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Global habitat degradation heightens the need to better understand patterns of genetic connectivity and diversity of marine biota across geographical ranges to guide conservation efforts. Corals across the Red Sea are subject to pronounced environmental differences, but studies so far suggest that animal populations are largely connected, excepting evidence for a genetic break between the northern-central and southern regions. Here, we investigated population structure and holobiont assemblage of two common pocilloporid corals, Pocillopora verrucosa and Stylophora pistillata, across the Red Sea. We found little evidence for population differentiation in P. verrucosa, except for the southernmost site. Conversely, S. pistillata exhibited a complex population structure with evidence for within-reef and regional genetic differentiation, in line with differences in their reproductive mode (P. verrucosa is a broadcast spawner and S. pistillata is a brooder). Analysis for genomic loci under positive selection identified 85 sites (18 of which were in coding sequences) that distinguished the southern P. verrucosa population from the remainder of the Red Sea population. By comparison, we found 128 loci (24 of which were residing in coding sequences) in S. pistillata with evidence for local adaptation at various sites. Functional annotation of the underlying proteins revealed putative roles in the response to stress, lipid metabolism, transport, cytoskeletal rearrangement, and ciliary function (among others). Microbial assemblages of both coral species showed pervasive association with microalgal symbionts from the genus Symbiodinium (former clade A) and bacteria from the genus Endozoicomonas that exhibited significant differences according to host genotype and environment. The disparity of population genetic and holobiont assemblage patterns even between closely related species (family Pocilloporidae) highlights the need for multispecies investigations to better understand the role of the environment in shaping evolutionary trajectories. It further emphasizes the importance of networks of reef reserves to achieve conservation of genetic variants critical to the future survival of coral ecosystems. published

The precise identification of mutualistic dinoflagellates is critical for understanding the physiology, ecology and evolution of their mutualisms with animals. Cotylorhiza tuberculata (Macri 1778) is a common scyphozoan endemic to the Mediterranean Sea and relies in part on endosymbiotic dinoflagellates (zooxanthellae) for survival and growth. To further study the diversity of symbionts associated with these animals, we analyzed specimens of C. tuberculata collected across the western Mediterranean Sea and from public aquaria, using a combination of next generation sequencing (NGS) of ITS2 rDNA and direct Sanger sequencing of partial 28 S rRNA and mitochondrial cob genes. Two diagnostic ITS2 profiles were characterized during our analysis of NGS data. Combined with information from additional genetic markers, each profile corresponds to a single species of symbiont, not diverse community assemblages as are sometimes inferred. Breviolum psygmophilum was common in all specimens, while Philozoon medusarum occurred at lower abundances in many individuals. The ribosomal array of B. psygmophilum was highly heterogeneous and contained ~ 15 co-occurring sequence variants found in the same relative proportions across all samples obtained in this study, while the ribosomal array in the genomes of P. medusarum was relatively homogeneous represented mostly by one abundant sequence variant. This precise interpretation of rDNA data improves understanding of the ecology and evolution of these mutualisms. Cotylorhiza tuberculata’s association with dinoflagellate symbionts from different genera is consistent with previous findings and suggests that evolutionary divergent symbionts with dissimilar niches are better able to coexist in hospite.

[poster presented at iMarCo congress, abstract from the Molecular Ecology related article] All metazoans are in fact holobionts, resulting from the association of several organisms, and organismal adaptation is then due to the composite response of this association to the environment. Deciphering the mechanisms of symbiont acquisition in a holobiont is therefore essential to understanding the extent of its adaptive capacities. In cnidarians, some species acquire their photosynthetic symbionts directly from their parents (vertical transmission) but may also acquire symbionts from the environment (horizontal acquisition) at the adult stage. The Mediterranean snakelocks sea anemone, Anemonia viridis (Forskål, 1775), passes down symbionts from one generation to the next by vertical transmission, but the capacity for such horizontal acquisition is still unexplored. To unravel the flexibility of the association between the different host lineages identified in A.viridis and its Symbiodiniaceae, we genotyped both the animal hosts and their symbiont communities in members of host clones in five different locations in the North Western Mediterranean Sea. The composition of within-host–symbiont populations was more dependent on the geographical origin of the hosts than their membership to a given lineage or even to a given clone. Additionally, similarities in host–symbiont communities were greater among genets (i.e. among different clones) than among ramets (i.e. among members of the same given clonal genotype). Taken together, our results demonstrate that A.viridis may form associations with a range of symbiotic dinoflagellates and suggest a capacity for horizontal acquisition. A mixed-mode transmission strategy in A.viridis, as we posit here, may help explain the large phenotypic plasticity that characterizes this anemone.

Tropical coral reefs are among the worst affected ecosystems by climate change with predictions ranging between a 70-90% loss of reefs in the coming decades. Effective conservation strategies that maximize ecosystem resilience, and potential for recovery, must be informed by the accurate characterization of extant genetic diversity and population structure together with an understanding of the adaptive potential of keystone species. Here, we analyzed samples from theTaraPacific Expedition (2016 to 2018) that completed an 18,000 km longitudinal transect of the Pacific Ocean sampling three widespread corals –Pocillopora meandrina, Porites lobata, andMilleporacf.platyphylla– across 33 sites from 11 islands. Using deep metagenomic sequencing of 269 colonies in conjunction with morphological analyses and climate variability data we can show that the sampled transect encompasses multiple morphologically cryptic species that exhibit disparate biogeographic patterns, and most importantly, distinct evolutionary patterns, despite exposure to identical environmental regimes. Our findings demonstrate on a basin-scale that evolutionary trajectories are species-specific and complex, and can only in part be predicted from the environment. This highlights that conservation strategies must integrate multi-species investigations to consider the distinct genomic footprints shaped by selection as well as the genetic potential for adaptive change.

The global decline in malaria has stalled1, emphasizing the need for vaccines that induce durable sterilizing immunity. Here we optimized regimens for chemoprophylaxis vaccination (CVac), for which aseptic, purified, cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ) were inoculated under prophylactic cover with pyrimethamine (PYR) (Sanaria PfSPZ-CVac(PYR)) or chloroquine (CQ) (PfSPZ-CVac(CQ))—which kill liver-stage and blood-stage parasites, respectively—and we assessed vaccine efficacy against homologous (that is, the same strain as the vaccine) and heterologous (a different strain) controlled human malaria infection (CHMI) three months after immunization ( https://clinicaltrials.gov/ , NCT02511054 and NCT03083847). We report that a fourfold increase in the dose of PfSPZ-CVac(PYR) from 5.12 × 104 to 2 × 105 PfSPZs transformed a minimal vaccine efficacy (low dose, two out of nine (22.2%) participants protected against homologous CHMI), to a high-level vaccine efficacy with seven out of eight (87.5%) individuals protected against homologous and seven out of nine (77.8%) protected against heterologous CHMI. Increased protection was associated with Vδ2 γδ T cell and antibody responses. At the higher dose, PfSPZ-CVac(CQ) protected six out of six (100%) participants against heterologous CHMI three months after immunization. All homologous (four out of four) and heterologous (eight out of eight) infectivity control participants showed parasitaemia. PfSPZ-CVac(CQ) and PfSPZ-CVac(PYR) induced a durable, sterile vaccine efficacy against a heterologous South American strain of P. falciparum, which has a genome and predicted CD8 T cell immunome that differs more strongly from the African vaccine strain than other analysed African P. falciparum strains. Two malaria vaccines comprising Plasmodium falciparum sporozoites and treatment with either pyrimethamine or chloroquine induced durable protective responses against both the African vaccine strain and a heterologous South American strain of P. falciparum.

Giant clams (Tridacninae) are important members of Indo‐Pacific coral reefs and among the few bivalve groups that live in symbiosis with unicellular algae (Symbiodiniaceae). Despite the importance of these endosymbiotic dinoflagellates for clam ecology, the diversity and specificity of these associations remain relatively poorly studied, especially in the Red Sea. Here, we used the internal transcribed spacer 2 (ITS2) rDNA gene region to investigate Symbiodiniaceae communities associated with Red Sea Tridacna maxima clams. We sampled five sites spanning 1,300 km (10° of latitude, from the Gulf of Aqaba, 29°N, to the Farasan Banks, 18°N) along the Red Sea's North‐South environmental gradient. We detected a diverse and structured assembly of host‐associated algae with communities demonstrating region and site‐specificity. Specimens from the Gulf of Aqaba harbored three genera of Symbiodiniaceae, Cladocopium, Durusdinium, and Symbiodinium, while at all other sites clams associated exclusively with algae from the Symbiodinium genus. Of these exclusively Symbiodinium‐associating sites, the more northern (27° and 22°) and more southern sites (20° and 18°) formed two separate groupings despite site‐specific algal genotypes being resolved at each site. These groupings were congruent with the genetic break seen across multiple marine taxa in the Red Sea at approximately 19°, and along with our documented site‐specificity of algal communities, contrasted the panmictic distribution of the T. maxima host. As such, our findings indicate flexibility in T. maxima‐Symbiodiniaceae associations that may explain its relatively high environmental plasticity and offers a mechanism for environmental niche adaptation., Here we demonstrate site‐ and region‐specific structuring of giant clam‐Symbiodiniaceae associations across 10° of latitude in the Red Sea. This structuring indicates a flexibility in clam‐algal associations that may explain their high environmental plasticity and offer a strategy for niche adaptation.

Coral bleaching is one of the main drivers of reef degradation. Most corals bleach and suffer mortality at just 1–2°C above their maximum monthly mean temperatures, but some species and genotypes resist or recover better than others. Here, we conducted a series of 18‐hr short‐term acute heat stress assays side‐by‐side with a 21‐day long‐term heat stress experiment to assess the ability of both approaches to resolve coral thermotolerance differences reflective of in situ reef temperature thresholds. Using a suite of physiological parameters (photosynthetic efficiency, coral whitening, chlorophyll a , host protein, algal symbiont counts, and algal type association), we assessed bleaching susceptibility of Stylophora pistillata colonies from the windward/exposed and leeward/protected sites of a nearshore coral reef in the central Red Sea, which had previously shown differential mortality during a natural bleaching event. Photosynthetic efficiency was most indicative of the expected higher thermal tolerance in corals from the protected reef site, denoted by an increased retention of dark‐adapted maximum quantum yields at higher temperatures. These differences were resolved using both experimental setups, as corroborated by a positive linear relationship, not observed for the other parameters. Notably, short‐term acute heat stress assays resolved per‐colony (genotype) differences that may have been masked by acclimation effects in the long‐term experiment. Using our newly developed portable experimental system termed the Coral Bleaching Automated Stress System (CBASS), we thus highlight the potential of mobile, standardized short‐term acute heat stress assays to resolve fine‐scale differences in coral thermotolerance. Accordingly, such a system may be suitable for large‐scale determination and complement existing approaches to identify resilient genotypes/reefs for downstream experimental examination and prioritization of reef sites for conservation/restoration. Development of such a framework is consistent with the recommendations of the National Academy of Sciences and the Reef Restoration and Adaptation Program committees for new intervention and restoration strategies. published

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships.

Endogenous viral elements (EVEs) offer insight into the evolutionary histories and hosts of contemporary viruses. This study leveraged DNA metagenomics and genomics to detect and infer the host of a non-retroviral dinoflagellate-infecting +ssRNA virus (dinoRNAV) common in coral reefs. As part of the Tara Pacific Expedition, this study surveyed 269 newly sequenced cnidarians and their resident symbiotic dinoflagellates (Symbiodiniaceae), associated metabarcodes, and publicly available metagenomes, revealing 178 dinoRNAV EVEs, predominantly among hydrocoral-dinoflagellate metagenomes. Putative associations between Symbiodiniaceae and dinoRNAV EVEs were corroborated by the characterization of dinoRNAV-like sequences in 17 of 18 scaffold-scale and one chromosome-scale dinoflagellate genome assembly, flanked by characteristically cellular sequences and in proximity to retroelements, suggesting potential mechanisms of integration. EVEs were not detected in dinoflagellate-free (aposymbiotic) cnidarian genome assemblies, including stony corals, hydrocorals, jellyfish, or seawater. The pervasive nature of dinoRNAV EVEs within dinoflagellate genomes (especially Symbiodinium), as well as their inconsistent within-genome distribution and fragmented nature, suggest ancestral or recurrent integration of this virus with variable conservation. Broadly, these findings illustrate how +ssRNA viruses may obscure their genomes as members of nested symbioses, with implications for host evolution, exaptation, and immunity in the context of reef health and disease.

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 processed in situ, during the Tara Pacific 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 h. 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.

Widespread coral bleaching occurred in the central Red Sea in 2010 and 2015. During both events, a cross-shelf and depth gradient of bleaching severity was identified within the Thuwal reef system, central Red Sea, Saudi Arabia. While bleaching and survival of coral taxa were monitored, neither in situ reef temperatures nor coral-associated algal communities (family Symbiodiniaceae) were characterized. Here, we determined coral host-associated Symbiodiniaceae communities and monitored temperatures along the same cross-shelf and depth gradient on six reefs in 2017 to better understand the role of these factors in the observed bleaching patterns and to generate a baseline for further studies. We characterized > 600 coral–algal associations across winter and summer in six genera of scleractinian coral (Pocillopora, Stylophora, Seriatopora, Galaxea, Gardineroseris, and Porites) and one fire coral (Family Milleporidae) using ITS2 next-generation sequencing in conjunction with the SymPortal analytical framework. We show that previous bleaching patterns correlate poorly with the largely coral host-specific structure of the 2017 Symbiodiniaceae community and are in better agreement with absolute and intraday sea water temperature variations monitored on the reefs. We demonstrate a greater distinctiveness of Symbiodiniaceae communities at the more severely bleached inshore reefs compared to those reefs further offshore. However, the potential Symbiodiniaceae community changes at these reefs prior to our sampling prevent us from evaluating this distinctiveness as determinative of the differences in bleaching severities. Based on our analyses, we discuss how fine-scale delineation of algal genotypes, including host-specific putative genotypes of Durusdinium trenchii that represent alluring targets for further taxonomic identification, corroborate a niche-adapted rather than generalist character of many coral–Symbiodiniaceae associations. In conclusion, as studies such as this one continue to build the global catalogue of coral–Symbiodiniaceae associations, we may be afforded a better oversight of how specialized coral–algal associations really are and how restricted their modification may be, both of which are critical considerations in predicting the adaptive potential of corals and the reef ecosystems they build.

Reef-building corals are at risk of extinction from ocean warming. While some corals can enhance their thermal limits by associating with dinoflagellate photosymbionts of superior stress tolerance, the extent to which symbiont communities will reorganize under increased warming pressure remains unclear. Here we show that corals in the hottest reefs in the world in the Persian Gulf maintain associations with the same symbionts across 1.5 years despite extreme seasonal warming and acute heat stress (≥35°C). Persian Gulf corals predominantly associated with Cladocopium (clade C) and most also hosted Symbiodinium (clade A) and/or Durusdinium (clade D). This is in contrast to the neighbouring and milder Oman Sea, where corals associated with Durusdinium and only a minority hosted background levels of Cladocopium. During acute heat stress, the higher prevalence of Symbiodinium and Durusdinium in bleached versus nonbleached Persian Gulf corals indicates that genotypes of these background genera did not confer bleaching resistance. Within symbiont genera, the majority of ITS2 rDNA type profiles were unique to their respective coral species, confirming the existence of host-specific symbiont lineages. Notably, further differentiation among Persian Gulf sites demonstrates that symbiont populations are either isolated or specialized over tens to hundreds of kilometres. Thermal tolerance across coral species was associated with the prevalence of a single ITS2 intragenomic sequence variant (C3gulf), definitive of the Cladocopium thermophilum group. The abundance of C3gulf was highest in bleaching-resistant corals and at warmer sites, potentially indicating a specific symbiont genotype (or set of genotypes) that may play a role in thermal tolerance that warrants further investigation. Together, our findings indicate that co-evolution of host-Symbiodiniaceae partnerships favours fidelity rather than flexibility in extreme environments and under future warming.

How can we explain morphological variations in a holobiont? The genetic determinism of phenotypes is not always obvious and could be circumstantial in complex organisms. In symbiotic cnidarians, it is known that morphology or colour can misrepresent a complex genetic and symbiotic diversity. Anemonia viridis is a symbiotic sea anemone from temperate seas. This species displays different colour morphs based on pigment content and lives in a wide geographical range. Here, we investigated whether colour morph differentiation correlated with host genetic diversity or associated symbiotic genetic diversity by using RAD sequencing and symbiotic dinoflagellate typing of 140 sea anemones from the English Channel and the Mediterranean Sea. We did not observe genetic differentiation among colour morphs of A. viridis at the animal host or symbiont level, rejecting the hypothesis that A. viridis colour morphs correspond to species level differences. Interestingly, we however identified at least four independent animal host genetic lineages in A. viridis that differed in their associated symbiont populations. In conclusion, although the functional role of the different morphotypes of A. viridis remains to be determined, our approach provides new insights on the existence of cryptic species within A. viridis. published

Corals from the northern Red Sea, in particular the Gulf of Aqaba (GoA), have exceptionally high bleaching thresholds approaching >5°C above their maximum monthly mean (MMM) temperatures. These elevated thresholds are thought to be due to historical selection, as corals passed through the warmer Southern Red Sea during re-colonization from the Arabian Sea. To test this hypothesis, we determined thermal tolerance thresholds of GoA versus Central Red Sea (CRS) Stylophora pistillata corals using the Coral Bleaching Automated Stress System (CBASS) to run a series of standardized acute thermal stress assays. Relative thermal thresholds of GoA and CRS corals were indeed similar and exceptionally high (~7°C above MMM). However, absolute thermal thresholds of CRS corals were on average 3°C above those of GoA corals. To explore the mechanistic underpinnings, we determined gene expression response and microbiome dynamics of coral holobiont compartments. Transcriptomic responses differed markedly, with a strong response to the thermal stress in GoA corals versus a remarkably muted response in corals from the CRS. This pattern was recapitulated in the algal symbionts that showed site-specific genetic differentiation. Concomitant to this, a subset of coral and algal genes showed temperature-induced expression in GoA corals, while exhibiting fixed high expression, i.e. front-loading, in CRS corals. Bacterial community composition of GoA corals changed dramatically under heat stress, whereas CRS corals displayed consistent assemblages, indicating distinct microbial response patterns. Our work demonstrates distinct patterns underlying thermal tolerance across spatial scales, even for the same species and ocean basin. We interpret the response of GoA corals as that of a resilient population approaching a tipping point in contrast to a pattern of consistently elevated thermal resistance in CRS corals that cannot further attune. Such response differences suggest distinct thermal tolerance mechanisms that affect the response of coral populations to ocean warming. published

Background The recent expansion of tools designed to accurately quantify malaria parasite-produced antigens has enabled us to evaluate the performance of rapid diagnostic tests (RDTs) as a function of the antigens they detect—typically histidine rich protein 2 (HRP2) or lactate dehydrogenase (LDH). Methods For this analysis, whole blood specimens from a longitudinal study in Bancoumana, Mali were used to evaluate the performance of the ultra-sensitive HRP2-based Alere™ Malaria Ag P.f RDT (uRDT). The samples were collected as part of a transmission-blocking vaccine trial in a high transmission region for Plasmodium falciparum malaria. Furthermore, antigen dynamics after successful anti-malarial drug treatment were evaluated in these samples using the Q-Plex Human Malaria Array (4-Plex) to quantify antigen concentrations. Results The uRDT had a 50% probability of a positive result at 207 pg/mL HRP2 [95% credible interval (CrI) 160–268]. Individuals with symptomatic infection remained positive by uRDT for a median of 33 days [95% confidence interval (CI) 28–47] post anti-malarial drug treatment. Biphasic exponential decay models accurately captured the population level post-treatment dynamics of both HRP2 and Plasmodium LDH (pLDH), with the latter decaying more rapidly. Motivated by these differences in rates of decay, a novel algorithm that used HRP2:pLDH ratios to predict if an individual had active versus recently cleared P. falciparum infection was developed. The algorithm had 77.5% accuracy in correctly classifying antigen-positive individuals as those with and without active infection. Conclusions These results characterize the performance of the ultra-sensitive RDT and demonstrate the potential for emerging antigen-quantifying technologies in the field of malaria diagnostics to be helpful tools in distinguishing between active versus recently cleared malaria infections.

The coastal waters of Singapore support coral reefs that are biodiverse but characterized by high turbidity and sedimentation. Here, we used internal transcribed spacer two (ITS2) amplicon sequencing to investigate the Symbiodiniaceae communities associated with this marginal reef system, as turbid reefs may serve as potential refugia from future thermal stress. Using the analytical framework SymPortal, we identified a predominance of Cladocopium among the five coral species studied across six reef sites. Durusdinium was present in comparatively lower abundances and was composed of multiple Durusdinium trenchii strains. In contrast to other marginal environments, the Cladocopium communities exhibited low diversity and lacked the host-specificity of strains reported elsewhere. Nevertheless, we identified a site-specific strain across three species, which was supported by sequencing of the non-coding region of the psbA minicircle (psbAncr). The overall low diversity of the symbiont communities suggests that, although Singapore’s reefs may provide habitat for a diverse coral assemblage, the strong selective pressure exerted by the prevalent turbidity likely limits the diversity of the associated symbiont community. published

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 communities of the Persian Gulf (PG) withstand maximum and annual ranges of water temperatures that surpass those found on the majority of reefs elsewhere. As such, these communities may inform on how coral reefs might adapt to the warmer waters of the future. Depending on the algal symbiont (genus Symbiodinium) harboured, advantages in stress tolerance may be conferred to the coral holobiont. Characterizing the algal component is therefore critical in determining the coral holobiont's tolerance phenotype. Coral associations off the Arabian coastline of the PG have been characterized as biogeographically unique, containing a rare group of taxonomically ancestral and thermotolerant algal symbionts, the Symbiodinium thermophilum group. In contrast, waters off the Iranian coast remain poorly characterized with a notable lack of S. thermophilum group symbionts identified in coral associations to date. Here, we characterize the algal component of two reef building corals (Porites harrisoni and Cyphastrea microphthalma) predominant at three sites spanning almost the entire length of the PG's Iranian coast. Genotyping using the chloroplastic 23S and nuclear ITS2 genes, we demonstrate the presence of S. thermophilum group symbionts. We discuss the probable physical drivers of these associations and highlight the need for further research in these relatively understudied waters.

Climate change threatens the survival of scleractinian coral from exposure to concurrent ocean warming, acidification and deoxygenation; how corals can potentially adapt to this trio of stressors is currently unknown. This study investigates three coral species (Acropora muricata, Acropora pulchra and Porites lutea) dominant in an extreme mangrove lagoon (Bouraké, New Caledonia) where abiotic conditions exceed those predicted for many reef sites over the next 100 years under climate change and compared them to conspecifics from an environmentally more benign reef habitat. We studied holobiont physiology as well as plasticity in coral-associated microorganisms (Symbiodiniaceae and bacteria) through ITS2 and 16S rRNA sequencing, respectively. We hypothesised that differences in coral-associated microorganisms (Symbiodiniaceae and bacteria) between the lagoonal and adjacent reef habitats may support coral host productivity and ultimately the ability of corals to live in extreme environments. In the lagoon, all coral species exhibited a metabolic adjustment of reduced photosynthesis-to-respiration ratios (P/R), but this was accompanied by highly divergent coral host-specific microbial associations. This was substantiated by the absence of shared ITS2-type profiles (proxies for Symbiodiniaceae genotypes). We observed that ITS2 profiles originating from Durusdinium taxa made up

Background Chemoprophylaxis vaccination with sporozoites (CVac) with chloroquine induces protection against a homologous Plasmodium falciparum sporozoite (PfSPZ) challenge, but whether blood-stage parasite exposure is required for protection remains unclear. Chloroquine suppresses and clears blood-stage parasitemia, while other antimalarial drugs, such as primaquine, act against liver-stage parasites. Here, we evaluated CVac regimens using primaquine and/or chloroquine as the partner drug to discern whether blood-stage parasite exposure impacts protection against homologous controlled human malaria infection. Methods In a Phase I, randomized, partial double-blind, placebo-controlled study of 36 malaria-naive adults, all CVac subjects received chloroquine prophylaxis and bites from 12–15 P. falciparum–infected mosquitoes (CVac-chloroquine arm) at 3 monthly iterations, and some received postexposure primaquine (CVac-primaquine/chloroquine arm). Drug control subjects received primaquine, chloroquine, and uninfected mosquito bites. After a chloroquine washout, subjects, including treatment-naive infectivity controls, underwent homologous, PfSPZ controlled human malaria infection and were monitored for parasitemia for 21 days. Results No serious adverse events occurred. During CVac, all but 1 subject in the study remained blood-smear negative, while only 1 subject (primaquine/chloroquine arm) remained polymerase chain reaction–negative. Upon challenge, compared to infectivity controls, 3/3 chloroquine arm subjects displayed delayed patent parasitemia (P = .01) but not sterile protection, while 3/11 primaquine/chloroquine subjects remained blood-smear negative. Conclusions CVac-primaquine/chloroquine is safe and induces sterile immunity to P. falciparum in some recipients, but a single 45 mg dose of primaquine postexposure does not completely prevent blood-stage parasitemia. Unlike previous studies, CVac-chloroquine did not produce sterile immunity. Clinical Trials Registration NCT01500980.

Shifting the malaria priorities from a paradigm of control and elimination to a goal of global eradication calls for renewed attention to the interruption of malaria transmission. Sustained progress toward eradication will require both improved understanding of infectious reservoirs and efficient development of novel transmission-blocking interventions, such as rapidly acting and highly efficacious therapeutics and vaccines. Here, we review the direct skin feeding assay (DSF), which has been proposed as a valuable tool for measuring the in natura transmission of malaria parasites from human hosts to mosquito vectors across heterogeneous populations. To capture the methodological breadth of this assay’s use, we first systematically review and qualitatively synthesize previously published investigations using DSFs to study malaria transmission in humans. Then, using a recent Phase 1 trial in Mali of the Pfs25H-EPA/Alhydrogel® vaccine candidate (NCT01867463) designed to interrupt Plasmodium falciparum transmission as a case study, we describe the potential opportunities and current limitations of utilizing the endpoints measured by DSF in making early clinical decisions for individually randomized transmission-interrupting intervention candidates. Using simulations based on the data collected in the clinical trial, we demonstrate that the capacity of the DSF to serve as an evaluative tool is limited by the statistical power constraints of the “effective sample size” (i.e. the number of subjects that are capable of transmitting at the time of feeding). Altogether, our findings suggest DSFs have great potential utility for assessing the public health impacts of emerging antimalarial tools, but additional research is needed to address issues of scalability and to establish correlation with community-wide clinical endpoints as well as complementary in vitro measures, such as standard membrane feeding assays.

AimThe aim of the study was to assess the diversity of algal symbionts of the family Symbiodiniaceae associated with the coral genus Porites in the Red Sea, and to test for host‐specificity and environmental variables driving biogeographical patterns of algal symbiont distribution.LocationSaudi Arabian Red Sea.TaxonEndosymbiotic dinoflagellates of the family Symbiodiniaceae in association with the reef‐building coral genus Porites.MethodsEighty Porites coral specimens were collected along the Saudi Arabian Red Sea coast. Species boundaries were assessed morphologically and genetically (putative Control Region – mtCR; ITS region – ITS). Community composition of symbiotic dinoflagellates of the family Symbiodiniaceae was also assessed. Using the ITS2 marker with the SymPortal framework, Symbiodiniaceae data at the genus, majority ITS2 sequence and ITS2 type profile were used to assess symbiont diversity and distribution patterns. These were analysed in relation to coral host diversity, geographic location and environmental variables.ResultsAmong the 80 Porites samples, 10 morphologies were identified. These corals were clustered into five lineages (clades I–V) by each of the markers independently. Clades I, II and III each comprised of a single Porites morphology, while clades IV and V contained up to five distinct morphologies. The diversity of Symbiodiniaceae associated with Porites was high and latitudinal differentiation was observed. In particular, a shift from a Cladocopium‐dominated to a Durusdinium‐dominated community was found along the north–south gradient. Symbiont diversity showed the patterns of geographic‐specific association at Symbiodiniaceae genus, majority ITS2 sequence and ITS2 type profile level. Specific associations with host genotypes (but not morphological species) were also recovered when considering Symbiodiniaceae majority ITS2 sequence and ITS2 type profiles.Main conclusionsThis study provides the first large‐scale molecular characterization of Symbiodiniaceae communities associated with Porites corals from the Saudi Arabian Red Sea. The use of intragenomic diversity data enabled the resolution of host‐symbiont specificity and biogeographical patterns of distribution, previously unachievable with the ITS2 marker alone. Finally, correlation among symbiont diversity and Red Sea environmental gradients was documented. published

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 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.

The adaptation of tropical coral communities to the world's hottest sea, the Persian/Arabian Gulf (PAG), has recently been associated with ecological selection acting on a group of coral-associated algal symbionts, the Symbiodinium thermophilum group. Previous studies have shown that considerable genetic diversity exists within the group and that group members found within the PAG are significantly differentiated from those found externally, in the Gulf of Oman and wider waters. However, little is known about this genetic diversity. As an initial step towards understanding whether this diversity could represent niche adapted, selectable populations within the S. thermophilum group that may act as natural sources of stress tolerant associations to Indo-Pacific reefs, we investigate whether the diversity is structured between populations and where the location of the internal-external genetic partition lies. We use regions of the nuclear ribosomal DNA (ITS1-5.8S-ITS2) and chloroplastic psbA gene (non-coding region) from > 100 S. thermophilum group-harbouring Porites spp. (P. lobata, P. lutea, and P. harrisoni) sampled across steep temperature and salinity gradients to conduct analyses of variance and create maximum parsimony networks to assess genetic structure and (dis)similarity within and between populations of S. thermophilum found within the PAG and externally in the Gulf of Oman. Our analyses resolve a sharp genetic boundary between Symbiodinium populations in the western Strait of Hormuz and identify significant genetic structure between populations with as little as 20 km between them demonstrating that differentiation between populations is likely due to factors other than limited connectivity. Further, we hypothesize that genotypes identified outside of the PAG in the Gulf of Oman existing in near-oceanic salinities, yet thermally challenging waters, putatively represent candidates for stress-tolerant symbionts that could act as natural seed populations of stress tolerant genotypes to the wider indo-Pacific.

The binary designation Symbiodinium thermophilum was invalid due to the absence of an illustration as required by Article 44.2 of the ICN. Herein, it is validated. This species is the most common symbiont in reef corals in the southern Persian/Arabian Gulf, the world's hottest body of water sustaining reef coral growth.

Malaria transmission-blocking vaccines (TBV) have been evaluated in field trials in Mali since 2013. However, the assays currently used to measure serum antibody TB activity (TBA) after vaccination are highly variable, in part due to the lack of optimization and standardization for field assays in which mosquitoes feed on gametocytemic blood. Herein, we report a study conducted in Bancoumana village, Mali, where we identify and optimize the parameters that contribute to successful mosquito feeding outcomes in both direct skin feeds (DSFs) and direct membrane feeding assays (DMFA). These parameters include: 1) mosquito age, 2) duration of mosquito starvation prior to feeding, 3) membrane selection for DMFA, 4) anatomical location of DSF feeding (arm, calf, and ankle), and 5) time of day for DSF (dawn or dusk). We found that younger mosquitoes were significantly associated with higher feeding, survival, and infection rates. Longer starvation times were positively, but not significantly, associated with higher infection rates, but were negatively associated with feeding and survival. Membrane type and body location did not affect infection outcome significantly. Although dusk was found to be associated with higher infection rates, this may be confounded by the time from positive blood smear. Based on these findings, we make specific recommendations for optimal feeding parameters in the different assay types to maximize the chance of detecting parasite transmission in a standardized manner.

Malaria remains a major health concern for a large percentage of the world's population. While great strides have been made in reducing mortality due to malaria, new strategies and therapies are still needed. Therapies that are capable of blocking the transmission of Plasmodium parasites are particularly attractive, but only primaquine accomplishes this, and toxicity issues hamper its widespread use. In this study, we describe a series of pyrazolopyrimidine- and imidazopyrazine-based compounds that are potent inhibitors of PfCDPK4, which is a calcium-activated Plasmodium protein kinase that is essential for exflagellation of male gametocytes. Thus, PfCDPK4 is essential for the sexual development of Plasmodium parasites and their ability to infect mosquitoes. We demonstrate that two structural features in the ATP-binding site of PfCDPK4 can be exploited in order to obtain potent and selective inhibitors of this enzyme. Furthermore, we demonstrate that pyrazolopyrimidine-based inhibitors that are potent inhibitors of the in vitro activity of PfCDPK4 are also able to block Plasmodium falciparum exflagellation with no observable toxicity to human cells. This medicinal chemistry effort serves as a valuable starting point in the development of safe, transmission-blocking agents for the control of malaria.

Fatal infestations of land-based Acropora cultures with so-called Acropora-eating flatworms (AEFWs) are a global phenomenon. We evaluate the hypothesis that AEFWs represent a risk to coral reefs by studying the biology and the invasive potential of an AEFW strain from the UK. Molecular analyses identified this strain as Amakusaplana acroporae, a new species described from two US aquaria and one natural location in Australia. Our molecular data together with life history strategies described here suggest that this species accounts for most reported cases of AEFW infestations. We show that local parasitic activity impairs the light-acclimation capacity of the whole host colony. A. acroporae acquires excellent camouflage by harbouring photosynthetically competent, host-derived zooxanthellae and pigments of the green-fluorescent protein family. It shows a preference for Acropora valida but accepts a broad host range. Parasite survival in isolation (5–7 d) potentially allows for an invasion when introduced as non-native species in coral reefs.

International audience; Malaria transmission-blocking vaccines (TBV) have been evaluated in field trials in Mali since 2013. However, the assays currently used to measure serum antibody TB activity (TBA) after vaccination are highly variable, in part due to the lack of optimization and standardization for field assays in which mosquitoes feed on gametocytemic blood. Herein, we report a study conducted in Bancoumana village, Mali, where we identify and optimize the parameters that contribute to successful mosquito feeding outcomes in both direct skin feeds (DSFs) and direct membrane feeding assays (DMFA). These parameters include: 1) mosquito age, 2) duration of mosquito starvation prior to feeding, 3) membrane selection for DMFA, 4) anatomical location of DSF feeding (arm, calf, and ankle), and 5) time of day for DSF (dawn or dusk). We found that younger mosquitoes were significantly associated with higher feeding, survival, and infection rates. Longer starvation times were positively, but not significantly, associated with higher infection rates, but were negatively associated with feeding and survival. Membrane type and body location did not affect infection outcome significantly. Although dusk was found to be associated with higher infection rates, this may be confounded by the time from positive blood smear. Based on these findings, we make specific recommendations for optimal feeding parameters in the different assay types to maximize the chance of detecting parasite transmission in a standardized manner.

Corals in the Arabian/Persian Gulf (PAG) survive extreme sea temperatures (summer mean: >34°C), and it is unclear whether these corals have genetically adapted or physiologically acclimated to these conditions. In order to elucidate the processes involved in the thermal tolerance of PAG corals, it is essential to understand the connectivity between reefs within and outside of the PAG. To this end, this study set out to investigate the genetic structure of the coral, Platygyra daedalea, and its symbiotic algae in the PAG and neighbouring Gulf of Oman. Using nuclear markers (the ITS region and an intron of the Pax-C gene), this study demonstrates genetic divergence of P. daedalea on reefs within the thermally extreme PAG compared with those in the neighbouring Gulf of Oman. Isolation by distance of P. daedalea was supported by the ITS dataset but not the Pax-C intron. In addition, the symbiont community within the PAG was dominated by C3 symbionts, while the purportedly thermotolerant clade D was extremely rare and was common only at sites outside of the PAG. Analysis of the psbAncr indicates that the C3 variant hosted by P. daedalea in the PAG belongs to the newly described species, Symbiodinium thermophilum. The structuring of the coral and symbiont populations suggests that both partners of the symbiosis may contribute to the high bleaching thresholds of PAG corals. While limited gene flow has likely played a role in local adaptation within the PAG, it also indicates limited potential for natural export of thermal tolerance traits to reefs elsewhere in the Indian Ocean threatened by climate change.