Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma.

Apicomplexans are ubiquitous intracellular parasites of animals. These parasites use a programmed sequence of secretory events to find, invade, and then re-engineer their host cells to enable parasite growth and proliferation. The secretory organelles micronemes and rhoptries mediate the first steps of invasion. Both secrete their contents through the apical complex which provides an apical opening in the parasite's elaborate inner membrane complex (IMC) - an extensive subpellicular system of flattened membrane cisternae and proteinaceous meshwork that otherwise limits access of the cytoplasm to the plasma membrane for material exchange with the cell exterior. After invasion, a second secretion programme drives host cell remodelling and occurs from dense granules. The site(s) of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii, small subapical annular structures that are embedded in the IMC have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown. Here, we determined that integral membrane proteins of the plasma membrane occur specifically at these apical annular sites, that these proteins include SNARE proteins, and that the apical annuli are sites of vesicle fusion and exocytosis. Specifically, we show that dense granules require these structures for the secretion of their cargo proteins. When secretion is perturbed at the apical annuli, parasite growth is strongly impaired. The apical annuli, therefore, represent a second type of IMC-embedded structure to the apical complex that is specialised for protein secretion, and reveal that in Toxoplasma there is a physical separation of the processes of pre- and post-invasion secretion that mediate host-parasite interactions.

A Bioconductor workflow for processing, evaluating, and interpreting expression proteomics data.

Background: Expression proteomics involves the global evaluation of protein abundances within a system. In turn, differential expression analysis can be used to investigate changes in protein abundance upon perturbation to such a system. Methods: Here, we provide a workflow for the processing, analysis and interpretation of quantitative mass spectrometry-based expression proteomics data. This workflow utilizes open-source R software packages from the Bioconductor project and guides users end-to-end and step-by-step through every stage of the analyses. As a use-case we generated expression proteomics data from HEK293 cells with and without a treatment. Of note, the experiment included cellular proteins labelled using tandem mass tag (TMT) technology and secreted proteins quantified using label-free quantitation (LFQ). Results: The workflow explains the software infrastructure before focusing on data import, pre-processing and quality control. This is done individually for TMT and LFQ datasets. The application of statistical differential expression analysis is demonstrated, followed by interpretation via gene ontology enrichment analysis. Conclusions: A comprehensive workflow for the processing, analysis and interpretation of expression proteomics is presented. The workflow is a valuable resource for the proteomics community and specifically beginners who are at least familiar with R who wish to understand and make data-driven decisions with regards to their analyses.

Coral-bleaching responses to climate change across biological scales.

The global impacts of climate change are evident in every marine ecosystem. On coral reefs, mass coral bleaching and mortality have emerged as ubiquitous responses to ocean warming, yet one of the greatest challenges of this epiphenomenon is linking information across scientific disciplines and spatial and temporal scales. Here we review some of the seminal and recent coral-bleaching discoveries from an ecological, physiological, and molecular perspective. We also evaluate which data and processes can improve predictive models and provide a conceptual framework that integrates measurements across biological scales. Taking an integrative approach across biological and spatial scales, using for example hierarchical models to estimate major coral-reef processes, will not only rapidly advance coral-reef science but will also provide necessary information to guide decision-making and conservation efforts. To conserve reefs, we encourage implementing mesoscale sanctuaries (thousands of km2 ) that transcend national boundaries. Such networks of protected reefs will provide reef connectivity, through larval dispersal that transverse thermal environments, and genotypic repositories that may become essential units of selection for environmentally diverse locations. Together, multinational networks may be the best chance corals have to persist through climate change, while humanity struggles to reduce emissions of greenhouse gases to net zero.

Correlation of fluorescence microscopy, electron microscopy, and NanoSIMS stable isotope imaging on a single tissue section.

Correlative light and electron microscopy allows localization of specific molecules at the ultrastructural level in biological tissue but does not provide information about metabolic turnover or the distribution of labile molecules, such as micronutrients. We present a method to directly correlate (immuno)fluorescent microscopy, (immuno)TEM imaging and NanoSIMS isotopic mapping of the same tissue section, with nanometer-scale spatial precision. The process involves chemical fixation of the tissue, cryo sectioning, thawing, and air-drying under a thin film of polyvinyl alcohol. It permits to effectively retain labile compounds and strongly increases NanoSIMS sensitivity for 13C-enrichment. The method is illustrated here with correlated distribution maps of a carbonic anhydrase enzyme isotype, ß-tubulin proteins, and 13C- and 15N-labeled labile micronutrients (and their anabolic derivates) within the tissue of a reef-building symbiotic coral. This broadly applicable workflow expands the wealth of information that can be obtained from multi-modal, sub-cellular observation of biological tissue.

Sub-cellular imaging shows reduced photosynthetic carbon and increased nitrogen assimilation by the non-native endosymbiont Durusdinium trenchii in the model cnidarian Aiptasia.

Hosting different symbiont species can affect inter-partner nutritional fluxes within the cnidarian-dinoflagellate symbiosis. Using nanoscale secondary ion mass spectrometry (NanoSIMS), we measured the spatial incorporation of photosynthetically fixed 13 C and heterotrophically derived 15 N into host and symbiont cells of the model symbiotic cnidarian Aiptasia (Exaiptasia pallida) when colonized with its native symbiont Breviolum minutum or the non-native Durusdinium trenchii. Breviolum minutum exhibited high photosynthetic carbon assimilation per cell and translocation to host tissue throughout symbiosis establishment, whereas D. trenchii assimilated significantly less carbon, but obtained more host nitrogen. These findings suggest that D. trenchii has less potential to provide photosynthetically fixed carbon to the host despite obtaining considerable amounts of heterotrophically derived nitrogen. These sub-cellular events help explain previous observations that demonstrate differential effects of D. trenchii compared to B. minutum on the host transcriptome, proteome, metabolome and host growth and asexual reproduction. Together, these differential effects suggest that the non-native host-symbiont pairing is sub-optimal with respect to the host's nutritional benefits under normal environmental conditions. This contributes to our understanding of the ways in which metabolic integration impacts the benefits of a symbiotic association, and the potential evolution of novel host-symbiont pairings.

Intracellular competition for nitrogen controls dinoflagellate population density in corals.

The density of dinoflagellate microalgae in the tissue of symbiotic corals is an important determinant for health and productivity of the coral animal. Yet, the specific mechanism for their regulation and the consequence for coral nutrition are insufficiently understood due to past methodological limitations to resolve the fine-scale metabolic consequences of fluctuating densities. Here, we characterized the physiological and nutritional consequences of symbiont density variations on the colony and tissue level in Stylophora pistillata from the Red Sea. Alterations in symbiont photophysiology maintained coral productivity and host nutrition across a broad range of symbiont densities. However, we demonstrate that density-dependent nutrient competition between individual symbiont cells, manifested as reduced nitrogen assimilation and cell biomass, probably creates the negative feedback mechanism for symbiont population growth that ultimately defines the steady-state density. Despite fundamental changes in symbiont nitrogen assimilation, we found no density-related metabolic optimum beyond which host nutrient assimilation or tissue biomass declined, indicating that host nutrient demand is sufficiently met across the typically observed range of symbiont densities under ambient conditions.

Developmental carryover effects of ocean warming and acidification in corals from a potential climate refugium, the Gulf of Aqaba.

Coral reefs are degrading from the effects of anthropogenic activities, including climate change. Under these stressors, their ability to survive depends upon existing phenotypic plasticity, but also transgenerational adaptation. Parental effects are ubiquitous in nature, yet empirical studies of these effects in corals are scarce, particularly in the context of climate change. This study exposed mature colonies of the common reef-building coral Stylophora pistillata from the Gulf of Aqaba to seawater conditions likely to occur just beyond the end of this century during the peak planulae brooding season (Representative Concentration Pathway 8.5: pH -0.4 and +5°C beyond present day). Parent and planulae physiology were assessed at multiple time points during the experimental incubation. After 5 weeks of incubation, the physiology of the parent colonies exhibited limited treatment-induced changes. All significant time-dependent changes in physiology occurred in both ambient and treatment conditions. Planulae were also resistant to future ocean conditions, with protein content, symbiont density, photochemistry, survival and settlement success not significantly different compared with under ambient conditions. High variability in offspring physiology was independent of parental or offspring treatments and indicate the use of a bet-hedging strategy in this population. This study thus demonstrates weak climate-change-associated carryover effects. Furthermore, planulae display temperature and pH resistance similar to those of adult colonies and therefore do not represent a larger future population size bottleneck. The findings add support to the emerging hypothesis that the Gulf of Aqaba may serve as a coral climate change refugium aided by these corals' inherent broad physiological resistance.

Temperature and feeding induce tissue level changes in autotrophic and heterotrophic nutrient allocation in the coral symbiosis - A NanoSIMS study.

Corals access inorganic seawater nutrients through their autotrophic endosymbiotic dinoflagellates, but also capture planktonic prey through heterotrophic feeding. Correlating NanoSIMS and TEM imaging, we visualized and quantified the subcellular fate of autotrophic and heterotrophic C and N in the coral Stylophora pistillata using stable isotopes. Six scenarios were compared after 6 h: autotrophic pulse (13C-bicarbonate, 15N-nitrate) in either unfed or regularly fed corals, and heterotrophic pulse (13C-, 15N-labelled brine shrimps) in regularly fed corals; each at ambient and elevated temperature. Host assimilation of photosynthates was similar under fed and unfed conditions, but symbionts assimilated 10% more C in fed corals. Photoautotrophic C was primarily channelled into host lipid bodies, whereas heterotrophic C and N were generally co-allocated to the tissue. Food-derived label was detected in some subcellular structures associated with the remobilisation of host lipid stores. While heterotrophic input generally exceeded autotrophic input, it was more negatively affected by elevated temperature. The reduced input from both modes of nutrition at elevated temperature was accompanied by a shift in the partitioning of C and N, benefiting epidermis and symbionts. This study provides a unique view into the nutrient partitioning in corals and highlights the tight connection of nutrient fluxes in symbiotic partners.

Measuring coral calcification under ocean acidification: methodological considerations for the 45Ca-uptake and total alkalinity anomaly technique.

As the oceans become less alkaline due to rising CO2 levels, deleterious consequences are expected for calcifying corals. Predicting how coral calcification will be affected by on-going ocean acidification (OA) requires an accurate assessment of CaCO3 deposition and an understanding of the relative importance that decreasing calcification and/or increasing dissolution play for the overall calcification budget of individual corals. Here, we assessed the compatibility of the 45Ca-uptake and total alkalinity (TA) anomaly techniques as measures of gross and net calcification (GC, NC), respectively, to determine coral calcification at pHT 8.1 and 7.5. Considering the differing buffering capacity of seawater at both pH values, we were also interested in how strongly coral calcification alters the seawater carbonate chemistry under prolonged incubation in sealed chambers, potentially interfering with physiological functioning. Our data indicate that NC estimates by TA are erroneously ∼5% and ∼21% higher than GC estimates from 45Ca for ambient and reduced pH, respectively. Considering also previous data, we show that the consistent discrepancy between both techniques across studies is not constant, but largely depends on the absolute value of CaCO3 deposition. Deriving rates of coral dissolution from the difference between NC and GC was not possible and we advocate a more direct approach for the future by simultaneously measuring skeletal calcium influx and efflux. Substantial changes in carbonate system parameters for incubation times beyond two hours in our experiment demonstrate the necessity to test and optimize experimental incubation setups when measuring coral calcification in closed systems, especially under OA conditions.

Common reef-building coral in the Northern Red Sea resistant to elevated temperature and acidification.

Coral reefs are currently experiencing substantial ecological impoverishment as a result of anthropogenic stressors, and the majority of reefs are facing immediate risk. Increasing ocean surface temperatures induce frequent coral mass bleaching events-the breakdown of the nutritional photo-symbiosis with intracellular algae (genus: Symbiodinium). Here, we report that Stylophora pistillata from a highly diverse reef in the Gulf of Aqaba showed no signs of bleaching despite spending 1.5 months at 1-2°C above their long-term summer maximum (amounting to 11 degree heating weeks) and a seawater pH of 7.8. Instead, their symbiotic dinoflagellates exhibited improved photochemistry, higher pigmentation and a doubling in net oxygen production, leading to a 51% increase in primary productivity. Nanoscale secondary ion mass spectrometry imaging revealed subtle cellular-level shifts in carbon and nitrogen metabolism under elevated temperatures, but overall host and symbiont biomass proxies were not significantly affected. Now living well below their thermal threshold in the Gulf of Aqaba, these corals have been evolutionarily selected for heat tolerance during their migration through the warm Southern Red Sea after the last ice age. This may allow them to withstand future warming for a longer period of time, provided that successful environmental conservation measures are enacted across national boundaries in the region.

Photoacclimatory and photoprotective responses to cold versus heat stress in high latitude reef corals.

Corals at the world's southernmost coral reef of Lord Howe Island (LHI) experience large temperature and light fluctuations and need to deal with periods of cold temperature (<18°C), but few studies have investigated how corals are able to cope with these conditions. Our study characterized the response of key photophysiological parameters, as well as photoacclimatory and photoprotective pigments (chlorophylls, xanthophylls, and ß-carotene), to short-term (5-d) cold stress (~15°C; 7°C below control) in three LHI coral species hosting distinct Symbiodinium ITS2 types, and compared the coral-symbiont response to that under elevated temperature (~29°C; 7°C above control). Under cold stress, Stylophora sp. hosting Symbiodinium C118 showed the strongest effects with regard to losses of photochemical performance and symbionts. Pocillopora damicornis hosting Symbiodinium C100/C118 showed less severe bleaching responses to reduced temperature than to elevated temperature, while Porites heronensis hosting Symbiodinium C111* withstood both reduced and elevated temperature. Under cold stress, photoprotection in the form of xanthophyll de-epoxidation increased in unbleached P. heronensis (by 178%) and bleached Stylophora sp. (by 225%), while under heat stress this parameter increased in unbleached P. heronensis (by 182%) and in bleached P. damicornis (by 286%). The xanthophyll pool size was stable in all species at all temperatures. Our comparative study demonstrates high variability in the bleaching vulnerability of these coral species to low and high thermal extremes and shows that this variability is not solely determined by the ability to activate xanthophyll de-epoxidation.

Differential coral bleaching-Contrasting the activity and response of enzymatic antioxidants in symbiotic partners under thermal stress.

Mass coral bleaching due to thermal stress represents a major threat to the integrity and functioning of coral reefs. Thermal thresholds vary, however, between corals, partly as a result of the specific type of endosymbiotic dinoflagellate (Symbiodinium sp.) they harbour. The production of reactive oxygen species (ROS) in corals under thermal and light stress has been recognised as one mechanism that can lead to cellular damage and the loss of their symbiont population (Oxidative Theory of Coral Bleaching). Here, we compared the response of symbiont and host enzymatic antioxidants in the coral species Acropora millepora and Montipora digitata at 28°C and 33°C. A. millepora at 33°C showed a decrease in photochemical efficiency of photosystem II (PSII) and increase in maximum midday excitation pressure on PSII, with subsequent bleaching (declining photosynthetic pigment and symbiont density). M. digitata exhibited no bleaching response and photochemical changes in its symbionts were minor. The symbiont antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and catalase peroxidase showed no significant upregulation to elevated temperatures in either coral, while only catalase was significantly elevated in both coral hosts at 33°C. Increased host catalase activity in the susceptible coral after 5days at 33°C was independent of antioxidant responses in the symbiont and preceded significant declines in PSII photochemical efficiencies. This finding suggests a potential decoupling of host redox mechanisms from symbiont photophysiology and raises questions about the importance of symbiont-derived ROS in initiating coral bleaching.

Transcriptomic characterization of the enzymatic antioxidants FeSOD, MnSOD, APX and KatG in the dinoflagellate genus Symbiodinium.

BACKGROUND: The diversity of the symbiotic dinoflagellate Symbiodinium sp., as assessed by genetic markers, is well established. To what extent this diversity is reflected on the amino acid level of functional genes such as enzymatic antioxidants that play an important role in thermal stress tolerance of the coral-Symbiodinium symbiosis is, however, unknown. Here we present a predicted structural analysis and phylogenetic characterization of the enzymatic antioxidant repertoire of the genus Symbiodinium. We also report gene expression and enzymatic activity under short-term thermal stress in Symbiodinium of the B1 genotype. RESULTS: Based on eight different ITS2 types, covering six clades, multiple protein isoforms for three of the four investigated antioxidants (ascorbate peroxidase [APX], catalase peroxidase [KatG], manganese superoxide dismutase [MnSOD]) are present in the genus Symbiodinium. Amino acid sequences of both SOD metalloforms (Fe/Mn), as well as KatG, exhibited a number of prokaryotic characteristics that were also supported by the protein phylogeny. In contrast to the bacterial form, KatG in Symbiodinium is characterized by extended functionally important loops and a shortened C-terminal domain. Intercladal sequence variations were found to be much higher in both peroxidases, compared to SODs. For APX, these variable residues involve binding sites for substrates and cofactors, and might therefore differentially affect the catalytic properties of this enzyme between clades. While expression of antioxidant genes was successfully measured in Symbiodinium B1, it was not possible to assess the link between gene expression and protein activity due to high variability in expression between replicates, and little response in their enzymatic activity over the three-day experimental period. CONCLUSIONS: The genus Symbiodinium has a diverse enzymatic antioxidant repertoire that has similarities to prokaryotes, potentially as a result of horizontal gene transfer or events of secondary endosymbiosis. Different degrees of sequence evolution between SODs and peroxidases might be the result of potential selective pressure on the conserved molecular function of SODs as the first line of defence. In contrast, genetic redundancy of hydrogen peroxide scavenging enzymes might permit the observed variations in peroxidase sequences. Our data and successful measurement of antioxidant gene expression in Symbiodinium will serve as basis for further studies of coral health.

Antioxidant plasticity and thermal sensitivity in four types of Symbiodinium sp.

Warmer than average summer sea surface temperature is one of the main drivers for coral bleaching, which describes the loss of endosymbiotic dinoflagellates (genus: Symbiodinium) in reef-building corals. Past research has established that oxidative stress in the symbiont plays an important part in the bleaching cascade. Corals hosting different genotypes of Symbiodinium may have varying thermal bleaching thresholds, but changes in the symbiont's antioxidant system that may accompany these differences have received less attention. This study shows that constitutive activity and up-regulation of different parts of the antioxidant network under thermal stress differs between four Symbiodinium types in culture and that thermal susceptibility can be linked to glutathione redox homeostasis. In Symbiodinium B1, C1 and E, declining maximum quantum yield of PSII (Fv /Fm ) and death at 33°C were generally associated with elevated superoxide dismutase (SOD) activity and a more oxidized glutathione pool. Symbiodinium F1 exhibited no decline in Fv /Fm or growth, but showed proportionally larger increases in ascorbate peroxidase (APX) activity and glutathione content (GSx), while maintaining GSx in a reduced state. Depressed growth in Symbiodinium B1 at a sublethal temperature of 29°C was associated with transiently increased APX activity and glutathione pool size, and an overall increase in glutathione reductase (GR) activity. The collapse of GR activity at 33°C, together with increased SOD, APX and glutathione S-transferase activity, contributed to a strong oxidation of the glutathione pool with subsequent death. Integrating responses of multiple components of the antioxidant network highlights the importance of antioxidant plasticity in explaining type-specific temperature responses in Symbiodinium.

Synthesis of (1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid vinyl-ACCA) derivatives: key intermediates for the preparation of inhibitors of the hepatitis C virus NS3 protease.

(1R,2S)-1-Amino-2-vinylcyclopropanecarboxylic acid (vinyl-ACCA) is a key building block in the synthesis of potent inhibitors of the hepatitis C virus NS3 protease such as BILN 2061, which was recently shown to dramatically reduce viral load after administration to patients infected with HCV genotype 1. We have developed a scalable process that delivers derivatives of this unusual amino acid in >99% ee. The strategy was based on the dialkylation of a glycine Schiff base using trans-1,4-dibromo-2-butene as an electrophile to produce racemic vinyl-ACCA, which was subsequently resolved using a readily available, inexpensive esterase enzyme (Alcalase 2.4L). Factors that affect diastereoselection in the initial dialkylation steps were examined and the conditions optimized to deliver the desired diastereomer selectively. Product inhibition, which was encountered during the enzymatic resolution step, initially resulted in prolonged cycle times. Enrichment of racemic vinyl-ACCA through a chemical resolution via diastereomeric salt formation or the use of forcing conditions in the enzymatic reaction both led to improvements in throughput and the development of a viable process. The chemistry described herein was scaled up to produce multikilogram quantities of this building block.