Bacterial nutritional transfer to a hydrothermal vent shrimp

Endosymbiotic relationships between invertebrates and chemoautotrophic bacteria are regularly encountered in deep sea hydrothermal vents. Although most are regarded as nutritional endosymbioses, there has been little direct evidence of the bacteria-host interactions to date. Ponsard et al. (2013) investigated the bacteria-host interactions of the shrimp Rimicaris exoculata, which dominates several Mid-Atlantic ridge hydrothermal vent sites and possesses a large bacterial community in their enlarged gill chamber. They aimed to find out whether there was transfer across the gill integument of inorganic carbon fixed by the chemoautotrophic metabolisms of the bacterial epibionts and to do this the shrimp that were collected from a deep sea site were incubated in pressurized, temperature controlled vessels, with different isotope labeled molecules as well as different electron donors. They then looked at the concentrations of the different isotope-labeled molecules in different tissues of the shrimp as well as in the bacterial biofilms within the shrimp.

Ponsard et al. (2013) found that the bacterial mats of the branchiostegites and the mouthparts showed high incorporation rates of both ¹³C- and ¹⁴C-bicarbonate, with much lower rates in the digestive tract and abdominal muscles. The rate of incorporation was found to be higher when the shrimp were incubated in the presence of either iron or thiosulphate than when they were incubated in seawater alone, without the additional presence of an electron donor, however incorporation of the ¹³C- and ¹⁴C-bicarbonate occurred whatever the incubation conditions were. The digestive gland showed the lowest incorporation rates. When the shrimp were incubated with ¹⁴C-acetate the incorporation rates were once again highest in the mouthparts and inner bacterial biofilms, indicating that acetate is a suitable carbon source for the bacteria. Incorporation rates for ³H-lysine were much lower than for acetate, but were significant and suggest that the bacteria can also use lysine. This analytical data was confirmed by autoradiography (pictures taken of segments of the shrimp showing the radiolabeled areas).

The results of this study produce strong evidence for a mutualistic relationship between R. exoculata and their chemosynthetic epibiotic bacteria, although the fixation rates here are lower than for other chemosynthetic symbionts of other hydrothermal vent invertebrates, so it is suggested that the shrimp may also feed on molecules and bacteria from the chimney walls of the vents. They also provide evidence disproving a previous hypothesis that crustaceans cannot take up dissolved organic matter or dissolved organic carbon as the shrimp appear to be able to take up dissolved organic molecules such as acetate and lysine across their gill integument. Further research should focus on what the dominant energy (carbon) source of these bacteria is as well as whether they are capable of mixotrophic metabolism or whether a consortia of bacteria is involved depending on the prevailing conditions.

It was really interesting to read a study that actually used a deep sea organism in the lab, as previously there have been issues in maintaining them in a pressurized environment, however, I found the method for this review a little difficult to follow as it’s not a method I’m familiar with at all! I also thought it was really interesting to read of direct evidence for the bacterial transfer of nutrition to an organism, and not just the consumption of the bacteria by the shrimp.

Ponsard, J., Cambon-Bonavita, M., Zbinden, M., Lepoint, G., Joassin, A., Corbari, L., Shillito, B. et al. (2013) Inorganic carbon fixation by chemosynthetic ectosymbionts and nutritional transfers to the hydrothermal vent host shrimp Rimicaris exoculata. The ISME Journal. 7, 96-109

Coral Dinoflagellate's have gone VIRAL!

A healthy coral consists of a symbiosis between the coral animal and a bacterium, for example dinoflagellate algae. A coral can also consist of microbes from Archaea or fungi which exist on the mucosal layer. In a healthy reef ecosystem different corals consist of different combinations of host and symbiont genotypes. The different phenotypes expressed from these different combinations contributes to the survivability of the reef, corals having variability in their tolerances to; temperature, light, growth rates, and disease resistance. Coral bleaching is a major environmental issue, the causes of which have been attributed to long term environmental stress or bacterial induced. Much work has also been done on the effects of viruses on bleaching which is still currently inconclusive. Thus far there is evidence that indicates herpes-like viruses target the coral animal, but less is known if any viruses target the dinoflagellate partner within the holobiont.

In this study they looked for evidence for viral infection in the symbiotic dinoflagellate Symbiodinium within the Montastraea cavernosa coral through generating a cDNA library from a Symbiodinium culture and comparing this with isolated DNA from Symbiodinium within a heat-stressed coral and a normal coral. It was found that large DNA viruses associate with both the free-living algae as well as the symbiont within the coral, which is the first genomic evidence of this. Viral transcripts were found which are similar to known viruses which infect planktonic dinoflagellate’s, suggesting that novel viruses target symbiotic dinoflagellate’s. More research is needed to expand upon the known viromes for marine ecosystems as shown by the identification of novel dsDNA and ssRNA viruses within this study. If these viruses’ are lytic to the dinoflagellate partner then they are an important factor when assessing coral health.

This paper was intriguing in its approach to isolating viral cDNA librarys, although the accuracy of the data could have been improved by increasing the number of samples pyrosequenced, as for both heat-stressed and control corals N=1. Despite this, the paper does highlight another aspect to the assessment of coral health which can be further studied.

Correa, A.M.S., Welsh, R.M. & Thurber, R.L.V. (2013) Unique nucleocytoplasmic dsDNA and þssRNA viruses are associated with the dinoflagellate endosymbionts of corals. The ISME journal 7, 13-27.

Macroalgal competitors cause shifts in coral-associated bacteria

Corals harbour diverse eukaryotic and prokaryotic microorganisms that form dynamic associations with their coral host. The ecological roles of coral associated microbes are not, at present, fully understood but it is clear that they are of significant importance to coral health as shifts in these microbiota have been shown to cause detrimental effects on coral health. As a result of the plight facing corals and the potential significance of their associated microbes, many scientists have begun studying the shifts in microbiota in relation to anthropogenic change, such as increased sea temperatures and lowered pH levels. However, anthropogenic change is also causing shifts in the dominant species in coral reefs themselves. Raised nutrient levels have led to an influx of macroalgae to colonise and compete with reef-building corals, macroalgae are known to produce a wide range of metabolites to defend against bacterial colonisation, either by inhibiting behaviour or causing bacterial lysis, such metabolites are also likely to indirectly affect coral-associated bacteria.

Morrow et al. (2012) aimed to study what effect macroalgae had on coral-associated bacteria by applying crude extracts from three common Caribbean macroalgae directly on to two species of reef-building coral found in Florida and Belize. Denaturing gel electrophoresis (DGGE) was used to examine changes in the bacteria assemblages associated with the surface mucus layer (SML) of both coral species.

Results showed that bacterial communities within the SML of both coral species were significantly altered from their initial communities by some macroalgal extracts on both Florida and Belize reefs. The extent to which extracts shifted the bacterial assemblages depended on both the coral host and type of macroalgae tested. Furthermore, there was also a significant increase in glutathione S-transferase activity (an essential enzyme for detoxification) within coral tissues exposed to macroalgal species, suggesting a heightened level of stress.

The findings from this study are an important first step toward examining the impact of macroalgal compounds on coral-associated bacteria and could be another potential stressor effecting corals, especially in areas with low herbivore activity. Although authors show the presence of shifts in the coral-associated bacteria, they fail to isolate the associated bacteria, and so, the bacteria which are affected remain unknown. Furthermore, the structure of the coral host provides a heterogenous habitat, with distinct and diverse communities but the authors of this study only look at one (SML), this could of hidden further shifts in coral-associated bacterial assemblages. Future research should include the identification of the coral-associated bacteria and also study the communities associated with the coral tissue surface and SML.

It seems obvious to me that anthropogenic shifts may favour coral competitors causing stress to corals through competition for light, nutrients and space. However, I had completely missed the potential effect of metabolite defences produced by competitors on coral-associated bacteria, a consideration which I believe many scientists studying anthropogenic change on corals have also overlooked, and so, is my reason for reviewing this paper.

I welcome any questions.

Morrow, K.M., Ritson-Williams, R., Ross, C.,  Liles, M.R. and Paul, V.J. (2012) Macroalgal Extracts Induce Bacterial Assemblage Shifts and Sublethal Tissue Stress in Caribbean Corals. PLOS one 7, e44859. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0044859

Precence of mobile genetic elements in Vibrios

Integrating conjugative elements (ICE’s) are mobile genetic elements that integrate and replicate along with the host chromosome. ICE’s of the SXT/R391 family have been studied quite extensively, though most of the research done to date has been on strains of Vibrio cholerae. ICE’s of the SXT/R391 family were first documented to confer resistance to antibiotics and heavy metals, however in since then they have also been found to help regulate motility and biofilm formation. The SXT/R391 ICE’s were also found to contain Restriction-Modification systems (RM). restriction (R) enzymes recognise specific DNA sequences cut the DNA, which the modification (M) enzymes then methylate. This functions as a defence mechanism against invading genetic material. Balado et al (2013) set out to examine the distribution and ecology of these particular genetic elements in the aquatic ecosystem. 

Balado et al (2013) collected 203 strains of 23 distinct species of Vibrio, which were screened for the presence of ICE’s. The strains were isolated from internal organs collected from 18 separate aquaculture facilities of the coast of Spain and Portugal. Cultures were grown at 25^oC on tryptic soy agar or broth, with added NaCl. Resistance to HgCl₂ and tetracycline was tested on strains found to contain ICE’s. Cultures were screened for ICE’s using PCR methods with primers targeting the integrase gene, resulting amplified ICE’s where then sequenced. ICE’s were also tested for motility, this was done by mixing both donor and recipient cultures and growing the mixture on LB agar plates. After initial incubation donor and recipient bacteria were separated growing on antibiotic containing agar using known variations resistance between the bacteria. Strains containing ICE’s were also subjected to a phage infection assay, to determine the effectiveness of RM systems found to be present. 

Initial PCR amplification uncovered the presence of two ICE’s of the SXT/R391 isolated from V. splendidus and V. alginolyticus, which were distinctly different from previously documented ICE’s from this family. It was found that these particular ICE’s are mobile, and may be transferred to a wide range of Gamaproteobacteria, and may confer resistance to both tetracycline and heavy metals. After testing for phage protection it was also concluded that both the of the new ICE’s conferred some degree of protection to bacteriophage infection. 

Overall Balado et al (2013) suggest that the presence of ICE’s is much more widespread in the marine environment than previously thought. The ability to spread to Gamaproteobacteria , which are often found in aquaculture, suggests that these genetic elements may have a much wider distribution than was previously expected. This link to aquaculture may have further effects as it may spread to pathogens and provide resistance to antibiotics. Finally the authors suggest that due to the presence of the RM systems spread and maintenance of these particular genes would be favourable, and these ICE’s may be maintained due to functionality past the antibiotic and heavy metal resistance already associated with the SXT/R391 family of ICE’s.

Balado, M., Lemos, M., L., Osorio, C., R.. (2013). Integrating conugative elements of the SXT/R391 family from fish-isolated Vibrios encode restriction-modification systems that confer resistance to bacteriophages. FEMS Microbial Ecology. 83 (1), 457-467.

The VBNC state and resistance to environemental stress factors.

As a common method to survive harsh conditions bacteria enter a viable but non culturable state (VBNC). In this state metabolic activity is at a minimal level, but as soon at favourable conditions return they revert to a normal growth. Vibrio vulnificus is recognised as one of the major sea-food associated pathogens, and it is well known that when water temperatures are below 15 ^oC they enter a VBNC state. While it is generally accepted that bacteria enter this VBNC state, the effects this has on resistance to environmental stress factors is less well known. Nowakowska and Oliver (2013) set out to document the effect of several environmental factors on the normal culturable and VBNC state of V. vulnificus. 

For this study they used two different V. vulnificus strains: C7184 as a clinical strain and JY170 as an environmental strain. Cultures were either used for direct exposure to environmental stresses or stored at 5 ^oC to induce the VBNC state, after which they were exposed to the stress factors. Immediately before and after exposure to the challenges a viability assay was conducted , by determining cell membrane integrity, to determine the effects of the stresses on the culture. For cells from the VBNC state that were exposed to the stress factors, they resuscitated by culturing overnight at 22 ^oC and then plated onto agar to determine viability. Stress factors applied were: Temperature, Oxidative stress, Osmotic stress, pH, ethanol, antibiotic and heavy metals. 

Overall results indicate that cells in the logarithmic growth phase appear to be highly susceptible to the tested environmental stresses, but that cells from the VBNC state appear to be mostly resistant. Both the clinical and environmental strains lost culturabillity when exposed to temperatures over 42 ^oC, but VBNC cells were able to recover after overnight resuscitation. The same was found to be the case for cells exposed to ethanol. When exposed to hypersaline environments log phase cells for both the clinical and environmental strain lost culturability, but interestingly the VBNC cells for the clinical were found to be highly sensitive, where as the environmental strain was found to be significant resistance. A similar result to the salinity stress was found in response to oxidative stress and both high and low pH, with the environmental VBNC cells exhibiting a higher tolerance compared to the other cultures. When cultures were exposed to antibiotics and heavy metals it was found that yet again the VBNC cells exhibited a higher level of resistance. Though it appears that there is a difference depending on the heavy metal compound, with several copper containing compounds being resisted but others causing cell death.

This study is of interest as many of the cleaning methods used in the food industry rely on cleaning with highly acidic or alkaline substances, pasteurisation, or similar methods. This would suggest that these cleaning methods are ineffective. While the growing cells may be removed, the VBNC cells may be able to return to a normal logarithmic growth and cause infection even after supposed cleaning and sterilisation. Overall this means that while we think we may be safe and the food industry is clean, we may still be subject to many dangerous pathogens. 

Nowakowska, J., Oliver, J., D.. (2013). Resistance to environmental stresses by Vibrio vulnificus in the viable but nonculturable state. FEMS Microbial Ecology. 84, 213-222.

Bivalve symbionts. (exam relevant)

This paper is relevant to a commonly occurring exam question regarding symbiosis and I also found it an extremely interesting read. 

Symbionts have been noted and observed in all types of ecosystems, a lot of them marine with the corals and their zooxanthellae to jellyfish and their symbionts as well.  This study looked at marine bivalves, more specifically the species, deep sea mussel- Bathymodiolus- which has for years had an association with chemosynthetic bacteria.  It has been clearly noted that from these studies the bacteria are usually only associated/ restricted to the adults except hosts that linearly transfer them.

These mussels occur commonly and globally at hydrothermal vents and cold seeps and harbor their intracellular symbionts in their gills and these are known as bacteriocytes. The individuals are just like the Euprymna- bobtail squid- these mussels acquire their symbionts from the external environment even if the process is still unknown specifically. TEM studies have demonstrated that juveniles as small as 0.12mm appear to already harbor symbionts.

This study by Wentrup et al (2013) wanted to observe the process and specificity of the infection process and they saw a shift from widespread infection to a specific colonization of the gills in these juvenile mussels, hence the title of the paper being this.

In this study the authors essentially just used FISH to observe the bacteria from beginning to end of the infection process to see whether symbiotic acquirement was specific or whether it was just from a whole body colonisation and then specific development of certain bacteria. They were able to look at both symbionts and the other eubacterial probes, as they used FISH specific signals for these symbionts.

What they saw was that in the juvenile individuals gills at a certain size there were only symbiont specific bacteria and no eubacterial probes present thus indicating that only the specific symbionts can colonize and enter the gills, from further analysis of all host tissues they saw a combination of both regular bacteria and symbionts thus demonstrating that all round colonisation of host tissues does infact then lead to specific symbiont colonisation of the gills. This specific colonisation of just the gills and not any other areas with symbionts on top of eubacteria is most probably due to the fact that their presence does not outweigh their nutritional input. This is what was concluded by the authors.

This paper is very interesting because once again it shows that FISH can be applied to numerous areas and has once again been used to show colonisation (specific) of symbionts within a host. I’m sure after this more studies will be done to see exactly how the developmental process occurs.

Hope you enjoyed this read, the paper is available at:

http://www.nature.com/ismej/journal/vaop/ncurrent/pdf/ismej20135a.pdf

 

 

 

Reference:

Wentrup,C., Wendeberg,A., Huang,J., Borowski, C. & Dubilier. N. (2013). Shift from widespread symbiont infection of host tissues to specific colonization of gills in juvenile deep-sea mussels. International society for Microbial ecology.

The “Extended” Coral Holobiont: Truth or Fiction?

The “Extended” Coral Holobiont: Truth or Fiction?

The term “coral holobiont” refers to the mutualistic relationship between coral animals and a diverse range of microorganisms (dinoflagellates, bacteria, archaea, fungi and viruses). As discussed in lectures and many previous blogs, there is a growing appreciation in science for the fundamental dependence of all macroscopic organisms on microbes. In particular there is an increasing opinion that a healthy coral reef has a certain signature of associated microbial life, and in shifting into a diseased state, the associated microbial consortium also changes. A major contribution to the growing appreciation of microbial life is the rapid development of molecular and genetic tools to assess diversity, population numbers and metabolic interactions within ecosystems. As powerful molecular survey techniques have taken over the literature, unfortunately the limitations, and potential contaminations, of such methods in many cases have been forgotten. The reviewed paper does not use fashionable molecular techniques, but instead aimed to visualise any adherent microbial presence on the surface of normal, presumed healthy, Porites compressa using scanning electron microscopy (SEM).

Johnston & Rohwer (2007) collected coral fragments from a reef around Coconut Island, Hawaii. Fragments were mounted onto plastic splints and kept in aquaria for several days for observations (polyps extended or contracted), before being fixed for electron microscopy. The fixation technique used was particularly important as it was exceptionally fast; the modified Parducz fixative was fast-acting enough to fix expanded polyps in a state of only partial shortening of the tentacles. Other traditional fixatives cause living corals to contract into their skeleton before the coral’s structural and metabolic components are inactivated and chemically preserved. In contrast to many previous reports, Johnston & Rohwer found no evidence for microbial life living directly on coral epidermal cells. Specifically the authors report that corals with extended polyps were clean of directly adhering microbes and evidence of adhering material was limited to clumps, or flocs, of detrital material on the occasional sample. Samples which had permanently retracted polyps had developed a stable mucous sheet which became heavily colonized by both prokaryotic and eukaryotic microbes. However, the microbes did not penetrate the mucous sheet and the animal’s epidermal cell surfaces remained sterile.

In 2003 Nancy Knowlton and Forest Rohwer published a key paper proposing corals as a more extensive holobiont. In this important publication the authors recognised microbial associates of reef corals are largely unknown, but describe preliminary studies which indicate that individual coral colonies host diverse assemblages of bacteria. Since 2003 there has been a huge explosion of coral-microbe investigations. The results reported by Johnston & Rohwer (2007) in no way rule out the theory of coral-microbe mutualisms, however they conceivably change the way we think of the overall holobiont. Perhaps the more extensive holobiont consists of a dynamic community of microorganisms hovering in the boundary layers of water immediately above the epidermis rather than in the tissues or directly adhered to cell surfaces. It is difficult to understand just how the reviewed work fits into the coral holobiont literature, it is hard to imagine that hundreds of molecular surveys are incorrect and merely an artefact of contamination or mucus associated bacteria alone. In my opinion the most important conclusion of Johnston & Rohwer’s work is that molecular techniques should not proceed in isolation, methods such as SEM and FISH should be included as protocol to increase understanding of the spatial distribution of microorganisms in the coral holobiont. I think the study would have been more complete if accompanied by a molecular survey of a subset of samples; this would have provided direct comparisons between techniques and given the contradictory results more validity.

Johnston, I. S., & Rohwer, F. (2007). Microbial landscapes on the outer tissue surfaces of the reef-building coral Porites compressa. Coral Reefs, 26(2), 375–383.

 http://link.springer.com/article/10.1007%2Fs00338-007-0208-z