Search

Abstract Next‐generation DNA sequencing has shown that the great plate count anomaly, that is, the difference between bacteria present in the environment and those that can be obtained in culture from that environment, is even greater and more persisting than initially thought. This hampers fundamental understanding of bacterial physiology and biotechnological application of the unculture majority. With big sequence data as foundation, artificial intelligence (AI) may be a game changer in bacterial isolation efforts and provide directions for the cultivation media and conditions that are most promising and as such be used to canalize limited human and financial resources. This opinion paper discusses how AI is or can be used to improve the success of bacterial isolation.

Abstract The potential of sponge-derived chemicals for pharmaceutical applications remains largely unexploited due to limited available biomass. Although many have attempted to culture marine sponge cells in vitro to create a scalable production platform for such biopharmaceuticals, these efforts have been mostly unsuccessful. We recently showed that Geodia barretti sponge cells could divide rapidly in M1 medium. In this study we established the first continuous marine sponge cell line, originating from G. barretti. G. barretti cells cultured in OpM1 medium, a modification of M1, grew more rapidly and to a higher density than in M1. Cells in OpM1 reached 1.74 population doublings after 30 min, more than twofold higher than the already rapid growth rate of 0.74 population doublings in 30 min in M1. The maximum number of population doublings increased from 5 doublings in M1 to at least 98 doublings in OpM1. Subcultured cells could be cryopreserved and used to inoculate new cultures. With these results, we have overcome a major obstacle that has blocked the path to producing biopharmaceuticals with sponge cells at industrial scale for decades.

Abstract Background Prebiotic feed additives aim to improve gut health by influencing the microbiota and the gut barrier. Most studies on feed additives concentrate on one or two (monodisciplinary) outcome parameters, such as immunity, growth, microbiota or intestinal architecture. A combinatorial and comprehensive approach to disclose the complex and multifaceted effects of feed additives is needed to understand their underlying mechanisms before making health benefit claims. Here, we used juvenile zebrafish as a model species to study effects of feed additives by integrating gut microbiota composition data and host gut transcriptomics with high-throughput quantitative histological analysis. Zebrafish received either control, sodium butyrate or saponin-supplemented feed. Butyrate-derived components such as butyric acid or sodium butyrate have been widely used in animal feeds due to their immunostimulant properties, thereby supporting intestinal health. Soy saponin is an antinutritional factor from soybean meal that promotes inflammation due to its amphipathic nature. Results We observed distinct microbial profiles associated with each diet, discovering that butyrate (and saponin to a lesser extent) affected gut microbial composition by reducing the degree of community-structure (co-occurrence network analysis) compared to controls. Analogously, butyrate and saponin supplementation impacted the transcription of numerous canonical pathways compared to control-fed fish. For example, both butyrate and saponin increased the expression of genes associated with immune response and inflammatory response, as well as oxidoreductase activity, compared to controls. Furthermore, butyrate decreased the expression of genes associated with histone modification, mitotic processes and G-coupled receptor activity. High-throughput quantitative histological analysis depicted an increase of eosinophils and rodlet cells in the gut tissue of fish receiving butyrate after one week of feeding and a depletion of mucus-producing cells after 3 weeks of feeding this diet. Combination of all datasets indicated that in juvenile zebrafish, butyrate supplementation increases the immune and the inflammatory response to a greater extent than the established inflammation-inducing anti-nutritional factor saponin. Such comprehensive analysis was supplemented by in vivo imaging of neutrophil and macrophage transgenic reporter zebrafish (mpeg1:mCherry/mpx:eGFPi114) larvae. Upon exposure to butyrate and saponin, these larvae displayed a dose-dependent increase of neutrophils and macrophages in the gut area. Conclusion The omics and imaging combinatorial approach provided an integrated evaluation of the effect of butyrate on fish gut health and unraveled inflammatory-like features not previously reported that question the usage of butyrate supplementation to enhance fish gut health under basal conditions. The zebrafish model, due to its unique advantages, provides researchers with an invaluable tool to investigate effects of feed components on fish gut health throughout life.

This study presents a large-scale analysis of microbial diversity in deep-sea sponges. They show that sponge microbial abundance status, geographic distance, sponge phylogeny and the physical-biogeochemical environment drive microbiome composition, in descending order of relevance. The uniqueness of each deep-sea sponge ground stresses the need for their strategic preservation.

Abstract Marine sponges (phylum Porifera) are leading organisms for the discovery of bioactive compounds from nature. Their often rich and species-specific microbiota is hypothesised to be producing many of these compounds. Yet, environmental influences on the sponge-associated microbiota and bioactive compound production remain elusive. Here, we investigated the changes of microbiota and metabolomes in sponges along a depth range of 1232 m. Using 16S rRNA gene amplicon sequencing and untargeted metabolomics, we assessed prokaryotic and chemical diversities in three deep-sea sponge species: Geodia barretti, Stryphnus fortis, and Weberella bursa. Both prokaryotic communities and metabolome varied significantly with depth, which we hypothesized to be the effect of different water masses. Up to 35.5% of microbial ASVs (amplicon sequence variants) showed significant changes with depth while phylum-level composition of host microbiome remained unchanged. The metabolome varied with depth, with relative quantities of known bioactive compounds increasing or decreasing strongly. Other metabolites varying with depth were compatible solutes regulating osmolarity of the cells. Correlations between prokaryotic community and the bioactive compounds in G. barretti suggested members of Acidobacteria, Proteobacteria, Chloroflexi, or an unclassified prokaryote as potential producers.

ABSTRACT Marine sponges and their microbial symbiotic communities are rich sources of diverse natural products (NPs) that often display biological activity, yet little is known about the global distribution of NPs and the symbionts that produce them. Since the majority of sponge symbionts remain uncultured, it is a challenge to characterize their NP biosynthetic pathways, assess their prevalence within the holobiont, and measure the diversity of NP biosynthetic gene clusters (BGCs) across sponge taxa and environments. Here, we explore the microbial biosynthetic landscapes of three high-microbial-abundance (HMA) sponges from the Atlantic Ocean and the Mediterranean Sea. This data set reveals striking novelty, with

Summary Marine photosynthetic microalgae are ubiquitously associated with bacteria in nature. However, the influence of these bacteria on algal cultures in bioreactors is still largely unknown. In this study, eighteen different bacterial strains were isolated from cultures of Nannochloropsis sp. CCAP211/78 in two outdoor pilot‐scale tubular photobioreactors. The majority of isolates was affiliated with the classes Alphaproteobacteria and Flavobacteriia. To assess the impact of the eighteen strains on the growth of Nannochloropsis sp. CCAP211/78, 24‐well plates coupled with custom‐made LED boxes were used to simultaneously compare replicate axenic microalgal cultures with addition of individual bacterial isolates. Co‐culturing of Nannochloropsis sp. CCAP211/78 with these strains demonstrated distinct responses, which shows that the technique we developed is an efficient method for screening the influence of harmful/beneficial bacteria. Two of the tested strains, namely a strain of Maritalea porphyrae (DMSP31) and a Labrenzia aggregata strain (YP26), significantly enhanced microalgal growth with a 14% and 12% increase of the chlorophyll concentration, respectively, whereas flavobacterial strain YP206 greatly inhibited the growth of the microalga with 28% reduction of the chlorophyll concentration. Our study suggests that algal production systems represent a ‘natural’ source to isolate and study microorganisms that can either benefit or harm algal cultures.

Abstract Background Members of the bacterial family Flavobacteriaceae are widely distributed in the marine environment and often found associated with algae, fish, detritus or marine invertebrates. Yet, little is known about the characteristics that drive their ubiquity in diverse ecological niches. Here, we provide an overview of functional traits common to taxonomically diverse members of the family Flavobacteriaceae from different environmental sources, with a focus on the Marine clade. We include seven newly sequenced marine sponge-derived strains that were also tested for gliding motility and antimicrobial activity. Results Comparative genomics revealed that genome similarities appeared to be correlated to 16S rRNA gene- and genome-based phylogeny, while differences were mostly associated with nutrient acquisition, such as carbohydrate metabolism and gliding motility. The high frequency and diversity of genes encoding polymer-degrading enzymes, often arranged in polysaccharide utilization loci (PULs), support the capacity of marine Flavobacteriaceae to utilize diverse carbon sources. Homologs of gliding proteins were widespread among all studied Flavobacteriaceae in contrast to members of other phyla, highlighting the particular presence of this feature within the Bacteroidetes. Notably, not all bacteria predicted to glide formed spreading colonies. Genome mining uncovered a diverse secondary metabolite biosynthesis arsenal of Flavobacteriaceae with high prevalence of gene clusters encoding pathways for the production of antimicrobial, antioxidant and cytotoxic compounds. Antimicrobial activity tests showed, however, that the phenotype differed from the genome-derived predictions for the seven tested strains. Conclusions Our study elucidates the functional repertoire of marine Flavobacteriaceae and highlights the need to combine genomic and experimental data while using the appropriate stimuli to unlock their uncharted metabolic potential.

Recent reviews have reinforced sponge-associated bacteria as a valuable source of structurally diverse secondary metabolites with potent biological properties, which makes these microbial communities promising sources of new drug candidates. However, the overall diversity of secondary metabolite biosynthetic potential present in bacteria is difficult to access due to the fact that the majority of bacteria are not readily cultured in the laboratory. Thus, use of cultivation-independent approaches may allow accessing “silent” and “cryptic” secondary metabolite biosynthetic gene clusters present in bacteria that cannot yet be cultured. In the present study, we investigated the diversity of secondary metabolite biosynthetic gene clusters (BGCs) in metagenomes of bacterial communities associated with three sponge species: Clathria reinwardti, Rhabdastrella globostellata, and Spheciospongia sp. The results reveal that the three metagenomes contain a high number of predicted BGCs, ranging from 282 to 463 BGCs per metagenome. The types of BGCs were diverse and represented 12 different cluster types. Clusters predicted to encode fatty acid synthases and polyketide synthases (PKS) were the most dominant BGC types, followed by clusters encoding synthesis of terpenes and bacteriocins. Based on BGC sequence similarity analysis, 363 gene cluster families (GCFs) were identified. Interestingly, no GCFs were assigned to pathways responsible for the production of known compounds, implying that the clusters detected might be responsible for production of several novel compounds. The KS gene sequences from PKS clusters were used to predict the taxonomic origin of the clusters involved. The KS sequences were related to 12 bacterial phyla with Actinobacteria, Proteobacteria, and Firmicutes as the most predominant. At the genus level, the KSs were most related to those found in the genera Mycolicibacterium, Mycobacterium, Burkholderia, and Streptomyces. Phylogenetic analysis of KS sequences resulted in detection of two known ‘sponge-specific’ BGCs, i.e., SupA and SwfA, as well as a new ‘sponge-specific’ cluster related to fatty acid synthesis in the phylum Candidatus Poribacteria and composed only by KS sequences of the three sponge-associated bacterial communities assessed here.

The application of high-throughput microbial community profiling as well as “omics” approaches unveiled high diversity and host-specificity of bacteria associated with marine sponges, which are renowned for their wide range of bioactive natural products. However, exploration and exploitation of bioactive compounds from sponge-associated bacteria have been limited because the majority of the bacteria remains recalcitrant to cultivation. In this review, we (i) discuss recent/novel cultivation techniques that have been used to isolate sponge-associated bacteria, (ii) provide an overview of bacteria isolated from sponges until 2017 and the associated culture conditions and identify the bacteria not yet cultured from sponges, and (iii) outline promising cultivation strategies for cultivating the uncultivated majority of bacteria from sponges in the future. Despite intensive cultivation attempts, the diversity of bacteria obtained through cultivation remains much lower than that seen through cultivation-independent methods, which is particularly noticeable for those taxa that were previously marked as “sponge-specific” and “sponge-enriched.” This poses an urgent need for more efficient cultivation methods. Refining cultivation media and conditions based on information obtained from metagenomic datasets and cultivation under simulated natural conditions are the most promising strategies to isolate the most wanted sponge-associated bacteria.

Sponge-associated bacteria possess biotechnologically interesting properties but as yet have largely evaded cultivation. Thus, “omics”-based information on the ecology and functional potential of sponge symbionts is awaiting its integration into the design of innovative cultivation approaches. To cultivate bacteria derived from the marine sponge Aplysina aerophoba, nine novel media formulations were created based on the predicted genomic potential of the prevalent sponge symbiont lineage Poribacteria. In addition, to maintain potential microbial metabolic interactions in vitro, a Liquid-Solid cultivation approach and a Winogradsky-column approach were applied. The vast majority of microorganisms in the inoculum appeared viable after cryopreservation of sponge specimen as determined by selective propidium monoazide DNA modification of membrane-compromised cells, however, only 2% of the initial prokaryotic diversity could be recovered through cultivation. In total, 256 OTUs encompassing seven prokaryotic phyla were cultivated. The diversity of the cultivated community was influenced by the addition of the antibiotic aeroplysinin-1 as well as by medium dilution, rather than carbon source. Furthermore, the Winogradsky-column approach reproducibly enriched distinct communities at different column depths, amongst which were numerous Clostridia and OTUs that could not be assigned to a known phylum. While some bacterial taxa such as Pseudovibrio and Ruegeria were recovered from nearly all applied cultivation conditions, others such as Bacteroidetes were specific to certain medium types. Predominant sponge-associated prokaryotic taxa remained uncultured, nonetheless, alternative cultivation approaches applied here enriched for previously uncultivated microbes.

Aquafeed companies aim to provide solutions to the various challenges related to nutrition and health in aquaculture. Solutions to promote feed efficiency and growth, as well as improving the fish health or protect the fish gut from inflammation may include dietary additives such as prebiotics and probiotics. The general assumption is that feed additives can alter the fish microbiota which, in turn, interacts with the host immune system. However, the exact mechanisms by which feed influences host-microbe-immune interactions in fish still remain largely unexplored. Zebrafish rapidly have become a well-recognized animal model to study host-microbe-immune interactions because of the diverse set of research tools available for these small cyprinids. Genome editing technologies can create specific gene-deficient zebrafish that may contribute to our understanding of immune functions. Zebrafish larvae are optically transparent, which allows for in vivo imaging of specific (immune) cell populations in whole transgenic organisms. Germ-free individuals can be reared to study host-microbe interactions. Altogether, these unique zebrafish features may help shed light on the mechanisms by which feed influences host-microbe-immune interactions and ultimately fish health. In this review, we first describe the anatomy and function of the zebrafish gut: the main surface where feed influences host-microbe-immune interactions. Then, we further describe what is currently known about the molecular pathways that underlie this interaction in the zebrafish gut. Finally, we summarize and critically review most of the recent research on prebiotics and probiotics in relation to alterations of zebrafish microbiota and immune responses. We discuss the advantages and disadvantages of the zebrafish as an animal model for other fish species to study feed effects on host-microbe-immune interactions.

This study aimed to assess the diversity and antimicrobial activity of cultivable bacteria associated with Vietnamese sponges. In total, 460 bacterial isolates were obtained from 18 marine sponges. Of these, 58.3% belonged to Proteobacteria, 16.5% to Actinobacteria, 18.0% to Firmicutes, and 7.2% to Bacteroidetes. At the genus level, isolated strains belonged to 55 genera, of which several genera, such as Bacillus, Pseudovibrio, Ruegeria, Vibrio, and Streptomyces, were the most predominant. Culture media influenced the cultivable bacterial composition, whereas, from different sponge species, similar cultivable bacteria were recovered. Interestingly, there was little overlap of bacterial composition associated with sponges when the taxa isolated were compared to cultivation-independent data. Subsequent antimicrobial assays showed that 90 isolated strains exhibited antimicrobial activity against at least one of seven indicator microorganisms. From the culture broth of the isolated strain with the strongest activity (Bacillus sp. M1_CRV_171), four secondary metabolites were isolated and identified, including cyclo(L-Pro-L-Tyr) (1), macrolactin A (2), macrolactin H (3), and 15,17-epoxy-16-hydroxy macrolactin A (4). Of these, compounds 2-4 exhibited antimicrobial activity against a broad spectrum of reference microorganisms.

Abstract Host‐associated microbiota undergoes a continuous transition, from the birth to adulthood of the host. These developmental stage‐related transitions could lead to specific microbial signatures that could impact the host biological processes. In this study, the succession of early‐life and intestinal bacterial communities of Atlantic salmon (starting from embryonic stages to 80‐week post hatch; wph) was studied using amplicon sequencing of 16S rRNA. Stage‐specific bacterial community compositions and the progressive transitions of the communities were evident in both the early life and the intestine. The embryonic communities showed lower richness and diversity (Shannon and PD whole tree) compared to the hatchlings. A marked transition of the intestinal communities also occurred during the development; Proteobacteria were dominant in the early stages (both embryonic and intestinal), though the abundant genera under this phylum were stage‐specific. Firmicutes were the most abundant group in the intestine of late freshwater; Weissella being the dominant genus at 20 wph and Anaerofilum at 62 wph. Proteobacteria regained its dominance after the fish entered seawater. Furthermore, LEfSe analysis identified genera under the above ‐ mentioned phyla that are significant features of specific stages. The environmental (water) bacterial community was significantly different from that of the fish, indicating that the host is a determinant of microbial assemblage. Overall the study demonstrated the community dynamics during the development of Atlantic salmon.

Botryococcus braunii (Chlorophyta) is a green microalga known for producing hydrocarbons and exopolysaccharides (EPS). Improving the biomass productivity of B. braunii and hence, the productivity of the hydrocarbons and of the EPS, will make B. braunii more attractive for industries. Microalgae usually cohabit with bacteria which leads to the formation of species-specific communities with environmental and biological advantages. Bacteria have been found and identified with a few B. braunii strains, but little is known about the bacterial community across the different strains. A better knowledge of the bacterial community of B. braunii will help to optimize the biomass productivity, hydrocarbons, and EPS accumulation. To better understand the bacterial community diversity of B. braunii, we screened 12 strains from culture collections. Using 16S rRNA gene analysis by MiSeq we described the bacterial diversity across 12 B. braunii strains and identified possible shared communities. We found three bacterial families common to all strains: Rhizobiaceae, Bradyrhizobiaceae, and Comamonadaceae. Additionally, the results also suggest that each strain has its own specific bacteria that may be the result of long-term isolated culture.

Organic and inorganic chlorine compounds are formed by a broad range of natural geochemical, photochemical and biological processes. In addition, chlorine compounds are produced in large quantities for industrial, agricultural and pharmaceutical purposes, which has led to widespread environmental pollution. Abiotic transformations and microbial metabolism of inorganic and organic chlorine compounds combined with human activities constitute the chlorine cycle on Earth. Naturally occurring organochlorines compounds are synthesized and transformed by diverse groups of (micro)organisms in the presence or absence of oxygen. In turn, anthropogenic chlorine contaminants may be degraded under natural or stimulated conditions. Here, we review phylogeny, biochemistry and ecology of microorganisms mediating chlorination and dechlorination processes. In addition, the co-occurrence and potential interdependency of catabolic and anabolic transformations of natural and synthetic chlorine compounds are discussed for selected microorganisms and particular ecosystems.

Marine sponges harbor diverse microbial communities that represent a significant source of natural products. In the present study, extracts of 21 sponge-associated bacteria were screened for their antimicrobial and anticancer activity, and their genomes were mined for secondary metabolite biosynthetic gene clusters (BGCs). Phylogenetic analysis assigned the strains to four major phyla in the sponge microbiome, namely Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Bioassays identified one extract with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and more than 70% of the total extracts had a moderate to high cytotoxicity. The most active extracts were derived from the Proteobacteria and Actinobacteria, prominent for producing bioactive substances. The strong bioactivity potential of the aforementioned strains was also evident in the abundance of BGCs, which encoded mainly beta-lactones, bacteriocins, non-ribosomal peptide synthetases (NRPS), terpenes, and siderophores. Gene-trait matching was performed for the most active strains, aiming at linking their biosynthetic potential with the experimental results. Genetic associations were established for the anti-MRSA and cytotoxic phenotypes based on the similarity of the detected BGCs with BGCs encoding natural products with known bioactivity. Overall, our study highlights the significance of combining in vitro and in silico approaches in the search of novel natural products of pharmaceutical interest.

Chloroform (CF) is an environmental contaminant that can be naturally formed in various environments ranging from forest soils to salt lakes. Here we investigated CF removal potential in sediments obtained from hypersaline lakes in Western Australia. Reductive dechlorination of CF to dichloromethane (DCM) was observed in enrichment cultures derived from sediments of Lake Strawbridge, which has been reported as a natural source of CF. No CF removal was observed in abiotic control cultures without artificial electron donors, indicating biotic CF dechlorination in the enrichment cultures. Increasing vitamin B12 concentration from 0.04 to 4 µM in enrichment cultures enhanced CF removal and reduced DCM formation. In cultures amended with 4 µM vitamin B12 and 13C labelled CF, formation of 13CO2 was detected. Known organohalide-respiring bacteria and reductive dehalogenase genes were neither detected using quantitative PCR nor metagenomic analysis of the enrichment cultures. Rather, members of the order Clostridiales, known to co-metabolically transform CF to DCM and CO2, were detected. Accordingly, metagenome-assembled genomes of Clostridiales encoded enzymatic repertoires for the Wood-Ljungdahl pathway and cobalamin biosynthesis, which are known to be involved in fortuitous and nonspecific CF transformation. This study indicates that hypersaline lake microbiomes may act as a filter to reduce CF emission to the atmosphere.

Background: Massive high-throughput sequencing of short, hypervariable segments of the 16S ribosomal RNA (rRNA) gene has transformed the methodological landscape describing microbial diversity within and across complex biomes. However, several studies have shown that the methodology rather than the biological variation is responsible for the observed sample composition and distribution. This compromises meta-analyses, although this fact is often disregarded. Results: To facilitate true meta-analysis of microbiome studies, we developed NG-Tax, a pipeline for 16S rRNA gene amplicon sequence analysis that was validated with different mock communities and benchmarked against QIIME as a frequently used pipeline. The microbial composition of 49 independently amplified mock samples was characterized by sequencing two variable 16S rRNA gene regions, V4 and V5-V6, in three separate sequencing runs on Illumina’s HiSeq2000 platform. This allowed for the evaluation of important causes of technical bias in taxonomic classification: 1) run-to-run sequencing variation, 2) PCR–error, and 3) region/primer specific amplification bias. Despite the short read length (~140 nt) and all technical biases, the average specificity of the taxonomic assignment for the phylotypes included in the mock communities was 97.78%. On average 99.95% and 88.43% of the reads could be assigned to at least family or genus level, respectively, while assignment to ‘spurious genera’ represented on average only 0.21% of the reads per sample. Analysis of α- and β-diversity confirmed conclusions guided by biology rather than the aforementioned methodological aspects, which was not achieved with QIIME. Conclusions: Different biological outcomes are commonly observed due to 16S rRNA region-specific performance. NG-Tax demonstrated high robustness against choice of region and other technical biases associated with 16S rRNA gene amplicon sequencing studies, diminishing their impact and providing accurate qualitative and quantitative representation of the true sample composition. This will improve comparability between studies and facilitate efforts towards standardization.

Sponge-associated prokaryotic diversity has been studied from a wide range of marine environments across the globe. However, for certain regions, e.g., Vietnam, Thailand, Cambodia, and Singapore, an overview of the sponge-associated prokaryotic communities is still pending. In this study we characterized the prokaryotic communities from 27 specimens, comprising 18 marine sponge species, sampled from the central coastal region of Vietnam. Illumina MiSeq sequencing of 16S ribosomal RNA (rRNA) gene fragments was used to investigate sponge-associated bacterial and archaeal diversity. Overall, 14 bacterial phyla and one archaeal phylum were identified among all 27 samples. The phylum Proteobacteria was present in all sponges and the most prevalent phylum in 15 out of 18 sponge species, albeit with pronounced differences at the class level. In contrast, Chloroflexi was the most abundant phylum in Halichondria sp., whereas Spirastrella sp. and Dactylospongia sp. were dominated by Actinobacteria. Several bacterial phyla such as Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Deferribacteres, Gemmatimonadetes, and Nitrospirae were found in two-thirds of the sponge species. Moreover, the phylum Thaumarchaeota (Archaea), which is known to comprise nitrifying archaea, was highly abundant among the majority of the 18 investigated sponge species. Altogether, this study demonstrates that the diversity of prokaryotic communities associated with Vietnamese sponges is comparable to sponge-prokaryotic assemblages from well-documented regions. Furthermore, the phylogenetically divergent sponges hosted species-specific prokaryotic communities, thus demonstrating the influence of host identity on the composition and diversity of the associated communities. Therefore, this high-throughput 16S rRNA gene amplicon analysis of Vietnamese sponge-prokaryotic communities provides a foundation for future studies on sponge symbiont function and sponge-derived bioactive compounds from this region.

Sponges are engaged in intimate symbioses with a diversity of microorganisms from all three domains of life, namely Bacteria, Archaea and Eukarya. Sponges have been well studied and categorized for their bacterial communities, some displaying a high microbial abundance (HMA), while others show low microbial abundance (LMA). However, the associated Archaea and Eukarya have remained relatively understudied. We assessed the bacterial, archaeal and eukaryotic diversities in the LMA sponge species Dysidea avara and Dysidea etheria by deep amplicon sequencing, and compared the results to those in the HMA sponges Aplysina aerophoba and Aplysina cauliformis. D. avara and A. aerophoba are sympatric in the Mediterranean Sea, while D. etheria and A. cauliformis are sympatric in the Caribbean Sea. The bacterial communities followed a host-specific pattern, with host species identity explaining most of the variation among samples. We identified OTUs shared by the Aplysina species that support a more ancient association of these microbes, before the split of the two species studied here. These shared OTUs are suitable targets for future studies of the microbial traits that mediate interactions with their hosts. Even though the archaeal communities were not as rich as the bacterial ones, we found a remarkable diversification and specificity of OTUs of the family Cenarchaeaceae and the genus Nitrosopumilus in all four sponge species studied. Similarly, the differences in fungal communities were driven by sponge identity. The structures of the communities of small eukaryotes such as dinophytes and ciliophores (alveolates), and stramenopiles, could not be explained by either sponge host, sponge genus or geographic location. Our analyses suggest that the host specificity that was previously described for sponge bacterial communities also extends to the archaeal and fungal communities, but not to other microbial eukaryotes.

Fungi and other eukaryotes represent one of the last frontiers of microbial diversity in the sponge holobiont. In this study we employed pyrosequencing of 18S ribosomal RNA gene amplicons containing the V7 and V8 hypervariable regions to explore the fungal diversity of seven sponge species from the North Sea and the Mediterranean Sea. For most sponges, fungi were present at a low relative abundance averaging 0.75% of the 18S rRNA gene reads. In total, 44 fungal OTUs (operational taxonomic units) were detected in sponges, and 28 of these OTUs were also found in seawater. Twenty-two of the sponge-associated OTUs were identified as yeasts (mainly Malasseziales), representing 84% of the fungal reads. Several OTUs were related to fungal sequences previously retrieved from other sponges, but all OTUs were also related to fungi from other biological sources, such as seawater, sediments, lakes and anaerobic digesters. Therefore our data, supported by currently available data, point in the direction of mostly accidental presence of fungi in sponges and do not support the existence of a sponge-specific fungal community.

Sponge-associated bacteria have been mostly cultured from shallow water (≤30 m) sponges, whereas only few studies targeted specimens from below 30 m. This study assessed the cultivability of bacteria from two marine sponges Xestospongia muta and Agelas sventres collected from shallow (

Rare actinomycetes are prolific in the marine environment; however, knowledge about their diversity, distribution and biochemistry is limited. Marine rare actinomycetes represent a rather untapped source of chemically diverse secondary metabolites and novel bioactive compounds. In this review, we aim to summarize the present knowledge on the isolation, diversity, distribution and natural product discovery of marine rare actinomycetes reported from mid-2013 to 2017. A total of 97 new species, representing 9 novel genera and belonging to 27 families of marine rare actinomycetes have been reported, with the highest numbers of novel isolates from the families Pseudonocardiaceae, Demequinaceae, Micromonosporaceae and Nocardioidaceae. Additionally, this study reviewed 167 new bioactive compounds produced by 58 different rare actinomycete species representing 24 genera. Most of the compounds produced by the marine rare actinomycetes present antibacterial, antifungal, antiparasitic, anticancer or antimalarial activities. The highest numbers of natural products were derived from the genera Nocardiopsis, Micromonospora, Salinispora and Pseudonocardia. Members of the genus Micromonospora were revealed to be the richest source of chemically diverse and unique bioactive natural products.

Wide application of antibiotics has contributed to the evolution of multi-drug resistant human pathogens, resulting in poorer treatment outcomes for infections. In the marine environment, seawater samples have been investigated as a resistance reservoir; however, no studies have methodically examined sponges as a reservoir of antibiotic resistance. Sponges could be important in this respect because they often contain diverse microbial communities that have the capacity to produce bioactive metabolites. Here, we applied functional metagenomics to study the presence and diversity of functional resistance genes in the sponges Aplysina aerophoba, Petrosia ficiformis and Corticium candelabrum. We obtained 37 insert sequences facilitating resistance to D-cycloserine (n=6), gentamicin (n=1), amikacin (n=7), trimethoprim (n=17), chloramphenicol (n=1), rifampicin (n=2) and ampicillin (n=3). Fifteen of 37 inserts harboured resistance genes that shared

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes.

The gut microbiota of fish larvae evolves fast towards a complex community. Both host and environment affect the development of the gut microbiota; however, the relative importance of both is poorly understood. Determining specific changes in gut microbial populations in response to a change in an environmental factor is very complicated. Interactions between factors are difficult to separate and any response could be masked due to high inter-individual variation even for individuals that share a common environment. In this study we characterized and quantified the spatio-temporal variation in the gut microbiota of tilapia larvae, reared in recirculating aquaculture systems (RAS) or active suspension tanks (AS). Our results showed that variation in gut microbiota between replicate tanks was not significantly higher than within tank variation, suggesting that there is no tank effect on water and gut microbiota. However, when individuals were reared in replicate RAS, gut microbiota differed significantly. The highest variation was observed between individuals reared in different types of system (RAS vs. AS). Our data suggest that under experimental conditions in which the roles of deterministic and stochastic factors have not been precisely determined, compositional replication of the microbial communities of an ecosystem is not predictable.

Sponges are the most prolific marine organisms with respect to their arsenal of bioactive compounds including antimicrobials. However, the majority of these substances are probably not produced by the sponge itself, but rather by bacteria or fungi that are associated with their host. This review for the first time provides a comprehensive overview of antimicrobial compounds that are known to be produced by sponge-associated microbes. We discuss the current state-of-the-art by grouping the bioactive compounds produced by sponge-associated microorganisms in four categories: antiviral, antibacterial, antifungal and antiprotozoal compounds. Based on in vitro activity tests, identified targets of potent antimicrobial substances derived from sponge-associated microbes include: human immunodeficiency virus 1 (HIV-1) (2-undecyl-4-quinolone, sorbicillactone A and chartarutine B); influenza A (H1N1) virus (truncateol M); nosocomial Gram positive bacteria (thiopeptide YM-266183, YM-266184, mayamycin and kocurin); Escherichia coli (sydonic acid), Chlamydia trachomatis (naphthacene glycoside SF2446A2); Plasmodium spp. (manzamine A and quinolone 1); Leishmania donovani (manzamine A and valinomycin); Trypanosoma brucei (valinomycin and staurosporine); Candida albicans and dermatophytic fungi (saadamycin, 5,7-dimethoxy-4-p-methoxylphenylcoumarin and YM-202204). Thirty-five bacterial and 12 fungal genera associated with sponges that produce antimicrobials were identified, with Streptomyces, Pseudovibrio, Bacillus, Aspergillus and Penicillium as the prominent producers of antimicrobial compounds. Furthemore culture-independent approaches to more comprehensively exploit the genetic richness of antimicrobial compound-producing pathways from sponge-associated bacteria are addressed.

Background Massive high-throughput sequencing of short, hypervariable segments of the 16S ribosomal RNA (rRNA) gene has transformed the methodological landscape describing microbial diversity within and across complex biomes. However, several studies have shown that the methodology rather than the biological variation is responsible for the observed sample composition and distribution. This compromises true meta-analyses, although this fact is often disregarded. Results To facilitate true meta-analysis of microbiome studies, we developed NG-Tax, a pipeline for 16S rRNA gene amplicon sequence analysis that was validated with different mock communities and benchmarked against QIIME as the currently most frequently used pipeline. The microbial composition of 49 independently amplified mock samples was characterized by sequencing two variable 16S rRNA gene regions, V4 and V5-V6, in three separate sequencing runs on Illumina’s HiSeq2000 platform. This allowed evaluating important factors of technical bias in taxonomic classification: 1) run-to-run sequencing variation, 2) PCR–error, and 3) region/primer specific amplification bias. Despite the short read length (~140 nt) and all technical biases, the average specificity of the taxonomic assignment for the phylotypes included in the mock communities was 96%. On average 99.94% and 92.02% of the reads could be assigned to at least family or genus level, respectively, while assignment to ‘spurious genera’ represented on average only 0.02% of the reads per sample. Analysis of α- and β-diversity confirmed conclusions guided by biology rather than the aforementioned methodological aspects, which was not the case when samples were analysed using QIIME. Conclusions Different biological outcomes are commonly observed due to 16S rRNA region-specific performance. NG-Tax demonstrated high robustness against choice of region and other technical biases associated with 16S rRNA gene amplicon sequencing studies, diminishing their impact and providing accurate qualitative and quantitative representation of the true sample composition. This will improve comparability between studies and facilitate efforts towards standardization.