconstructions of ITS2 sequences amplified from Symbiodiniaceae nrDNA in the selected coral samples collected in LHT. Fig. S6. Proportion of photosynthetically fixed carbon translocated to host at control and elevated temperatures. Fig. S7. Principal component analysis (PCA) of physiological traits mediating overall coral response to heat stress in P. damicornis. Table S1. The mean, maximum, and minimum sea surface temperatures (SST) in the two sampling sites. Table S2. Water quality parameters in the two sampling sites. Table S3. Effect of temperature on bleaching rate, Symbiodiniaceae density, photochemical efficiency, and calcification rate in P. damicornis. Table S4. Generalized linear mixed-effects model comparing the effects of fixed and random factors on physiological traits. Table S5. One-way ANOVA assessing the impact of heat stress on physiological traits. Table S6. Three-way ANOVA comparing the impacts of temperature, time and symbiont genotype on Symbiodiniaceae density

DataSheet_6_Transcriptional responses indicate acclimation to prolonged deoxygenation in the coral Stylophora pistillata.xlsx

The current decrease in oceanic dissolved oxygen is a widespread and pressing problem that raises concern as to how marine biota in general, and coral reefs in particular will be affected. However, the molecular response underlying tolerance of corals to prolonged severe deoxygenation where acclimation to hypoxia can accrue is not yet known. Here, we investigated the effect of two weeks of continuous exposure to conditions of extreme deoxygenation, not hitherto exerted under laboratory conditions (~ 0.35 mg L−1 dissolved oxygen), on the physiology and the diurnal gene expression of the coral, Stylophora pistillata. Deoxygenation had no physiologically significant effect on tissue loss, calcification rates, symbiont numbers, symbiont chlorophyll-a content and symbiont photosynthesis rate. However, deoxygenation evoked a significant transcriptional response that was much stronger at night, showing an acute early response followed by acclimation after two weeks. Acclimation included increased mitochondria DNA copy numbers, possibly increasing energy production. Gene expression indicated that the uptake of symbiosis-derived components was increased together with a decrease in nematocyst formation, suggesting that prolonged deoxygenation could enhance the corals’ need for symbiosis-derived components and reduces its predation abilities. Coral orthologs of the conserved hypoxia pathway, including oxygen sensors, hypoxia-inducible factor (HIF) and its target genes were differentially expressed in a similar temporal sequence as observed in other metazoans including other species of corals. Overall, our studies show that by utilizing highly conserved and coral–specific response mechanisms, S. pistillata can acclimate to deoxygenation and possibly survive under climate change-driven oceanic deoxygenation. On the other hand, the critical importance of algal symbionts in this acclimation suggests that any environmental perturbations that disrupt such symbiosis might negatively affect the ability of corals to withstand ocean oxygen depletion.</p

DataSheet_4_Transcriptional responses indicate acclimation to prolonged deoxygenation in the coral Stylophora pistillata.xlsx

The current decrease in oceanic dissolved oxygen is a widespread and pressing problem that raises concern as to how marine biota in general, and coral reefs in particular will be affected. However, the molecular response underlying tolerance of corals to prolonged severe deoxygenation where acclimation to hypoxia can accrue is not yet known. Here, we investigated the effect of two weeks of continuous exposure to conditions of extreme deoxygenation, not hitherto exerted under laboratory conditions (~ 0.35 mg L−1 dissolved oxygen), on the physiology and the diurnal gene expression of the coral, Stylophora pistillata. Deoxygenation had no physiologically significant effect on tissue loss, calcification rates, symbiont numbers, symbiont chlorophyll-a content and symbiont photosynthesis rate. However, deoxygenation evoked a significant transcriptional response that was much stronger at night, showing an acute early response followed by acclimation after two weeks. Acclimation included increased mitochondria DNA copy numbers, possibly increasing energy production. Gene expression indicated that the uptake of symbiosis-derived components was increased together with a decrease in nematocyst formation, suggesting that prolonged deoxygenation could enhance the corals’ need for symbiosis-derived components and reduces its predation abilities. Coral orthologs of the conserved hypoxia pathway, including oxygen sensors, hypoxia-inducible factor (HIF) and its target genes were differentially expressed in a similar temporal sequence as observed in other metazoans including other species of corals. Overall, our studies show that by utilizing highly conserved and coral–specific response mechanisms, S. pistillata can acclimate to deoxygenation and possibly survive under climate change-driven oceanic deoxygenation. On the other hand, the critical importance of algal symbionts in this acclimation suggests that any environmental perturbations that disrupt such symbiosis might negatively affect the ability of corals to withstand ocean oxygen depletion.</p

DataSheet_7_Transcriptional responses indicate acclimation to prolonged deoxygenation in the coral Stylophora pistillata.xlsx

The current decrease in oceanic dissolved oxygen is a widespread and pressing problem that raises concern as to how marine biota in general, and coral reefs in particular will be affected. However, the molecular response underlying tolerance of corals to prolonged severe deoxygenation where acclimation to hypoxia can accrue is not yet known. Here, we investigated the effect of two weeks of continuous exposure to conditions of extreme deoxygenation, not hitherto exerted under laboratory conditions (~ 0.35 mg L−1 dissolved oxygen), on the physiology and the diurnal gene expression of the coral, Stylophora pistillata. Deoxygenation had no physiologically significant effect on tissue loss, calcification rates, symbiont numbers, symbiont chlorophyll-a content and symbiont photosynthesis rate. However, deoxygenation evoked a significant transcriptional response that was much stronger at night, showing an acute early response followed by acclimation after two weeks. Acclimation included increased mitochondria DNA copy numbers, possibly increasing energy production. Gene expression indicated that the uptake of symbiosis-derived components was increased together with a decrease in nematocyst formation, suggesting that prolonged deoxygenation could enhance the corals’ need for symbiosis-derived components and reduces its predation abilities. Coral orthologs of the conserved hypoxia pathway, including oxygen sensors, hypoxia-inducible factor (HIF) and its target genes were differentially expressed in a similar temporal sequence as observed in other metazoans including other species of corals. Overall, our studies show that by utilizing highly conserved and coral–specific response mechanisms, S. pistillata can acclimate to deoxygenation and possibly survive under climate change-driven oceanic deoxygenation. On the other hand, the critical importance of algal symbionts in this acclimation suggests that any environmental perturbations that disrupt such symbiosis might negatively affect the ability of corals to withstand ocean oxygen depletion.</p

DataSheet_5_Transcriptional responses indicate acclimation to prolonged deoxygenation in the coral Stylophora pistillata.xlsx

The current decrease in oceanic dissolved oxygen is a widespread and pressing problem that raises concern as to how marine biota in general, and coral reefs in particular will be affected. However, the molecular response underlying tolerance of corals to prolonged severe deoxygenation where acclimation to hypoxia can accrue is not yet known. Here, we investigated the effect of two weeks of continuous exposure to conditions of extreme deoxygenation, not hitherto exerted under laboratory conditions (~ 0.35 mg L−1 dissolved oxygen), on the physiology and the diurnal gene expression of the coral, Stylophora pistillata. Deoxygenation had no physiologically significant effect on tissue loss, calcification rates, symbiont numbers, symbiont chlorophyll-a content and symbiont photosynthesis rate. However, deoxygenation evoked a significant transcriptional response that was much stronger at night, showing an acute early response followed by acclimation after two weeks. Acclimation included increased mitochondria DNA copy numbers, possibly increasing energy production. Gene expression indicated that the uptake of symbiosis-derived components was increased together with a decrease in nematocyst formation, suggesting that prolonged deoxygenation could enhance the corals’ need for symbiosis-derived components and reduces its predation abilities. Coral orthologs of the conserved hypoxia pathway, including oxygen sensors, hypoxia-inducible factor (HIF) and its target genes were differentially expressed in a similar temporal sequence as observed in other metazoans including other species of corals. Overall, our studies show that by utilizing highly conserved and coral–specific response mechanisms, S. pistillata can acclimate to deoxygenation and possibly survive under climate change-driven oceanic deoxygenation. On the other hand, the critical importance of algal symbionts in this acclimation suggests that any environmental perturbations that disrupt such symbiosis might negatively affect the ability of corals to withstand ocean oxygen depletion.</p