Novel T4 bacteriophages associated with black band disease in corals.

Research into causative agents underlying coral disease have focused primarily on bacteria, whereas potential roles of viruses have been largely unaddressed. Bacteriophages may contribute to diseases through the lysogenic introduction of virulence genes into bacteria, or prevent diseases through lysis of bacterial pathogens. To identify candidate phages that may influence the pathogenicity of black band disease (BBD), communities of bacteria (16S rRNA) and T4-bacteriophages (gp23) were simultaneously profiled with amplicon sequencing among BBD-lesions and healthy-coral-tissue of Montipora hispida, as well as seawater (study site: the central Great Barrier Reef). Bacterial community compositions were distinct among BBD-lesions, healthy coral tissue and seawater samples, as observed in previous studies. Surprisingly, however, viral beta diversities based on both operational taxonomic unit (OTU)-compositions and overall viral community compositions of assigned taxa did not differ statistically between the BBD-lesions and healthy coral tissue. Nonetheless, relative abundances of three bacteriophage OTUs, affiliated to Cyanophage PRSM6 and Prochlorococcus phages P-SSM2, were significantly higher in BBD-lesions than in healthy tissue. These OTUs associated with BBD samples suggest the presence of bacteriophages that infect members of the cyanobacteria-dominated BBD community, and thus have potential roles in BBD pathogenicity.

Predation scars may influence host susceptibility to pathogens: evaluating the role of corallivores as vectors of coral disease.

Infectious diseases not regulated by host density, such as vector-borne diseases, have the potential to drive population declines and extinctions. Here we test the vector potential of the snail Drupella sp. and butterflyfish Chaetodon plebeius for two coral diseases, black band (BBD) and brown band (BrB) disease. Drupella transmitted BrB to healthy corals in 40% of cases immediately following feeding on infected corals, and even in 12% of cases 12 and 24 hours following feeding. However, Drupella was unable to transmit BBD in either transmission treatment. In a field experiment testing the vector potential of naturally-occurring fish assemblages, equivalent numbers of caged and uncaged coral fragments became infected with either BrB, BBD or skeletal eroding band, indicating that corallivorous fish were unlikely to have caused transmission. In aquaria, C. plebeius did not transmit either BBD or BrB, even following extended feeding on both infected and healthy nubbins. A literature review confirmed only four known coral disease vectors, all invertebrates, corroborating our conclusion that polyp-feeding fishes are unlikely to be vectors of coral diseases. This potentially because polyp-feeding fishes produce shallow lesions, not allowing pathogens to invade coral tissues. In contrast, corallivorous invertebrates that create deeper feeding scars increase pathogens transmission.

Effects of depression, metabolic syndrome, and cardiorespiratory fitness on mortality: results from the Cooper Center Longitudinal Study.

BACKGROUND: Depression and metabolic syndrome (MetS) are frequently comorbid disorders that are independently associated with premature mortality. Conversely, cardiorespiratory fitness (CRF) is associated with reduced mortality risk. These factors may interact to impact mortality; however, their effects have not been assessed concurrently. This analysis assessed the mortality risk of comorbid depression/MetS and the effect of CRF on mortality in those with depression/MetS. METHODS: Prospective study of 47 702 adults in the Cooper Center Longitudinal Study. Mortality status was attained from the National Death Index. History of depression was determined by patient response (yes or no) to a standardized medical history questionnaire. MetS was categorized using the American Heart Association/National Heart, Lung, and Blood Institute criteria. CRF was estimated from the final speed/grade of a treadmill graded exercise test. RESULTS: 13.9% reported a history of depression, 21.4% met criteria for MetS, and 3.0% met criteria for both MetS and history of depression. History of depression (HR = 1.24, p = 0.003) and MetS (HR = 1.28, p < 0.001) were independently associated with an increased mortality risk, with the greatest mortality risk among individuals with both a history of depression and MetS (HR = 1.59, p < 0.001). Higher CRF was associated with a significantly lower risk of mortality (p < 0.001) in all individuals, including those with MetS and/or a history of depression. CONCLUSIONS: Those with higher levels CRF had reduced mortality risk in the context of depression/MetS. Interventions that improve CRF could have substantial impact on the health of persons with depression/MetS.

Microsatellite allele sizes alone are insufficient to delineate species boundaries in Symbiodinium.

Symbiodinium are a diverse group of unicellular dinoflagellates that are important nutritional symbionts of reef-building corals. Symbiodinium putative species ('types') are commonly identified with genetic markers, mostly nuclear and chloroplast encoded ribosomal DNA regions. Population genetic analyses using microsatellite loci have provided insights into Symbiodinium biogeography, connectivity and phenotypic plasticity, but are complicated by: (i) a lack of consensus criteria used to delineate inter- vs. intragenomic variation within species; and (ii) the high density of Symbiodinium in host tissues, which results in single samples comprising thousands of individuals. To address this problem, Wham & LaJeunesse (2016) present a method for identifying cryptic Symbiodinium species from microsatellite data based on correlations between allele size distributions and nongeographic genetic structure. Multilocus genotypes that potentially do not recombine in sympatry are interpreted as secondary 'species' to be discarded from downstream population genetic analyses. However, Symbiodinium species delineations should ideally incorporate multiple physiological, ecological and molecular criteria. This is because recombination tests may be a poor indicator of species boundaries in Symbiodinium due to their predominantly asexual mode of reproduction. Furthermore, discontinuous microsatellite allele sizes in sympatry may be explained by secondary contact between previously isolated populations and by mutations that occur in a nonstepwise manner. Limitations of using microsatellites alone to delineate species are highlighted in earlier studies that demonstrate occasional bimodal distributions of allele sizes within Symbiodinium species and considerable allele size sharing among Symbiodinium species. We outline these issues and discuss the validity of reinterpretations of our previously published microsatellite data from Symbiodinium populations on the Great Barrier Reef (Howells et al. 2013).

The transcriptomic response of the coral Acropora digitifera to a competent Symbiodinium strain: the symbiosome as an arrested early phagosome.

Despite the ecological significance of the relationship between reef-building corals and intracellular photosynthetic dinoflagellates of the genus Symbiodinium, very little is known about the molecular mechanisms involved in its establishment. Indeed, microarray-based analyses point to the conclusion that host gene expression is largely or completely unresponsive during the establishment of symbiosis with a competent strain of Symbiodinium. In this study, the use of Illumina RNA-Seq technology allowed detection of a transient period of differential expression involving a small number of genes (1073 transcripts; <3% of the transcriptome) 4 h after the exposure of Acropora digitifera planulae to a competent strain of Symbiodinium (a clade B strain). This phenomenon has not previously been detected as a consequence of both the lower sensitivity of the microarray approaches used and the sampling times used. The results indicate that complex changes occur, including transient suppression of mitochondrial metabolism and protein synthesis, but are also consistent with the hypothesis that the symbiosome is a phagosome that has undergone early arrest, raising the possibility of common mechanisms in the symbiotic interactions of corals and symbiotic sea anemones with their endosymbionts.

Genetic assignment of recruits reveals short- and long-distance larval dispersal in Pocillopora damicornis on the Great Barrier Reef.

Understanding connectivity of coral populations among and within reefs over ecologically significant timescales is essential for developing evidence-based management strategies, including the design of marineprotected areas. Here, we present the first assessment of contemporary connectivity among populations of two Molecular Operational Taxonomic Units (MOTUs) of the brooding coral Pocillopora damicornis. We used individual-based genetic assignment methods to identify the proportions of philopatric and migrant larval recruits, settling over 12 months at sites around Lizard Island (northern Great Barrier Reef [GBR]) and over 24 months at sites around the Palms Islands (central GBR). Overall, we found spatially and temporally variable rates of self-recruitment and dispersal, demonstrating the importance of variation in local physical characteristics in driving dispersal processes. Recruitment patterns and inferred dispersal distances differed between the two P. damicornis MOTUs, with type α recruits exhibiting predominantly philopatric recruitment, while the majority of type ß recruits were either migrants from identified putative source populations or assumed migrants based on genetic exclusion from all known populations. While P. damicornis invests much energy into brooding clonal larvae, we found that only 15% and 7% of type α and type ß recruits, respectively, were clones of sampled adult colonies or other recruits, challenging the hypothesis that reproduction is predominantly asexual in this species on the GBR. We explain high rates of self-recruitment and low rates of clonality in these MOTUs by suggesting that locally retained larvae originate predominantly from spawned gametes, while brooded larvae are mainly vagabonds.

Revisiting the connectivity puzzle of the common coral Pocillopora damicornis.

Understanding levels of connectivity among scleractinian coral populations over a range of temporal and spatial scales is vital for managing tropical coral reef ecosystems. Here, we use multilocus microsatellite genotypes to assess the spatial genetic structure of two molecular operational taxonomic units (MOTUs, types α and ß) of the widespread coral Pocillopora damicornis on the Great Barrier Reef (GBR) and infer the extent of connectivity on spatial scales spanning from local habitat types to latitudinal sectors of the GBR. We found high genetic similarities over large spatial scales spanning > 1000 km from the northern to the southern GBR, but also strong genetic differentiation at local scales in both MOTUs. The presence of a considerable number of first-generation migrants within the populations sampled (12% and 27% for types α and ß, respectively) suggests that genetic differentiation over small spatial scales is probably a consequence of stochastic recruitment from different genetic pools into recently opened up spaces on the reef, for example, following major disturbance events. We explain high genetic similarity among populations over hundreds of kilometres by long competency periods of brooded zooxanthellate larvae and multiple larval release events each year, combined with strong longshore currents typical along the GBR. The lack of genetic evidence for predominantly clonal reproduction in adult populations of P. damicornis, which broods predominantly asexually produced larvae, further undermines the paradigm that brooded larvae settle close to parent colonies shortly after the release.

Enzyme activity demonstrates multiple pathways of innate immunity in Indo-Pacific anthozoans.

Coral reefs are threatened by increasing levels of coral disease and the functional loss of obligate algal symbionts (bleaching). Levels of immunity relate directly to susceptibility to these threats; however, our understanding of fundamental aspects of coral immunology is lacking. We show that three melanin-synthesis pathway components (mono-phenoloxidase, ortho-diphenoloxidase (tyrosinase-type pathway) and para-diphenoloxidase (laccase-type pathway)) are present in both their active (phenoloxidase, PO) and inactive (prophenoloxidase, PPO) forms across a diverse range of 22 species of healthy Indo-Pacific anthozoans. We also demonstrate transglutaminase activity of the coagulation cascade for, to our knowledge, the first time in a coral. Melanin-synthesis enzyme activities varied among taxa, although they were generally lowest in the coral family Acroporidae and highest in the Poritidae and Oculinidae. Inactive tyrosinase-type activity (PPO) and active laccase-type activity (PO) correlate with taxonomic patterns in disease resistance, whereas the converse pattern in activity levels correlates with bleaching resistance. Overall, we demonstrate the presence of several melanin-synthesis pathways in Indo-Pacific corals, co-regulation among some pathway components, and highlight their potential roles in coral health.

High potential for formation and persistence of chimeras following aggregated larval settlement in the broadcast spawning coral, Acropora millepora.

In sessile modular marine invertebrates, chimeras can originate from fusions of closely settling larvae or of colonies that come into contact through growth or movement. While it has been shown that juveniles of brooding corals fuse under experimental conditions, chimera formation in broadcast spawning corals, the most abundant group of reef corals, has not been examined. This study explores the capacity of the broadcast spawning coral Acropora millepora to form chimeras under experimental conditions and to persist as chimeras in the field. Under experimental conditions, 1.5-fold more larvae settled in aggregations than solitarily, and analyses of nine microsatellite loci revealed that 50 per cent of juveniles tested harboured different genotypes within the same colony. Significantly, some chimeric colonies persisted for 23 months post-settlement, when the study ended. Genotypes within persisting chimeric colonies all showed a high level of relatedness, whereas rejecting colonies displayed variable levels of relatedness. The nearly threefold greater sizes of chimeras compared with solitary juveniles, from settlement through to at least three months, suggest that chimerism is likely to be an important strategy for maximizing survival of vulnerable early life-history stages of corals, although longer-term studies are required to more fully explore the potential benefits of chimerism.

A comparative study of phenoloxidase activity in diseased and bleached colonies of the coral Acropora millepora.

In scleractinian (hard) corals, immune responses involving phenoloxidase (PO) activity are known to play a role in coral wound healing, but there have been no studies investigating their roles in mitigating either disease or bleaching in an Indo-Pacific coral. PO activity induces the release of reactive oxygen species leading to a cytotoxic cellular environment, which enhances resistance against pathogens, but is also likely to compound oxidative stress induced during bleaching. Antioxidants such as melanin, whose synthesis is activated by PO activity, and peroxidase are potentially important for mitigating the effects of oxidative stress. Therefore, PO activity was investigated in healthy and diseased colonies of Acropora millepora. PO activity levels were compared among tissues bordering white syndrome lesions (WS) and at two locations (mid and outer) at increasing distances from lesions. Equivalent locations were sampled for PO activity on visibly healthy colonies. Additionally, PO and peroxidase activity were compared between severely bleached and healthy colonies of A. millepora. Overall, PO activity of diseased colonies was significantly lower than that of healthy colonies, but with relatively higher activity at the WS lesion border. Severely bleached colonies had significantly lower PO activity than healthy colonies, and peroxidase was also lower, but not significantly. Lower PO activity in unhealthy colonies supports earlier suggestions that lower immune activity leads to increased susceptibility to disease and bleaching. Additionally, low enzyme activity levels may indicate a depletion of colony resources. Increased PO activity at lesion borders in diseased colonies confirms the relative up-regulation of a key coral immune defense in response to WS in A. millepora.

Newly characterized distinct phases of the coral disease 'atramentous necrosis' on the Great Barrier Reef.

Previously undetected earlier phases of the coral disease 'atramentous necrosis' are documented and described. New observations relating to the occurrence of initial stages and progression of the disease are reported, and potential cause(s) are examined. In direct contrast to earlier published findings, temperature data indicated that occurrence of early bleached stages of atramentous necrosis is not correlated with warmer water temperatures; however, the relationship between temperature and disease prevalence is still unclear.

Unexpected patterns of genetic structuring among locations but not colour morphs in Acropora nasuta (Cnidaria; Scleractinia).

Symbiotic relationships have contributed greatly to the evolution and maintenance of biological diversity. On the Great Barrier Reef, species of obligate coral-dwelling fishes (genus Gobiodon) coexist by selectively recruiting to colonies of Acropora nasuta that differ in branch-tip colour. In this study, we investigate genetic variability among sympatric populations of two colour morphs of A. nasuta ('blue-tip' and 'brown-tip') living in symbiosis with two fish species, Gobiodon histrio and G. quinquestrigatus, respectively, to determine whether gobies are selecting between intraspecific colour polymorphisms or cryptic coral species. We also examine genetic differentiation among coral populations containing both these colour morphs that are separated by metres between local sites, tens of kilometres across the continental shelf and hundreds of kilometres along the Great Barrier Reef. We use three nuclear DNA loci, two of which we present here for the first time for Acropora. No significant genetic differentiation was detected between sympatric colour morphs at these three loci. Hence, symbiotic gobies are selecting among colour morphs of A. nasuta, rather than cryptic species. Significant genetic geographical structuring was observed among populations, independent of colour, at regional (i.e. latitudinal separation by < 500 km) and cross-shelf (< 50 km) scales, alongside relative homogeneity between local populations on within reef scales (< 5 km). This contrasts with the reported absence of large-scale genetic structuring in A. valida, which is a member of the same species group as A. nasuta. Apparent differences in biogeographical structuring between species within the A. nasuta group emphasize the need for comparative sampling across both spatial (i.e. within reefs, between reefs and between regions) and taxonomic scales (i.e. within and between closely related species).

The highly cross-fertile coral species, Acropora hyacinthus and Acropora cytherea, constitute statistically distinguishable lineages.

A major challenge for understanding the evolutionary genetics of mass-spawning corals is to explain the maintenance of discrete morphospecies in view of high rates of interspecific fertilization in vitro and nonmonophyletic patterns in molecular phylogenies. In this study, we focused on Acropora cytherea and A. hyacinthus, which have one of the highest potentials for interspecific fertilization. Using sequences of a nuclear intron, we performed phylogenetic and nested clade analyses (NCA). Both species were polyphyletic in molecular phylogenies, but the NCA indicated that they constitute statistically distinguishable lineages. Phylogenetic analysis using an intergenic region of the mitochondrial DNA (mtDNA), was inconclusive because of low levels of variability in this marker. The position of these two species differed between the nuclear DNA (nDNA) and mtDNA phylogenies and was also at odds with a cladistic analysis based on morphology. We conclude that despite the potential for high levels of hybridization and introgression, A. cytherea and A. hyacinthus constitute statistically distinguishable lineages and their taxonomic status is consistent with the cohesion species concept.

Sympatric populations of the highly cross-fertile coral species Acropora hyacinthus and Acropora cytherea are genetically distinct.

High cross-fertilization rates in vitro and non-monophyletic patterns in molecular phylogenies challenge the taxonomic status of species in the coral genus Acropora. We present data from eight polymorphic allozyme loci that indicate small, but significant, differentiation between sympatric populations of Acropora cytherea and Acropora hyacinthus (F(ST) = 0.025-0.068, p < 0.05), a pair of acroporid corals with very high interspecific fertilization rates in vitro. Although no fixed allelic differences were found between these species, the absence of genetic differentiation between widely allopatric populations suggests that allele frequency differences between A. cytherea and A. hyacinthus in sympatry are biologically significant. By contrast, populations of Acropora tenuis, a species which spawns 2-3 hours earlier and shows low cross-fertilization rates with congeners in vitro, were clearly distinct from A. cytherea and A. hyacinthus (F(ST) = 0.427-0.465, p < 0.05). Moreover, allopatric populations of A. tenuis differed significantly, possibly as a consequence of its relatively short period of larval competency. Our results effectively rule out the possibility that A. hyacinthus and A. cytherea are morphotypes within a single species, and indicate that hybridization occurs relatively infrequently between these taxa in nature.

Examination of species boundaries in the Acropora cervicornis group (Scleractinia, cnidaria) using nuclear DNA sequence analyses.

Although Acropora is the most species-rich genus of the scleractinian (stony) corals, only three species occur in the Caribbean: A. cervicornis, A. palmata and A. prolifera. Based on overall coral morphology, abundance and distribution patterns, it has been suggested that A. prolifera may be a hybrid between A. cervicornis and A. palmata. The species boundaries among these three morphospecies were examined using DNA sequence analyses of the nuclear Pax-C 46/47 intron and the ribosomal DNA Internal Transcribed Spacer (ITS1 and ITS2) and 5.8S regions. Moderate levels of sequence variability were observed in the ITS and 5.8S sequences (up to 5.2% overall sequence difference), but variability within species was as large as between species and all three species carried similar sequences. Since this is unlikely to represent a shared ancestral polymorphism, the data suggest that introgressive hybridization occurs among the three species. For the Pax-C intron, A. cervicornis and A. palmata had very distinct allele frequencies and A. cervicornis carried a unique allele at a frequency of 0.769 (although sequence differences between alleles were small). All A. prolifera colonies examined were heterozygous for the Pax-C intron, whereas heterozygosity was only 0.286 and 0.333 for A. cervicornis and A. palmata, respectively. These data support the hypothesis that A. prolifera is the product of hybridization between two species that have a different allelic composition for the Pax-C intron, i.e. A. cervicornis and A. palmata. We therefore suggest that A. prolifera is a hybrid between A. cervicornis and A. palmata, which backcrosses with the parental species at low frequency.

Atypically low rate of cytochrome b evolution in the scleractinian coral genus Acropora.

Unexpectedly low levels of mitochondrial DNA (mtDNA) cytochrome b sequence divergence are found between species of the scleractinian coral genus Acropora. Comparison of 964 positions of the cytochrome b gene of two out of the three Caribbean Acropora species with seven of their Pacific congeners shows only 0.3-0.8% sequence difference. Species in these biogeographic regions have been evolving independently for at least three million years (since the rise of the Isthmus of Panama) and this geological date is used to estimate nucleotide divergence rates. The results indicate that the Acropora cytochrome b gene is evolving at least 10-20 times slower than the 'standard' vertebrate mtDNA clock and is one of the most slowly evolving animal mitochondrial genes described to date. The possibility is discussed that, unlike higher animals, cnidarians may have a functional mtDNA mismatch repair system.

Mass spawning in tropical reef corals.

Synchronous multispecific spawning by a total of 32 coral species occurred a few nights after late spring full moons in 1981 and 1982 at three locations on the Great Barrier Reef, Australia. The data invalidate the generalization that most corals have internally fertilized, brooded planula larvae. In every species observed, gametes were released; external fertilization and development then followed. The developmental rates of externally fertilized eggs and longevities of planulae indicate that planulae may be dispersed between reefs.