The dispersant Corexit 9500 and (dispersed) oil are lethal to coral endosymbionts.

Endosymbionts (Symbiodiniaceae) play a vital role in the health of corals. Seawater pollution can harm these endosymbionts and dispersants used during oil spill cleanup can be extremely toxic to these organisms. Here, we examined the impact of oil and a specific dispersant, Corexit-9500, on two representative endosymbionts - Symbiodinium and Cladocopium - from the Southwestern endemic coral Mussismilia braziliensis. The survival and photosynthetic potential of the endosymbionts decreased dramatically after exposure to the dispersant and oil by ~25 % after 2 h and ~50 % after 7 days. Low concentrations of dispersant (0.005 ml/l) and dispersed oil (Polycyclic Aromatic Hydrocarbons, 1132 µg/l; Total Petroleum Hydrocarbons, 595 µg/l) proved highly toxic to both Symbiodinium and Cladocopium. These levels triggered a reduction in growth rate, cell size, and cell wall thickness. After a few hours of exposure, cellular organelles were damaged or destroyed. These acute toxic effects underline the fragile nature of coral endosymbionts.

Microbiome diversity from sponges biogeographically distributed between South America and Antarctica.

Sponges from South America and Antarctica are evolutionarily closely related. Specific symbiont signatures that could differentiate these two geographic regions are unknown. This study aimed to investigate the microbiome diversity of sponges from South America and Antarctica. In total 71 sponge specimens were analyzed (Antarctica: N = 59, 13 different species; South America: N = 12, 6 different species). Illumina 16S rRNA sequences were generated (2.88 million sequences; 40K ± 29K/sample). The most abundant symbionts were heterotrophic (94.8 %) and belonged mainly to Proteobacteria and Bacteroidota. EC94 was the most abundant symbiont and dominated the microbiome of some species (70-87 %), comprising at least 10 phylogroups. Each of the EC94 phylogroups was specific to one genus or species of sponge. Furthermore, South America sponges had higher abundance of photosynthetic microorganisms (2.3 %) and sponges from Antarctica, the highest abundance of chemosynthetic (5.5 %). Sponge symbionts may contribute to the function of their hosts. The unique features from each of these two regions (e.g., light, temperature, and nutrients) possibly stimulate distinct microbiome diversity from sponges biogeographically distributed across continents.

Diversity of Microbiomes Across a 13,000-Year-Old Amazon Sediment.

The microbiome is fundamental for understanding bacterial activities in sediments. However, only a limited number of studies have addressed the microbial diversity of Amazonian sediments. Here, we studied the microbiome of sediments from a 13,000-year BP core retrieved in a floodplain lake in Amazonia using metagenomics and biogeochemistry. Our aim was to evaluate the possible environmental influence over a river to a lake transition using a core sample. To this end, we sampled a core in the Airo Lake, a floodplain lake in the Negro River basin. The Negro River is the largest tributary of the Amazon River. The obtained core was divided into three strata: (i) surface, almost complete separation of the Airo Lake from the Negro River when the environment becomes more lentic with greater deposition of organic matter (black-colored sediment); (ii) transitional environment (reddish brown); and (iii) deep, environment with a tendency for greater past influence of the Negro River (brown color). The deepest sample possibly had the greatest influence of the Negro River as it represented the bottom of this river in the past, while the surface sample is the current Airo Lake bottom. In total, six metagenomes were obtained from the three different depth strata (total number of reads: 10.560.701; sequence length: 538 ± 24, mean ± standard deviation). The older (deeper) sediment strata contained a higher abundance of Burkholderia, Chitinophaga, Mucilaginibacter, and Geobacter, which represented ~ 25% of the metagenomic sequences. On the other hand, the more recent sediment strata had mainly Thermococcus, Termophilum, Sulfolobus, Archaeoglobus, and Methanosarcina (in total 11% of the metagenomic sequences). The sequence data were binned into metagenome-assembled genomes (MAGs). The majority of the obtained MAGs (n = 16) corresponded to unknown taxa, suggesting they may belong to new species. The older strata sediment microbiome was enriched with sulfur cycle genes, TCA cycle, YgfZ, and ATP-dependent proteolysis in bacteria. Meanwhile, serine-glyoxylate cycle, stress response genes, bacterial cell division, cell division-ribosomal stress protein cluster, and oxidative stress increased in the younger strata. Metal resistance and antimicrobial resistance genes were found across the entire core, including genes coding for fluoroquinolones, polymyxin, vancomycin, and multidrug resistance transporters. These findings depict the possible microbial diversity during the depositional past events and provided clues of the past microbial metabolism throughout time.

Vibrio fluminensis sp. nov. isolated from the skin of Southern Atlantic sharpnose-pufferfish (Canthigaster figueiredoi).

This manuscript provides the description of the bacterial strain A621T characterized by Gram negative motile rods, presenting green circular colonies on TCBS. It was obtained from the skin of the sharpnose pufferfish Canthigaster figueredoi (Tetraodontidae Family), collected in Arraial do Cabo, located in the Rio de Janeiro region, Brazil. Optimum growth occurs at 20-28 °C in the presence of 3% NaCl. The Genome sequence of the novel isolate consisted of 4.224 Mb, 4431 coding genes and G + C content of 44.5%. Genomic taxonomy analysis based on average amino acid (AAI), genome-to-genome-distance (GGDH) and phylogenetic reconstruction placed (A621T= CBAS 741T = CAIM 1945T = CCMR 150T) into a new species of the genus Vibrio (Vibrio fluminensis sp. nov). The genome of the novel species contains four gene clusters (~ 56.17 Kbp in total) coding for different types of bioactive compounds that hint to several possible ecological roles in the sharpnose pufferfish host.

Insights on the biochemical and cellular changes induced by heat stress in the Cladocopium isolated from coral Mussismilia braziliensis.

Corals are treatened by global warming. Bleaching is one immediate effect of global warming, resulting from the loss of photosynthetic endosymbiont dinoflagellates. Understanding host-symbiont associations are critical for assessing coral's habitat requirements and its response to environmental changes. Cladocopium (formerly family Symbiodiniaceae clade C) are dominant endosymbionts in the reef-building coral, Mussismilia braziliensis. This study aimed to investigate the effect of temperature on the biochemical and cellular features of Cladocopium. Heat stress increased oxygen (O2) and decreased proteins, pigments (Chla + Chlc2), hexadecanoic acid- methyl ester, methyl stearate, and octadecenoic acid (Z)- methyl ester molecules. In addition, there was an increase in neutral lipids such as esterified cholesterol and a decrease in free fatty acids that may have been incorporated for the production of lipid droplets. Transmission electron microscopy (TEM) demonstrated that Cladocopium cells subjected to heat stress had thinner cell walls, deformation of chloroplasts, and increased lipid droplets after 3 days at 28°C. These findings indicate that thermal stress negatively affects isolated Cladocopium spp. from Mussismilia host coral.

Fluxes of the Amazon River plume nutrients and microbes into marine sponges.

Sponges have co-evolved with microbes for over 400 myr. Previous studies have demonstrated that sponges can be classified according to the abundance of microbes in their tissues as Low Microbial Abundance (LMA) and High Microbial Abundance (HMA). While LMA sponges rely mainly on water column microbes, HMA appear to rely much more on symbiotic fermentative and autotrophic microbes maintained in their tissues. However, it is unclear if this pattern holds when comparing different species of tropical sponges under extreme nutrient conditions and sediment loads in the water column, such as the Great Amazon Reef System (GARS), which covers an area of ~56,000 km2 off the Amazon River mouth. Sponges are the major GARS benthic components. However, these sponges' microbiome across the GARS is still unknown. Here, we investigated water quality, isotopic values (δ13C and δ15N), metagenomic and lipidomic profiles of sponges obtained from different sectors throughout the GARS. >180 million shotgun metagenomic reads were annotated, covering 22 sponge species. Isotopic and lipidomic analyses suggested LMA sponges rely on the Amazon River Plume for nutrition. HMA sponges (N = 15) had higher Roseiflexus and Nitrospira abundance, whereas LMA sponges (N = 7) had higher Prochlorococcus and Pelagibacter abundance. Functional data revealed that the LMA sponge microbiomes had greater number of sequences related to phages and prophages as well as electron transport and photophosphorylation which may be related to photosynthetic processes associated with the Prochlorococcus and Synechococcus found in the LMA. The higher phages abundance in LMA sponges could be related to these holobionts' reduced defense towards phage infection. Meanwhile, HMA sponge microbiomes had higher Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR abundance, which may be involved in defense against phage infection. This study sheds light on the nutrient fluxes and microbes from the Amazon River plume into the sponge holobionts.

Breviolum and Cladocopium Are Dominant Among Symbiodiniaceae of the Coral Holobiont Madracis decactis.

The scleractinian reef building coral Madracis decactis is a cosmopolitan species. Understanding host-symbiont associations is critical for assessing coral's habitat requirements and its response to environmental changes. In this study, we performed a fine grained phylogenetic analyses of Symbiodiniaceae associated with Madracis in two locations in the Southwest Atlantic Ocean (Abrolhos Bank and St. Peter and St. Paul Archipelago). Previous studies have argued that Madracis is a specialist coral, with colonies harboring a single symbiont from the genus Breviolum (formerly clade B). However, these previous studies have not precisely addressed if Madracis is colonized by several types of Symbiodiniaceae simultaneously or whether this coral is a specialist. The hypothesis that Madracis is a generalist coral host was evaluated in the present study. A total of 1.9 million reads of ITS2 nuclear ribosomal DNA were obtained by Illumina MiSeq sequencing. While Symbiodiniaceae ITS2 sequences between two sampling depths were almost entirely (62%) from the genus Breviolum (formerly clade B), shallow (10-15 m) populations in Abrolhos had a greater diversity of ITS2 sequences in comparison to deeper (25-35 m) populations of St. Peter and St. Paul Archipelago. Cladocopium (formerly clade C) and Symbiodinium (formerly clade A) were also found in Abrolhos. A single Madracis colony can host different symbiont types with > 30 Symbiodiniaceae ITS2-type profiles. Abrolhos corals presented a higher photosynthetic potential as a possible result of co-occurrence of multiple Symbiodiniaceae in a single coral colony. Multiple genera/clades of Symbiodiniaceae possibly confer coral hosts with broader environmental tolerance and ability to occupy diverse or changing habitats.

Mangrove microbiome reveals importance of sulfur metabolism in tropical coastal waters.

Mangroves under macro-tidal regimes are global carbon sequestration hotspots but the microbial drivers of biogeochemical cycles remain poorly understood. Here, we investigate the drivers of mangrove microbial community composition across a porewater-creek-estuary-ocean continuum. Observations were performed on the Amazon region in one of the largest mangrove systems worldwide with effective sequestration of organic carbon buried in soils and dissolved carbon via outwelling to the ocean. The potential export to the adjacent oceanic region ranged from 57 to 380 kg of dissolved and particulate organic carbon per second (up to 33 thousand tons C per day). Macro tides modulated microbial communities and their metabolic processes, e.g., anoxygenic phototrophy, sulfur, and nitrogen cycling. Respiration, sulfur metabolism and dissolved organic carbon (DOC) levels were linked to functional groups and microbial cell counts. Total microbial counts decreased and cyanobacteria counts peaked in the spring tide. The microbial groups driving carbon, nitrogen, sulfur and methane cycles were consistent across all spatial scales. Taxonomic groups engaged in sulfur cycling (Allochromatium, Desulfovibrio, and Thibacillus) within mangroves were abundant at all scales. Tidally-driven porewater exchange within mangroves drove a progressive increase of sulfur cycle taxonomic groups and their functional genes both temporally (tidal cycles) and spatially (from mangrove porewater to continental shelf). Overall, we revealed a unified and consistent response of microbiomes at different spatial and temporal scales to tidally-driven mangrove porewater exchange.

Microbiome associated with the tetrodotoxin-bearing anuran Brachycephalus pitanga.

The genus Brachycephalus includes small species of aposematic anurans known as microendemic, occurring in the mountains of the Atlantic Forest. Brachycephalus ephippium, B. nodoterga and B. pernix have been reported to contain the neurotoxin tetrodotoxin in skin and viscera. The biological conservation of several Brachycephalus species is currently threatened by climate change, deforestation, and the pandemic caused by the fungus Batrachochytrium dendrobatidis (Bd). Despite the well-known importance of amphibians' associated bacteria in the defensive role against pathogens, there is still a poor understanding of amphibian microbiome composition. The present study investigated the composition of B. pitanga microbial community and the presence of TTX in the host and in cultures of bacterial isolates, using a combination of metagenomics, bacterial culture isolation, mass spectrometry and metabolomic analyses. Results of culture-dependent and -independent analyses characterized the microbial communities associated with the skin and viscera of B. pitanga. Mass spectrometry analysis indicated the presence of TTX in host tissues, while bacterial production of TTX was not observed under the experimental conditions used in this investigation. This is the first report confirming the occurrence of TTX in B. pitanga.

Genome sequence of Vibrio fluvialis 362.3 isolated from coral Mussismilia braziliensis reveals genes related to marine environment adaptation.

Vibrio fluvialis is a halophilic bacterium frequently found in estuarine and coastal waters environments. The strain 362.3 was isolated from Mussismilia braziliensis coral of Abrolhos Bank. In this study, to gain insights into the marine adaptation in V. fluvialis, we sequenced the genome of 362.3 strain, which comprised 4,607,294 bp with a G + C content of 50.2%. In silico analysis showed that V. fluvialis 362.2 encodes genes related to chitin catabolic pathway, iron metabolism, osmotic stress and membrane transport.

Vibrio tetraodonis sp. nov.: genomic insights on the secondary metabolites repertoire.

Description of a Gram-negative, motile, circular-shaped bacterial strain, designated A511T obtained from the skin of the pufferfish Sphoeroides spengleri (Family Tetraodontidae), collected in Arraial do Cabo, Brazil. Optimum growth occurs at 20-28 °C in the presence of 3% NaCl. The genome sequence of the novel isolate consisted of 4.36 Mb, 3,976 coding genes and G + C content of 42.5%. Genomic taxonomy analyses based on average amino acid (AAI), genome-to-genome-distance (GGDH) and phylogenetic reconstruction placed A511T (= CBAS 712T = CAIM 1939T) into a new species of the genus Vibrio (Vibrio tetraodonis sp. nov.). The genome of the novel species contains eight genes clusters (~ 183.9 Kbp in total) coding for different types of bioactive compounds that hint to several possible ecological roles in the pufferfish host.

A new genomic taxonomy system for the Synechococcus collective.

Cyanobacteria of the genus Synechococcus are major contributors to global primary productivity and are found in a wide range of aquatic ecosystems. This Synechococcus collective (SC) is metabolically diverse, with some lineages thriving in polar and nutrient-rich locations and others in tropical or riverine waters. Although many studies have discussed the ecology and evolution of the SC, there is a paucity of knowledge on its taxonomic structure. Thus, we present a new taxonomic classification framework for the SC based on recent advances in microbial genomic taxonomy. Phylogenomic analyses of 1085 cyanobacterial genomes demonstrate that organisms classified as Synechococcus are polyphyletic at the order rank. The SC is classified into 15 genera, which are placed into five distinct orders within the phylum Cyanobacteria: (i) Synechococcales (Cyanobium, Inmanicoccus, Lacustricoccus gen. Nov., Parasynechococcus, Pseudosynechococcus, Regnicoccus, Synechospongium gen. nov., Synechococcus and Vulcanococcus); (ii) Cyanobacteriales (Limnothrix); (iii) Leptococcales (Brevicoccus and Leptococcus); (iv) Thermosynechococcales (Stenotopis and Thermosynechococcus) and (v) Neosynechococcales (Neosynechococcus). The newly proposed classification is consistent with habitat distribution patterns (seawater, freshwater, brackish and thermal environments) and reflects the ecological and evolutionary relationships of the SC.

Rapid screening of marine bacterial symbionts using MALDI-TOF MS.

Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) is a rapid, cost-effective and high-throughput method for bacteria characterization. However, most previous studies focused on clinical isolates. In this study, we evaluated the use of MALDI-TOF MS as a rapid screening tool for marine bacterial symbionts. A set of 255 isolates from different marine sources (corals, sponge, fish and seawater) was analyzed using cell lysates to obtain a rapid grouping. Cluster analysis of mass spectra and 16S rRNA showed 18 groups, including Vibrio, Bacillus, Pseudovibrio, Alteromonas and Ruegeria. MALDI-TOF distance similarity scores ≥ 60% and ≥ 70% correspond to ≥ 98.7% 16S rRNA gene sequence similarity and ≥ 95% pyrH gene sequence similarity, respectively. MALDI-TOF MS is a useful tool for Vibrio species groups' identification.

Oil leakage induces changes in microbiomes of deep-sea sediments of Campos Basin (Brazil).

The Campos Basin (100,000 km2) is located on the continental shelf of southeastern Brazil. Despite the significant oil and gas industrial activities underway in the Campos Basin, scarce information is available regarding the hydrocarbon contents and microbial communities in the deep-sea sediments. To gain new insights on these aspects, we first obtained deep-sea sediment samples with different degrees of oil exposure. We obtained samples from a seabed fissure (N = 28), surroundings (250 m to 500 m from the fissure; N = 24), and a control area (N = 4). We used shotgun metagenomics to characterize the taxonomic and metabolic diversity and analyzed biogeochemical parameters (metal and oil concentration) of all samples. The high levels of unresolved complex mixture of hydrocarbons in the fissure indicate a potentially recent petrogenic contribution in these sediments. The fissure area was found to have a higher abundance of hydrocarbonoclastic bacterial genera and hydrocarbon degradation genes. These bacteria may be used as biosensors of sediment contamination. The effects of oil contamination, mainly around the fissure, are less clear at 250 m and 500 m, suggesting that the surroundings may not have been heavily affected by the oil leakage. Our study demonstrates that metagenomics can disclose biosensors for environmental monitoring.

New bacterial and archaeal lineages discovered in organic rich sediments of a large tropical Bay.

The nutrient and oxygen gradient present in marine sediments promotes high levels of microbial diversity. We applied metagenomics and biogeochemical tools to analyze microbial communities in different sediment depths (0-4 m below sea floor, mbsf) from Guanabara Bay, Brazil, a brackish tropical ecosystem with a history of massive anthropogenic impacts, and a largely unknown sediment microbial diversity. Methanogens (e.g. Methanosarcinales, Methanomicrobiales) were more abundant at 1 mbsf, while sulphate-reducing microbes (Desulfurococcales, Thermoprotales, and Sulfolobales) were more abundant at deeper layers (4 mbsf; corresponding to 3 K Radiocarbon years before present, Holocene Epoch). Taxonomic analyzes and functional gene identification associated with anaerobic methane oxidation (e.g. monomethylamine methyltransferase (mtmB), trimethylamine methyltransferase (mttB) and CO dehydrogenase/acetyl-CoA synthase delta subunit) and sulfate reduction indicated the dominance of Campylobacteria (Sulfurimonas) at deeper sediment layers. Gene sequences related to assimilation of inorganic sulfur increased with depth, while organic sulfur related sequences decrease, accompanying the clear reduction in the concentration of sulfur, organic carbon and chla torwards deeper layers. Analyzes of metagenome assembled genomes also led to the discovery of a novel order within the phylum Acidobacteriota, named Guanabacteria. This novel order had several in silico phenotyping features that differentiate it from closely related phylogenetic neighbors (e.g. Acidobacteria, Aminicenantes, and Thermoanaerobaculum), including several genes (carbon monoxide dehydrogenase, CO dehydrogenase/CO-methylating acetyl-CoA synthase complex subunit beta, heterodisulfide reductase, sulfite exporter TauE/SafE family protein, sulfurtransferase) that relevant for the S and C cycles. Furthermore, the recovered Bathyarchaeota genome SS9 illustrates the methanogenic potential in deeper sediment layer.

Ecogenomics of the Marine Benthic Filamentous Cyanobacterium Adonisia.

Turfs are among the major benthic components of reef systems worldwide. The nearly complete genome sequences, basic physiological characteristics, and phylogenomic reconstruction of two phycobiliprotein-rich filamentous cyanobacteria strains isolated from turf assemblages from the Abrolhos Bank (Brazil) are investigated. Both Adonisia turfae CCMR0081T (= CBAS 745T) and CCMR0082 contain approximately 8 Mbp in genome size and experiments identified that both strains exhibit chromatic acclimation. Whereas CCMR0081T exhibits chromatic acclimation type 3 (CA3) regulating both phycocyanin (PC) and phycoerythrin (PE), CCMR0082 strain exhibits chromatic acclimation type 2 (CA2), in correspondence with genes encoding specific photosensors and regulators for PC and PE. Furthermore, a high number and diversity of secondary metabolite synthesis gene clusters were identified in both genomes, and they were able to grow at high temperatures (28 °C, with scant growth at 30 °C). These characteristics provide insights into their widespread distribution in reef systems.

Genomic and ecological attributes of marine bacteriophages encoding bacterial virulence genes.

BACKGROUND: Bacteriophages encode genes that modify bacterial functions during infection. The acquisition of phage-encoded virulence genes is a major mechanism for the rise of bacterial pathogens. In coral reefs, high bacterial density and lysogeny has been proposed to exacerbate reef decline through the transfer of phage-encoded virulence genes. However, the functions and distribution of these genes in phage virions on the reef remain unknown. RESULTS: Here, over 28,000 assembled viral genomes from the free viral community in Atlantic and Pacific Ocean coral reefs were queried against a curated database of virulence genes. The diversity of virulence genes encoded in the viral genomes was tested for relationships with host taxonomy and bacterial density in the environment. These analyses showed that bacterial density predicted the profile of virulence genes encoded by phages. The Shannon diversity of virulence-encoding phages was negatively related with bacterial density, leading to dominance of fewer genes at high bacterial abundances. A statistical learning analysis showed that reefs with high microbial density were enriched in viruses encoding genes enabling bacterial recognition and invasion of metazoan epithelium. Over 60% of phages could not have their hosts identified due to limitations of host prediction tools; for those which hosts were identified, host taxonomy was not an indicator of the presence of virulence genes. CONCLUSIONS: This study described bacterial virulence factors encoded in the genomes of bacteriophages at the community level. The results showed that the increase in microbial densities that occurs during coral reef degradation is associated with a change in the genomic repertoire of bacteriophages, specifically in the diversity and distribution of bacterial virulence genes. This suggests that phages are implicated in the rise of pathogens in disturbed marine ecosystems.

New tetrodotoxin analogs in Brazilian pufferfishes tissues and microbiome.

While tetrodotoxin (TTX) is commonly found in pufferfish tissues, it is unclear if bacterial symbionts isolated from pufferfish tissues can produce TTX. In this investigation, UPLC qTOF-MS/MS analysis of tissue extracts obtained from Sphoeroides spengleri and Canthigaster figuereidoi identified TTX in their composition, indicating their consumption is unsafe. UPLC qTOF-MS/MS analysis coupled with Molecular Networking indicated new TTX analogs (methyl-TTX, TTX-acetate, hydroxypropyl-TTX and glycerol-TTX). Bacterial extracts from sixteen strains revealed a compound with a [M+H]+ ion at m/z 320.1088, identical to TTX. However, TTX itself was not detected in these cultures by UPLC-MS/MS. Neurotoxicity of Vibrio A665 purified fraction 2 (with precursor [M+H]+ ion at m/z 320.1088) was significant in human neural stem cells (hNSCs), but the Nav blockage activity was not confirmed by the veratridine/ouabain essays, indicating a possible difference in the mechanism of action between the bacterium A665 purified fraction 2 and TTX. Vibrios symbionts of pufferfish point out involving in the production of TTX precursors.

Genome sequence of Shewanella corallii strain A687 isolated from pufferfish (Sphoeroides spengleri).

We present here the genome sequence of Shewanella corallii strain A687 isolated from pufferfish Sphoeroides spengleri (Family Tetraodontidae). The assembly consists of 5,215,037 bp and contains 284 contigs, with a G+C content of 50.3%.

Enterovibrio baiacu sp. nov.

We report here the novel species to encompass the isolate A649T (=CBAS 716T = CBRVS P1061T) obtained from viscera of the healthy pufferfish Sphoeroides spengleri (Family Tetraodontidae). Genomic taxonomy analysis demonstrates that the novel strain A649T had < 95% average amino acid identity/average nucleotide identity (AAI/ANI) and < 70% similarity of genome-to-genome distance (GGDH) towards its closest neighbors which places A649T into a new Enterovibrio species (Enterovibrio baiacu sp nov.). In silico phenotyping disclosed several features that may be used to differentiate related Enterovibrio species. The nearly complete genome assembly of strain A649T consisted of 5.4 Mbp and 4826 coding genes.