Machine-Learning-Based Proteomic Predictive Modeling with Thermally-Challenged Caribbean Reef Corals

Coral health is currently diagnosed retroactively; colonies are deemed “stressed” upon succumbing to bleaching or disease. Ideally, health inferences would instead be made on a pre-death timescale that would enable, for instance, environmental mitigation that could promote coral resilience. To this end, diverse Caribbean coral (Orbicella faveolata) genotypes of varying resilience to high temperatures along the Florida Reef Tract were exposed herein to elevated temperatures in the laboratory, and a proteomic analysis was taken with a subset of 20 samples via iTRAQ labeling followed by nano-liquid chromatography + mass spectrometry; 46 host coral and 40 Symbiodiniaceae dinoflagellate proteins passed all stringent quality control criteria, and the partial proteomes of biopsies of (1) healthy controls, (2) sub-lethally stressed samples, and (3) actively bleaching corals differed significantly from one another. The proteomic data were then used to train predictive models of coral colony bleaching susceptibility, and both generalized regression and machine-learning-based neural networks were capable of accurately forecasting the bleaching susceptibility of coral samples based on their protein signatures. Successful future testing of the predictive power of these models in situ could establish the capacity to proactively monitor coral health.

Effects of Ocean Acidification on Resident and Active Microbial Communities of Stylophora pistillata

Ocean warming and ocean acidification (OA) are direct consequences of climate change and affect coral reefs worldwide. While the effect of ocean warming manifests itself in increased frequency and severity of coral bleaching, the effects of ocean acidification on corals are less clear. In particular, long-term effects of OA on the bacterial communities associated with corals are largely unknown. In this study, we investigated the effects of ocean acidification on the resident and active microbiome of long-term aquaria-maintained Stylophora pistillata colonies by assessing 16S rRNA gene diversity on the DNA (resident community) and RNA level (active community). Coral colony fragments of S. pistillata were kept in aquaria for 2 years at four different pCO2 levels ranging from current pH conditions to increased acidification scenarios (i.e., pH 7.2, 7.4, 7.8, and 8). We identified 154 bacterial families encompassing 2,047 taxa (OTUs) in the resident and 89 bacterial families including 1,659 OTUs in the active communities. Resident communities were dominated by members of Alteromonadaceae, Flavobacteriaceae, and Colwelliaceae, while active communities were dominated by families Cyclobacteriacea and Amoebophilaceae. Besides the overall differences between resident and active community composition, significant differences were seen between the control (pH 8) and the two lower pH treatments (7.2 and 7.4) in the active community, but only between pH 8 and 7.2 in the resident community. Our analyses revealed profound differences between the resident and active microbial communities, and we found that OA exerted stronger effects on the active community. Further, our results suggest that rDNA- and rRNA-based sequencing should be considered complementary tools to investigate the effects of environmental change on microbial assemblage structure and activity.

Characterization of the Coral Disease 'Porites Bleaching with Tissue Loss' (PBTL) From Hawaii

<p>Coral reefs around the world are facing many threats and have sustained severe losses in coral cover over the past few decades. Coral bleaching and disease outbreaks have contributed substantially to this reef decline, however our understanding of factors contributing to the increase in coral disease prevalence are poorly understood. Information on the disease dynamics of different diseases affecting a reef system is essential for the development of effective management strategies. The aim of this research was to characterise and build a case study of a bleaching response affecting Porites compressa in Kaneohoe Bay, Oahu, Hawaii. It manifests as a localised, discrete area on the coral colony with a bleached coenenchyme and pigmented polyps, giving the affected area a “speckled” appearance. A disease by definition is any interruption, cessation or disorder of body functions, systems or organs. Results of this study showed that this localised bleaching causes tissue loss and a reduction in the number of gametes, and hence harm to the host. It was therefore classified as a disease and named Porites bleaching with tissue loss (PBTL). In addition, PBTL does not appear to represent a common thermal bleaching response as it was present throughout the year during times when seawater temperature was well within the coral’s thermal threshold. Symbiodinium cell density in PBTL-affected areas of the coral colony was reduced by 65%, and examination of affected host tissue using light microscopy showed fragmentation and necrosis. However, no potential pathogen was observed. Transmission electron microscopy (TEM) revealed a high occurrence of potential apoptotic Symbiodinium cells and a potential increase in the abundance of virus-like particles (VLPs) in PBTL-affected tissue. However a causal relationship remains to be established. Long-term monitoring showed spatio-temporal variations in PBTL prevalence. Temporal variations in prevalence reflected a seasonal trend with a peak during the summer months, linked to increasing seawater temperature. Spatial variations in disease prevalence were correlated with parrotfish density, turbidity and water motion. Of these, a negative correlation with variability (SD) in turbidity explained most of the variability in PBTL prevalence (12.8%). A positive correlation with water motion explained 9% and a positive correlation with the variability in parrotfish density explained 4.4%. Overall, only a relatively small proportion of variability in PBTL prevalence could be explained by these three factors (26.2%), suggesting that other factors, not investigated in this study, play a more important role in explaining PBTL patterns or that temporal variation in temperature is the overall major driving force. Monitoring of individually tagged P. compressa colonies showed that >80% of affected colonies sustained partial colony mortality (tissue loss) within two months; on average, one third of the colony is lost. The amount of tissue loss sustained was correlated to lesion size but not colony size. Case fatality (total mortality) was low (2.6%), however this disease can affect the same colonies repeatedly, suggesting a potential for progressive damage which could cause increased tissue loss over time. PBTL was not transmissible through direct contact or the water column in controlled aquaria experiments, suggesting that this disease might not be caused by a pathogen, is not highly infectious, or perhaps requires a vector for transmission. At present, PBTL has only been observed within Kaneohe Bay. An investigation of the potential role of host and Symbiodinium genetics in disease susceptibility revealed the same Symbiodinium sub-clade (C15) in healthy and PBTL-affected colonies, suggesting no involvement of Symbiodinium type in disease etiology. Results regarding host genetics remained inconclusive; however a difference in allele frequency at one microsatellite locus was observed between healthy and diseased samples. This difference could, however, be due to a lower amplification of PBTL-affected samples at this locus and needs to be regarded with some caution. The results of this study provide a case definition of PBTL which can be used as a baseline in further studies. P. compressa is the main framework building species in Kaneohe Bay, and the information gathered here on disease dynamics and virulence suggests that PBTL has the potential to negatively impact the resilience of reefs within the bay. Further research into the etiology of PBTL is necessary to fully understand the impact that this disease could have on coral reefs in Hawaii.</p>

Characterization of the Coral Disease 'Porites Bleaching with Tissue Loss' (PBTL) From Hawaii

<p>Coral reefs around the world are facing many threats and have sustained severe losses in coral cover over the past few decades. Coral bleaching and disease outbreaks have contributed substantially to this reef decline, however our understanding of factors contributing to the increase in coral disease prevalence are poorly understood. Information on the disease dynamics of different diseases affecting a reef system is essential for the development of effective management strategies. The aim of this research was to characterise and build a case study of a bleaching response affecting Porites compressa in Kaneohoe Bay, Oahu, Hawaii. It manifests as a localised, discrete area on the coral colony with a bleached coenenchyme and pigmented polyps, giving the affected area a “speckled” appearance. A disease by definition is any interruption, cessation or disorder of body functions, systems or organs. Results of this study showed that this localised bleaching causes tissue loss and a reduction in the number of gametes, and hence harm to the host. It was therefore classified as a disease and named Porites bleaching with tissue loss (PBTL). In addition, PBTL does not appear to represent a common thermal bleaching response as it was present throughout the year during times when seawater temperature was well within the coral’s thermal threshold. Symbiodinium cell density in PBTL-affected areas of the coral colony was reduced by 65%, and examination of affected host tissue using light microscopy showed fragmentation and necrosis. However, no potential pathogen was observed. Transmission electron microscopy (TEM) revealed a high occurrence of potential apoptotic Symbiodinium cells and a potential increase in the abundance of virus-like particles (VLPs) in PBTL-affected tissue. However a causal relationship remains to be established. Long-term monitoring showed spatio-temporal variations in PBTL prevalence. Temporal variations in prevalence reflected a seasonal trend with a peak during the summer months, linked to increasing seawater temperature. Spatial variations in disease prevalence were correlated with parrotfish density, turbidity and water motion. Of these, a negative correlation with variability (SD) in turbidity explained most of the variability in PBTL prevalence (12.8%). A positive correlation with water motion explained 9% and a positive correlation with the variability in parrotfish density explained 4.4%. Overall, only a relatively small proportion of variability in PBTL prevalence could be explained by these three factors (26.2%), suggesting that other factors, not investigated in this study, play a more important role in explaining PBTL patterns or that temporal variation in temperature is the overall major driving force. Monitoring of individually tagged P. compressa colonies showed that >80% of affected colonies sustained partial colony mortality (tissue loss) within two months; on average, one third of the colony is lost. The amount of tissue loss sustained was correlated to lesion size but not colony size. Case fatality (total mortality) was low (2.6%), however this disease can affect the same colonies repeatedly, suggesting a potential for progressive damage which could cause increased tissue loss over time. PBTL was not transmissible through direct contact or the water column in controlled aquaria experiments, suggesting that this disease might not be caused by a pathogen, is not highly infectious, or perhaps requires a vector for transmission. At present, PBTL has only been observed within Kaneohe Bay. An investigation of the potential role of host and Symbiodinium genetics in disease susceptibility revealed the same Symbiodinium sub-clade (C15) in healthy and PBTL-affected colonies, suggesting no involvement of Symbiodinium type in disease etiology. Results regarding host genetics remained inconclusive; however a difference in allele frequency at one microsatellite locus was observed between healthy and diseased samples. This difference could, however, be due to a lower amplification of PBTL-affected samples at this locus and needs to be regarded with some caution. The results of this study provide a case definition of PBTL which can be used as a baseline in further studies. P. compressa is the main framework building species in Kaneohe Bay, and the information gathered here on disease dynamics and virulence suggests that PBTL has the potential to negatively impact the resilience of reefs within the bay. Further research into the etiology of PBTL is necessary to fully understand the impact that this disease could have on coral reefs in Hawaii.</p>

Investigating the Prevailing Hydrodynamics Around a Cold-Water Coral Colony Using a Physical and a Numerical Approach

Framework-forming cold-water corals provide a refuge for numerous organisms and, consequently, the ecosystems formed by these corals can be considered as impressive deep-sea biodiversity hotspots. If suitable environmental conditions for coral growth persist over sufficiently long periods of time in equilibrium with continuous sediment input, substantial accumulations of coral mound deposits consisting of coral fragments and baffled sediments can form. Although this conceptual approach is widely accepted, little is known about the prevailing hydrodynamics in their close proximity, which potentially affect sedimentation patterns. In order to refine the current understanding about the hydrodynamic mechanisms in the direct vicinity of a model cold-water coral colony, a twofold approach of a laboratory flume experiment and a numerical model was set up. In both approaches the flow dynamics around a simplified cold-water coral colony used as current obstacle were investigated. The flow measurements of the flume provided a dataset that served as the basis for validation of the numerical model. The numerical model revealed data from the vicinity of the simplified cold-water coral, such as the pressure field, velocity field, or the turbulent kinetic energy (TKE) in high resolution. Features of the flow like the turbulent wake and streamlines were also processed to provide a more complete picture of the flow that passes the simplified cold-water coral colony. The results show that a cold-water coral colony strongly affects the flow field and eventually the sediment dynamics. The observed decrease in flow velocities around the cold water-coral hints to a decrease in the sediment carrying potential of the flowing water with consequences for sediment deposition.

A Long-Term Symbiotic Relationship: Recruitment and Fidelity of the Crab Trapezia on Its Coral Host Pocillopora

The symbiotic relationship between the crab Trapezia spp. and pocilloporid corals has been characterized as obligate. Although this relationship is considered common and has been widely registered within the distribution areas of these corals, the initiation of this symbiotic relation and its potential persistence throughout the life cycle of the crustacean is still poorly described. To understand the Trapezia–Pocillopora symbiosis, determining the time and conditions when Trapezia recruits a coral colony and the factors influencing this process are key. Thus, in the present study, healthy, small and unrecruited coral fragments were attached to the substrates (using cable ties) of nearby adult Pocillopora colonies. All fragments were monitored for two years to measure their growth and size at the first evidence of Trapezia crab recruitment, as well as the abundance and permanence of the crabs on the coral fragments. Results showed a relation between the space available (coral volume) and crab recruitment as an increase in substrate complexity is required to provide protection for the crabs and hence maintain the symbiosis, while abiotic conditions such as sea temperature and the distance of the fragments from the adult coral colonies seemingly did not affect the recruitment process. In addition, crabs are able to move between colonies, thus discarding the theory that once recruited, crabs are obligate residents on this specific colony.

Image Analysis to Quantify Coral Bleaching Using Greyscale Model v1

This protocol outlines a method of quantitatively measuring the degree of bleaching of a coral colony non-destructively in the field using image analysis. Previous studies have shown that mean intensity grey (MIG), also known as percent whiteness, is highly correlated with chlorophyll a and Symbiodiniaceae density (Chow et al. 2016, Amid et al. 2018), and therefore can be used to quantify the bleaching intensity of a coral colony. Color analysis can be done using digital photographs of live coral colonies either in situ (e.g., Maguire et al. 2003) or ex-situ in the lab (Amid et al. 2018; this protocol). Photographs must be taken prior to any preservation or processing of tissue, such as freezing, use of preservatives or fixatives, airbrushing etc., to ensure no alteration of the original coral color occurs. In this protocol, corals are photographed in front of a white reference standard and the resulting color images are subsequently converted to 8-bit greyscale and analyzed. There are two steps to this protocol: 1) Photographing live coral fragments 2) Image analysis of mean grey value This protocol was written by Dr. Rowan McLachlan and was reviewed by Dr. Andréa Grottoli. Acknowledgments I would like to thank Dr. Eugene Katrukha for kindly taking the time to teach me this method, and providing me feedback on how to produce higher quality images for analysis.

Calcium Transport along the Axial Canal in Acropora

In Acropora, the complex canals in a coral colony connect all polyps to a holistic network, enabling them to collaborate in performing biological processes. There are various types of canals, including calice, axial canals, and other internal canals, with structures that are dynamically altered during different coral growth states due to internal calcium transport. In this study, we investigated the morphological changes in the corallite of six Acropora muricata samples by high resolution micro-computed tomography, observing the patterns of calcium carbonate deposition within axial corallite during processes of new branch formation and truncated tip repair. We visualized the formation of a new branch from a calice and the calcium carbonate deposition in the axial canal. Furthermore, the diameter and volume changes of the axial canal in truncated branches during rebuilding processes were calculated, revealing that the volume ratio of calcareous deposits in the axial canal exhibit significant increases within the first three weeks, returning to levels in the initial state in the following week. This work demonstrates that calcium carbonate can be stored temporarily and then remobilized as needed for rapid growth. The results of this study shed light on the control of calcium carbonate deposition and growth of the axial corallite in Acropora.

The coral symbiont Candidatus Aquarickettsia is variably abundant in threatened Caribbean acroporids and transmitted horizontally

The symbiont “Candidatus Aquarickettsia rohweri” infects a diversity of aquatic hosts. In the threatened Caribbean coral, Acropora cervicornis, Aquarickettsia proliferates in response to increased nutrient exposure, resulting in suppressed growth and increased disease susceptibility and mortality of coral. This study evaluated the extent, as well as the ecology and evolution of Aquarickettsia infecting threatened corals, Ac. cervicornis, and Ac. palmata and their hybrid (“Ac. prolifera”). Aquarickettsia was found in all acroporids, with coral host and geographic location impacting the infection magnitude. Phylogenomic and genome-wide single-nucleotide variant analysis of Aquarickettsia found phylogenetic clustering by geographic region, not by coral taxon. Analysis of Aquarickettsia fixation indices suggests multiple sequential infections of the same coral colony are unlikely. Furthermore, relative to other Rickettsiales species, Aquarickettsia is undergoing positive selection, with Florida populations experiencing greater positive selection relative to other Caribbean locations. This may be due in part to Aquarickettsia proliferating in response to greater nutrient stress in Florida, as indicated by greater in situ replication rates in these corals. Aquarickettsia was not found to significantly codiversify with either the coral animal or the coral’s algal symbiont (Symbiodinium “fitti”). Quantitative PCR analysis showed that gametes, larvae, recruits, and juveniles from susceptible, captive-reared coral genets were not infected with Aquarickettsia. Thus, horizontal transmission of Aquarickettsia via coral mucocytes or an unidentified host is more likely. The prevalence of Aquarickettsia in Ac. cervicornis and its high abundance in the Florida coral population suggests that coral disease mitigation efforts focus on preventing early infection via horizontal transmission.

Calcium Transport along the Axial Canal in Acropora

In Acropora, the complex canals in a coral colony connect all polyps into a holistic network to collaborate in performing biological processes. There are various types of canals, including calice, axial canals, and other internal canals, with structures that are dynamically altered during different coral growth states due to internal calcium transport. However, few studies have considered the regulation of calcium transport in Acropora. In this study, we investigated the morphological changes of the axial canal in six Acropora muricata samples by high resolution micro-computed tomography, observing the patterns of the axial canal during the processes of new branch formation and truncated branch rebuilding. We visualized the formation of a new branch from a calice and deposition of the iconic hexactin skeletons in the axial canal. Furthermore, the diameter and volume changes of the axial canal in truncated branches during rebuilding processes were calculated, revealing that the volume ratio of calcareous deposits in the axial canal exhibit significant increases within the first three weeks, returning to levels in the initial state in the following week. This work indicates that the axial canal can transport calcium to form hexactin skeletons in a new branch and rebuild the tip of a truncated branch. The calcium transport along canal network regulates various growth processes, including budding, branching, skeleton forming, and self-rebuilding of an Acropora colony. Understanding the changes in canal function under normal and extreme conditions will provide theoretical guidance for restoration and protection of coral reefs.