vacuuming for virus viromes

Deep sea sediments are one of the largest biomes on earth containing most of the microbial life in the sea. Viruses are one of the most abundant biological components in the oceans. Viruses play a key role in biogeochemical cycles and ecosystem functioning at a global scale by infecting their hosts. However, the diversity of viruses in benthic systems is unknown.

There have been previous studies considering virus diversity but most papers focus on viral assemblages found in the water column. There are four steps taken to investigate the viral diversity in benthic sediments. 1) recovery and concentration of viral particles, 2) extraction and purification of viral DNA, 3) high throughput sequencing of viral DNA and 4) bioinformatic analysis.

Samples were taken from the Black Sea, two sites in the NE Atlantic (NE Atlantic sites 1 & 2), the Arctic ocean and the Mediterranean Sea. Three independent replicates were taken from each site using a multiple corer. Samples were recovered from the sediment using a Physical chemical (PC) treatment. Samples were stained with SYBER Gold and analysed with epifluorescence microscopy to assess the recovery efficiency of the PC procedure and to check for contamination by eukaryote and bacteria cells. Next the viruses were concentrated using a vacuuming step to filter viruses from any contaminants. qPCR was used to check for contamination by looking for 16s rRNA bacteria and 18s rRNA eukaryote genes. Bioinformatic analysis was used to determine the taxonomic annotation of sequences down to the domain level.

The study found that the black sea and NE Atlantic sea site 2 were mostly viral associated sequences. The Mediterranean and Arctic sites had bacterial associated sequences and the NE Atlantic 1 site had the highest amount of eukaryotic sequences. The composition of the viral assemblages at each site showed relationships with environmental factors at those sites. The black sea had viral compositions related to the organic Carbon load in the sediment. The Mediterranean was related to temperature and the Arctic ocean site had assemblages relating to salinity.

 In the deep-sea viromes, viral genotypes belonged mostly to the order Caudovirales (48–66%). The most represented families, from the order Caudovirales, were Siphoviridae, Myoviridae and Podoviridae. The cluster analysis revealed a low similarity among the viromes generated from different benthic deep-sea sites. The fraction of reads annotated in the viromes were highly variable suggesting that a proportion of the viral sequences are still unknown and could be of novel viral genotypes.

The putative viral functions were very different, suggesting that benthic deep-sea ecosystems are characterized by the different ecological settings. Thus, leading to taxonomically diverse viral genotypes with distinct functions which can give viruses advantages when interacting with their hosts.

This paper was difficult to read in places and seemed out of logical order. This paper was only accepted in August of 2017 but was received November 2015 and has yet to be cited. I feel perhaps this paper should have been two separate studies.

The authors used high throughput sequencing and epifluorescence microscopy to evaluate and compare different methods of collecting, purifying and analysing viral assemblages. The washing based (WB) procedure is commonly used to collect microbes from sediments however the physical chemical (PC) procedure created for this study collected up to 2x as much viral particles. Tangential flow filtration (TFF) is commonly used to concentrate viral samples however they found that 80% of viruses were lost in this step compared to using a vacuum procedure which uses a filter to collect viral particles.

When comparing physical chemical (PC) procedure to the washing based (WB) procedure they found a higher level of viral genotypes using the PC procedure. Some families were found only with the PC procedure. The results from this study show that the WB procedure can lead to a major under estimation of viral diversity in deep sea sediments, this means that the efficiency of viral DNA recovery can influence viral genotype richness and assemblage composition. This comparison of the two procedures and the use of sequencing to assess the efficiency of both could be useful for future studies of viral assemblages.

Corinaldesi et al., (2017) ‘From virus isolation to metagenome generation for investigating viral diversity in deep-sea sediments’,  Scientific Reports, 7(1). doi:10.1038/s41598-017-08783-4

Symbiodinium photosymbiosis in the open ocean

Symbiosis is central to the ecology of many ecosystems. The close relationship that occurs with a photosynthetic partner is defined as photosymbiosis, which has led to many eukaryotic lineages acquiring transient and even sometimes permanent photosynthesis. This type of symbiosis is common in both marine and freshwater ecosystems and it understood to be mutualistic. Symbiodinium is known to be one of the most common photosynthetic symbiont in marine environments. The genus Symbiodinium is genetically diverse and has evolved into nine different clades (A to I), which all have distinct physiological capacities, spatial distribution and host spectra. Despite several photosynthetic dinoflagellate taxa being identified as common symbionts in oceanic plankton, to date Symbiodinium, which is a common symbiont is coastal waters, has not been found in symbiosis in pelagic plankton. Ciliates are known to acquire phototrophy through photosymbiosis with eukaryotic or prokaryotic microalgal cells, benthic ciliates Maristentor dinoferus and Euplotes uncinatus host Symbiodinium endosymbionts in coral reefs and some Oligotrichida ciliates associate with prasinophytes in estuarine environments. Photosymbiotic ciliates have been found almost exclusive in coastal or benthic habitats. This study used a combination of microscopy and molecular tools to characterize a novel pelagic photosymbiosis between a calcifying ciliate host and Symbiodinium endosymbionts. By using the worldwide Tara Oceans expedition samples and metabarcoding data set allowed for the study of global specificity, biogeography and ecology.

Consistent microscopy observations on multiple specimens, systematic PCR detection of Symbiodinium within the ciliate and the geographic distribution in surface oceans together provide evidence in favour of a long-term mutualistic symbiosis. The study identified and characterised a novel widespread photosymbiosis between the dinoflagellate Symbiodinium and an undescribed calcifying ciliate. This newly described relationship is relevant not only because the host is previously unknown, but also because the occurrence of Symbiodinium as a pelagic symbiont was unknown, despite it being one of the most extensively studied microalgal genera. Within Symbiodinium clade A it was revealed that 8 subclade types are endemic to pelagic waters as they have not previously been reported in benthic coastal habitats. The selective pressures within the pelagic realm have generated these host specializations and may have created distinct ecological niches as well as the diversification of Symbiodinium.  This symbiosis likely plays many biogeochemical roles in pelagic ecosystems through contribution to primary productivity and calcium cycling. As the cilia-bearing host has the ability to actively move through the environment, unlike the ‘passive’ photosymbiotic rhizarian Radiolaria and Foraminifera, may indicate a chemotactic behaviour for finding symbiotic partners, suitable light conditions, and food or nutrient patches. The benefits to Tiarina sp. can be understood through the metabolic capacities of Symbiodinium in reef ecosystems, these likely include the benefits of a significant source of carbon, nitrogen, phosphate and other key nutrients for growth which would have otherwise been limited in the open ocean. Another benefit may also be the possibility that Symbiodinium plays a crucial role in the calcification of the ciliate’s skeleton (similar to scleractinian corals) and in protection from ultraviolet radiation. Clade A has been known to produce a significant amount of UV-absorbing amino acids which explains why it would be the clade that is represented in the transparent open ocean waters.

Future studies should aim to add to the understanding the nature, ecological role and life cycle of this novel photosymbiosis, and explore the possibility that other Symbiodinium clades can be found in the open ocean. Also in future automatic and in situ imaging techniques could be used to properly quantify this interaction, thus determining its ecological impact to the environment.

Reviewed Paper: Mordret, S., Romac, S., Henry, N., Colin, S., Carmichael, M., Berney, C., Audic, S., Richter, D., Pochon, X., de Vargas, C. and Decelle, J. (2015). The symbiotic life of Symbiodinium in the open ocean within a new species of calcifying ciliate (Tiarina sp.). The ISME Journal, 10(6), pp.1424-1436. 

Fish microbiomes vary in time and space

               

Microbial symbionts play an important role in the survival of multicellular organisms. Specifically, bacteria found in the gut are important for host nutrition, immune functions and behaviour. While gut microbiomes have been studied in terrestrial animals and economically valuable marine species, less attention has been paid to ecologically valuable marine species and our understanding of fish gut microbiomes is limited.

Most studies of fish microbiomes involve sampling faeces or simultaneously sampling the entire gastrointestinal track. These methods cannot discriminate between autochthonous and allochthonous microbes or assess spatial distribution; as diet has a strong influence on gut microbiomes and compartmentalisation has been seen in terrestrial species, these sampling methods are only telling us part of the story. Nielson et al. (2017) aimed to gather data on the gut microbiome of Siganus fuscescens (an ecologically important species in coastal ecosystems), while sampling in a way that allowed these factors to be considered.

The intestinal tract of four individuals were removed and separated into the midgut and hindgut; DNA was then extracted from the gut content and wall of these two sections separately. The V4 region of the 16S rRNA gene was then amplified using PCR; the OTUs produced were used to assess alpha- and beta- diversity of the bacterial community as well as taxa present. Species richness and species diversity (measures of alpha-diversity) were found to vary between individuals and were higher in the gut wall than the gut content. The authors suggest that the large variation between individuals is due to the variable diet of this species, a hypothesis supported by the larger individual variation seen in midgut content than other gut locations. Beta-diversity assessments showed that the midgut and hindgut contain different bacterial communities as do the gut wall and gut lumen.  The taxa present also changed between gut locations, which was suggested to be a result of whether the taxa are autochthonous or allochthonous and the environment in each gut location. For example, the Synechococcaceae family was only found in the midgut content, suggesting it is an autochthonous member of the microbiome, either from their diet or being ingested from the environment, while the enrichment of Desulfovibrio and Clostridium in the hindgut implies an anoxic, sulfate rich environment, indicating compartmentalisation due to host/environmental factors.

While the method used in this paper highlights the importance of spatial distribution of microbiome species along the gastrointestinal tract as well as the contribution of microorganisms obtained from the environment, there are limitations. A sample size of four individuals makes interpretations difficult; for example, species diversity of one individual was very different to the others, however with only four samples it is difficult to know how unusual this is. Such a small sample size also reduces statistical power, so some observed trends could not be supported statistically. There were also slight differences in sample collection which the authors suggest may have affected results. While the method used in this paper does allow greater insights into fish microbiomes, it is perhaps not such a widely-used method due to ethical considerations and difficulty in standardising sample collection; the use of faeces could be less invasive, allowing a larger sample size, and the use of whole gut analysis could make sample collection easier, especially in smaller species.

Reviewed Article:

Nielsen, S., Walburn, J. W., Vergés, A., Thomas, T., & Egan, S. (2017). Microbiome patterns across the gastrointestinal tract of the rabbitfish Siganus fuscescens. PeerJ, 5, e3317.

https://peerj.com/articles/3317/

Fish gill mucus is a bacterial oasis in tropical seas

Bacteria form symbioses with a range of eukaryotic hosts, including humans and a vast array of marine invertebrates. Coral bacterial symbionts have received particular attention, while fish microbiomes have been comparatively less studied; however, this is now changing due to recent efforts to characterise the “fish gastrointestinal microbiome”, with a focus on commercially important species and the hope to increase their health and nutritional status by targeting their microbial symbionts (Tarnecki et al., 2017). Moreover, a number of studies have been published on other less exploited fish species. For instance, Miriam Reverter and her colleagues recently described the abundance and diversity of bacteria associated with the gill mucus in four butterflyfish species, drawing insightful links with the ecology of their animal hosts (Reverter et al., 2017). In the mentioned study, the V1-V3 16S rDNA barcodes were sequenced from 4 sympatric butterflyfish species (Chaetodon lunulatus, C. ornatissimus, C. vagabundus and C. reticulatus) in order to identify their gill mucus-associated bacteria. The sequences were quality checked and diversity was estimated with a range of indices from a rarefied OTU table.

The “core microbiome”, i.e. bacteria found in all four species of fish, comprised only 26 OTUs (~ 2.5% of the total OTUs) belonging to the Proteobacteria, Firmicutes and Actinobacteria, with an additional Cyanobacteria OTU. The former three phyla were also the most abundant in terms of OTUs, though the authors were rightly cautious by recognising that OTU and cellular abundances do not always coincide. C. lunulatus was shown to have the highest abundance of unique OTUs, and the authors related this finding to the absence of monogean parasitic flatworms in this fish species; however, the study did not assess the presence of the said parasites on the remaining fish species.

The phyla identified as the most abundant in the four butterflyfishes’ gill mucus also dominated gastrointestinal microbiomes of most other fishes studied to date (Tarnecki et al., 2017). In light of this, the authors acknowledged the often-seen correlation between microbiome composition and diet, and the study offers further support for this: in fact, C. lunulatus and C. ornatissimus, both corallivores, had the most taxonomically similar gill mucus-associated bacterial assemblage. The two species are also the least phylogenetically distant, therefore the authors propose phylogeny as another possible factor influencing the composition of their bacterial microbiomes, though it is unclear how this might result. Arguably, diet and host ecology would be expected to play a more significant role in sympatric close relatives, such as the four chaetodontids under investigation. At the family level, there was a relatively high abundance of Vibrionaceae in C. lunulatus and C. ornatissimus, and Rhodobacteraceae in C. reticulatus and C. vagabundus. Representatives from both bacterial families are known and potential coral pathogens respectively (Sunagawa et al., 2009), and, considering the opportunistic nature of many coral pathogens (Rohwer & Youle, 2010), it would be interesting to assess the composition changes of the two groups in diseased butterflyfish.

Perhaps most remarkably, the diversity and abundance of bacterial symbionts associated with fish gill mucus were comparable to coral-associated bacterial communities. The hypothesis made by the authors, that fish mucus represents a “reservoir for coral reef bacterial diversity”, awaits further investigation, as does the role of host phylogeny in shaping symbiotic bacterial communities. Future experiments aiming to determine the mode of host “colonisation” used by bacteria, and encompassing a more evolutionary distant set of host species might help answer these questions and improve our understanding of bacterial symbionts of marine fishes.

Reviewed article:

Reverter, M., Sasal, P., Tapissier-Bontemps, N., Lecchini, D. & Suzuki, M. (2017) 'Characterisation of the gill mucosal bacterial communities of four butterflyfish species: a reservoir of bacterial diversity in coral reef ecosystems'. FEMS Microbiol Ecol, 93 (6).

References

Rohwer, F. & Youle, M. (2010) 'Coral Diseases', Coral Reefs in the Microbial Seas. Plaid Press.

Sunagawa, S., DeSantis, T. Z., Piceno, Y. M., Brodie, E. L., DeSalvo, M. K., Voolstra, C. R., Weil, E., Andersen, G. L. & Medina, M. (2009) 'Bacterial diversity and White Plague Disease-associated community changes in the Caribbean coral Montastraea faveolata'. ISME J, 3 (5), pp. 512-521.

Tarnecki, A. M., Burgos, F. A., Ray, C. L. & Arias, C. R. (2017) 'Fish intestinal microbiome: diversity and symbiosis unravelled by metagenomics'. Journal of Applied Microbiology, 123 (1), pp. 2-17.

The curious case of planctomycetes: a bacterium with a “nucleus” challenges eukaryotic evolutionary theory

When picturing a bacterial cell, one might envisage a cytoplasm with free floating ribosomes, plasmids and nucleoid, encapsulated in a peptidoglycan cell wall. Far different from the eukaryote, characterised by a membrane bound nucleus and organelles. However, defying bacterial stereotypes is the planctomycetes, which at first appears as a curious crossover between the two cell types. Possessing phenotypic features such as a lack of peptidoglycan cell wall and compartmentalization of the cell via internal membranes, including the formation of a membrane bound nucleoid. In addition, all species reproduce via budding, with the exception of one marine genera (Phycisphaera) still utilising binary fission. The Planctomycetes form a distinct phylum of the domain Bacteria and their unique cell biology challenges concepts of evolutionary history of eukaryotes and the bacterial cell plan.

Planctomycetes are a very diverse phylum, found in fresh and marine waters and sediments globally, from your garden pond to hydrothermal vents (surviving at 85°C!), existing as a range of nutritional types. Noteworthy are the anammox species, autotrophic anaerobes that oxidize ammonia to dinitrogen, contributing to 50% of all atmospheric N₂. In recent years full scale anammox plants utilize this species for nitrogen-rich-wastewater remediation. Further research of microbial usage in this way could lead to energy generating sewage treatment in the future.  

Arguably the most unusual species of this phylum is Gemmata obscuriglobus. Its nucleoid DNA is encased in a double membrane envelope surrounded by ribosomes, forming a nuclear body analogous to the eukaryotic nucleus. If that wasn’t enough, G. obscuriglobus can also “endocytose”, taking up environmental protein via the formation of internal vesicles, a process analogous to receptor- and clathrin mediated endocytosis of eukaryotes. This nutritional mode and membrane coat-like proteins have only been found in eukaryotes and members of the PVC superphylum (of which planctomycetes is a part).

This cross over of characteristics between the two domains can be explained by four different models:

1) Planctomycetes descended from a complex eukaryote like common ancestor that possessed membrane coat-like proteins (perhaps even from LUCA itself).

2) Planctomycetes evolved these characteristics and provided the gene transfer to a proto-eukaryote lineage which became the common ancestor to all modern eukaryotes.

3) These traits are simply a product of parallel convergent evolution resulting from similar adaptive needs. There is not homology only analogy in structure.

4) Ancient and extensive lateral gene-transfer from evolved eukaryotes occurred.

Regardless of which model (if any) is correct, this suggestion of autogenous development of internal membranes resulting in endomebrane systems and endocytosis like mechanism, discounts the need for symbiotic fusion between archaeal and bacterial cells to produce the modern eukaryote. Further research is needed to understand if planctomycetes possess homologues of genes associated with membrane formation in eukaryotes or if the functions of these eukaryotic genes are performed by functional analogues in planctomycetes.

One thing is for sure, future study of this species is needed to help understand our own evolutionary past.  

Reference paper:

Fuerst, J. and Sangulenko, E. (2011). Beyond the bacterium: planctomycetes challenge our concepts of microbial structure and function. Nature Reviews Microbiology, 9(6), pp.403-413.