Multiparameter Sensing of Oxygen and pH at Biological Interfaces via Hyperspectral Imaging of Luminescent Sensor Nanoparticles.

Chemical dynamics in biological samples are seldom stand-alone processes but represent the outcome of complicated cascades of interlinked reaction chains. In order to understand these processes and how they correlate, it is important to monitor several parameters simultaneously at high spatial and temporal resolution. Hyperspectral imaging is a promising tool for this, as it provides broad-range spectral information in each pixel, enabling the use of multiple luminescent indicator dyes, while simultaneously providing information on sample structures and optical properties. In this study, we first characterized pH- and O2-sensitive indicator dyes incorporated in different polymer matrices as optical sensor nanoparticles to provide a library for (hyperspectral) chemical imaging. We then demonstrate the successful combination of a pH-sensitive indicator dye (HPTS(DHA)3), an O2-sensitive indicator dye (PtTPTBPF), and two reference dyes (perylene and TFPP), incorporated in polymer nanoparticles for multiparameter chemical imaging of complex natural samples such as green algal biofilms (Chlorella sorokiniana) and seagrass leaves (Zostera marina) with high background fluorescence. We discuss the system-specific challenges and limitations of our approach and further optimization possibilities. Our study illustrates how multiparameter chemical imaging with hyperspectral read-out can now be applied on natural samples, enabling the alignment of several chemical parameters to sample structures.

Green fluorescent protein-like pigments optimise the internal light environment in symbiotic reef-building corals.

Pigments homologous to the green fluorescent protein (GFP) have been proposed to fine-tune the internal light microclimate of corals, facilitating photoacclimation of photosynthetic coral symbionts (Symbiodiniaceae) to life in different reef habitats and environmental conditions. However, direct measurements of the in vivo light conditions inside the coral tissue supporting this conclusion are lacking. Here, we quantified the intra-tissue spectral light environment of corals expressing GFP-like proteins from widely different light regimes. We focus on: (1) photoconvertible red fluorescent proteins (pcRFPs), thought to enhance photosynthesis in mesophotic habitats via wavelength conversion, and (2) chromoproteins (CPs), which provide photoprotection to the symbionts in shallow water via light absorption. Optical microsensor measurements indicated that both pigment groups strongly alter the coral intra-tissue light environment. Estimates derived from light spectra measured in pcRFP-containing corals showed that fluorescence emission can contribute to >50% of orange-red light available to the photosynthetic symbionts at mesophotic depths. We further show that upregulation of pink CPs in shallow-water corals during bleaching leads to a reduction of orange light by 10-20% compared to low-CP tissue. Thus, screening by CPs has an important role in mitigating the light-enhancing effect of coral tissue scattering and skeletal reflection during bleaching. Our results provide the first experimental quantification of the importance of GFP-like proteins in fine-tuning the light microclimate of corals during photoacclimation.

Untangling the molecular basis of coral response to sedimentation.

Urbanized coral reefs are often chronically affected by sedimentation and reduced light levels, yet many species of corals appear to be able to thrive under these highly disturbed conditions. Recently, these marginal ecosystems have gained attention as potential climate change refugia due to the shading effect of suspended sediment, as well as potential reservoirs for stress-tolerant species. However, little research exists on the impact of sedimentation on coral physiology, particularly at the molecular level. Here, we investigated the transcriptomic response to sediment stress in corals of the family Merulinidae from a chronically turbid reef (one genet each of Goniastrea pectinata and Mycedium elephantotus from Singapore) and a clear-water reef (multiple genets of G. pectinata from the Gulf of Aqaba/Eilat). In two ex-situ experiments, we exposed corals to either natural sediment or artificial sediment enriched with organic matter and used whole-transcriptome sequencing (RNA sequencing) to quantify gene expression. Analysis revealed a shared basis for the coral transcriptomic response to sediment stress, which involves the expression of genes broadly related to energy metabolism and immune response. In particular, sediment exposure induced upregulation of anaerobic glycolysis and glyoxylate bypass enzymes, as well as genes involved in hydrogen sulphide metabolism and in pathogen pattern recognition. Our results point towards hypoxia as a probable driver of this transcriptomic response, providing a molecular basis to previous work that identified hypoxia as a primary cause of tissue necrosis in sediment-stressed corals. Potential metabolic and immunity trade-offs of corals living under chronic sedimentation should be considered in future studies on the ecology and conservation of turbid reefs.

The Microbiome of the Reef Macroalga Sargassum ilicifolium in Singapore.

The large canopy-forming macroalga, Sargassum ilicifolium, provides shelter and food for numerous coral reef species, but it can also be detrimental at high abundances where it outcompetes other benthic organisms for light and space. Here, we investigate the microbial communities associated with S. ilicifolium in Singapore, where it is an abundant and important member of coral reef communities. We collected eight complete S. ilicifolium thalli from eight island locations along an approximate 14 km east-to-west transect. Each thallus was dissected into three separate parts: holdfast, vesicles, and leaves. We then characterized the bacterial communities associated with each part via polymerase chain reaction (PCR) amplification of the 16S rRNA gene V4 region. We then inferred predicted metagenome functions using METAGENassist. Despite the comparatively short distances between sample sites, we show significant differences in microbial community composition, with communities further differentiated by part sampled. Holdfast, vesicles and leaves all harbor distinct microbial communities. Functional predictions reveal some separation between holdfast and leaf communities, with higher representation of sulphur cycling taxa in the holdfast and higher representation of nitrogen cycling taxa in the leaves. This study provides valuable baseline data that can be used to monitor microbial change, and helps lay the foundation upon which we can begin to understand the complexities of reef-associated microbial communities and the roles they play in the functioning and diversity of marine ecosystems.

Optical Feedback Loop Involving Dinoflagellate Symbiont and Scleractinian Host Drives Colorful Coral Bleaching.

Coral bleaching, caused by the loss of brownish-colored dinoflagellate photosymbionts from the host tissue of reef-building corals, is a major threat to reef survival. Occasionally, bleached corals become exceptionally colorful rather than white. These colors derive from photoprotective green fluorescent protein (GFP)-like pigments produced by the coral host. There is currently no consensus regarding what causes colorful bleaching events and what the consequences for the corals are. Here, we document that colorful bleaching events are a recurring phenomenon in reef regions around the globe. Our analysis of temperature conditions associated with colorful bleaching events suggests that corals develop extreme coloration within 2 to 3 weeks after exposure to mild or temporary heat stress. We demonstrate that the increase of light fluxes in symbiont-depleted tissue promoted by reflection of the incident light from the coral skeleton induces strong expression of the photoprotective coral host pigments. We describe an optical feedback loop involving both partners of the association, discussing that the mitigation of light stress offered by host pigments could facilitate recolonization of bleached tissue by symbionts. Our data indicate that colorful bleaching has the potential to identify local environmental factors, such as nutrient stress, that can exacerbate the impact of elevated temperatures on corals, to indicate the severity of heat stress experienced by corals and to gauge their post-stress recovery potential. VIDEO ABSTRACT.

FRET-Mediated Long-Range Wavelength Transformation by Photoconvertible Fluorescent Proteins as an Efficient Mechanism to Generate Orange-Red Light in Symbiotic Deep Water Corals.

Photoconvertible fluorescent proteins (pcRFPs) are a group of fluorophores that undergo an irreversible green-to-red shift in emission colour upon irradiation with near-ultraviolet (near-UV) light. Despite their wide application in biotechnology, the high-level expression of pcRFPs in mesophotic and depth-generalist coral species currently lacks a biological explanation. Additionally, reduced penetration of near-UV wavelengths in water poses the question whether light-driven photoconversion is relevant in the mesophotic zone, or whether a different mechanism is involved in the post-translational pigment modification in vivo. Here, we show in a long-term mesocosm experiment that photoconversion in vivo is entirely dependent on near-UV wavelengths. However, a near-UV intensity equivalent to the mesophotic underwater light field at 80 m depth is sufficient to drive the process in vitro, suggesting that photoconversion can occur near the lower distribution limits of these corals. Furthermore, live coral colonies showed evidence of efficient Förster Resonance Energy Transfer (FRET). Our simulated mesophotic light field maintained the pcRFP pool in a partially photoconverted state in vivo, maximising intra-tetrameric FRET and creating a long-range wavelength conversion system with higher quantum yield than other native RFPs. We hypothesise that efficient conversion of blue wavelengths, abundant at depth, into orange-red light could constitute an adaptation of corals to life in light-limited environments.