Much recent marine microbial research has focused on sponges, but very little is known about how the sponge microbiome fits in the greater coral reef microbial metacommunity. Here, we present an extensive survey of the prokaryote communities of a wide range of biotopes from Indo-Pacific coral reef environments. We find a large variation in operational taxonomic unit (OTU) richness, with algae, chitons, stony corals and sea cucumbers housing the most diverse prokaryote communities. These biotopes share a higher percentage and number of OTUs with sediment and are particularly enriched in members of the phylum Planctomycetes. Despite having lower OTU richness, sponges share the greatest percentage (>90%) of OTUs with >100 sequences with the environment (sediment and/or seawater) although there is considerable variation among sponge species. Our results, furthermore, highlight that prokaryote microorganisms are shared among multiple coral reef biotopes, and that, although compositionally distinct, the sponge prokaryote community does not appear to be as sponge-specific as previously thought.
In recent years, high-throughput sequencing methods have generated an unprecedented amount of information on the structural and functional diversity of microbial communities1. Marine host-associated prokaryote communities, particularly those associated with sponges, have been reported to be highly diverse2. Despite the constant influx of seawater, sponges are able to sustain dense and diverse symbiotic communities, which can comprise up to 35% of sponge biomass3,4. These associations, furthermore, appear to be consistent over different geographical areas and under different environmental conditions5,6,7,8,9,10.
Much like the human gut, sponges are considered to be an important model to study host–prokaryote associations4. Although much recent research has characterised the phylogenetic diversity and biogeography of sponge-associated microorganisms, relatively little is known about a range of other hosts in coral reef ecosystems. If, and to what extent, sponge-associated microorganisms occur in these other hosts is still largely unknown. This is an important hiatus in our understanding of coral reef microbial ecology given that the prokaryote communities of sponges are part of a wider prokaryote 'metacommunity' of host-associated and free-living (in sediment and seawater) microorganisms11. This metacommunity forms the regional pool of prokaryote species from which local (within a single host) host-associated communities of microorganisms are assembled. These local communities are presumably linked by dispersal, mainly between host organisms and the external environment, thus maintaining the intricate structure of the metacommunity12. Occasionally, direct contact between different host taxa may also induce dispersal and shape the microbial community. Pratte et al.13, for example, showed that direct contact between turf algae and the coral species Porites sp. had a strong influence on the coral (but not the algal) bacterial community.
In the present study, we assess and compare prokaryote communities from a range of host taxa and the abiotic environment (sediment and seawater) in Indo-Pacific coral reef habitats. Our samples include high and lower diversity hosts. High diversity hosts include samples of algae, chitons, stony corals and the sea cucumber gut and mantle. Samples from these hosts are compositionally similar, and have relatively high abundances of operational taxonomic units (OTUs) assigned to the phylum Planctomycetes and relatively high OTU richness and evenness. Prokaryote communities of this group also share significantly more OTUs100 (OTUs with >100 sequences) with sediment (i.e. OTUs found in sediment but not seawater) than other biotopes. The lower diversity host group includes sponges, sponge denizens and the nudibranch gut and mantle biotopes. Compared to the first group, samples of this group have a relatively low OTU richness and evenness (with the exception of high microbial abundance [HMA] sponges) and a relatively low percentage of sediment OTUs100. The mean percentage of total environmental OTUs100 (OTUs recorded in sediment and/or seawater), however, is highest in sponges. The main compositional differences observed in the present study appear to be driven by the apparent permeability of certain taxa (namely algae, sea cucumbers and stony corals) to sediment prokaryotes and the concomitant high prokaryote richness found in these taxa. In turn, sponges, nudibranchs, flatworms and sponge denizens have much fewer sediment prokaryotes OTUs100 and a concomitantly lower prokaryote richness, despite having a sometimes very high contribution of environmental OTUs100 to total OTUs100 richness.
In this study, we applied high-throughput 16S rRNA gene sequencing analysis to simultaneously assess the diversity of 216 prokaryote communities (Supplementary Data 1) from the following 14 biotopes: algae, chitons, stony corals, sea cucumber gut, sea cucumber mantle, sponge denizens (organisms that live on or within sponges), flatworms, nudibranch gut, nudibranch mantle, soft corals, sponges, sea urchins, seawater and sediment (Fig. 1). All host-associated biotopes consisted of multiple species, with the exception of chitons (only included the species Liolophura japonica), soft corals (only included the species Cladiella sp.) and sea urchins (only included the species Diadema savignyi). Samples were collected from coral reef sites in Taiwan and Thailand (Supplementary Data 1).
We recorded 30,725 OTUs assigned to 68 phyla over 2,160,000 sequences (after rarefying to 10,000 sequences per sample). The number of OTUs recorded per sample varied from only 103 for a gut sample of the nudibranch Phyllidia picta to 3704 for a sediment sample (Supplementary Data 1). The richest host-associated sample (2997 OTUs) was from the gut of the sea cucumber Holothuria hilla. The richest (in terms of OTUs) and most abundant (in terms of sequences) prokaryote phyla sampled in the present study included Proteobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Chloroflexi and Actinobacteria. Abundant phyla with relatively few OTUs, but numerous sequence reads, included Tenericutes, Cyanobacteria, Spirochaetae, Thaumarchaeota and Nitrospirae (Supplementary Fig. 1 and Supplementary Table 1).
The relative abundance of 18 of the most abundant phyla (with the exception of Proteobacteria) and the four most abundant proteobacterial classes (with the exception of Gammaproteobacteria), varied significantly among biotopes (Fig. 2; pairwise comparisons between pairs of biotopes are presented in Supplementary Data 2). Some biotopes were strongly enriched by specific prokaryote phyla. The abundance of Planctomycetes, for example, was significantly higher in sediment, and the sea cucumber gut and mantle than the nudibranch gut and mantle and sponge biotopes (Fig. 2i and Supplementary Data 2). The relative abundance of Chloroflexi, in turn, was highest in the sponge, sponge denizen and nudibranch mantle biotopes and significantly higher than in the algae and nudibranch gut biotopes. There was, however, pronounced variation in Chloroflexi abundance within these biotopes as shown by the large standard deviations in Fig. 2d. For example, the sponge species Aaptos lobata, Hyrtios erectus and Xestospongia testudinaria, which have been previously identified as HMA sponges or have been shown to house prokaryote communities very similar to those found in HMA sponges7,14,15,16,17,18, had higher relative abundances of Chloroflexi, and other taxa including SBR1093 (Fig. 2p) and Poribacteria (Fig. 2r), than all other sponge species (Supplementary Data 1). At the class level, alphaproteobacterial abundance was highest in the nudibranch mantle and significantly higher than in the sea cucumber gut, soft coral, sponge and sea urchin biotopes (Fig. 2t and Supplementary Data 2). Deltaproteobacterial abundance was highest in the stony coral, sea cucumber gut and mantle, sediment and sea urchin biotopes and significantly higher than in the algal, sponge denizen, nudibranch gut and mantle, flatworm, soft coral, sponge and seawater biotopes (Fig. 2u). Betaproteobacterial abundance was highest in the sponge and sponge denizen biotopes and significantly more so than in the algae, sea cucumber gut and nudibranch gut and mantle biotopes (Fig. 2v and Supplementary Data 2).
OTU sample richness was highest in the sediment, chiton, algae, stony coral and sea cucumber gut and mantle biotopes and lowest in the flatworm, sponge, nudibranch gut and mantle, soft coral, sea urchin and seawater biotopes (Fig. 2x and Supplementary Data 1). This same pattern also applied to cumulative OTU richness (Supplementary Fig. 2). Histograms of OTU richness also showed largely non-overlapping distributions with samples of sponges and the nudibranch mantle clustered at low OTU richness values while samples of algae, the sea cucumber gut and sediment were spread out over a larger range at higher OTU richness values (Supplementary Fig. 3). This distinction also held after removing all OTUs <100 sequences (Supplementary Fig. 4). Singletons are sometimes removed due to possible problems with sequencing errors associated with Illumina and other next-generation sequencing platforms19. Removing all OTUs <100 sequences shows the robustness of the pattern and, thus, the apparent prevalence of high diversity and low diversity hosts in coral reef habitat.
Evenness was also high in biotopes with the highest richness and was lowest in the flatworm and nudibranch gut biotopes. Evenness was particularly low in prokaryote communities of the soft coral Cladiella sp. (Fig. 2w). For example, 95.5 ± 2.9% (mean ± standard deviation; n = 4) of the prokaryote community of Cladiella sp. consisted of just three OTUs (OTUs 4, 14 and 17).
There was a highly significant compositional difference among biotopes (Adonis test: F13, 201 = 6.64, R2 = 0.293, P < 0.001; Fig. 3a). The factor biotope, thus, explained almost 30% of the variation in OTU composition. The main axis of variation (axis 1) separated samples of algae, chitons, sediment, stony corals and the sea cucumber gut and mantle from samples of sponges, sponge denizens, seawater and the nudibranch gut and mantle. Samples from the flatworm, soft coral and sea urchin biotopes were intermediate. The second axis of variation (axis 2 in Fig. 3a) separated a cluster of sponge and seawater samples at high axis 2 values from a cluster of sponge, nudibranch gut and mantle and sponge denizen samples at low axis 2 values. OTUs that significantly discriminated between pairs of biotopes are presented in Fig. 4 and Supplementary Data 3.
The most abundant OTUs observed in the present study were OTUs 1, 2, 9 and 25, all with >30,000 sequence reads. With the exception of OTU-25, the most abundant OTUs were not the most widespread (in terms of their occurrence in samples), but rather were very abundant in selected hosts (Fig. 4). OTU-2, assigned to Mycoplasma sp., and with only 92% sequence similarity to an OTU obtained from the oyster Crassostrea gigas from Australia (Gb-Acc: JF827444; Supplementary Data 4), was mainly found in the nudibranch species Halgerda willeyi (although it was a rare constituent of the sea cucumber gut and mantle and stony coral biotopes). OTU-9, assigned to the Rhodospirillales order, and with 96% sequence similarity to an OTU obtained from seawater in the Northeast subarctic Pacific Ocean (Gb-Acc: HQ672247), was most abundant in the nudibranch species Hypselodoris maritima and Mexichromis multituberculata. OTU-1, assigned to the Rhizobiales order, and with 99% sequence similarity to an OTU obtained from the sponge Tethya californiana (Gb-Acc: EU290221), was abundant in various Phyllidia species. OTU-25, assigned to the genus Synechococcus, and with 100% sequence similarity to an OTU obtained from seawater in the Mediterranean Sea (Gb-Acc: MH076976), was the most widespread OTU and was found in 209 (of 216; 96.8% of all samples) samples and was most abundant in seawater samples (Fig. 4).
As can be seen in Fig. 4 and Supplementary Figs. 5, 6 and 7, most of the abundant OTUs, including significantly discriminating OTUs, were recorded in multiple biotopes, albeit oftentimes a rare component of these biotopes. Notable exceptions to this pattern were OTUs assigned to the phylum Tenericutes (e.g. OTU-2), which were highly abundant in selected biotopes and often absent in other biotopes. OTUs found across a range of biotopes included OTUs assigned to phyla that have been deemed to be indicator phyla of HMA sponges, such as Chloroflexi, Acidobacteria and Poribacteria18,20. Despite, for example, the relatively high abundance of Chloroflexi in HMA sponges (Fig. 3b and Supplementary Data 1), the most abundant Chloroflexi OTUs were also present in most biotopes, albeit at lower relative abundances (Supplementary Fig. 5). This same pattern held for other abundant phyla, e.g. Acidobacteria and Actinobacteria, but also for less abundant phyla, including Poribacteria, of which OTUs were found in relatively low numbers in a large number of biotopes (Supplementary Fig. 7). In the present study, OTUs assigned to phyla including Chloroflexi, Acidobacteria, Actinobacteria and Poribacteria were present in most biotopes, although they were particularly abundant in HMA sponges, sponge denizens and nudibranchs (Supplementary Data 1).
A large amount of variation in the adonis analysis (~70%) remained unexplained. This is, in part, due to the pronounced overlap among samples from different biotopes or a separation between different groups or species within the same biotope. Within algae, for example, specimens of Halimeda sp. were compositionally distinct from other algal species and had lower OTU richness and evenness (Supplementary Data 1). Sponges, in turn, included samples of the species Acanthella cavernosa, Echinodictyum asperum, Ptilocaulis spiculifer and Stylissa carteri that clustered with seawater samples (high axis 1 and low axis 2 values; Fig. 4). Species of these genera have been previously identified as low microbial abundance (LMA) sponges14. Other sponge samples clustered together with a subset of samples from the sponge denizens and nudibranch gut and mantle biotopes (high axis 1 and high axis 2 values). These were all from the HMA sponges A. lobata, H. erectus and X. testudinaria. Other samples of sponges appeared to house prokaryote communities intermediate in composition between these two previous clusters (high axis 1 and intermediate axis 2 values). These included the agelasids Agelas nemoechinata and Acanthostylotella cornuta. Finally, a number of sponge samples were compositionally similar to samples from other host taxa with intermediate axis 1 and 2 values (Fig. 3 and Supplementary Data 1). These included samples of Haliclona cymaeformis, Suberites diversicolor and Hymeniacidon sp. (Supplementary Data 1).
In general, there was a positive linear relationship between richness and evenness, among biotopes but also within biotopes (Fig. 5a). This figure also highlights that, although there was a continuous variation in prokaryote OTU richness among samples, there appear to be high and low diversity host species, in addition to host species of intermediate diversity. Species hosting some of the richest prokaryote communities included the sea cucumber H. hilla (2260 ± 383 OTUs; mantle; n = 7), the chiton L. japonica (2001 ± 439 OTUs; n = 3) and the alga Padina sp. (2099 ± 267 OTUs; n = 3). In contrast, some of the least diverse prokaryote communities were found in the soft coral Cladiella sp. (170 ± 58 OTUs; n = 4) and the gut (218 ± 182 OTUs; n = 3) and mantle (311 ± 114 OTUs; n = 4) of the nudibranch P. picta. Species of intermediate diversity included the sponge E. asperum (801 ± 311 OTUs; n = 3) and the sea urchin D. savignyi (764 ± 113 OTUs; n = 5). The large standard deviations in richness values within species, particularly in high diversity hosts, highlights that there was also substantial variation within host species. Certain species also deviated from the general trend of increasing richness and evenness. This was most apparent with species in the 'HMA' cluster, A. lobata, H. erectus and X. testudinaria, in addition to certain nudibranch and sponge denizen samples, that were characterised by relatively low richness, but high evenness (Fig. 5a; encircled in red; Supplementary Data 1).
In order to study the distribution of OTUs among biotopes, we created a subset of the total dataset only including OTUs with >100 sequences (hereafter called OTUs100; Supplementary Data 5). This subset included 1731 OTUs100 and 1,922,781 sequences (89% of all sequences). In this subset, only a very small percentage (1.2%; 21 OTUs100) of OTUs100 were restricted to a single biotope and less than 3.9% (69 OTUs100) were restricted to one or two biotopes (Fig. 5b and Supplementary Data 5). Of the 21 OTUs100 restricted to a single biotope, all except three (restricted to the sea cucumber mantle) were only found in sponges. Thirty-four of the 48 OTUs100 restricted to two biotopes were also found in sponges and another biotope. An additional 11 were found in the sea cucumber gut and/or mantle biotopes (Supplementary Data 5).
The 21 OTUs100 restricted to a single biotope, give us a new look into the rare members of the coral reef prokaryote metacommunity. The total abundance of those OTUs100 varied from 102 (0.005% of OTUs100 sequences) to 905 (0.11%) sequences (Supplementary Data 5). The most abundant of these (OTUs-579) was restricted to sponges and assigned to the Latescibacteria phylum with only 81% sequence similarity to an organism previously obtained from a deep-sea octocoral (Gb-Acc: DQ395794). The most abundant OTUs100 restricted to two biotopes included OTUs 71, 550 and 762. OTUs 550 and 762 were restricted to the sponge and sediment biotopes while OTU-71 was restricted to the sea cucumber and nudibranch gut biotopes. OTU-71, assigned to the gammaproteobacterial order HTA4, had 92% sequence similarity to an organism obtained from black deposit in a lava tube from a cave in the Canary Islands (Gb-Acc: LT702969). OTU-550, assigned to the Caldilineaceae (Chloroflexi), had 95% sequence similarity with an organism obtained from the sponge Agelas dilatata (Gb-Acc: EF076192). OTU-762, assigned to the Gemmatimonadetes, had 98% sequence similarity with an organism obtained from the sponge Amphimedon compressa (Gb-Acc: GU984210).
In Fig. 5c, it can be seen that there is both a wide variation in the number of OTUs100 found in a single biotope, and a rapid increase in the number of total OTUs100 sampled as biotopes are added. To explore this further, we assessed the number of OTUs100 shared among biotopes (Fig. 5d). Figure 5d shows the numbers of OTUs100 shared among five biotopes, namely, sediment, the sea cucumber gut, algae, sponge and nudibranch mantle biotopes. All but 4 OTUs100 (99.8% of all OTUs100; Fig. 5d) were found in these five biotopes. These five biotopes shared 867 OTUs100, while 2 OTUs100 were only found in sediment, 0 in algae, 9 in the nudibranch mantle, 32 in sponges and 46 in the sea cucumber gut. Note that these OTUs100 may be present in other biotopes. Sponges shared 59 OTUs100 with sediment, which were not shared with the other biotopes compared to 9 OTUs100 shared between sediment and the sea cucumber gut. Note that just three biotopes, namely, the sea cucumber gut, sponges and nudibranch mantle encompassed all but 6 OTUs100 (99.7% of all OTUs100).
In order to study the influence of seawater, sediment and the broader surrounding environment (sediment and seawater) on prokaryote composition in our host biotopes, we assessed the number and percentage of OTUs100 in each host that were also found in (1) sediment but not seawater (hereafter known as sediment OTUs100), (2) seawater but not sediment (hereafter known as seawater OTUs100) and (3) sediment and/or seawater (hereafter known as environmental OTUs100). Note that category 3 (sediment and/or seawater) also includes all OTUs100 of categories 1 and 2.
Significantly more sediment OTUs100 were recorded in algae, chitons, stony corals and the sea cucumber gut and mantle than all other biotopes with the exception of the sponge denizen and sea urchin biotopes (Fig. 6a and Supplementary Data 2). This also held as a percentage of total OTUs100 (Fig. 6d). The number of seawater OTUs100 was highest in the algae and chiton biotopes and significantly more so than in the nudibranch gut and mantle, sponge and soft coral biotopes (Fig. 6b and Supplementary Data 2). Algae, chitons, stony corals and the sea cucumber gut and mantle also housed significantly more environmental OTUs100 than the nudibranch gut and mantle, flatworm, soft coral and sponge biotopes (Fig. 6c). However, the percentage of environmental OTUs100 was significantly higher in sponges than all other biotopes, except chitons (Fig. 6f). Sponges housed a mean of 93.8 ± 3.5% (representing 91.1 ± 18.3% of OTUs100 sequences; Fig. 6l; n = 63) environmental OTUs100 compared, for example, to just 71.0 ± 5.2% (50.8 ± 37.2% of OTUs100 sequences; n = 4) for soft corals, 74.2 ± 3.5% (62.9 ± 12.7% of OTUs100 sequences; n = 5) for sea urchins and 79.8 ± 3.9% (24.6 ± 18.9% of OTUs100 sequences; n = 7) for flatworms. Four of the most abundant OTUs in flatworms (OTUs 33, 40, 126 and 1761) and two in soft corals (OTUs 14 and 17) were only found in host-associated biotopes and were not found in seawater or sediment, explaining the low percentages of environmental sequences in both biotopes (Fig. 4).
Although, on average, almost 94% of the OTUs100 recorded in sponges were found in the surrounding environment (whether sediment or seawater), there was pronounced variation among sponge species. More than 97% of the OTUs100 of E. asperum, and S. carteri were present in the surrounding environment compared to just 79.7 ± 5.2% of H. cymaeformis (n = 4), 86.0 ± 0.3% of Paratetilla sp. (n = 2) and 86.6 ± 2.6% of Hymeniacidon sp. (n = 4) OTUs100. For the HMA sponges, 95.8 ± 1.9% of A. lobata (n = 2), 96.3 ± 1.1% of H. erectus (n = 9) and 91.5 ± 1.1% of X. testudinaria (n = 9) OTUs100 were found in the surrounding environment. The very high prevalence of 'environmental' OTUs in certain sponge species would appear to support the prevalence of horizontal transmission in sponge-prokaryote dynamics. However, sponges may also seed the abiotic environment with their prokaryote symbionts.
The present study revealed pronounced differences in composition and diversity among host-associated biotopes. The great majority of OTUs100, however, were recorded in multiple biotopes and a large percentage of OTUs were shared with environmental samples (sediment and/or seawater) with the highest percentage found in sponges. Despite the prevalence of environmental OTUs in sponges, there was pronounced compositional variation between sponges and other host taxa and among sponge species. Certain species, for example, housed prokaryote communities similar to seawater (LMA sponges) while others (HMA sponges) housed communities similar to those found in certain samples of nudibranchs and sponge denizens.
A number of studies have previously remarked on the greater compositional similarity of the prokaryote communities of HMA as opposed to LMA sponges and the greater prevalence of transient (seawater) bacteria in the latter21,22,23,24. LMA sponges have also been shown to be dominated by different sets of highly abundant OTUs and sometimes even a single dominant OTU8,15,16,25,26,27,28. Compare this to the prokaryote communities of the HMA sponge species X. testudinaria where the core community of 44 specimens sampled across the vast expanse of the Indo-Pacific region consisted of 71 OTUs representing 57.5% of sequences on average29.
The greater evenness of HMA sponge species observed in the present study and other studies may help to explain the greater similarity and limited prevalence of transient bacteria in these sponges23. Importantly, species evenness has been shown to be positively related to invasion resistance, presumably by limiting the invaders access to available resources30. The question remains, however, as to why HMA sponges house more even (and compositionally similar) prokaryote communities than LMA sponges. Previous studies have shown that certain sponge species are able to transmit microorganisms through their larvae (vertical transmission) and suggested that this plays an important role in structuring the prokaryote community3,31,32. Other studies have focused on horizontal transmission, e.g. from water column to sponge33 and the ability of sponges to selectively recruit specific microbial symbionts from seawater3,12,34. The actual degree to which the sponge prokaryote community is shaped by both forms of transmission, however, remains largely unknown.
The compositional similarity between certain sponge samples and samples of nudibranchs and sponge denizens suggests that sponges may influence the prokaryote composition of organisms that live on or within them or that feed on them. The sponge denizen biotope included sea cucumbers and barnacles that lived within or on the sponge, presumably for much of their life35,36. Nudibranchs, however, are more mobile and may represent vectors carrying microorganisms from one sponge to the other. Nudibranchs also come into intimate contact with their sponge prey during feeding whereby certain species evert and extend their pharyngeal bulb deep into the sponge37. During this process, they are also able to sequester toxins from the sponge for their own defence38. Our results indicated that the gut and/or mantle prokaryote communities of specimens from certain nudibranch species (Doriprismatica atromarginata, Phyllidiella pustulosa, Phyllidiella nigra, Phyllidia ocellata and Phyllidia elegans) closely resembled that of sponge prokaryote communities. All of these nudibranch species have been recorded feeding on sponges37,39,40,41. A number of these specimens were also collected from sponges while diving. Specimens of the sea cucumber Synaptula sp., a sponge denizen sampled from X. testudinaria, housed a prokaryote community similar to that of the 'HMA' sponge cluster, which included X. testudinaria. Members of the genus Synaptula are often common in coral reef habitat, particularly in association with sponges and can sometimes be so abundant that they cover the sponge's surface. They have also been shown to be able to exploit sponge exudates35. Interestingly, the barnacle Acasta sp., which was collected within X. testudinaria, was the only sponge denizen barnacle that also housed a prokaryote community similar to that of members of the 'HMA' sponge cluster. The other sponge barnacles were collected within samples of the sponge species Dasychalina fragilis, Agelas cavernosa and Cinachyrella sp.
The similarity between the prokaryote communities of sponges and the guts of certain nudibranch samples, may be an indication that the nudibranch gut communities are dominated by transient microorganisms derived from their preferred food source, namely sponges37,38. An individual's diet can have a profound effect on gut prokaryote composition42,43. This difference can extend to species, whereby there are marked differences in gut microbiome composition among mammal species with different diets44,45. This distinction appears to apply to nudibranchs, whereby the gut and mantle prokaryote communities of species known to feed on sponges closely resembled that of certain sponge species (Fig. 3). It would be interesting to test how different diets (e.g. different sponge species) affect the nudibranch prokaryote community.
The very high number of OTUs shared among different biotopes would appear to lend support to the 'everything is everywhere but the environment selects' hypothesis of Baas Becking46. In line with this, the very high richness and evenness of sediment suggest that it may function as a microbial seed bank. There was also considerable compositional similarity between sediment samples and high diversity host samples of algae, stony corals and sea cucumbers among others. In contrast, seawater samples were only compositionally similar to samples of certain sponge species. Previously, Cleary and Polónia47 also showed that populations of mussels inhabiting Indonesian marine lakes and mangroves shared much more OTUs with sediment than with seawater and were compositionally more similar to sediment than to seawater. Gibbons et al.48 previously suggested that the marine biosphere maintains a persistent microbial seed bank. In their scenario, all microbes are found everywhere due to the immensity and persistence of this seed bank, and apparent local or host-associated endemism is merely a result of insufficient sequencing. Community structure is, thus, a function of relative abundance rather than the presence or absence of certain microbial taxa. The presence of such a seed bank has repercussions for ecological theory, given the limited importance of long-distance dispersal and the ability of low abundance populations to rapidly expand when the appropriate environment is encountered48. In the global marine environment, hydrographic parameters of seawater masses, furthermore, greatly contribute to the dispersion of sediment microbial communities at regional and global scales, although microbial cell dispersion is highly dependent on the ability to tolerate stress49,50.
Although wide in scope, the present study only represents a small fraction of marine species in the coral reef environment and even in this dataset, there was considerable variation among species within biotopes. Much more research is needed to understand the variation in microbial composition of taxa such as sea cucumbers, flatworms, algae and nudibranchs. A large amount of time and resources have been spent studying the prokaryote communities of a limited number of taxa leaving large gaps in our knowledge of the coral reef metacommunity. Sponges have been deemed major contributors to total microbial diversity in the world's oceans, and are considered to be reservoirs of exceptional microbial diversity2 without, however, having actually studied other host taxa in detail. In coral reefs, sponges do not appear to stand alone as the main contributors to total prokaryote diversity as this study highlights; other biotopes host more diverse prokaryote communities, e.g. sea cucumbers. The present study shows that sponges are only one, albeit an interesting, component of a much larger coral reef metacommunity.
All host-associated, sediment and seawater samples were collected from various sites in Taiwan and Thailand (Supplementary Data 1). All locations were coral reef habitat. A detailed description of the Taiwanese sampling sites can be found in Coelho et al.51 and Huang et al.52 and meta data for all samples including the sampling location and time of sampling can be found in Supplementary Data 1. Fragments of host individuals were collected using SCUBA diving, or snorkelling, including the surface and interior or the whole organism (depending on the size) in order to sample as much as possible of the whole prokaryote community. Sediment was collected from the upper 5 cm surface layer using a plastic disposable syringe from which the end had been cut in order to facilitate sampling. Seawater was collected between the depths of 1–2 m with a 1.5 L bottle and subsequently 1 L (±50 ml) of water was filtered through a Millipore® White Isopore Membrane Filter (0.22 µm pore size) to obtain seawater prokaryote communities. All samples were subsequently preserved in 96% EtOH. All samples were kept cool (<4 °C) immediately after collection and during transport. In the laboratory, samples were stored at −20 °C until DNA extraction.
A total of 216 samples belonging to algae, chitons, stony corals, sea cucumbers, sponge denizens (organisms that live on or within sponges), nudibranchs, flatworms, soft corals, sponges, sea urchins, water and sediment were collected. In the present study, all samples were assigned to 14 biotopes, which included the guts and mantles of sea cucumbers and nudibranchs as separate biotopes. Certain biotopes were well represented, e.g. sponges (63 samples from 18 species) and nudibranchs (48 samples from 13 species) while others only consisted of a just few samples and/or a single species., e.g. soft corals (4 samples from the species Cladiella sp.), chitons (3 samples from the species L. japonica) and sea urchins (5 samples from the species D. savignyi). All the samples used in the present study can be found in Supplementary Data 1 including the sampling site and taxonomic identification.
PCR-ready genomic DNA was isolated from all samples using the FastDNA® SPIN soil Kit (MPbiomedicals) following the manufacturer's instructions. Briefly, the whole membrane filter (for seawater samples) and ±500 mg of sediment and host specimens (including parts of the surface and/or interior) were cut into small pieces (in the case of the membrane filter and host specimens) and transferred to Lysing Matrix E tubes containing a mixture of ceramic and silica particles. A blank control, in which no tissue was added to the Lysing Matrix E tubes, was also included. The microbial cell lysis was performed in the FastPrep® Instrument (Q Biogene) for 80 s at 6.0 ms−1. The extracted DNA was eluted into DNase/Pyrogen-Free Water to a final volume of 50 μl and stored at −20 °C until use. The 16S rRNA gene V3V4 variable region PCR primers 341F 5′-CCTACGGGNGGCWGCAG-3′ and 785R 5′-GACTACHVGGGTATCTAATCC-3′ 53 with barcode on the forward primer were used in a 30 cycle PCR assay using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands; the blank control did not yield any bands. Multiple samples were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. Pooled and purified PCR product was used to prepare the DNA library following the Illumina TruSeq DNA library preparation protocol. Next-generation, paired-end sequencing was performed at MrDNA (Molecular Research LP; http://www.mrdnalab.com/; last checked 18 November 2016) on an Illumina MiSeq device (Illumina Inc., San Diego, CA, USA) following the manufacturer's guidelines. Sequences from each end were joined following Q25 quality trimming of the ends followed by reorienting any 3′–5′ reads back into 5′–3′ and removal of short reads (<150 bp). The resultant files were analysed using the QIIME (Quantitative Insights Into Microbial Ecology)54 software package (http://www.qiime.org/) and USEARCH10 19.
For a detailed description of the sequence analysis, see Coelho et al.51 and Cleary et al.55. Briefly, in QIIME, fasta and qual files were used as input for the split_libraries.py script in QIIME. Default arguments were used except for the minimum sequence length, which was set at 250 base pairs (bps) after removal of forward primers and barcodes. Using USEARCH10 (https://www.drive5.com/usearch/; last checked 2019 02 11), reads were filtered with the -fastq_filter command and the following arguments: -fastq_trunclen 250 -fastq_maxee 0.5 -fastq_truncqual 15. Sequences were then dereplicated and sorted using the -derep_fulllength and -sortbysize commands. OTU clustering (97% sequence similarity threshold) was performed using the -cluster_otus command of USEARCH10 yielding 12025383 sequences assigned to 48880 OTUs. Potential contaminants were removed from the OTU table if they occurred at least two times in the blank control. This conservative measure was chosen because of observations of bleeding between samples from Illumina sequencing and the appearance of abundant reads in blank controls with very low counts56,57. Based on this procedure, 958995 sequences and 77 OTUs were removed from the non-rarefied OTU table. OTUs not classified as Bacteria or Archaea or classified as chloroplasts and mitochondria were also removed. Taxonomy was assigned to reference sequences of OTUs using default arguments in the assign_taxonomy.py script in QIIME using the SILVA_128_QIIME_release database and the uclust classifier method58. The make_otu_table.py script in QIIME was used to generate a square matrix of OTUs × SAMPLES and subsequently rarefied to 10,000 sequences per sample with the single_rarefaction.py script in QIIME yielding 2,160,000 sequences and 30,725 OTUs. This rarefied table was used as input for further analyses using the R language for statistical computing and has been included as a source data file (https://www.r-project.org/; last checked 2018–07–17).
A data matrix containing OTU counts per sample was imported into R using the read.csv() function. This table was used to compare community composition, estimate richness and assess the relative abundance of selected higher taxa and is included as a Source Data file. The OTU abundance matrix was loge (x + 1) transformed (in order to normalise the distribution of the data) and a distance matrix constructed using the Bray–Curtis index with the vegdist() function in the vegan package59. The Bray–Curtis index is one of the most frequently applied (dis)similarity indices used in ecology60,61,62,63. Variation in prokaryote composition among biotopes was assessed with Principal Coordinates Analysis (PCO) using the cmdscale() function in R with the Bray–Curtis distance matrix as input. Variation among biotopes was tested for significance using the adonis() function in vegan. In the adonis analysis, the Bray–Curtis distance matrix of species composition was the response variable with biotope as independent variable. The number of permutations was set at 999; all other arguments used the default values set in the function. Weighted average scores were computed for OTUs on the first four PCO axes using the wascores() function in the vegan package. The simper() function in vegan was used to identify significantly discriminating OTUs between pairs of biotopes based on the loge (x + 1) transformed OTU table and 999 permutations. The discriminating OTUs contribute the most to differences between pairs of biotopes.
We tested for significant differences in the relative abundance of 18 of the most abundant phyla, the four most abundant proteobacterial classes, and the count and relative abundance of sediment and environmental OTUs among biotopes with an analysis of deviance using the glm() function in R. For the most abundant phyla, proteobacterial classes, and the relative abundance of sediment and environmental OTUs, we first applied a generalized linear model (GLM) with the family argument set to binomial. The ratio, however, of residual deviance to residual d.f. in the models substantially exceeded 1 so we set family to 'quasibinomial'. In the 'quasibinomial' family, the dispersion parameter is not fixed at one so that it can model over-dispersion. For the counts of sediment and environmental OTUs, we set the family argument to 'quasipoisson'. For the least abundant phyla and the two least abundant proteobacterial classes, which included zero counts in the samples, we set the family argument to 'tweedie'64 with var.power = 1.5 and link.power = 0 (a compound Poisson–gamma distribution). Using the glm models, we tested for significant variation among biotopes using the anova() function in R with the F test, which is most appropriate when dispersion is estimated by moments as is the case with quasibinomial fits. We subsequently used the emmeans() function in the emmeans library65 to perform multiple comparisons of mean abundance among biotopes using the false discovery rate (fdr) method in the adjust argument. Additional graphs were produced using the ggplot66 and limma67 packages. Detailed descriptions of the functions used here can be found in R (e.g.?cmdscale) and online in reference manuals (http://cran.r-project.org/web/packages/vegan/index.html).
The DNA sequences generated in this study can be downloaded from NCBI BioProject IDs: PRJNA382576, PRJNA397173, PRJNA397177 and PRJNA397178. The source data underlying Figs. 2–6 and Supplementary Figs. 1–7 are provided as a Source Data file.
Journal peer review information: Nature Communications thanks Christian Voolstra and Cristina Diez-Vives for their contribution to the peer review of this work. Peer reviewer reports are available.
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van Alphen, J., de Voogd, N. J. & Hoeksema, B. W. Differential feeding strategies in phyllidiid nudibranchs on coral reefs at Halmahera, northern Moluccas. Coral Reefs 30, 59 (2011).
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Fusetani, N. Two sesquiterpene isocyanides and a sesquiterpene thiocyanate from the marine sponge Acanthella cf. cavernosa and the nudibranch Phyllidia ocellata. Tetrahedron Lett. 33, 6823–6826 (1992).
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This work was supported by European Funds through COMPETE [FCOMP-01-0124-FEDER-008657] and by National Funds through the Portuguese Foundation for Science and Technology (FCT) within the LESS CORAL [PTDC/AAC-AMB/115304/2009] and Ecotech-Sponge (PTDC/BIAMIC/6473/2014 – POCI-01-0145-FEDER-016531) projects. This work is also part of the research programmes NWO-VIDI with project number 16.161.301 and ASPASIA (015.010.030), which are both (partly) financed by the Netherlands Organisation for Scientific Research (NWO). Thanks are due for financial support to CESAM (UID/AMB/50017 - POCI-01-0145-FEDER-007638), to FCT/MCTES through national funds (PIDDAC), and co-funding by FEDER, within the PT2020 Partnership Agreement and Compete 2020. Francisco J.R.C. Coelho (postdoctoral scholarship: SFRH/BPD/92366/2013), Ana R.M. Polónia (postdoctoral scholarship: SFRH/BPD/117563/2016) and Marina R.S. Ferreira (Ph.D. scholarship: SFRH/BD/114809/2016), were supported by scholarships funded by FCT, Portugal within the Human Capital Operational Programme (HCOP), subsidised by the European Social Fund (ESF) and national funds (MCTES). This research was facilitated by the generous support of the Ministry of Science and Technology (MOST) and the Marine National Parks Headquarter (MNPH), Taiwan to Y.M.H. (MOST 105-2621-B-346-002 and MNPH 104403). All specimens collected in Taiwan were under a permit (No. 20160316364) issued by MNPH. The authors would also like to acknowledge Ana Cecilia Pires, Helder Gomes, Bastian Reijnen and Niels van der Windt for their support in the laboratory and Devrim Gunsel Zahir, Chad Scott, Gregory Hanigan, Floris Cleary, Julian Cleary, Yi-Chin Wu, Yuan-Hao Lin, Ming-Hong Chang, Miao-Yin Syu and You-Hua Lin for their help in the field.
These authors contributed equally: Daniel F.R. Cleary, Thomas Swierts.
Fieldwork: D.F.R.C., N.J.d.V., Y.M.H. and S.P. Labwork: T.S., E.v.d.E., M.R.S.F., A.R.M.P., D.F.R.C. and N.C.M.G. Data analysis: D.F.R.C., F.J.R.C.C. and L.C. Writing: D.F.R.C., N.J.d.V., Y.M.H., S.P., T.S., E.v.d.E., M.R.S.F., A.R.M.P., N.C.M.G., F.J.R.C.C., L.C. and J.-P.U.
Correspondence to Daniel F. R. Cleary.


text:
Home > Get Support > Grants Search > The Roger Edwards Educational Trust
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redpajama_set_name: RedPajamaCommonCrawl


LIVERPOOL look set to finally win the Premier League this season but previous title races prove it is not quite over yet.
That is the opinion of former Arsenal and England goalkeeper David Seaman.
Liverpool are currently four points clear of second-placed Manchester City at the top of the table.
City suffered a shock defeat away at Newcastle on Tuesday and Liverpool have their game in hand at home to Leicester this evening [Wednesday].
A victory would extend the Reds' lead to seven points with 14 games remaining.
Seaman admits Liverpool are overwhelming favourites to win their first ever Premier League title.
However, he has warned Jurgen Klopp's side larger gaps have been pulled back in the past.
Seaman's Arsenal side trailed Manchester United by 11 points back in the 1997/98 campaign.
But the Gunners fought back and finished one point clear of United at the top.
"Yeah, it certainly looks like that [it's Liverpool's time]," Seaman said on talkSPORT.
"With City having four defeats now, you can sometimes just get by by having five defeats.
"They struggled last night but before that you wouldn't believe they'd get beat at Newcastle.
"Liverpool have got a great chance tonight.
"But it's tricky, I remember us chasing Man United down when they were 11 points clear and winning it.
Virgil van Dijk is a doubt for the visit of Leicester due to illness.
Trent Alexander-Arnold has not recovered in time from his knee injury.


text:
Ashley Phillips may refer to:

 Ashley Phillips (footballer, born 1986), American women's soccer goalkeeper
 Ashley Phillips (footballer, born 2005), English football centre-back

redpajama_set_name: RedPajamaWikipedia


text:
package com.restfb;

import static org.junit.Assert.assertTrue;

import java.math.BigDecimal;
import java.math.BigInteger;
import java.util.ArrayList;
import java.util.HashMap;
import java.util.List;
import java.util.Map;

import org.junit.Assert;

import org.junit.Test;

/**
 * Unit tests that exercise {@link JsonMapper} implementations, specifically the "convert Java to JSON" functionality.
 *
 * @author Mark Allen
 */
public class JsonMapperToJsonTest extends AbstractJsonMapperTests {
  /**
   * Can we handle null?
   */
  @Test
  public void nullObject() {
    String json = createJsonMapper().toJson(null);
    Assert.assertTrue("null".equals(json));
  }

  /**
   * Can we handle the empty list?
   */
  @Test
  public void emptyList() {
    String json = createJsonMapper().toJson(new ArrayList<Object>());
    Assert.assertTrue("[]".equals(json));
  }

  /**
   * Can we handle the empty object?
   */
  @Test
  public void emptyObject() {
    String json = createJsonMapper().toJson(new Object());
    Assert.assertTrue("{}".equals(json));
  }

  /**
   * Can we handle primitives?
   */
  @Test
  public void primitives() {
    // Close your eyes and pretend that string is a primitive here
    Assert.assertTrue("Testing".equals(createJsonMapper().toJson("Testing")));
    Assert.assertTrue("true".equals(createJsonMapper().toJson(true)));
    Assert.assertTrue("1".equals(createJsonMapper().toJson(1)));
    Assert.assertTrue("1".equals(createJsonMapper().toJson(1L)));
    Assert.assertTrue("1.0".equals(createJsonMapper().toJson(1F)));
    Assert.assertTrue("1.0".equals(createJsonMapper().toJson(1D)));
    Assert.assertTrue("1".equals(createJsonMapper().toJson(new BigInteger("1"))));
    Assert.assertTrue("1.0".equals(createJsonMapper().toJson(new BigDecimal("1"))));
  }

  /**
   * Can we handle a basic Javabean?
   */
  @Test
  public void basicJavabean() {
    BasicUser basicUser = new BasicUser();
    basicUser.uid = 12345L;
    basicUser.name = "Fred";
    String json = createJsonMapper().toJson(basicUser);
    Assert.assertTrue("{\"uid\":12345,\"name\":\"Fred\"}".equals(json));
  }

  /**
   * Can we handle a more complex Javabean?
   */
  @Test
  public void complexJavabean() {
    UserWithPhotos userWithPhotos = new UserWithPhotos();
    userWithPhotos.uid = 12345L;
    userWithPhotos.name = null;
    userWithPhotos.photos = new ArrayList<Photo>();
    userWithPhotos.photos.add(new Photo());
    Photo photo = new Photo();
    photo.photoId = 5678L;
    photo.location = "Las Vegas";
    userWithPhotos.photos.add(photo);

    String json = createJsonMapper().toJson(userWithPhotos);

    Assert
      .assertTrue("{\"uid\":12345,\"photos\":[{\"id\":null,\"location\":null},{\"id\":5678,\"location\":\"Las Vegas\"}],\"name\":null}"
        .equals(json));
  }

  /**
   * Can we handle a full stream.publish example?
   *

   * See http://wiki.developers.facebook.com/index.php/Attachment_(Streams).
   */
  @Test
  public void streamPublish() {
    ActionLink category = new ActionLink();
    category.href = "http://bit.ly/KYbaN";
    category.text = "humor";

    Properties properties = new Properties();
    properties.category = category;
    properties.ratings = "5 stars";

    Medium medium = new Medium();
    medium.href = "http://bit.ly/187gO1";
    medium.src =
        "http://icanhascheezburger.files.wordpress.com/2009/03/funny-pictures-your-cat-is-bursting-with-joy1.jpg";
    medium.type = "image";

    List<Medium> media = new ArrayList<Medium>();
    media.add(medium);

    Attachment attachment = new Attachment();
    attachment.name = "i'm bursting with joy";
    attachment.href = "http://bit.ly/187gO1";
    attachment.caption = "{*actor*} rated the lolcat 5 stars";
    attachment.description = "a funny looking cat";
    attachment.properties = properties;
    attachment.media = media;

    String json = createJsonMapper().toJson(attachment);
    Assert
      .assertTrue("{\"description\":\"a funny looking cat\",\"name\":\"i'm bursting with joy\",\"caption\":\"{*actor*} rated the lolcat 5 stars\",\"properties\":{\"category\":{\"text\":\"humor\",\"href\":\"http://bit.ly/KYbaN\"},\"ratings\":\"5 stars\"},\"media\":[{\"src\":\"http://icanhascheezburger.files.wordpress.com/2009/03/funny-pictures-your-cat-is-bursting-with-joy1.jpg\",\"type\":\"image\",\"href\":\"http://bit.ly/187gO1\"}],\"href\":\"http://bit.ly/187gO1\"}"
        .equals(json));
  }

  /**
   * Can we handle an empty Map?
   */
  @Test
  public void emptyMap() {
    Assert.assertTrue("{}".equals(createJsonMapper().toJson(new HashMap<String, Object>())));
  }

  /**
   * Can we handle a Map?
   */
  @Test
  public void map() {
    UserWithPhotos basicUser = new UserWithPhotos();
    basicUser.uid = 12345L;
    basicUser.name = "Fred";

    Map<String, Object> map = new HashMap<String, Object>();
    map.put("testId", new BigInteger("412"));
    map.put("floatId", Float.valueOf(123.45F));
    map.put("basicUser", basicUser);

    String json = createJsonMapper().toJson(map);
    Assert
      .assertTrue("{\"floatId\":123.45,\"testId\":412,\"basicUser\":{\"uid\":12345,\"photos\":null,\"name\":\"Fred\"}}"
        .equals(json));
  }

  @Test
  public void duplicateAnnotations() {
    DuplicateAnnotationsUser user = new DuplicateAnnotationsUser();
    user.hometown = "Philadelphia, PA";
    user.hometownObject = new HashMap<String, Object>() {
      {
        put("id", "123");
        put("name", "Philly");
      }
    };

    String json = createJsonMapper().toJson(user);

    // The current behavior is that it's luck of the draw which field is
    // selected to marshal to JSON if there are multiple fields with the same
    // mapping
    assertTrue(json.contains("Philly") || json.contains("Philadelphia"));
  }

  static class BasicUser {
    @Facebook
    Long uid;

    @Facebook
    String name;

    Byte ignored;
  }

  static class Photo {
    @Facebook("id")
    Long photoId;

    @Facebook
    String location;
  }

  static class DuplicateAnnotationsUser {
    @Facebook
    String hometown;

    @Facebook("hometown")
    Map<String, Object> hometownObject;
  }

  static class UserWithPhotos extends BasicUser {
    @Facebook
    List<Photo> photos;
  }

  static class ActionLink {
    @Facebook
    String text;

    @Facebook
    String href;
  }

  static class Medium {
    @Facebook
    String type;

    @Facebook
    String src;

    @Facebook
    String href;
  }

  static class Properties {
    @Facebook
    ActionLink category;

    @Facebook
    String ratings;
  }

  static class Attachment {
    @Facebook
    String name;

    @Facebook
    String href;

    @Facebook
    String caption;

    @Facebook
    String description;

    @Facebook
    Properties properties;

    @Facebook
    List<Medium> media;
  }
}

redpajama_set_name: RedPajamaGithub


However, if you have valid reasons which demand you to travel at a short notice, such as seeking medical treatment in Singapore, you may provide us with a formal letter from the hospital/doctor in Singapore together with your visa application.... Get the most up-to-date information for Singapore related to Singapore travel visas, Singapore visa requirements and applications, embassy and consulate addresses, foreign relations information, travel advisories, entry and exit restrictions, and travel tips from the US State Department's website.
Knowing full well that my in-laws had a valid multiple entry visa to Singapore and Indonesia would issue a visa upon arrival, I made sure that we carried all associated paperwork (including air tickets back to China that they used when applying for their SG visas) with us to avoid any unnecessary delays or concerns over the travel plans and my in-laws' intentions around their stay in Singapore.... Holders of a Visit Pass are not permitted to engage in any form of employment, business or occupation without a valid work pass issued under the Employment of Foreign Manpower Act (Cap. 91A). Kindly note that the extension application's approval, rejection and duration is considered on its own merits and is at the discretion of the Immigration and Checkpoints Authority.
Holders of a Visit Pass are not permitted to engage in any form of employment, business or occupation without a valid work pass issued under the Employment of Foreign Manpower Act (Cap. 91A). Kindly note that the extension application's approval, rejection and duration is considered on its own merits and is at the discretion of the Immigration and Checkpoints Authority.
The ICA website does have full instructions for extension of a social visit pass in a variety of circumstances. If she is arriving on a standard social visit pass which has a standard 90day maximum then she might get longer than 30days on request.
However, if you have valid reasons which demand you to travel at a short notice, such as seeking medical treatment in Singapore, you may provide us with a formal letter from the hospital/doctor in Singapore together with your visa application.


text:
<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN" "http://www.w3.org/TR/html4/loose.dtd">

<title>MultiThreadUTFoundation.ThreadLocalData</title>
<meta name="date" content="2015-12-01">
<link rel="stylesheet" type="text/css" href="../../../../stylesheet.css" title="Style">

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picklesjar.pickle.ut.runtime

Class MultiThreadUTFoundation.ThreadLocalData

java.lang.Object

picklesjar.pickle.ut.runtime.MultiThreadUTFoundation.ThreadLocalData

Enclosing class:

MultiThreadUTFoundation

private class MultiThreadUTFoundation.ThreadLocalData
extends java.lang.Object

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java.lang.String
lockKey

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result

java.lang.String
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private
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clone, equals, finalize, getClass, hashCode, notify, notifyAll, toString, wait, wait, wait

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public java.lang.String lockKey

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public java.lang.String scenarioName

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private ThreadLocalData()

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text:
import EpubEngine from './../../src/index';

describe('jsFile-epub', () => {
    describe('Library imports', () => {
        it('should import JS module', () => {
            assert.isFunction(EpubEngine);
        });

        it('should exist in global scope', () => {
            assert.isFunction(window.JsFileEpub.default);
        });
    });
});


text:
Learn more about getting involved with Reasons to Believe through events, communities, and online education.
Honolulu Christian Church
February 26, 2023 at 10:30 am - 12:00 pm (Pacific/Honolulu)
Join Hugh Ross at Honolulu Christian Church on Sunday, February 26, where he will speak on the topic Faith and Science.
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Join Hugh Ross for The Christianity and Science Conference taking place February 24–25, where he will speak on the topic Connecting the Cosmos with...
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Join us for a two-day conference exploring why Christianity still resonates with humanity. RESONATE brings together music, philosophy, science, and art to display the...
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Our Monthly Partner community, affectionately known as Advocates for Truth, is a group of mission-minded people dedicated to spreading God's truth through sound reason and scientific research. These faithful monthly givers are the financial foundation of RTB's ministry, resulting in lives transformed and faith in Christ strengthened.
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RTB Chapters exist to connect the ministry of RTB with communities around the world, uplifting and equipping fellow Christians for productive dialogue that invites skeptics of the Christian faith to follow Jesus Christ.
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Kufri is a famous hill station just 16 kms from Shimla in Himachal Pradesh well known for its ski slopes. Kufri Tourism gives paranomic view of the deep valleys, dense forests and lofty mountains. National Snow Statue Competition is held at Kufri every year in January.
Kufri along with Shimla and Chail forms the Golden Triangle of Himachal Pradesh. Tourists are fascinated by the breathtaking views of Kufri, Shimla and Chail along with the luxurious resorts like Toshali Royal View available for the tour to Shimla, Kufri and Chail.
Standing at an altitude of 2510 meters above sea level at Southern Himachal, Kufri was discovered in 1918 by the British people.
A small hill station of Himachal Pradesh, Kufri is 19-kms from Shimla connecting by national highway no. 22. Popular for its hiking and skiing trails in Himachal Pradesh, Kufri is at its natural best during winters. Skiing and tobogganing can be enjoyed along snow-capped slopes.
Skiing buffs and adventure seekers look forward to the annual winter sports festival held in February at Kufri to participate in it.
The Himalayan Nature Park is one of the major tourist Places in Kufri. This Park offers numerous endangered birds and animals such as goral, serow, musk deer, leopard, black bears and so on.
A walk through Mahasu Peak, crowned by a small temple, can be an ideal option for fun lovers as they have to cross a forest of spruce and deodar on the way. The Mahasu Ridge avails good slopes for skiing.
Kufri Tourism - Get all information on Kufri tourist places and sightseeing tours. Our website helps you plan your next tour and gives you great ideas on what to do, where to eat, where to stay and when to go there. We give the best information from the web to give you precise, meaningful and useful travel guide for leading places to visit in India and from across the world. Our travel information on KUFRI tourist places, tourist attractions, major sightseeing, the best time to visit, the top places to visit in Kufri, the must-see things to see and do, maps, restaurants, hotels, nearby tourist destinations, local public information and more. To start with check out information on how to reach Kufri.


text:
Q: HTML table styling not setting the outside colors I have this table in HTML and I'm trying to make it look prettier but no matter where I insert the "bgcolor='black'" it won't fill out the sides and make it an "enclosed" table so to speak. Here's what I'm working with:
It turns out like this:

body {
  padding: 8px;
}

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redpajama_set_name: RedPajamaStackExchange


MAKING THE WORLD A GREENER PLACE.
For the 2018 Rooted in Goodness Tour, we joined forces with Mrs. Meyer's Clean Day to spread brand awareness and create partnerships with key retailers. We focused on four main core experiences during the tour, including retail stops, guerrilla sampling, product drops and special events. At each of our 19 stops on the road, consumers pledged to help "make the world a greener place" by placing a symbolic thumbprint on our Pledge Wall Mural. In addition, brand ambassadors handed out samples on colorful bikes and did random acts of cleanliness, such as picking up trash in community gardens, speaking to Mrs. Meyer's neighborly actions. In every market, we dropped a tote chock full of products to 1-2 lucky businesses or individuals who could use a little Mrs. Meyer's lovin'. Lastly, we built tailored events unique to each retailer in key markets to help Mrs. Meyer's show their appreciation for some of their greatest retail partnerships.
The Results: Exceeded hand wash goals by 279% & bottle refills by 293% at retail locations. 10k+ pledges received. 820K+ mobile impressions.


text:
Turkish author Günday receives French award
November 06 2015 17:01:00
Hakan Günday received one of the most prestigious literary awards, Le Prix medicis Etranger, in Paris.
One of the world's most prestigious literary awards, France's Le Prix Medicis Etranger, was presented to Turkish author Hakan Günday for his novel "Daha," which is about immigrants. The award is presented each year to the best foreign novel in France.
Günday, 39, attended the award ceremony held in Paris. His book, which was translated into French with the title "Encore," is based on the life of a nine-year-old boy named Gaza whose father is a human trafficker.
Günday, who entered the Turkish literary world at age 24 with his novel "Kinyas and Kayra," has so far published eight novels.
Günday's style is known as underground literature.
Turkish Nobel laureate novelist Orhan Pamuk also received the same award for his novel "Kar" (Snow) in 2005.
Numerous world-renowned authors such as Umberto Eco and Paul Auster have been named for this prestigious award.
One Turkish consulate employee dead in shooting in Erbil: Foreign Ministry
Surf's up for Turkey's "calm city"
Italian author Camilleri, creator of 'Inspector Montalbano', dies
Painter's life to be featured in film
Salt Lake welcomes flamingo chicks


The Faithfull 3 Piece Hexagon Key Set is manufactured from fully hardened chrome vanadium steel and supplied in a fold out non-slip, soft grip coated case.
The Key Set contains 3 individual professional folding key sets covering the most commonly used Metric, A/F and Star keys.
Each set is clearly marked with the key sizes for easy identification.


text:
The Politics of World Heritage Negotiating Tourism and Conservation Edited by: David Harrison, Michael Hitchcock
Current Themes In Tourism
Channel View Publications
- Addresses the meaning of World Heritage to the citizens of different nations
- Of interest to NGOs, funding bodies, government and the educators and managers of heritage
- Foreword from the Director of the World Heritage Centre, UNESCO
This collection of papers discuss World Trade Law and focus on the contested nature of World Heritage at sites as diverse as The Netherlands, Ellis Island (USA), post-colonial Mesoamerica, Cambodia, Fiji, Kyrgyzstan, and Vietnam. In addition, eight research notes explore heritage interpretation in the USA, Lebanon, Peru, Indonesia, Singapore, Tasmania and India.
This book is a solid addition to the scholarly literature on heritage tourism and, in particular, the geopolitics of the management of UNESCO World Heritage Sites. The editors have done an excellent job in providing both a geographically wide-ranging set of case studies and critical scholarly research. This is a fine volume for practitioners and scholars in heritage tourism management and research. Those with serious interests in World Heritage Sites, the UNESCO selection process, heritage management and heritage tourism ought to consider this volume.
- Charles R. de Burlo, University of Vermont, in Journal of Heritage Tourism 2:1
David Harrison is a London educated Sociologist/Anthropologist who has taught at the University of Sussex, London Metropolitan University, and The University of the South Pacific in Fiji, from which he retired in 2014. The USP link continues, and he is also associated with St. Mary's University College in London and the University of Surrey. He has written extensively on tourism and development and has researched its impacts in Eastern Europe, Southern Africa, the Caribbean, the South Pacific and South-East Asia.
Michael Hitchcock is Director of the International Institute of Culture Tourism and Development, London Metropolitan University.


text:
The Embarrassing Product Recalls Businesses Want You to Forget
By Pauli Poisuo on June 27, 2019 Misc
When a product malfunctions, its buyer complains. When enough products malfunction and customers complain, the manufacturer may be prompted to issue a recall. Product recalls are rarely good news, but there's a world of difference between your average "there were 0.2% more bugs in this sausage than FDA regulations allow" recall and the ones we're talking about today. These are companies panickedly realizing that they have been manufacturing products that can explode, catch fire, emit poison or even kill people, and the unfortunate aftermath of those exact things happening. Let's take a look at some of the worst product recall cases of all time.
10. Dell notebook batteries
Mobile devices are everywhere, which means they need reliable batteries. However, every once in a while the occasional defective one manages to make it through the production line, leading to all those stories about exploding or otherwise malfunctioning batteries you occasionally read about in the news.
Between 2004 and 2006, Dell had a slightly less than lucky run with said defective batteries, when Sony delivered more than a few for their Inspiron, Latitude and Precision laptops and XPS units. Soon, reports of Dell notebooks catching fire or even exploding started surfacing, and the company was facing one of the largest recalls in the history of the electronics industry when it realized that the faulty batch was a whopping 4.1 million batteries that were a fire risk — almost 20% of Dell's computer sales during that time period.
After some high-profile scandals such as a Dell laptop bursting into flames at a conference, Dell and Sony ended up issuing a recall for all 4.1 million batteries.
9. Westland/Hallmark beef
February 2008 was a bad time for Westland/Hallmark Meat Packing Company in Chino, California. Not only was the company drawing accusations for treating its cows badly, the U.S. Department of Agriculture was also on their tail. They had slaughtered cows that had lost the ability to walk after they had already passed pre-processing inspections without having them examined for chronic illness, which was a bad move during a time when bovine spongiform encephalopathy — the Mad Cow Disease — was creating panic all over the Western world.
Westland/Hallmark paid a hefty price for their neglect when the Department of Agriculture forced them to recall the meat that had been potentially contaminated. All 143 million pounds of it. If you have a hard time wrapping your head around that number, it's enough to make two delicious burgers for every single man, woman and child living in the U.S. at the time.
8. Mattel toys
As the maker of iconic toys such as Hot Wheels and Barbie dolls, it's easy to assume that Mattel would pay pretty close attention to keeping its products safe for children. Unfortunately, not all of the company's business partners bother with such trivialities; In 2007, Mattel announced a recall of no less than 967,000 toys after finding out that they were covered in poisonous lead paint.
The toy giant managed to stop roughly two thirds of the batch, which had been made by a Chinese contract manufacturer. However, an estimated 300,000 toys still made it to the shelves of American toy stores. These weren't display action figures or over-12-years-old stuff, either — the 83 types of tainted products featured Nickelodeon and Sesame Street characters that were specifically aimed for toddlers.
The incident was particularly hurtful for Mattel because the Chinese company that had betrayed them was no cost-cutting spring chicken that had just started working for them. This was a manufacturer they had been working with for 15 years, which means they were in full knowledge of all the rules and regulations of safe toy-making … and yet, something went awry.
7. Ace Bayou bean bag chairs
Ace Bayou bean bag chairs were a popular and affordable piece of furniture that was sold widely by many popular retail stores and sites such as Walmart and Amazon. However, as you can probably guess by their presence on this list, their design had one serious safety flaw: The zipper. The Ace Bayou bean bag chairs were equipped with a zipper that was so easy to open that even small children could do it. Unfortunately, this exact thing happened more than once, and because an opened bean bag seems like an inviting miniature ball pit, kids climbed right in … and some of them closed the zipper behind them. We're not going to go into the depressing specifics, so let's just say that an airtight sack full of choking hazards is not the safest environment for a small child.
After two tragic deaths, Ace Bayou promptly issued a voluntary recall for its bean bags, and started to provide free repair kits that disabled the zipper.
6. The Hasbro Easy-Bake Oven
Hasbro's Easy-Bake Oven seems like a fairly simple toy … that is, until you remember that it's quite literally a miniature oven that young children can easily stick their fingers in. For everyone who has ever seen a toddler, let alone witnessed its parents frantically baby-proof the electrical outlets of the house, this seems like a pretty massive safety risk. After all, small children love nothing more than sticking their hands in every possible place, regardless of their danger factor.
With that in mind, please pretend to look surprised when we tell you that when Hasbro started using a genuine heating element (instead of the traditional light bulb) in the ovens in 2006, it took them less than a year to voluntarily issue a recall for a million easy-bake ovens, as toddlers kept sticking their hands inside the easy-bakey part of the toy, resulting in (occasionally serious) burns when their baby sausage fingers connected with the brand new heating element. Later in 2007, Hasbro repeated the process for another million toy ovens.
Between those two recalls, the toy company and the U.S. Consumer Product Safety Commission had been bombarded with a total of 249 complaints about kids getting their hands stuck in the oven, 77 of which reported burns. One 5-year-old received such bad burns that her finger had to be partially amputated.
5. Tylenol
Tylenol is a popular pain reliever that can nevertheless lead to massive complications — up to and including liver failure — when taken in large doses. It has also been the subject of several large-scale recalls over the years.
In 2009, the manufacturer had to recall several Tylenol brands because a wood-treating chemical had somehow made its way into the medicine and was causing diarrhea, vomiting and nausea to people who ingested the medicine. Another disaster struck in 2011, tens of thousands of Tylenol products had to be recalled over issues with quality control, and the problem became so large-scale that they had to close one of their manufacturing plants. In 2012, yet another 600,000 bottles of Tylenol for infants had to be recalled because dosing issues had turned the pill into a crapshoot of either too little or too much medicine. There have also been other, less significant recalls over mislabeling, development issues or a strange, uncharacteristically moldy odor.
However, the most infamous Tylenol product recall was no fault of the manufacturer. In 1984, someone in Chicago started tampering with bottles of Extra-Strength Tylenol, lacing them with poisonous cyanide. The poison killed seven people before Johnson & Johnson (the manufacturer) had time to send warnings to distributors and hospitals, but they were quick to stop both advertisements and production of the drug and swiftly recalled an estimated 31 million bottles.
The Chicago Tylenol Murders remain unsolved to this day.
4. Jensen Farms
Cantaloupe may or may not be your favorite fruit, but even if you hate it from the bottom of your heart, eating it is unlikely to kill you … that is, unless you bought Jensen Farms' sweet Rocky Ford cantaloupes in 2011. A batch of the farm's product was contaminated with listeria, but nevertheless made it to the stores, where the tainted cantaloupes proceeded to kill 33 people and making 147 people violently ill in an outbreak that spanned 28 states. The listeria-laced melons were also linked to at least one miscarriage.
After a hasty recall, the two brothers who owned of the farm were arrested (and later filed for bankruptcy). An investigation found that they had neglected to use a system that sprayed the melons with an antibacterial solution before packaging, despite the fact that they knew perfectly well this could mean that their product was contaminated. Unsurprisingly, federal charges followed, and the Jensens' farming career ended with six counts of adulteration of food and aiding and abetting.
In 2014, the brothers were each sentenced to five years probation and six months home detention, complete with 100 hours of community service and $150,000 of restitution payments. While this might seem like a slap on the wrist for men who were responsible for so many deaths, there were some mitigating factors at play. The case managed to prompt stricter food safety laws and liabities on producers, and many of the victims' family members also agreed that the apologetic Jensens should face no prison time. It's also fairly uncommon to actually charge food producers for food safety issues, and the FDA made it clear the Jensens' case was specifically meant to send a message to other food producers out there.
3. The Ford-Firestone tire recall
When hundreds of people die because of a defective product, you know that someone somewhere has messed up in a serious way. Unfortunately, Ford and tire manufacturer Firestone had a hard time agreeing on which one of them was the true culprit in 2000-2001, when Firestone's 15-inch Wilderness AT, radial ATX and ATX II tire treads started separating from the tires' cores, which led to many nasty and movie-worthy roll-overs and crashes … and, of course, countless human tragedies.
Most of the defective tires were custom equipment for the Ford Explorer, which happened to be the world's best-selling SUV at the time. Firestone immediately (and apparently genuinely) believed they had managed to sort the problem out and acted accordingly, but more and more accidents kept piling up, and Ford wasn't happy — especially when Firestone started claiming that the problem wasn't just the tires, but the design flaws in the Explorer itself. The back-and-forth between the two companies was a bitter one and reached epic levels that involved congressional hearings and the recall of 6.5 million tires. The situation came to a head in 2001, when the exasperated Ford promised to replace all 13 million Firestone Wilderness AT tires out there at their own expense. Firestone wasn't having any of it and severed their nearly 100-year-old business relationship.
The defective tires caused 200+ deaths and over 800 injuries and countless lawsuits. Executives resigned, new laws were forged, and in a development that shocked absolutely no one, the tire company's market value was cut in half.
2. Toyota's faulty floor mats
Cars have tons of parts that can cause a massive disaster if they don't function properly, but you wouldn't necessarily expect the floor mat to be one of them. As Toyota proved to the world in 2009, you'd be wrong to assume so when they announced the recall of almost four million Toyota and Lexus cars, including 2005-2009 editions of the popular hybrid model Prius. The problem was, as you can probably guess, the cars' floor mats, which thanks to a design flaw could become dislodged in multiple ways and get stuck under the accelerator, which could lead to every driver's worst nightmare: unintended, uncontrolled and dangerous acceleration. This ended up happening in the most dramatic way imaginable, when a Highway Patrolman and his family found to their horror that the doormat of their Lexus sent them speeding uncontrollably at over 100 miles per hour. The patrolman was able to call 911 and explain the terrifying situation, and was still on the phone when the fatal crash came.
As accidents started piling up, the FBI started to become interested. The ensuing investigation revealed that Toyota had downplayed the unintended acceleration problem, which also included issues with sticky pedals and plenty of allegations that they kept making cars despite being well aware of their potentially lethal issues. In 2014, the manufacturer agreed to pay a massive $1.2 billion in order to avoid prosecution for its safety issues. The agreement also forced Toyota to "admit" misleading American consumers and making deceptive statements about serious safety issues. By then, the car company's long-standing acceleration troubles had killed 89 people and injured a further 57, and the company had recalled over eight million vehicles.
1. Ford Pinto
Yes, it's Ford again, and if you know anything about bad cars, you might actually have seen this one coming. Ford Pinto was one of the best-selling cars of the 1970s right up until the moment it turned out that the car was basically a fireball waiting to happen. A massive and tragic flaw in the Pinto's design made the vehicle's gas tank vulnerable to rear-end collisions, which could create an explosion and turn the car into a fiery death trap. Unfortunately, many of the owners found this out in the worst possible way. Faulty Ford Pintos caused around 500 deaths and hundreds of injuries before 1978, when Ford agreed to recall 1.5 million of the vehicles, along with 30,000 Mercury Bobcats with similar design issues.
The product scandal left Ford's reputation in tatters, especially when it emerged that the fatal design flaw was the result of a very cynical cost-benefit analysis. Auto industry superstar Lee Iaocca, who had presided over Pinto's creation, was unceremoniously fired, and Ford's lawyers had to put in some serious overtime dealing with the 117 lawsuits by disgruntled customers. The recall also prompted a 1979 landmark case, "Indiana vs. Ford Motor Co.," which marked Ford as the first American corporation that was indicted and prosecuted for criminal homicide.
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