Metagenomic Analysis of Zinc Surface-Associated Marine Biofilms.

Biofilms are a significant source of marine biofouling. Marine biofilm communities are established when microorganisms adhere to immersed surfaces. Despite the microbe-inhibiting effect of zinc surfaces, microbes can still attach to the surface and form biofilms. However, the diversity of biofilm-forming microbes that can attach to zinc surfaces and their common functional features remain elusive. Here, by analyzing 9,000,000 16S rRNA gene amplicon sequences and 270 Gb of metagenomic data, we comprehensively explored the taxa and functions related to biofilm formation in subtidal zones of the Red Sea. A clear difference was observed between the biofilm and adjacent seawater microbial communities in terms of the taxonomic structure at phylum and genus levels, and a huge number of genera were only present in the biofilms. Saturated alpha-diversity curves suggested the existence of more than 14,000 operational taxonomic units in one biofilm sample, which is much higher than previous estimates. Remarkably, the biofilms contained abundant and diverse transposase genes, which were localized along microbial chromosomal segments and co-existed with genes related to metal ion transport and resistance. Genomic analyses of two cyanobacterial strains that were abundant in the biofilms revealed a variety of metal ion transporters and transposases. Our analyses revealed the high diversity of biofilm-forming microbes that can attach to zinc surfaces and the ubiquitous role of transposase genes in microbial adaptation to toxic metal surfaces.

Culture-dependent bacteria in commercial fishes: Qualitative assessment and molecular identification using 16S rRNA gene sequencing.

Fish contamination has been extensively investigated along the Saudi coasts, but studies pertaining to bacterial pathogens are scarce. We conducted qualitative assessment and molecular identification of culture-dependent bacteria in 13 fish species from three coastal sites and a local fish market in Jeddah, Saudi Arabia. Bacterial counts of gills, skin, gut and muscle were examined on agar plates of Macconkey's (Mac), Eosin Methylene Blue (EMB) and Thiosulfate Citrate Bile Salts (TCBS) culture media. Bacterial counts significantly differed between species, sources and feeding habits of examined fishes. Mugil cephalus exhibited higher counts on TCBS (all body parts), Mac (gills, muscle and gut) and EMB (gills and muscle). Fishes from Area I had higher bacterial loads, coinciding with those in seawater and sediment from the same site, indicating direct association between habitat conditions and the levels of bacterial contamination. By feeding habit, detritivorous fish harbored higher counts than herbivorous and carnivorous species. Bacterial counts of skin were higher in fish from market than field sites, and positively correlated with other body parts indicating the relation of surface bacterial load on the overall quality of fish. Rahnella aquatilis (Enterobacteriaceae) and Photobacterium damselae (Vibrionaceae) were among the dominant species from fish muscle based on 16S rRNA sequencing. These species are known human pathogens capable of causing foodborne illness with severe antibiotic resistance. Opportunistic pathogens, e.g. Hafnia sp. (Enterobacteriaceae) and Pseudomonas stutzeri (Pseudomonadaceae) also occurred in fish muscle. The inclusion of bacterial contamination in future monitoring efforts is thus crucial.

Congener-specific levels and patterns of polychlorinated biphenyls in edible fish tissue from the central Red Sea coast of Saudi Arabia.

All 209 congeners of polychlorinated biphenyls (PCBs) in edible fish tissue from the central Red Sea coast (Jeddah region) of Saudi Arabia were analyzed by isotope dilution high-resolution gas chromatography-mass spectrometry. The upper-bound total PCB (ΣPCB) levels in nine commonly consumed fish species from three areas were 0.2-82.5ng/g wet weight (17-8450ng/g lipid weight), which were at the lower end of reported global range and far below international tolerance limits (500-3000ng/g ww). Dioxin-like congeners contributed up to 12.8% (mean 6.5%) to ΣPCB in tissue samples, with the total PCB toxic equivalencies (TEQs) at a tolerable range (0.05-2.6pgTEQ/g ww or 2-238pgTEQ/g lw) for all species. PCB profiles were dominated by moderately chlorinated homologs, mainly hexachlorobiphenyls, but less chlorinated congeners were also consistently elevated, notably in Siganus rivulatus (Area III) and Mugil cephalus (Area I). It remains to be ascertained if the latter were breakdown products or due to fresh inputs. The top congeners based on dominance by both occurrence and abundance were identified as potential markers of ΣPCB in fish tissue, which can be used for future selective biomonitoring in case of reasonable constraints on full congener approach.

Zonation of Microbial Communities by a Hydrothermal Mound in the Atlantis II Deep (the Red Sea).

In deep-sea geothermal rift zones, the dispersal of hydrothermal fluids of moderately-high temperatures typically forms subseafloor mounds. Major mineral components of the crust covering the mound are barite and metal sulfides. As a result of the continental rifting along the Red Sea, metalliferous sediments accumulate on the seafloor of the Atlantis II Deep. In the present study, a barite crust was identified in a sediment core from the Atlantis II Deep, indicating the formation of a hydrothermal mound at the sampling site. Here, we examined how such a dense barite crust could affect the local environment and the distribution of microbial inhabitants. Our results demonstrate distinctive features of mineral components and microbial communities in the sediment layers separated by the barite crust. Within the mound, archaea accounted for 65% of the community. In contrast, the sediments above the barite boundary were overwhelmed by bacteria. The composition of microbial communities under the mound was similar to that in the sediments of the nearby Discovery Deep and marine cold seeps. This work reveals the zonation of microbial communities after the formation of the hydrothermal mound in the subsurface sediments of the rift basin.

Pyrosequencing revealed shifts of prokaryotic communities between healthy and disease-like tissues of the Red Sea sponge Crella cyathophora.

Sponge diseases have been widely reported, yet the causal factors and major pathogenic microbes remain elusive. In this study, two individuals of the sponge Crella cyathophora in total that showed similar disease-like characteristics were collected from two different locations along the Red Sea coast separated by more than 30 kilometers. The disease-like parts of the two individuals were both covered by green surfaces, and the body size was much smaller compared with adjacent healthy regions. Here, using high-throughput pyrosequencing technology, we investigated the prokaryotic communities in healthy and disease-like sponge tissues as well as adjacent seawater. Microbes in healthy tissues belonged mainly to the Proteobacteria, Cyanobacteria and Bacteroidetes, and were much more diverse at the phylum level than reported previously. Interestingly, the disease-like tissues from the two sponge individuals underwent shifts of prokaryotic communities and were both enriched with a novel clade affiliated with the phylum Verrucomicrobia, implying its intimate connection with the disease-like Red Sea sponge C. cyathophora. Enrichment of the phylum Verrucomicrobia was also considered to be correlated with the presence of algae assemblages forming the green surface of the disease-like sponge tissues. This finding represents an interesting case of sponge disease and is valuable for further study.

Species sorting during biofilm assembly by artificial substrates deployed in a cold seep system.

Studies focusing on biofilm assembly in deep-sea environments are rarely conducted. To examine the effects of substrate type on microbial community assembly, biofilms were developed on different substrates for different durations at two locations in the Red Sea: in a brine pool and in nearby bottom water (NBW) adjacent to the Thuwal cold seep II. The composition of the microbial communities in 51 biofilms and water samples were revealed by classification of pyrosequenced 16S rRNA gene amplicons. Together with the microscopic characteristics of the biofilms, the results indicate a stronger selection effect by the substrates on the microbial assembly in the brine pool compared with the NBW. Moreover, the selection effect by substrate type was stronger in the early stages compared with the later stages of the biofilm development. These results are consistent with the hypotheses proposed in the framework of species sorting theory, which states that the power of species sorting during microbial community assembly is dictated by habitat conditions, duration and the structure of the source community. Therefore, the results of this study shed light on the control strategy underlying biofilm-associated marine fouling and provide supporting evidence for ecological theories important for understanding the formation of deep-sea biofilms.

Interspecific and locational differences in metal levels in edible fish tissue from Saudi Arabia.

Metal levels in fish have been extensively studied, but little data currently exists for the Middle East. We examined the levels of metals and metalloids (aluminum, arsenic, copper, manganese, selenium, zinc, and mercury) in the flesh of 13 fish species collected from three fishing sites and a local fish market in Jeddah, Saudi Arabia. We tested the following null hypotheses: (1) there are no interspecific differences in metal levels, (2) there are no differences in metal levels in fishes between market and fishing sites, (3) there are no size-related differences in metal levels, and (4) there are no differences in selenium:mercury molar ratio among different fish species. There were significant interspecific differences in concentrations for all metals. There was an order of magnitude difference in the levels of aluminum, arsenic, mercury, manganese, and selenium, indicating wide variation in potential effects on the fish themselves and on their predators. Fishes from Area II, close to a large commercial port, had the highest levels of arsenic, mercury, and selenium, followed by market fishes. Mercury was positively correlated with body size in 6 of the 13 fish species examined. Mercury was correlated positively with arsenic and selenium, but negatively with aluminum, cobalt, copper, manganese, and zinc. Selenium:mercury molar ratios varied significantly among species, with Carangoides bajad, Cephalopholis argus, Variola louti, and Ephinephelus tauvina having ratios below 10:1. These findings can be used in risk assessments, design of mercury reduction plans, development of fish advisories to protect public health, and future management decision-making.

Fish consumption behavior and rates in native and non-native people in Saudi Arabia.

Fish are a healthy source of protein and nutrients, but contaminants in fish may provide health risks. Determining the risk from contaminants in fish requires site-specific information on consumption patterns. We examine consumption rates for resident and expatriates in the Jeddah region of Saudi Arabia, by species of fish and fishing location. For Saudis, 3.7% of males and 4.3% of females do not eat fish; for expatriates, the percent not eating fish is 6.6% and 6.1% respectively. Most people eat fish at home (over 90%), and many eat fish at restaurants (65% and 48%, respectively for Saudis and expatriates). Fish eaten at home comes from local fish markets, followed by supermarkets. Saudis included fish in their diets at an average of 1.4 ± 1.2 meals/week at home and 0.8 ± 0.7 meals/week at restaurants, while expats ate 2.0 ± 1.7 meals/week at home and 1.1 ± 1.1 meals/week in restaurants. Overall, Saudis ate 2.2 fish meals/week, while expats ate 3.1 meals/week. Grouper (Epinephelus and Cephalopholis) were eaten by 72% and 60% respectively. Plectropomus pessuliferus was the second favorite for both groups and Hipposcarus harid and Lethrinus lentjan were in 3rd and 4th place in terms of consumption. Average meal size was 68 g for Saudis and 128 g for expatriates. These data can be used by health professionals, risk assessors, and environmental regulators to examine potential risk from contaminants in fish, and to compare consumption rates with other sites.

Pyrosequencing reveals the microbial communities in the Red Sea sponge Carteriospongia foliascens and their impressive shifts in abnormal tissues.

Abnormality and disease in sponges have been widely reported, yet how sponge-associated microbes respond correspondingly remains inconclusive. Here, individuals of the sponge Carteriospongia foliascens under abnormal status were collected from the Rabigh Bay along the Red Sea coast. Microbial communities in both healthy and abnormal sponge tissues and adjacent seawater were compared to check the influences of these abnormalities on sponge-associated microbes. In healthy tissues, we revealed low microbial diversity with less than 100 operational taxonomic units (OTUs) per sample. Cyanobacteria, affiliated mainly with the sponge-specific species "Candidatus Synechococcus spongiarum," were the dominant bacteria, followed by Bacteroidetes and Proteobacteria. Intraspecies dynamics of microbial communities in healthy tissues were observed among sponge individuals, and potential anoxygenic phototrophic bacteria were found. In comparison with healthy tissues and the adjacent seawater, abnormal tissues showed dramatic increase in microbial diversity and decrease in the abundance of sponge-specific microbial clusters. The dominated cyanobacterial species Candidatus Synechococcus spongiarum decreased and shifted to unspecific cyanobacterial clades. OTUs that showed high similarity to sequences derived from diseased corals, such as Leptolyngbya sp., were found to be abundant in abnormal tissues. Heterotrophic Planctomycetes were also specifically enriched in abnormal tissues. Overall, we revealed the microbial communities of the cyanobacteria-rich sponge, C. foliascens, and their impressive shifts under abnormality.

Symbiotic adaptation drives genome streamlining of the cyanobacterial sponge symbiont "Candidatus Synechococcus spongiarum".

"Candidatus Synechococcus spongiarum" is a cyanobacterial symbiont widely distributed in sponges, but its functions at the genome level remain unknown. Here, we obtained the draft genome (1.66 Mbp, 90% estimated genome recovery) of "Ca. Synechococcus spongiarum" strain SH4 inhabiting the Red Sea sponge Carteriospongia foliascens. Phylogenomic analysis revealed a high dissimilarity between SH4 and free-living cyanobacterial strains. Essential functions, such as photosynthesis, the citric acid cycle, and DNA replication, were detected in SH4. Eukaryoticlike domains that play important roles in sponge-symbiont interactions were identified exclusively in the symbiont. However, SH4 could not biosynthesize methionine and polyamines and had lost partial genes encoding low-molecular-weight peptides of the photosynthesis complex, antioxidant enzymes, DNA repair enzymes, and proteins involved in resistance to environmental toxins and in biosynthesis of capsular and extracellular polysaccharides. These genetic modifications imply that "Ca. Synechococcus spongiarum" SH4 represents a low-light-adapted cyanobacterial symbiont and has undergone genome streamlining to adapt to the sponge's mild intercellular environment. IMPORTANCE Although the diversity of sponge-associated microbes has been widely studied, genome-level research on sponge symbionts and their symbiotic mechanisms is rare because they are unculturable. "Candidatus Synechococcus spongiarum" is a widely distributed uncultivated cyanobacterial sponge symbiont. The genome of this symbiont will help to characterize its evolutionary relationship and functional dissimilarity to closely related free-living cyanobacterial strains. Knowledge of its adaptive mechanism to the sponge host also depends on the genome-level research. The data presented here provided an alternative strategy to obtain the draft genome of "Ca. Synechococcus spongiarum" strain SH4 and provide insight into its evolutionary and functional features.

Diversity and distribution of eukaryotic microbes in and around a brine pool adjacent to the Thuwal cold seeps in the Red Sea.

A hypoxic/suboxic brine pool at a depth of about 850 m was discovered near the Thuwal cold seeps in the Red Sea. Filled with high concentrations of hydrogen sulfide and ammonia, such a brine pool might limit the spread of eukaryotic organisms. Here, we compared the communities of the eukaryotic microbes in a microbial mat, sediments and water samples distributed in 7 sites within and adjacent to the brine pool. Taxonomic classification of the pyrosequenced 18S rRNA amplicon reads showed that fungi highly similar to the species identified along the Arabic coast were almost ubiquitous in the water and sediment samples, supporting their wide distribution in various environments. The microbial mat displayed the highest species diversity and contained grazers and a considerable percentage of unclassified species. Phylogeny-based methods revealed novel lineages representing a majority of the reads from the interface between the sea water and brine pool. Phylogenetic relationships with more reference sequences suggest that the lineages were affiliated with novel Alveolata and Euglenozoa inhabiting the interface where chemosynthetic prokaryotes are highly proliferative due to the strong chemocline and halocline. The brine sediments harbored abundant species highly similar to invertebrate gregarine parasites identified in different oxygen-depleted sediments. Therefore, the present findings support the uniqueness of some microbial eukaryotic groups in this cold seep brine system.

In situ environment rather than substrate type dictates microbial community structure of biofilms in a cold seep system.

Using microscopic and molecular techniques combined with computational analysis, this study examined the structure and composition of microbial communities in biofilms that formed on different artificial substrates in a brine pool and on a seep vent of a cold seep in the Red Sea to test our hypothesis that initiation of the biofilm formation and spreading mode of microbial structures differs between the cold seep and the other aquatic environments. Biofilms on different substrates at two deployment sites differed morphologically, with the vent biofilms having higher microbial abundance and better structural features than the pool biofilms. Microbes in the pool biofilms were more taxonomically diverse and mainly composed of various sulfate-reducing bacteria whereas the vent biofilms were exclusively dominated by sulfur-oxidizing Thiomicrospira. These results suggest that the redox environments at the deployment sites might have exerted a strong selection on microbes in the biofilms at two sites whereas the types of substrates had limited effects on the biofilm development.

Spatial and species variations in bacterial communities associated with corals from the Red Sea as revealed by pyrosequencing.

Microbial associations with corals are common and are most likely symbiotic, although their diversity and relationships with environmental factors and host species remain unclear. In this study, we adopted a 16S rRNA gene tag-pyrosequencing technique to investigate the bacterial communities associated with three stony Scleractinea and two soft Octocorallia corals from three locations in the Red Sea. Our results revealed highly diverse bacterial communities in the Red Sea corals, with more than 600 ribotypes detected and up to 1,000 species estimated from a single coral species. Altogether, 21 bacterial phyla were recovered from the corals, of which Gammaproteobacteria was the most dominant group, and Chloroflexi, Chlamydiae, and the candidate phylum WS3 were reported in corals for the first time. The associated bacterial communities varied greatly with location, where environmental conditions differed significantly. Corals from disturbed areas appeared to share more similar bacterial communities, but larger variations in community structures were observed between different coral species from pristine waters. Ordination methods identified salinity and depth as the most influential parameters affecting the abundance of Vibrio, Pseudoalteromonas, Serratia, Stenotrophomonas, Pseudomonas, and Achromobacter in the corals. On the other hand, bacteria such as Chloracidobacterium and Endozoicomonas were more sensitive to the coral species, suggesting that the host species type may be influential in the associated bacterial community, as well. The combined influences of the coral host and environmental factors on the associated microbial communities are discussed. This study represents the first comparative study using tag-pyrosequencing technology to investigate the bacterial communities in Red Sea corals.