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Molecular characterization of the coral host and the microbial assemblages associated with it (referred to as the coral holobiont) is currently undertaken via marker gene sequencing. This requires bulky instruments and controlled laboratory conditions which are impractical for environmental experiments in remote areas. Recent advances in sequencing technologies now permit rapid sequencing in the field; however, development of specific protocols and pipelines for the effective processing of complex microbial systems are currently lacking. Here, we used a combination of 3 marker genes targeting the coral animal host, its symbiotic alga, and the associated bacterial microbiome to characterize 60 coral colonies collected and processedin situ, during theTaraPacific expedition. We used Oxford Nanopore Technologies to sequence marker gene amplicons and developed bioinformatics pipelines to analyze nanopore reads on a laptop, obtaining results in less than 24 hours. Reef scale network analysis of coral-associated bacteria reveals broadly distributed taxa, as well as host-specific associations. Protocols and tools used in this work may be applicable for rapid coral holobiont surveys, immediate adaptation of sampling strategy in the field, and to make informed and timely decisions in the context of the current challenges affecting coral reefs worldwide.

Coral microbiomes are critical to holobiont functioning, but much remains to be understood about how prevailing environment and host genotype affect microbial communities in ecosystems. Resembling human identical twin studies, we examined bacterial community differences of naturally occurring fire coral clones within and between contrasting reef habitats to assess the relative contribution of host genotype and environment to microbiome structure. Bacterial community composition of coral clones differed between reef habitats, highlighting the contribution of the environment. Similarly, but to a lesser extent, microbiomes varied across different genotypes in identical habitats, denoting the influence of host genotype. Predictions of genomic function based on taxonomic profiles suggest that environmentally determined taxa supported a functional restructuring of the microbial metabolic network. In contrast, bacteria determined by host genotype seemed to be functionally redundant. Our study suggests microbiome flexibility as a mechanism of environmental adaptation with association of different bacterial taxa partially dependent on host genotype., The microbiomes associated with reef corals are complex and diverse. Here, the authors investigate fire coral clones naturally occurring in distinct habitats as a model system to disentangle the contribution of host genotype and environment on their microbiome, and predict genomic functions based on taxonomic profiles.

Large-scale environmental disturbances may impact both partners in coral host-Symbiodinium systems. Elucidation of the assembly patterns in such complex and interdependent communities may enable better prediction of environmental impacts across coral reef ecosystems. In this study, we investigated how the community composition and diversity of dinoflagellate symbionts in the genus Symbiodinium were distributed among 12 host species from six taxonomic orders (Actinaria, Alcyonacea, Miliolida, Porifera, Rhizostoma, Scleractinia) and in the reef water and sediments at Lizard Island, Great Barrier Reef before the 3(rd) Global Coral Bleaching Event. 454 pyrosequencing of the ITS2 region of Symbiodinium yielded 83 Operational Taxonomic Units (OTUs) at a 97% similarity cut-off. Approximately half of the Symbiodinium OTUs from reef water or sediments were also present in symbio. OTUs belonged to six clades (A-D, F-G), but community structure was uneven. The two most abundant OTUs (100% matches to types C1 and A3) comprised 91% of reads and OTU C1 was shared by all species. However, sequence-based analysis of these dominant OTUs revealed host species-specificity, suggesting that genetic similarity cut-offs of Symbiodinium ITS2 data sets need careful evaluation. Of the less abundant OTUs, roughly half occurred at only one site or in one species and the background Symbiodinium communities were distinct between individual samples. We conclude that sampling multiple host taxa with differing life history traits will be critical to fully understand the symbiont diversity of a given system and to predict coral ecosystem responses to environmental change and disturbance considering the differential stress response of the taxa within.

Rare invasive fungal diseases (IFD) are challenging for the treating physicians because of their unspecific clinical presentation, as well as the lack of standardised diagnostic and effective treatment strategies. Late onset of treatment and inappropriate medication is associated with high mortality, thus, urging the need for a better understanding of these diseases. The purpose of FungiScope™ is to continuously collect clinical information and specimens to improve the knowledge on epidemiology and eventually improve patient management of these orphan diseases. FungiScope™ was founded in 2003, and today, collaborators from 66 countries support the registry. So far, clinical data of 794 cases have been entered using a web-based approach. Within the growing network of experts, new collaborations developed, leading to several publications of comprehensive analyses of patient subgroups identified from the registry. Data extracted from FungiScope™ have also been used as the sole control group for the approval of a new antifungal drug. Due to the rarity of these diseases, a global registry is an appropriate method of pooling the scarce and scattered information. Joining efforts across medical specialities and geographical borders is key for researching rare IFD. Here, we describe the structure and management of the FungiScope™ registry.

The productivity of coral reefs in oligotrophic tropical waters is sustained by an efficient uptake and recycling of nutrients. In reef-building corals, the engineers of these ecosystems, this nutrient recycling is facilitated by a constant exchange of nutrients between the animal host and endosymbiotic photosynthetic dinoflagellates (zooxanthellae), bacteria, and other microbes. Due to the complex interactions in this so-called coral holobiont, it has proven difficult to understand the environmental limitations of productivity in corals. Among others, the micronutrient iron has been proposed to limit primary productivity due to its essential role in photosynthesis and bacterial processes. Here, we tested the effect of iron enrichment on the physiology of the coral Pocillopora verrucosa from the central Red Sea during a 12-day experiment. Contrary to previous reports, we did not see an increase in zooxanthellae population density or gross photosynthesis. Conversely, respiration rates were significantly increased, and microbial nitrogen fixation was significantly decreased. Taken together, our data suggest that iron is not a limiting factor of primary productivity in Red Sea corals. Rather, increased metabolic demands in response to iron enrichment, as evidenced by increased respiration rates, may reduce carbon (i.e., energy) availability in the coral holobiont, resulting in reduced microbial nitrogen fixation. This decrease in nitrogen supply in turn may exacerbate the limitation of other nutrients, creating a negative feedback loop. Thereby, our results highlight that the effects of iron enrichment appear to be strongly dependent on local environmental conditions and ultimately may depend on the availability of other nutrients. published

It is now recognized that plastics represent a new challenge for the preservation of biodiversity and the associated resources. Plastic debris mainly disseminates from land and accumulate in the oceans where they represent the major part of marine litter. Global oceanic circulation allows a widespread distribution in surface waters, with the formation of garbage patches such as in the oceanic gyres. Plastics have also been found on the seabed of all seas and oceans across the planet, with higher concentration in areas of low circulation and high sediment accumulation.

The issues facing academic mothers have been discussed for decades. Coronavirus Disease 2019 (COVID-19) is further exposing these inequalities as womxn scientists who are parenting while also engaging in a combination of academic related duties are falling behind. These inequities can be solved by investing strategically in solutions. Here we describe strategies that would ensure a more equitable academy for working mothers now and in the future. While the data are clear that mothers are being disproportionately impacted by COVID-19, many groups could benefit from these strategies. Rather than rebuilding what we once knew, let us be the architects of a new world., The COVID-19 pandemic is highlighting the many long-standing inequalities that academic mothers face. This Essay describes solutions for a more equitable academia, now and in the future, maintaining that rather than rebuilding what we once knew, we should be the architects of a new world.

Animals and plants are metaorganisms and associate with microbes that affect their physiology, stress tolerance, and fitness. Here the hypothesis that alteration of the microbiome may constitute a fast‐response mechanism to environmental change is examined. This is supported by recent reciprocal transplant experiments with reef corals, which have shown that their microbiome adapts to thermally variable habitats and changes over time when transplanted into different environments. Further, inoculation of corals with beneficial bacteria increases their stress tolerance. But corals differ in their ability to flexibly associate with different bacteria. How scales of microbiome flexibility may reflect different metaorganism adaptation mechanisms is discussed and future directions for research are pinpointed. It is posited that microbiome flexibility is a broad phenomenon that contributes to the ability of organisms to respond to environmental change. Importantly, adapting with microbial help may provide an alternate route to organismal adaptation that facilitates rapid responses. published

Coral mucus is continuously released by most corals and acts as an important protective barrier and as a substrate for host-associated microbial communities due to its complex composition of carbohydrates, lipids, and proteins. On a reef scale, coral mucus functions as a particle trap, thereby retaining nutrients and energy in the ecosystem. Given the distinct environmental conditions in the Red Sea (high temperature, high salinity, high total alkalinity), we sought to investigate the carbohydrate composition of mucus from five corals from the central Red Sea. Our aim was to assess whether mucus from Red Sea corals is different from what is known from other corals and whether those differences could be aligned to putative beneficial functions with regard to the prevailing environment. Using gas chromatography/mass spectrometry, we detected nine sugars as the main prevalent carbohydrates. Although we detected significant differences between species with regard to the relative abundance of given carbohydrates, the identified sugars resembled those found in mucus from corals elsewhere, and we could corroborate high abundance of arabinose in acroporid corals. Taken together, our results suggest the presence of a common set of carbohydrates across a broad range of coral species from geographically diverse environments, highlighting the important role of mucus with regard to coral and reef ecosystem function. published

This chapter introduces Symbiodiniaceae, the diverse group of dinoflagellate microalgae, that form an obligate symbiosis with corals and other coral reef organisms. The Symbiodiniaceae cells reside within the coral tissue, their photosynthesis fuels the productivity and diversity of coral reef ecosystem, and the breakdown of this symbiosis leads to coral bleaching and may entail the death of the host. Here, we summarize Symbiodiniaceae taxonomy and phylogeny and the molecular tools that are used to study Symbiodiniaceae diversity in the Red Sea. We provide an overview over all described Symbiodiniaceae species and discuss the functional diversity within this phylogenetically diverse group as well as the implications of this diversity for coral-Symbiodiniaceae pairings and ecological niche partitioning in coral reef ecosystems. We review host-Symbiodiniaceae associations of 57 host genera in the Red Sea and discuss the emerging patterns in light of their wider biogeographic distribution. Last, we summarize how climate change-induced thermal anomalies have repeatedly led to coral bleaching and mortality in the Red Sea and how they threaten these reef ecosystems, otherwise thought to be comparatively resilient. We conclude with a perspective of important topics for Symbiodiniaceae research in the Red Sea that have the potential to contribute to a broader understanding of the basis of thermotolerance in this fragile symbiosis.

The Red Sea is one of the warmest and most saline seas on the planet. Yet, scleractinian corals have managed to flourish under these distinct conditions supporting one of the largest networks of coral reef ecosystems worldwide. Here, we summarize current knowledge on the ecophysiology of reef-building corals gained from 60 years of research in the Red Sea starting from insights in the 1960s to the most recent studies of the past few years. We provide a brief overview over seasonal dynamics and environmental gradients in the Red Sea that are used to study ecophysiological processes of corals under changing environmental and extreme conditions (i.e., temperature, salinity, nutrient, and light availability). We then focus on how this environmental variability shapes the central processes of coral physiology in the Red Sea covering the topics of photosynthesis, calcification, nutrient cycling, and reproduction. We continue by reporting the first physiological measurements of Red Sea deep-sea corals. Last, we discuss how, through the integration of traditional methods with recent developments in the omics field and model systems, we are now beginning to understand the complexity of processes that contribute to the ecological success of corals under these variable conditions. This synthesis may serve as a basis for future studies that aim to contribute to a better understanding of the impacts of environmental change on coral reefs in the Red Sea and the rest of the world.

Coral reefs in the Red Sea belong to the most diverse and productive reef ecosystems worldwide, although they are exposed to strong seasonal variability, high temperature, and high salinity. These factors are considered stressful for coral reef biota and challenge reef growth in other oceans, but coral reefs in the Red Sea thrive despite these challenges. In the central Red Sea high temperatures, high salinities, and low dissolved oxygen on the one hand reflect conditions that are predicted for ‘future oceans’ under global warming. On the other hand, alkalinity and other carbonate chemistry parameters are considered favourable for coral growth. In coral reefs of the central Red Sea, temperature and salinity follow a seasonal cycle, while chlorophyll and inorganic nutrients mostly vary spatially, and dissolved oxygen and pH fluctuate on the scale of hours to days. Within these strong environmental gradients micro- and macroscopic reef communities are dynamic and demonstrate plasticity and acclimatisation potential. Epilithic biofilm communities of bacteria and algae, crucial for the recruitment of reef-builders, undergo seasonal community shifts that are mainly driven by changes in temperature, salinity, and dissolved oxygen. These variables are predicted to change with the progression of global environmental change and suggest an immediate effect of climate change on the microbial community composition of biofilms. Corals are so-called holobionts and associate with a variety of microbial organisms that fulfill important functions in coral health and productivity. For instance, coral-associated bacterial communities are more specific and less diverse than those of marine biofilms, and in many coral species in the central Red Sea they are dominated by bacteria from the genus Endozoicomonas. Generally, coral microbiomes align with ecological differences between reef sites. They are similar at sites where these corals are abundant and successful. Coral microbiomes reveal a measurable footprint of anthropogenic influence at polluted sites. Coral-associated communities of endosymbiotic dinoflagellates in central Red Sea corals are dominated by Symbiodinium from clade C. Some corals harbour the same specific symbiont with a high physiological plasticity throughout their distribution range, while others maintain a more flexible association with varying symbionts of high physiological specificity over depths, seasons, or reef locations. The coral-Symbiodinium endosymbiosis drives calcification of the coral skeleton, which is a key process that provides maintenance and formation of the reef framework. Calcification rates and reef growth are not higher than in other coral reef regions, despite the beneficial carbonate chemistry in the central Red Sea. This may be related to the comparatively high temperatures, as indicated by reduced summer calcification and long-term slowing of growth rates that correlate with ocean warming trends. Indeed, thermal limits of abundant coral species in the central Red Sea may have been exceeded, as evidenced by repeated mass bleaching events during previous years. Recent comprehensive baseline data from central Red Sea reefs allow for insight into coral reef functioning and for quantification of the impacts of environmental change in the region.

We present SymPortal (SymPortal.org), a novel analytical framework and platform for genetically resolving the algal symbionts of reef corals using next-generation sequencing (NGS) data of the ITS2 rDNA. Although the ITS2 marker is widely used to genetically characterize taxa within the family Symbiodiniaceae (formerly the genus Symbiodinium), the multicopy nature of the marker complicates its use. Commonly, the intragenomic diversity resultant from this multicopy nature is collapsed by analytical approaches, thereby focusing on only the most abundant sequences. In contrast, SymPortal employs logic to identify within-sample informative intragenomic sequences, which we have termed 'defining intragenomic variants' (DIVs), to identify ITS2-type profiles representative of putative Symbiodiniaceae taxa. By making use of this intragenomic ITS2 diversity, SymPortal is able to resolve genetic delineations using the ITS2 marker at a level that was previously only possible by using additional genetic markers. We demonstrate this by comparing this novel approach to the most commonly used alternative approach for NGS ITS2 data, the 97% similarity clustering to operational taxonomic units (OTUs). The SymPortal platform accepts NGS raw sequencing data as input to provide an easy-to-use, standardization-enforced, and community-driven framework that integrates with a database to gain resolving power with increased use. We consider that SymPortal, in conjunction with ongoing large-scale sampling and sequencing efforts, should play an instrumental role in making future sampling efforts more comparable and in maximizing their efficacy in working towards the classification of the global Symbiodiniaceae diversity. published

The Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of Symbiodinium, a dinoflagellate genus of algal endosymbionts that is pervasively associated with marine invertebrates, and notably reef-building corals. Here we tested three commonly used ITS2 primer pairs (SYM_VAR_5.8S2/SYM_VAR_REV, ITSintfor2/ITSReverse, and ITS-DINO/ITS2Rev2) with regard to amplification specificity and sensitivity towards Symbiodinium, as well as sub-genera taxonomic bias. We tested these primers over a range of sample types including three coral species, coral surrounding water, reef surface water, and open ocean water to assess their suitability for use in large-scale next generation sequencing projects and to develop a standardized PCR protocol. We found the SYM_VAR_5.8S2/SYM_VAR_REV primers to perform superior to the other tested ITS2 primers. We therefore used this primer pair to develop a standardised PCR protocol. To do this, we tested the effect of PCR-to-PCR variation, annealing temperature, cycle number, and different polymerase systems on the PCR efficacy. The Symbiodinium ITS2 PCR protocol developed here delivers improved specificity and sensitivity towards Symbiodinium with apparent minimal sub-genera taxonomic bias across all sample types. In particular, the protocol’s ability to amplify Symbiodinium from a range of environmental sources will facilitate the study of Symbiodinium populations across biomes.

The Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of Symbiodinium, a dinoflagellate genus of algal endosymbionts that is pervasively associated with marine invertebrates, and notably reef-building corals. Here we tested three commonly used ITS2 primer pairs (SYM_VAR_5.8S2/SYM_VAR_REV, ITSintfor2/ITSReverse, and ITS-DINO/ITS2Rev2) with regard to amplification specificity and sensitivity towards Symbiodinium, as well as sub-genera taxonomic bias. We tested these primers over a range of sample types including three coral species, coral surrounding water, reef surface water, and open ocean water to assess their suitability for use in large-scale next generation sequencing projects and to develop a standardised PCR protocol. We found the SYM_VAR_5.8S2/SYM_VAR_REV primers to perform superior to the other tested ITS2 primers. We therefore used this primer pair to develop a standardised PCR protocol. To do this, we tested the effect of PCR-to-PCR variation, annealing temperature, cycle number, and different polymerase systems on the PCR efficacy. The Symbiodinium ITS2 PCR protocol developed here delivers improved specificity and sensitivity towards Symbiodinium with apparent minimal sub-genera taxonomic bias across all sample types. In particular, the protocol’s ability to amplify Symbiodinium from a range of environmental sources will facilitate the study of Symbiodinium populations across biomes.

International audience; Coral reef ecosystems worldwide are immediately threatened by the impacts of climate change. Here we report on the condition of coral reefs over 83 km of coastline at the island of Upolu, Samoa in the remote South West Pacific in 2016 during the Tara Pacific Expedition. Despite the distance to large urban centers, coral cover was extremely low (

From protists to humans, all animals and plants are inhabited by microbial organisms. There is an increasing appreciation that these resident microbes influence the fitness of their plant and animal hosts, ultimately forming a metaorganism consisting of a uni- or multicellular host and a community of associated microorganisms. Research on host-microbe interactions has become an emerging cross-disciplinary field. In both vertebrates and invertebrates a complex microbiome confers immunological, metabolic and behavioural benefits; conversely, its disturbance can contribute to the development of disease states. However, the molecular and cellular mechanisms controlling the interactions within a metaorganism are poorly understood and many key interactions between the associated organisms remain unknown. In this perspective article, we outline some of the issues in interspecies interactions and in particular address the question of how metaorganisms react and adapt to inputs from extreme environments such as deserts, the intertidal zone, oligothrophic seas, and hydrothermal vents. published

The association between corals and photosynthetic dinoflagellates (Symbiodinium spp.) is the key to the success of reef ecosystems in highly oligotrophic environments, but it is also their Achilles‘ heel due to its vulnerability to local stressors and the effects of climate change. Research during the last two decades has shaped a view that coral host–Symbiodinium pairings are diverse, but largely exclusive. Deep sequencing has now revealed the existence of a rare diversity of cryptic Symbiodinium assemblages within the coral holobiont, in addition to one or a few abundant algal members. While the contribution of the most abundant resident Symbiodinium species to coral physiology is widely recognized, the significance of the rare and low abundant background Symbiodinium remains a matter of debate. In this study, we assessed how coral–Symbiodinium communities assemble and how rare and abundant components together constitute the Symbiodinium community by analyzing 892 coral samples comprising >110 000 unique Symbiodinium ITS2 marker gene sequences. Using network modeling, we show that host–Symbiodinium communities assemble in non-random ‘clusters‘ of abundant and rare symbionts. Symbiodinium community structure follows the same principles as bacterial communities, for which the functional significance of rare members (the ‘rare bacterial biosphere’) has long been recognized. Importantly, the inclusion of rare Symbiodinium taxa in robustness analyses revealed a significant contribution to the stability of the host–symbiont community overall. As such, it highlights the potential functions rare symbionts may provide to environmental resilience of the coral holobiont., Research reported in this publication was supported by competitive research funding from King Abdullah University of Science and Technology (KAUST), Office of Sponsored Research (OSR) under Red Sea Research Center CCF Award No. FCC/1/1973-22-01 and baseline research funds to CRV.

Coral bleaching continues to be one of the most devastating and immediate impacts of climate change on coral reef ecosystems worldwide. In 2015, a major bleaching event was declared as the "3rd global coral bleaching event" by the United States National Oceanic and Atmospheric Administration, impacting a large number of reefs in every major ocean. The Red Sea was no exception, and we present herein in situ observations of the status of coral reefs in the central Saudi Arabian Red Sea from September 2015, following extended periods of high temperatures reaching upwards of 32.5°C in our study area. We examined eleven reefs using line-intercept transects at three different depths, including all reefs that were surveyed during a previous bleaching event in 2010. Bleaching was most prevalent on inshore reefs (55.6% ± 14.6% of live coral cover exhibited bleaching) and on shallower transects (41% ± 10.2% of live corals surveyed at 5m depth) within reefs. Similar taxonomic groups (e.g., Agariciidae) were affected in 2015 and in 2010. Most interestingly, Acropora and Porites had similar bleaching rates (~30% each) and similar relative coral cover (~7% each) across all reefs in 2015. Coral genera with the highest levels of bleaching (>60%) were also among the rarest (

Deserts, such as those found in Saudi Arabia, are one of the most hostile places for plant growth. However, desert plants are able to impact their surrounding microbial community and select beneficial microbes that promote their growth under these extreme conditions. In this study, we examined the soil, rhizosphere and endosphere bacterial communities of four native desert plants Tribulus terrestris, Zygophyllum simplex, Panicum turgidum and Euphorbia granulata from the Southwest (Jizan region), two of which were also found in the Midwest (Al Wahbah area) of Saudi Arabia. While the rhizosphere bacterial community mostly resembled that of the highly different surrounding soils, the endosphere composition was strongly correlated with its host plant phylogeny. In order to assess whether any of the native bacterial endophytes might have a role in plant growth under extreme conditions, we analyzed the properties of 116 cultured bacterial isolates that represent members of the phyla Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. Our analysis shows that different strains have highly different biochemical properties with respect to nutrient acquisition, hormone production and growth under stress conditions. More importantly, eleven of the isolated strains could confer salinity stress tolerance to the experimental model plant Arabidopsis thaliana suggesting some of these plant-associated bacteria might be useful for improving crop desert agriculture. published

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p

© 2017 John Wiley & Sons Ltd Tropical reefs have been impacted by thermal anomalies caused by global warming that induced coral bleaching and mortality events globally. However, there have only been very few recordings of bleaching within the Red Sea despite covering a latitudinal range of 15° and consequently it has been considered a region that is less sensitive to thermal anomalies. We therefore examined historical patterns of sea surface temperature (SST) and associated anomalies (1982–2012) and compared warming trends with a unique compilation of corresponding coral bleaching records from throughout the region. These data indicated that the northern Red Sea has not experienced mass bleaching despite intensive Degree Heating Weeks (DHW) of >15°C-weeks. Severe bleaching was restricted to the central and southern Red Sea where DHWs have been more frequent, but far less intense (DHWs 8°C-weeks), and bleaching was restricted to the central and southern Red Sea despite the lower thermal stress (DHWs

The “Dolly” based cloning (classical nuclear transfer, [CNT]) and the handmade cloning (HMC) are methods that are nowadays routinely used for somatic cloning of large domestic species. Both cloning protocols share several similarities, but differ with regard to the required in vitro culture, which in turn results in different time intervals until embryo transfer. It is not yet known whether the differences between cloned embryos from the two protocols are due to the cloning methods themselves or the in vitro culture, as some studies have shown detrimental effects of in vitro culture on conventionally produced embryos. The goal of this study was to unravel putative differences between two cloning methods, with regard to developmental competence, expression profile of a panel of developmentally important genes and epigenetic profile of porcine cloned embryos produced by either CNT or HMC, either with (D5 or D6) or without (D0) in vitro culture. Embryos cloned by these two methods had a similar morphological appearance on D0, but displayed different cleavage rates and different quality of blastocysts, with HMC embryos showing higher blastocyst rates (HMC vs. CNT: 35% vs. 10%, p

Coral reefs are subject to coral bleaching manifested by the loss of endosymbiotic algae from coral host tissue. Besides algae, corals associate with bacteria. In particular, bacteria residing in the surface mucus layer are thought to mediate coral health, but their role in coral bleaching is unknown. We collected mucus from bleached and healthy Porites lobata colonies in the Persian/Arabian Gulf (PAG) and the Red Sea (RS) to investigate bacterial microbiome composition using 16S rRNA gene amplicon sequencing. We found that bacterial community structure was notably similar in bleached and healthy corals, and the most abundant bacterial taxa were identical. However, fine-scale differences in bacterial community composition between the PAG and RS were present and aligned with predicted differences in sulfur- and nitrogen-cycling processes. Based on our data, we argue that bleached corals benefit from the stable composition of mucus bacteria that resemble their healthy coral counterparts and presumably provide a conserved suite of protective functions, but monitoring of post-bleaching survival is needed to further confirm this assumption. Conversely, fine-scale site-specific differences highlight flexibility of the bacterial microbiome that may underlie adjustment to local environmental conditions and contribute to the widespread success of Porites lobata.

Rare invasive fungal diseases (IFD) are challenging for the treating physicians because of their unspecific clinical presentation, as well as the lack of standardised diagnostic and effective treatment strategies. Late onset of treatment and inappropriate medication is associated with high mortality, thus, urging the need for a better understanding of these diseases. The purpose of FungiScope

Aim Coral reefs rely on the symbiosis between scleractinian corals and intracellular, photosynthetic dinoflagellates of the genus Symbiodinium making the assessment of symbiont diversity critical to our understanding of ecological resilience of these ecosystems. This study characterizes Symbiodinium diversity around the Arabian Peninsula, which contains some of the most thermally diverse and understudied reefs on Earth. Location Shallow water coral reefs throughout the Red Sea (RS), Sea of Oman (SO), and Persian/Arabian Gulf (PAG). Methods Next‐generation sequencing of the ITS2 marker gene was used to assess Symbiodinium community composition and diversity comprising 892 samples from 46 hard and soft coral genera. Results Corals were associated with a large diversity of Symbiodinium, which usually consisted of one or two prevalent symbiont types and many types at low abundance. Symbiodinium communities were strongly structured according to geographical region and to a lesser extent by coral host identity. Overall symbiont communities were composed primarily of species from clade A and C in the RS, clade A, C, and D in the SO, and clade C and D in the PAG, representing a gradual shift from C‐ to D‐dominated coral hosts. The analysis of symbiont diversity in an Operational Taxonomic Unit (OTU)‐based framework allowed the identification of differences in symbiont taxon richness over geographical regions and host genera. Main conclusions Our study represents a comprehensive overview over biogeography and molecular diversity of Symbiodinium in the Arabian Seas, where coral reefs thrive in one of the most extreme environmental settings on the planet. As such our data will serve as a baseline for further exploration into the effects of environmental change on host–symbiont pairings and the identification and ecological significance of Symbiodinium types from regions already experiencing ‘Future Ocean’ conditions.

In this study, we examined two non-scleractinian taxa, the rare nudibranch Phyllodesmium lizardensis and Bayerxenia sp., the octocoral on which the nudibranch lives and feeds, to investigate the effect of experimental heat stress on their symbioses with Symbiodinium. Bleaching has not been studied in nudibranchs. Bayerxenia sp. belongs to the alcyonacea family Xeniidae, members of which are known to be heat sensitive, but the genus has never been subject to heat stress experiments or bleaching observations. While qPCR did not reveal any changes to the symbiont community composition, the two host species responded differently to increased temperature. There were changes in the relative proportion of tissue types in Bayerxenia sp., but these were not attributable to the temperature treatment. Bayerxenia sp. exhibited no changes in cellular structure (apoptosis or cell necrosis), or symbiont functioning, cell size, density, or cladal community structure. On the other hand, the host, P. lizardensis, experienced tissue loss and symbiont densities decreased significantly with the majority of the remaining symbiont cells significantly degenerated after the heat stress. This decrease did not influence symbiont community composition, symbiont cell size, or photosynthetic efficiency. While the bleaching process in nudibranchs was demonstrated for the first time, the physiological and molecular pathways leading to this response still require attention.

Many coral species display changing distribution patterns across coral reef depths. While changes in the underwater light field and the ability to associate with different photosynthetic symbionts of the genus Symbiodinium explain some of the variation, the limits to physiological plasticity are unknown for most corals. In the central Red Sea, colonies of the branching coral Pocillopora verrucosa are most abundant in shallow high light environments and become less abundant in water depths below 10 m. To further understand what determines this narrow distribution, we conducted a cross-depths transplant experiment looking at physiological plasticity and acclimation in regard to depth. Colonies from 5, 10, and 20 m were collected, transplanted to all depths, and re-investigated after 30 and 210 d. All coral colonies transplanted downward from shallow to deep water displayed an increase in photosynthetic light-harvesting pigments, which resulted in higher photosynthetic efficiency. Shallow-water specimens transplanted to deeper water showed a significant decrease in total protein content after 30 and 210 d under low light conditions compared to specimens transplanted to shallow and medium depths. Stable isotope data suggest that heterotrophic input of carbon was not increased under low light, and consequently, decreasing protein levels were symptomatic of decreasing photosynthetic rates that could not be compensated for through higher light-harvesting efficiency. Our results provide insights into the physiological plasticity of P. verrucosa in changing light regimes and explain the observed depth distribution pattern. Despite its high abundance in shallow reef waters, P. verrucosa possesses limited heterotrophic acclimation potential, i.e., the ability to support its mainly photoautotrophic diet through heterotrophic feeding. We conclude that P. verrucosa might be a species vulnerable to sudden changes in underwater light fields resulting from processes such as increased turbidity caused by coastal development along the Saudi Arabian Red Sea coast. published

Summary Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases.

Aim:Coral reefs rely on the symbiosis between scleractinian corals and intracellular, photosynthetic dinoflagellates of the genus Symbiodinium making the assessment of symbiont diversity critical to our understanding of ecological resilience of these ecosystems. This study characterizes Symbiodinium diversity around the Arabian Peninsula, which contains some of the most thermally diverse and understudied reefs on Earth.Location:Shallow water coral reefs throughout the Red Sea (RS), Sea of Oman (SO), and Persian/Arabian Gulf (PAG).Methods:Next‐generation sequencing of the ITS2 marker gene was used to assess Symbiodinium community composition and diversity comprising 892 samples from 46 hard and soft coral genera.Results:Corals were associated with a large diversity of Symbiodinium, which usually consisted of one or two prevalent symbiont types and many types at low abundance. Symbiodinium communities were strongly structured according to geographical region and to a lesser extent by coral host identity. Overall symbiont communities were composed primarily of species from clade A and C in the RS, clade A, C, and D in the SO, and clade C and D in the PAG, representing a gradual shift from C‐ to D‐dominated coral hosts. The analysis of symbiont diversity in an Operational Taxonomic Unit (OTU)‐based framework allowed the identification of differences in symbiont taxon richness over geographical regions and host genera.Main conclusions:Our study represents a comprehensive overview over biogeography and molecular diversity of Symbiodinium in the Arabian Seas, where coral reefs thrive in one of the most extreme environmental settings on the planet. As such our data will serve as a baseline for further exploration into the effects of environmental change on host–symbiont pairings and the identification and ecological significance of Symbiodinium types from regions already experiencing ‘Future Ocean’ conditions. published

Ocean warming threatens corals and the coral reef ecosystem. Nevertheless, corals can be adapted to their thermal environment and inherit heat tolerance across generations. In addition, the diverse microbes that associate with corals have the capacity for more rapid change, potentially aiding the adaptation of long-lived corals. Here, we show that the microbiome of reef corals is different across thermally variable habitats and changes over time when corals are reciprocally transplanted. Exposing these corals to thermal bleaching conditions changes the microbiome for heat-sensitive corals, but not for heat-tolerant corals growing in habitats with natural high heat extremes. Importantly, particular bacterial taxa predict the coral host response in a short-term heat stress experiment. Such associations could result from parallel responses of the coral and the microbial community to living at high natural temperatures. A competing hypothesis is that the microbial community and coral heat tolerance are causally linked., Coral-associated microbes could enhance the capacity of their host organism to respond to environmental change. Ziegler and colleagues use a reciprocal transplant experiment to show that microbiomes of heat-tolerant corals are more resilient to change than those of heat-sensitive corals.

The endosymbiosis between dinoflagellate algae of the genus Symbiodinium and stony corals provides the foundation of coral reef ecosystems. Coral bleaching, the expulsion of endosymbionts from the coral host tissue as a consequence of heat or light stress, poses a threat to reef ecosystem functioning on a global scale. Hence, a better understanding of the factors contributing to heat stress susceptibility and tolerance is needed. In this regard, some of the most thermotolerant corals live in particularly saline habitats, but possible effects of high salinity on thermotolerance in corals are anecdotal. Here we test the hypothesis that high salinity may lead to increased thermotolerance. We conducted a heat stress experiment at low, intermediate, and high salinities using a set of host-endosymbiont combinations of the coral model Aiptasia. As expected, all host-endosymbiont combinations showed reduced photosynthetic efficiency and endosymbiont loss during heat stress, but the severity of bleaching was significantly reduced with increasing salinities for one of the host-endosymbiont combinations. Our results show that higher salinities can convey increased thermotolerance in Aiptasia, although this effect seems to be dependent on the particular host strain and/or associated symbiont type. This finding may help explain the extraordinarily high thermotolerance of corals in high salinity environments, such as the Red Sea and the Persian/Arabian Gulf, and provides novel insight regarding factors that contribute to thermotolerance. Since our results are based on a salinity effect in symbiotic sea anemones, it remains to be determined whether this salinity effect can also be observed in stony corals., Summary: High salinity can be a factor contributing to increased cnidarian thermal tolerance, as indicated by reduced algal endosymbiont loss and reduced photosynthetic impairment in the sea anemone Aiptasia.

Background Xenotransplantation is considered to be a promising solution to the growing demand for suitable donor organs for transplantation. Despite tremendous progress in the generation of pigs with multiple genetic modifications thought to be necessary to overcoming the severe rejection responses after pig-to-non-human primate xenotransplantation, the production of knockout pigs by somatic cell nuclear transfer (SCNT) is still an inefficient process. Producing genetically modified pigs by intracytoplasmic microinjection of porcine zygotes is an alluring alternative. The porcine GGTA1 gene encodes for the α1,3-galactosyltransferase that synthesizes the Gal epitopes on porcine cells which constitute the major antigen in a xenotransplantation setting. GGTA1-KO pigs have successfully been produced by transfecting somatic cells with zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or CRISPR/Cas targeting GGTA1, followed by SCNT. Methods Here, we microinjected a CRISPR/Cas9 vector coding for a single-guide RNA (sgRNA) targeting exon 8 of the GGTA1 gene into the cytoplasm of 97 in vivo-derived porcine zygotes and transferred 86 of the microinjected embryos into three hormonally synchronized recipients. Fetuses and piglets were analyzed by flow cytometry for remaining Gal epitopes. DNA was sequenced to detect mutations at the GGTA1 locus. Results Two of the recipients remained pregnant as determined by ultrasound scanning on day 25 of gestation. One pregnancy was terminated on day 26, and six healthy fetuses were recovered. The second pregnancy was allowed to go to term and resulted in the birth of six healthy piglets. Flow cytometry analysis revealed the absence of Gal epitopes in four of six fetuses (66%), indicating a biallelic KO of GGTA1. Additionally, three of the six live-born piglets (50%) did not express Gal epitopes on their cell surface. Two fetuses and two piglets showed a mosaicism with a mixed population of Gal-free and Gal-expressing cells. Only a single piglet did not have any genomic modifications. Genomic sequencing revealed indel formation at the GGTA1 locus ranging from +17 bp to −20 bp. Conclusions These results demonstrate the efficacy of CRISPR/Cas to generate genetic modifications in pigs by simplified technology, such as intracytoplasmic microinjection into zygotes, which would significantly facilitate the production of genetically modified pigs suitable for xenotransplantation. Importantly, this simplified injection protocol avoids the penetration of the vulnerable pronuclear membrane, and is thus compatible with higher survival rates of microinjected embryos, which in turn facilitates production of genetically modified piglets.

Larger benthic Foraminifera (LBF) are ecologically important coral reef protists that harbour a large diversity of symbionts from at least four algal phyla. In this study the photosynthetic plasticity of different endosymbiontic algae found within LBF was investigated using pulse amplitude modulated (PAM) fluorometry. Maximum quantum efficiencies of photosystem II (F v /F m ) obtained from foraminiferal specimens directly after field collection indicated several pronounced differences between species containing endosymbionts from different algal phyla and, to a lesser extent, also varied between species that contain the same phyla of endosymbiontic algae. Foraminiferal species retaining functional chloroplasts and rhodophyte-bearing species had distinctly lower F v /F m , than LBF with dinoflagellates, diatoms or chlorophytes. A laboratory experiment was conducted over 48 h exposing species occurring in high- (photophilic), medium- and low-light (sciaphilic) environments to three manipulated light levels. Photophysiological responses were monitored by measuring F v /F m at regular intervals and rapid light curves (RLCs) at the end of the experiment. This experiment demonstrated oscillation of maximum quantum efficiencies according to the light-dark cycle. Changes in the shape of the RLCs (e.g., higher α and lower E k under low light conditions) indicated that photosynthetic plasticity allows LBF to acclimatise rapidly (

We describe a new species of remipede crustacean from an anchialine cave system in the Exuma Cays, Bahamas. Pleomothra fragilis n. sp. is the second species assigned to the genus Pleomothra Yager, 1989. It is a relatively small species that occurs in sympatry with other remipedes, copepods, ostracods and amphipods. Pleomothra fragilis can easily be distinguished from Pleomothra apletocheles by the shape of its head shield, relatively long caudal rami, differently shaped, smaller maxillules, and a completely reduced lacinia mobilis on the left mandible. The reduction of the left lacinia and the sixth maxillular segment represent unique features that are only found in Pleomothra. At present, interpretations of these reductions are purely speculative since developmental data of remipedes are largely lacking. However, our recent collection of five larvae included a postnauplius that could be identified as belonging to the genus Pleomothra. The postnauplius exhibits transitional morphologic features that facilitate to evaluate autapomorphies in Pleomothra.

Transgenesis in the mouse is an essential tool for the understanding of gene function and genome organization. Here, we describe a simplified microinjection protocol for efficient germline transgenesis and sustained transgene expression in the mouse model employing binary Sleeping Beauty transposon constructs of different topology. The protocol is based on co‐injection of supercoiled plasmids or minicircles, encoding the Sleeping Beauty transposase and a transposon construct, into the cytoplasm of murine zygotes. Importantly, this simplified injection avoids the mechanical penetration of the vulnerable pronuclear membrane, resulting in higher survival rates of treated embryos and a more rapid pace of injections. Upon translation of the transposase, transposase‐catalyzed transposition into the genome results in stable transgenic animals carrying monomeric transgenes. In summary, cytoplasmic injection of binary transposon constructs is a feasible, plasmid‐based, and simplified microinjection method to generate genetically modified mice. The modular design of the components allows the multiplexing of different transposons, and the generation of multi‐transposon transgenic mice in a single step. Fil: Garrels, Wiebke. Friedrich Loeffler Institut; Alemania Fil: Talluri, Thirumala R.. Friedrich Loeffler Institut; Alemania Fil: Ziegler, Maren. Friedrich Loeffler Institut; Alemania Fil: Most, Ilka. Friedrich Loeffler Institut; Alemania Fil: Forcato, Diego Oscar. Friedrich Loeffler Institut; Alemania. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schmeer, Marco. PlasmidFactory; Alemania Fil: Schleef, Martin. PlasmidFactory; Alemania Fil: Ivics, Zoltán. Paul Ehrlich Institute; Alemania Fil: Kues, Wilfried A.. Friedrich Loeffler Institut; Alemania

Supplemental digital content is available in the text., Background Multiple modifications of the porcine genome are required to prevent rejection after pig-to-primate xenotransplantation. Here, we produced pigs with a knockout of the α1,3-galactosyltransferase gene (GGTA1-KO) combined with transgenic expression of the human anti-apoptotic/anti-inflammatory molecules heme oxygenase-1 and A20, and investigated their xenoprotective properties. Methods The GGTA1-KO/human heme oxygenase-1 (hHO-1)/human A20 (hA20) transgenic pigs were produced in a stepwise approach using zinc finger nuclease vectors targeting the GGTA1 gene and a Sleeping Beauty vector coding for hA20. Two piglets were analyzed by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and sequencing. The biological function of the genetic modifications was tested in a 51Chromium release assay and by ex vivo kidney perfusions with human blood. Results Disruption of the GGTA1 gene by deletion of few basepairs was demonstrated in GGTA1-KO/hHO-1/hA20 transgenic pigs. The hHO-1 and hA20 mRNA expression was confirmed by quantitative reverse-transcription polymerase chain reaction. Ex vivo perfusion of 2 transgenic kidneys was feasible for the maximum experimental time of 240 minutes without symptoms of rejection. Conclusions Results indicate that GGTA1-KO/hHO-1/hA20 transgenic pigs are a promising model to alleviate rejection and ischemia-reperfusion damage in porcine xenografts and could serve as a background for further genetic modifications toward the production of a donor pig that is clinically relevant for xenotransplantation.

Coral-associated bacteria play an increasingly recognized part in coral health. We investigated the effect of local anthropogenic impacts on coral microbial communities on reefs near Jeddah, the largest city on the Saudi Arabian coast of the central Red Sea. We analyzed the bacterial community structure of water and corals (Pocillopora verrucosa and Acropora hemprichii) at sites that were relatively unimpacted, exposed to sedimentation & local sewage, or in the discharge area of municipal wastewaters. Coral microbial communities were significantly different at impacted sites: in both corals the main symbiotic taxon decreased in abundance. In contrast, opportunistic bacterial families, such as e.g. Vibrionaceae and Rhodobacteraceae, were more abundant in corals at impacted sites. In conclusion, microbial community response revealed a measurable footprint of anthropogenic impacts to coral ecosystems close to Jeddah, even though the corals appeared visually healthy. published

Mesophotic coral ecosystems receive increasing attention owing to their potential as deep coral refuges in times of global environmental change. Here, the mechanisms of coral holobiont photoacclimatization over a 60 m depth gradient in the central Red Sea were examined for the four coral genera Porites, Leptoseris, Pachyseris, and Podabacia. General acclimatization strategies were common to all host-symbiont combinations, e.g., Symbiodinium cell densities and photoprotective (PP) to light-harvesting pigment ratios both significantly decreased with water depth. Porites harbored Symbiodinium type C15 over the whole 60 m depth range, while Pachyseris and Podabacia had limited vertical distributions and hosted mainly Symbiodinium type C1. Symbiodinium type C15 had generally higher xanthophyll de-epoxidation rates and lower maximum quantum yields than C1, and also exhibited a strong photoacclimatory signal over depth that relates to the large distribution range of Porites. Interestingly, the coral host had an effect on Symbiodinium pigment composition. When comparing Symbiodinium type C1 in Podabacia and Pachyseris, the ß-carotene chl a−1, the peridinin chl a−1, and diadinoxanthin chl a−1 ratios were significantly different between host species. Our data support a view that depth acclimatization of corals in the mesophotics is facilitated by Symbiodinium physiology, which in turn is host-specific. published

Background Pig-to-human xenotransplantation is promising to ameliorate the worldwide shortage of suitable human donor organs. The transplantation of xenografts evokes severe immunological rejection responses. Different approaches led to considerable progress in this respect: transgenic expression of human complement regulatory proteins such as human decay-accelerating factor (hCD55), membrane co-factor protein (hCD46, MCP) or hCD59 and/or knockout of the porcine α1,3-galactosyltransferase gene (GGTA1-KO) prevent the hyperacute rejection response (HAR) [1, 2]. Moreover, human anticoagulant and/or anti-apoptotic and anti-inflammatory transgenes, such as human A20 (hA20) and human heme oxygenase-1 (hHO-1), could protect porcine xenografts from being rejected by the acute vascular rejection response [3, 4]. Recently, we produced pigs transgenic for hA20 which provides cytoprotective properties in porcine aortic endothelial cells in vitro [5]. However, hA20 was expressed only at low levels in heart, skeletal muscle and porcine aortic endothelial cells. Here, we generated a new tri-cistronic hA20/hCD55/Puro vector based on the Sleeping Beauty system (kindly provided by Dr. Zoltan Ivics) and transfected it into porcine GGTA1-KO cells that served as donor cells for somatic cell nuclear transfer (SCNT). Methods We co-transfected GGTA1-KO [6] porcine fibroblasts with a tri-cistronic cDNA expression vector coding for hA20/hCD55/Puro and the SB transposase 100X plasmid. The advantage of this vector is a stable genomic integration without any plasmid-based vector backbone and the combined integration of all transgenes at a single genomic locus which avoids transgene segregation after mating. Following puromycin selection (5 μg/ml), cells were screened by PCR for genomic integration of the vector and used for SCNT. Results In total, 1,028 embryos were transferred to 13 recipients of which five remained pregnant. Two pregnancies were in too early stages for ultrasound diagnosis at the time of writing. One pregnancy was terminated on day 25 of gestation and eight fetuses were obtained. The hA20 mRNA levels of these fetuses were increased 21- to 44-fold compared to another line (hA20/hHO-1/GGTA1-KO) and even higher than in the first generation hA20 transgenic pigs. Preliminary immunofluorescence analysis data showed delta mean fluorescence intensities (MFI) for hCD55 (hCD55 MFI minus control antibody MFI) of 1,016 in average in transgenic fibroblasts in comparison to delta MFI 299 in HUVECs and 114 in PBMCs. The susceptibility of the cells to NK cell- and T cell-mediated cell death compared to wild-type fibroblasts will be determined in a 51chromium release assay. Three gilts are expected to deliver piglets in November of this year. The expression and organ distribution of hA20 and hCD55 will be characterised subsequently. Conclusions Our goal is the production of multi-transgenic pigs with an increased expression of xeno-relevant transgenes. The combination of ubiquitous, high expression of anti-apoptotic and-inflammatory transgenes such as hA20 together with a complement regulatory protein like hCD55 on a GGTA1-KO background has the potential to prolong the survival of porcine grafts after pig-to-primate xenotransplantation. References [1] Phelps CJ, Koike C, Vaught TD, et al. Production of alpha 1,3-Galactosyltransferase-Deficient Pigs. Science. 2002; 299: 411–4. [2] Cowan PJ, Aminan A, Barlow H, et al. Renal xenografts from triple-transgenic pigs are not hyperacutely rejected but cause coagulopathy in non-immunosuppressed baboons. Transplantation. 2000; 69: 2504–2515. [3] Bach FH, Ferran C, Hechenleitner P, et al. Accommodation of vascularized xenografts: expression of “protective genes” by donor endothelial cells in a host Th2 cytokine environment. Nat Med. 1997; 3: 196–204. [4] Petersen B, Ramackers W, Lucas-Hahn A, et al. Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. Xenotransplantation 2011; 18: 355–368. [5] Oropeza M, Petersen B, Carnwerth J W, et al. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation 2009; 16: 522–534. [6] Hausschild J, Peterssen B, Santiago Y, et al. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013–12027.

We followed the development of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) system during locust embryogenesis in whole mount nervous systems and brain sections by using various cytochemical techniques. We visualized NO-sensitive neurons by cGMP immunofluorescence after incubation with an NO donor in the presence of the soluble guanylyl cyclase (sGC) activator YC-1 and the phosphodiesterase-inhibitor isobutyl-methyl-xanthine (IBMX). Central nervous system (CNS) cells respond to NO as early as 38% embryogenesis. By using the NADPH-diaphorase technique, we identified somata and neurites of possible NO-synthesizing cells in the CNS. The first NADPH-diaphorase-positive cell bodies appear around 40% embryogenesis in the brain and at 47% in the ventral nerve cord. The number of positive cells reaches the full complement of adult cells at 80%. In the brain, some structures, e.g., the mushroom bodies acquire NADPH-diaphorase staining only postembryonically. Immunolocalization of L-citrulline confirmed the presence of NOS in NADPH-diaphorase-stained neurons and, in addition, indicated enzymatic activity in vivo. In whole mount ventral nerve cords, citrulline immunolabeling was present in varying subsets of NADPH-diaphorase-positive cells, but staining was very variable and often weak. However, in a regeneration paradigm in which one of the two connectives between ganglia had been crushed, strong, reliable staining was observed as early as 60% embryogenesis. Thus, citrulline immunolabeling appears to reflect specific activity of NOS. However, in younger embryos, NOS may not always be constitutively active or may be so at a very low level, below the citrulline antibody detection threshold. For the CNS, histochemical markers for NOS do not provide conclusive evidence for a developmental role of this enzyme.

Conventional “Dolly”-based cloned (CNT) embryos maintain zona pellucida and can be transferred early in development. Handmade cloned (HMC) embryos are zona free and are cultured to later stages for transfer. We have shown differences between HMC and CNT embryos (Rep. Fert. Dev. 26, 123), and both in vitro culture and cloning method (NT) are associated with alterations in histone acetylation. More studies are needed to clarify whether CNT and HMC embryos differ in epigenetic profiles due to NT method or culture condition. Here we investigated histone acetylation profile of NT embryos produced by CNT or HMC with or without 5 to 6 days in vitro culture, emphasising quality and gene expression in resulting embryos. Both NT methods were performed on Day 0 (D0) with same oocyte batch, donor cells, and culture medium (CNT in group, HMC in well of well). On D0, 5, and 6 after CNT (Clon. Stem Cells 10, 355) or HMC (Zygote 20, 61), all developed embryos of all morphological qualities were collected for immunostaining of H3K18ac, and on D0 and 6 for mRNA expression of the genes KAT2A/2B, EP300, HDAC1/2, DNMT1o/s, and GAPDH. Embryo quality was evaluated normal (clear inner cell mass, high cell number, no fragments) or bad (no clear inner cell mass, low cell number, fragments). Cell numbers per blastocyst were counted on D5 and 6. Differences in cell number and H3K18ac level between different groups and days were analysed by ANOVA; gene expression data were analysed by GLM (SAS version 9.3, SAS Institute Inc., Cary, NC, USA). Embryo development rates of both NT methods were reported previously (Rep. Fert. Dev. 26, 123). On D5 and 6, all HMC embryos were evaluated as normal, but the CNT group contained both normal and bad embryos. Regarding cell numbers (Table 1), on D5 there was no difference between normal CNT and HMC embryos, but numbers were lower in CNT bad embryos. On D6 the blastocyst cell number was lower in both normal and bad CNT embryos compared with HMC. Regarding H3K18ac levels (Table 1), no differences were found on D5 between normal CNT and HMC embryos, but on D6 both CNT normal and bad embryos had higher H3K18ac level compared with HMC. On D0, no difference was found in mRNA expression of all 8 genes. On D6, KAT2A expression was slight increased (1.8-fold) in CNT compared with HMC embryos (P

Induced pluripotent stem cells (iPSCs) are promising for developing novel cell-based medical treatments. One potential use of iPSCs is for xenotransplantation via production of chimeric organisms. The generation of organs originated from the iPSCs of one individual in a foreign organism could allow the production of immune compatible organ transplants. One of the major problems down this road is to define the different nutrient needs of chimeric embryos (i.e. iPSCs and the host embryos). Here, we evaluated different media for supporting development of chimeric embryos consisting of parthenogenetic porcine embryos and iPS cells, either from pig or nonhuman primate, to identify the optimal medium conditions for pig-monkey aggregation chimeras. First, we cultured 3-day-old porcine parthenogenetic embryos in porcine zygote medium (PZM), iPSC-medium, and mixtures of the two media to identify the most suitable culture conditions. Three-day-old parthenogenetic embryos developed poorly in pure iPSC-medium (6.3% blastocyst rate), but grew in a mixture of PZM and iPSC medium. The best results were achieved with PZM and PZM with 10% or 25% iPSC medium (38.8% and 30% blastocyst rates, respectively). Next, we checked aggregation results of chimeric embryos produced with two different iPSC lines in the respective media (PZM, PZM+10% iPSC medium, PZM+25% iPSC medium). The porcine iPSC line (piPSC) and cynomolgus monkey iPSC line (ciPSC) carry fluorescent markers (piPSC: GFP, ciPSC: venus), thus facilitating detection of integration into the host embryos. After aggregation of iPS cells between two Day-3 parthenogenetic embryos (sandwich technique), blastocyst rates at Day 6 were determined (see Table 1). While culture in PZM allowed for the highest blastocyst rate (>40%), the iPS cell participation was very low (0–11.1%). Cultivation in PZM+25% iPS medium was compatible with a high rate of embryo-iPSC chimeras (50–100%), but with a low blastocyst rate (7.7–16.7%). Possibly, the iPS cells proliferate more rapidly than blastomeres and the embryo is overgrown. The best results were obtained in medium mixture PZM+10% iPSC medium (25–40% blastocyst rate with 66.7–100% iPS cell participation). These results show that supplementation of the basic culture medium with 10% iPSC medium yields high blastocyst rates for chimeric embryos, and also ensures much higher iPS cell participation. This improves the use of 3-day-old embryos in aggregation chimeras. Table 1.Development of parthenogenetic porcine embryo aggregates with porcine (p) or cynomolgus monkey (c) iPS cells

Background The acute vascular rejection (AVR) is the major obstacle to successful pig-to-primate xenotransplantation. Changes of the endothelium to a procoagulant state which occur after xenotransplantation entail an increased expression of Tissue Factor (TF). This initiates the extrinsic coagulation cascade and results in microvascular thrombosis and rapid loss of organ function. Organs from pigs with a decreased expression of TF could be promising to overcome AVR. Since a homozygous TF knockout had shown to be lethal in mice [1], we decided to use siRNA-technology to knockdown TF to a basal level. Methods Different siRNAs directed against TF expression were tested in initial studies for their efficacy. A vector coding for the siRNA providing the highest decrease of TF mRNA levels was kindly provided by Dr. Joachim Denner, RKI Berlin. Porcine fetal fibroblasts were co-transfected with this plasmid and a vector (DsRed, Clontech) which confers neomycin resistance and red fluorescence. After 14 days of selection with 800 μg/ml Geneticin (G418), cells were screened for siRNA expression prior to somatic cell nuclear transfers (SCNTs). Subsequently, the reconstructed embryos were transferred to synchronised recipient sows. Liveborn offspring were analysed by real-time PCR for TF mRNA quantification. The production of the siRNA was detected via RT-PCR followed by real-time PCR. Results An average of 95 embryos per sow was transferred to 28 synchronized recipients. The pregnancy rate was 54%. One sow was sacrificed on day 25 of pregnancy and one fetus (#893/1) was obtained. The candidate siRNA could be detected fetal tissues. Real-time PCR analysis revealed decreased TF mRNA expression in fibroblasts of fetus #893/1 compared to wild-type fibroblasts. Therefore, somatic cells from fetus #893/1 was used for recloning. A total of 12 live born piglets could be obtained; five piglets died within few days after delivery. The mRNA levels and production of the siRNA are currently determined. Protein expression analysis by fluorescence cytometry and functionality assessment by coagulation assays are planned for the near future with collaborating laboratories at the Medical School Hannover. Conclusions The AVR is currently the bottleneck in porcine-to-primate organ xenotransplantation. Controlling TF-induced coagulation might provide protective effects for porcine xenografts. Thus, TF knockdown pigs could be critical in producing the ultimate multi-transgenic pig as organ donor for human patients. References [1] Toomey J, Kratzer K, Lasky N, Stanton J, Broze GJ. Targeted disruption of the murine tissue factor gene results in embryonic lethality. Blood. 1996; 88(5): 1583–1587.

Multi-transgenic pigs are required for prolonging survival of porcine xenografts after transplantation into primate recipients. A homozygous knockout of the alpha1,3-galactosyltransferase gene (GGTA-1, encoding for Gal-epitopes) is critical for controlling the hyperacute rejection response. Nevertheless, the additional expression of complement regulatory proteins such as CD55 (decay accelerating factor, DAF) on a GGTA-1 KO background is thought to further improve survival after xenotransplantation. The next hurdle is the acute vascular rejection (AVR), which is primarily caused by endothelial cell activation. Previous experiments had shown that expression of anti-apoptotic/anti-inflammatory genes, such as human heme oxygenase-1 (hHO-1) or human A20 in pig organs may overcome AVR (1, 2). Adult fibroblasts from previously generated hHO-1 transgenic pigs were transfected with Zinc-finger nuclease plasmids directed towards knockout of the GGTA-1 gene. Transfected cells were selected via a magnetic bead selection approach as previously described (3). GGTA-1-KO/hHO-1 cells were used as donor cells in somatic cell nuclear transfer (SCNT). The first pregnancy was terminated at day 25 of gestation and 11 healthy fetuses were obtained. All fetuses were GGTA-1-KO and expressed hHO-1 at similar levels as the hHO-1 transgenic cell donors. Next, fetal GGTA-1-KO/hHO-1 cells from one fetus (C6F5) were transfected with a Sleeping Beauty (SB) transposon (kindly provided by Dr. Zoltan Ivics) coding for hCD55. Two positive cell clones were pooled and used as donor cells for SCNT. One pregnancy was terminated on day 25 and four healthy fetuses were obtained which showed the desired GGTA-1 KO/HO-1/CD55 genotype. Real-time PCR detected a 0.9 fold expression of CD55 over the housekeeping gene GAPDH. Compared to human umbilical vein cells (HUVEC), GGTA-1 KO/HO-1/CD55 fetal fibroblasts expressed CD55 10–15 fold higher than HUVEC. Two fetuses showed a slightly higher expression of CD55 and are currently used as cell donors for recloning to produce live triple transgenic offspring. To solve the problem of transgene segregation occurring after mating of multitransgenic pigs, we generated two tri-cistronic SB vectors coding for A20 and CD55 separated by the self-cleaving peptide E2 site. To select for transfected cells, one vector carried a Tomato-fluorescence cassette whereas the second conferred puromycin resistance. Red fluorescent/puromycin resistant cells served as donor cells for SCNT. Six healthy fetuses were collected on day 25 of pregnancy; all showed high expression of A20 and CD55 as detected by Real-time PCR and FACS. One of the fetuses was used as cell donor for recloning. Currently, three pregnancies with A20/CD55 fetuses are going on and will be allowed to go to term. These results show that molecular scissors, including ZFNs and TALENS, and expression vectors such as the Sleeping beauty system are valuable tools to enhance and accelerate the generation of multi-transgenic pigs for xenotransplantation. References [1] Oropeza M, Petersen B, Carnwath JW et al. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation. Proc. Nat. Acad. Sci. USA 2009; 16: 522–534. [2] Petersen B, Ramackers W, Lucas-Hahn A et al. Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. Xenotransplantation 2011; in press. [3] Hauschild J, Petersen B, Santiago Y et al. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc. Nat. Acad. Sci. USA. 2011; 108: 12013–12017.

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients. Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1

Over this century, coral reefs will run the gauntlet of climate change, as marine heatwaves (MHWs) become more intense and frequent, and ocean acidification (OA) progresses. However, we still lack a quantitative assessment of how, and to what degree, OA will moderate the responses of corals to MHWs as they intensify throughout this century. Here, we first projected future MHW intensities for tropical regions under three future greenhouse gas emissions scenario (representative concentration pathways, RCP2.6, RCP4.5 and RCP8.5) for the near-term (2021-2040), mid-century (2041-2060) and late-century (2081-2100). We then combined these MHW intensity projections with a global data set of 1,788 experiments to assess coral attribute performance and survival under the three emissions scenarios for the near-term, mid-century and late-century in the presence and absence of OA. Although warming and OA had predominately additive impacts on the coral responses, the contribution of OA in affecting most coral attributes was minor relative to the dominant role of intensifying MHWs. However, the addition of OA led to greater decreases in photosynthesis and survival under intermediate and unrestricted emissions scenario for the mid- and late-century than if intensifying MHWs were considered as the only driver. These results show that role of OA in modulating coral responses to intensifying MHWs depended on the focal coral attribute and extremity of the scenario examined. Specifically, intensifying MHWs and OA will cause increasing instances of coral bleaching and substantial declines in coral productivity, calcification and survival within the next two decades under the low and intermediate emissions scenario. These projections suggest that corals must rapidly adapt or acclimatize to projected ocean conditions to persist, which is far more likely under a low emissions scenario and with increasing efforts to manage reefs to enhance resilience. published

Our understanding of the response of reef-building corals to changes in their physical environment is largely based on laboratory experiments, analysis of long-term field data, and model projections. Experimental data provide unique insights into how organisms respond to variation of environmental drivers. However, an assessment of how well experimental conditions cover the breadth of environmental conditions and variability where corals live successfully is missing. Here, we compiled and analyzed a globally distributed dataset of in-situ seasonal and diurnal variability of key environmental drivers (temperature, pCO2, and O2) critical for the growth and livelihood of reef-building corals. Using a meta-analysis approach, we compared the variability of environmental conditions assayed in coral experimental studies to current and projected conditions in their natural habitats. We found that annual temperature profiles projected for the end of the 21st century were characterized by distributional shifts in temperatures with warmer winters and longer warm periods in the summer, not just peak temperatures. Furthermore, short-term hourly fluctuations of temperature and pCO2 may regularly expose corals to conditions beyond the projected average increases for the end of the 21st century. Coral reef sites varied in the degree of coupling between temperature, pCO2, and dissolved O2, which warrants site-specific, differentiated experimental approaches depending on the local hydrography and influence of biological processes on the carbonate system and O2 availability. Our analysis highlights that a large portion of the natural environmental variability at short and long timescales is underexplored in experimental designs, which may provide a path to extend our understanding on the response of corals to global climate change., This study was conducted as part of a competitive research funding grant by the Red Sea Research Center of King Abdullah University of Science and Technology awarded to AA, NG, SK, SSR, and MZ. TLF was supported by the Swiss National Science Foundation (198897), the Swiss National Supercomputing Centre, and the European Union’s Horizon 2020 research and innovation program under grant agreement no. 820989 (project COMFORT, “Our common future ocean in the Earth system - quantifying coupled cycles of carbon, oxygen, and nutrients for determining and achieving safe operating spaces with respect to tipping points”) and grant agreement no. 862923 (project AtlantECO, “Atlantic Ecosystems Assessment, Forecasting & Sustainability”).