A mechanistic understanding of DNA uptake in Vibrio cholerae.

Despite being defined as the etiological agent of cholera in 1854, Vibrio cholerae still causes vast loss of life, with around 130,000 people killed in 2010. V. cholerae inhabits the small intestine of humans, and upon ingestion enters a hyper-virulent state triggered by the rapid change in temperature and pH. During this state, the cholera toxin which comprised of 2 subunits, a and b is produced. The a subunit penetrates the host membrane, and is an ADP ribosyl transferase allowing transfer of NAD to a regulatory G protein. This G protein then activates adenyl cyclase, which converts ATP to cAMP. cAMP stimulates excretion of chloride ions, owing to osmotic and electrochemical gradients potassium and sodium ions are also secreted, thus causing extreme water loss. When not infecting humans, V. cholerae can be found in close association with the chitin-rich exoskeleton of zooplankton; chitin enables external DNA to be taken up by V. cholerae. The transfer of external DNA is an integral part of the V. cholerae life cycle; the cholera toxin in encoded for by the CTX is a bacteriophage derived gene thus V. cholerae needs to acquire this somehow. The ability to uptake genetic material is termed competence. Generally, external DNA can be taken up by bacteria via conjugation, transduction and transformation; these mechanisms are generally well understood.  

Previously, it was thought that DNA acquisition in V. cholerae was achieved through the cyclic extension and retraction action of a pilus, structurally similar to the type IV pili (Tfp). Uncertainty as to how these proteins functioned still remained however. It has been suggested that other competence enabling proteins may be at work, such as ComEA, in spite of this the experimental evidence to support this was lacking until the publication of work by Seitz et al. (2014).

In order to elucidate the importance of ComEA in DNA acquisition, it was necessary to identify its location and activity within the cell. This was achieved through use of translational fusion between ComEA and mCherry (a monomeric fluorophore). The lack of stop codon between the fluorescent protein gene and the gene of interest enables the gene of interest to be tagged. Researchers found that ComEA was located at the cell periphery (see Fig 1)

                                   Fig 1. Location of ComEA protein in V. cholerae cells. Column 1

                                   represents ComEA-mCherry translational fusion, here localization
                                   around the cell periphery can be observed. Column 2 represents DAPI
                                  stained individuals. Column 3 displayed a merge between column 1 and 2,                                     and the localisation of ComEA at the cell periphery is further exemplified.                                     Column 4 depicts phase contrast images. 

Validation of these results was also undertaken by replacing the comEA allele with a beta-lactamase (bla)-comEA translational fusion; these individuals displayed a lesser transformation ability (2.5 x10^-5 ± 3.0 x10^-5 compared with 7.9 x10^-5 ± 2.5 x10^-5). Furthermore, individuals displayed resistance against ampicillin; beta-lactamase can only function in the periplasm of gram-negative bacteria further supporting the evidence of the periplasmic localisation of ComEA-bla. Using fluorescence loss in photobleaching (FLIP), the protein dynamics of cells were examined (see Fig 2). One pole of a cell was subject to bleaching, this prevents fluorescence recovery. Mobile proteins will move to this degraded area and a subsequent decline in fluorescence will be observed. It appears that ComEA is highly motile as observed within the periplasm as indicated by the rapidly declining fluorescence. No net changes in fluorescence were observed in control individuals.

                                     Fig 2. On the left, depletion of relative fluorescence can be observed                                                owing to bleaching, and the subsequent movement of proteins. No change
                                     in fluorescence can be observed for non-bleached individuals.                                                          On the right, the corresponding sites of bleaching, and measurement                                               location of florescence measurement can be observed.
                                     

To determine the necessity of ComEA presence for DNA uptake, all comEA genes were substituted with comEA-mCherry alleles. A transformation assay and localisation of the comEA-mCherry allele encoded protein confirmed the functionality of the chromosomally encoded ComEA. External transforming DNA  (tDNA) was supplemented, and ComEA-mCherry centred, protein clusters were formed. The size of these structures varied in accordance with the length of supplemented DNA. Again, control conditions were set up; periplasmic mCherry alone did not aggregate. Thus, it can be inferred that ComEA binds to transforming DNA within the periplasm, and may be the active agent in the uptake of environmental DNA.

To confirm the presence of external DNA in the peri- or cytoplasm of a cell, a whole cell duplex PCR-based DNA uptake assay was employed. No tDNA was observed in V. cholerae deficient in comEA, those with comEA displayed significant uptake of tDNA however.  That said ComEA has a secondary function, to protect tDNA from degradation by nucleases. Detailed analysis of transformants and translocated tDNA confirmed that protection against nucelases is not the main function of ComEA. Despite this, other nucleases may be at work (other than the few examined), thus it is better to conclude that translocation of tDNA should not be solely attributed to Tfp-like structures.

Predictions of the structural architecture of ComEA were made using in silico techniques; unlike the many other proteins which interact with DNA, ComEA did not possess a helix-loop-helix, or helix-turn-helix motifs. Instead a helix-hairpin-helix motif is thought to be present. This hypothesis was achieved through comparative analysis of protein sequences of helix-hairpin-helix motifs, from a range of bacterial species from which ComEA/ComE homologs were characterised; a large number of conserved regions were present.

A number of other analyses to confirm the role of ComEA were carried out, including examination of the co-operativity for DNA binding, site of tDNA entry and ComEA function in other competent bacteria. However, these will not be reported here.

To conclude, Seitz et al. (2014) presented detailed and convincing examination of ComEA and its role in tDNA uptake. Whilst further work is required to fully elucidate its role, ComEA seems to play an important role in the life-style of V. cholerae. As aforementioned, the gene encoding for the cholera toxin is derived from a bacteriophage, and inability to acquire this genetic information would likely be deleterious for V. cholerae. However, this type of research may have application in the field of medicine; not just to treat cholera but also other bacterial infections which may show similar life-histories and DNA uptake mechanisms.

 Jack 

References

Seitz, P., Modarres, H.P., Borgeaud, S., Bulushev, R.D., Steinbock, L.J., Radenovic, A., Peraro, M.D. and Blokesch, M. (2014) ComEA is essential for the transfer of external DNA into the periplasm in naturally transformable Vibrio cholerae cells. PLOS Genetics. 10; (1). 

Mum... I don’t like it... *spits out food*

Tetrodotoxin (TTX) is known as one of the most potent neurotoxins and is a specific blocker of voltage-gated sodium channels of excitable membranes of muscle and nerve tissues. It was originally believed that it occurred exclusively in pufferfish, however, it has been detected in an array of other species since; in the eggs of the California newt Taricha torosa, other fish such as gobies, and invertebrates including octopuses, crabs, shellfish, flat and ribbon worms. TTX is produced primarily by marine bacteria, such as Pseudoalteromonas tetraodonis, certain species of Pseudomonas and Vibrio, and it appears that it finds its way into pufferfish through the food chain. Tissue-specific distribution of TTX has been widely investigated with food hygiene as the main viewpoint, using mainly the genus Takifugu. It has been revealed that it is commonly distributed in the liver and ovaries, however, localisation in other tissues is species-specific; i.e., besides finding TTX in the liver and ovaries of Takifugu rubripes, it was found to be concentrated in the skin and intestine and marginally present in the testes and skeletal muscle in Takifugu niphobles. Previous work by Itoi et al., has revealed that tissue-specific distribution and the amount of TTX in the mature pufferfish T. niphobles were sex-dependent; female gonads and male liver showed the highest concentrations of TXT followed by male skin. Following this, suggestions that TTX may act as a chemical defence against predators has surfaced, additionally, it has been suggested that it may be used as a pheromone during spawning giving larvae an advantage for survival.

In this study, Itoi et al., (2014) conducted predation experiments, measurement, and immunohistochemical analysis to reveal the effect of TTX as a chemical defence in pufferfish larvae. Predation behaviour was observed using larvae of up to four days old post-hatch; juveniles of Japanese flounder Paralichthys olivaceus and sea bass Lateolabrax sp. were used as the predatory fish against T. rubripes larvae. For T. niphobles larvae, juveniles of fish such as Yatabe blennies (Parablennius yatabei and Omobranchus elegans), gobies (Chaenogobius annularis and Tridentiger trigonocephalus) and Smallscale blackfish (Girella punctata) were used as the predators. Juveniles of G. punctata were also used as predators against T. niphobles eggs. Adult brine shrimp (Artemia sp.) and medaka larvae were used as negative control for the prey. For TTX quantification, the method sounds quite complex and I can insert it is the comments field if anybody wants to review in more detail. However, the overall gist of it was quantifying the positive charged TTX via Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) from 60–100 specimens of pufferfish larvae. Immunohistochemistry followed, by labelling sections of pufferfish larvae with fluorescence and observing under an all-in-one fluorescence microscope (again, if anyone would like the complete method in detail, I can insert it in the comments). Difference in responses of predators (expelling vs swallowing) to TTX- pufferfish and to nontoxic organisms (medaka and Artemia sp.) was tested by the Pearson’s Chi-square test with Yates’ continuity correction.

In the predation experiments for T. rubripes larvae both of the predators ingested the pufferfish larvae but spat them out immediately; similar behaviour was observed in the predators for T. niphobles larvae. Negative control prey (Artemia sp. and medaka larvae) revealed significant differences between the responses of predators to that of the TTX-pufferfish.  LC-MSMS analysis revealed very small amounts of TTX in the egg and larvae of T. niphobles, and T. rubripes, suggesting that the amount of TTX in the pufferfish larvae does not constitute a lethal dose to the juvenile predator fish. However, the results suggest that the predators can sense even trace amounts of TTX in the larval pufferfish. Itoi et al., (2014) summarised that the female parent transfers TTX vertically to the eggs and larvae from the ovaries suggesting beneficial strategies for increasing the survival of egg and larvae in pufferfish. Itoi et al., (2014) went on to conclude that in a natural environment, it is easy to imagine rapid speciation of Takifugu could be a consequence of TTX, however, despite the toxicity of the pufferfish; overfishing is still an issue and can cause major decline in stocks.

Itoi, S., Yoshikawa, S., Asahina, K., Suzuki, M., Ishizuka, K., Takimoto, N., Mitsuoka, R., Yokoyama, N., Detake, A., Takayanagi, C., Eguchi, M., Tatsuno, R., Kawane, M., Kokubo, S., Takanashi, S., Miura, A., Suitoh, K., Takatani, T., Arakawa, O., Sakakura, Y., Sugita, H., 2014. Larval puffer fish protected by maternal tetrodotoxin. Toxicon. 78, 35-40.

Transmission virulence of SVCV in Caspian White Fish

The study describes infection of Caspian White Fish with Spring Viraemia of Carp Virus (SVCV) experimentally and describes the influence of different challenge routes on virulence of the virus.

Spring Viraemia of Carp Virus (SVCV), a member of the Rhabdoviridae Family, in capable of inducing an acute haermorrhagic and contagious viraemia in several cyprinid species such as carps, zebrafish and roach. SVCV account for up to 70% of carp’s mortality during the spring time outbreaks (Ahne et al., 2002). Caspian White Fish, Rutilus frisii kutum, accounts for 60% of the bony fish population in the Caspian Sea in the north of Iran. It is also a highly valued commercial fish.

The aim of the current study was to evaluate susceptibility of Caspian White Fish to SVCV and to test whether the outcome of the experimental infection is influenced by the challenge routes.

The purpose of this investigation was to understand the susceptibility of Caspian White Fish to SVCV and to prevent pandemic from happening. This is because Caspian white fish is highly valuable as a food source and is recently an important cultures species in Iran. Secondly, Caspian White Fish is a wild fish living in large areas of the Caspian Sea and its tributaries and infected fish may spread the virus to large aquatic areas. Thus, a functional SVCV infection model for this fish is vital to minimize SVCV-associated economic losses and to effort to control spread of the infection.

This study have shown that Caspian White Fish are susceptible to infection by SVCV and virulence of the virus could be influenced by the route of transmission. Infective route of transmission (from highest to lowest) – immersion, intra-peritonea (i.p.) injection, cohabitation and oral. They found that immersion was the best infectious route of transmission with the highest mortality of 85% of test subjects, whereas oral transmission showed the lowest mortality rate of at most 10%.

Transmission route through immersion is a natural route of infection, the experimental model shown that this is the transmission route that cause the highest mortality percentage, thus water could be regarded as the major abiotic factor of virus transmission. The authors suggest that this might be due to the entire body surface is potentially in contact with the virus, allowing transmission through gills, skin and fin bases simultaneously. Other reports have also suggested that gills are portal of entry and primary multiplication site of SVCV. Cohabitation model, on the other hand, shown that healthy fish could be infected by secondary infection via horizontal transmission as the mortality kinetics progressed slower than immersion or i.p. injection models.

Inoculation of homogenates from infected fish onto Epithelioma Papulosum Cyprini (EPC) cell monolayer from all treatment groups except oral transmission showed a complete cytopathic effect (CPE) of the original viral strain. This suggests that SVCV remained live during the experimental period in Caspian White Fish and indicates possibility of the development of carrier state in these fish in aquatic environments. Also supported by the RT-PCR results that shown that SVCV is detected in both dead and survive fish. Which thus indicate that survived fish may serve as a reservoir of the virus and transmit infection to healthy population of susceptible fish. They also have shown that SVCV did not lose infectivity potential during the experimental period. However, possibility of change of infectivity and virulence of SVCV following several passages in Caspian White Fish need more investigations.

This study has thus increased our understanding on the mode of transmission in fish disease. It gives an indication that virus infection, in particularly for SVCV, direct contact of virus with host caused highest infection and mortality rate.  

Ghasemi. M., Zamani. H., Hosseini. S. M., Haghighi Karsidani. S. and Bergmann. S. M. (2014). Caspian White Fish (Rutilus frisii kutum) as a host for Spring Viraemia of Carp Virus. Veterinary Microbiology. 170(3-4), 408-413.

Ahne. W., Bjorklund. H. V., Essbauer. S., Fijan. N., Kurath. G. and Winton. J. R. (2002). Spring viraemia od carp (SVC). Dis. Aquat. Org. 52,261-272.

Time to retire the CLAW hypothesis: evidence summarised

So we all know what the CLAW hypothesis is about, as there has been quite a lot of interest in posts on how dimethyl sulphide  (DMS) contributes to this phenomenon. However, over the past two decades, observations in the marine boundary layer (MBL), laboratory studies and modelling efforts have been conducted seeking evidence for the CLAW hypothesis. The results indicate that a DMS biological control over cloud condensation nuclei probably does not exist and that sources of these nuclei to the marine boundary layer and the response of clouds to changes in aerosol are much more complex than was recognised twenty years ago. These results indicate that it is time to retire the CLAW hypothesis, which has been nicely reviewed and put forward by Quinn & Bates (2011), which I highly recommend reading for some background knowledge.

What the CLAW hypothesis assumed

Particles that are less than 300 nm in diameter determine the cloud condensation nuclei (CCN) concentration in the remote MBL and have the potential to change cloud properties. The CLAW hypothesis was based on data available at the time that indicated that (1) non-sea-salt sulphate was ubiquitous in sub-micrometre marine aerosols and (2) concentrations of sodium containing particles at cloud height were negligible (thus ruling out sea salt particles as a source of CCN). Organic species were not considered because little was known about their concentration and composition in the marine atmosphere. The original proposed climate feedback loop (Fig. 1) requires (1) that DMS is a significant source of CCN to the MBL, (2) a change in DMS-derived CCN yields a change in cloud albedo, and (3) a change in cloud albedo, surface temperature, and/or incident solar radiation leads to a change in DMS production. If any one step in the feedback loop shown in Fig. 1 has a small response, the proposed bio-regulation of the climate with DMS will be minimal.

Fig. 1 The climate feedback loop proposed by Charlson et al. 1987 'The CLAW hypothesis'

The post- CLAW view of MBL CCN (there are other sources of CCN!)

Bubble bursting at the ocean surface is a major source of aerosol mass and aerosol number to the MBL. This process introduces both inorganic and organic components of sea-water to the atmosphere. Inorganic components are comprised of sea salt while the organic components are derived from phytoplankton and the large pool of organics in the ocean surface- including TEP which contains microbes (see post ‘The importance of the sea surface microlayer: influences on the atmosphere’). Hence, the concentration of CCN in the remote MBL is a result of emissions of sea salt and organics in sea spray (dependent upon biological activity and wind speed), subsidence of DMS-derived and continentally derived particulates from the free troposphere (dependent upon oxidation and entrainment rates), and particle growth (dependent upon condensation, coagulation and cloud processing). This updated view of the multiple sources of CCN to the MBL is shown in Fig. 2.

The evidence gained over the past 20 years of the significance of non- DMS sources of MBL CCN, the lack of observational evidence for a DMS-controlled marine biota–climate feedback, and the modelled low sensitivity between change and response in each step of the CLAW hypothesis feedback loop all indicate that it is time to retire the CLAW hypothesis. Please remember that retiring CLAW does not rule out a link between ocean-derived CCN and climate- it may just be on a smaller scale that climate regulation happens. It is only that now we have a much better appreciation of the complexity of biogeochemistry and climate physics than when the CLAW hypothesis was first put forward. The interdisciplinary research that it motivated is now needed to address the complexity of multiple sources of CCN to the MBL and potential impacts on climate.

Fig. 2 The new view- Major sources and production mechanisms for CCN in the remote MBL. DMS contributes to the MBL CCN population primarily via particle nucleation in the free troposphere in cloud outflow regions with subsequent subsidence. Sea salt and organics are emitted as a result of wind-driven bubble bursting. 

References:

Quinn, P. K., & Bates, T. S. (2011). The case against climate regulation via oceanic phytoplankton sulphur emissions. Nature, 480(7375), 51-56.

Charlson, R. J., Lovelock, J. E., Andreae, M. O. & Warren, S. G. Oceanic phytoplankton, atmospheric sulphur, cloud albedo, and climate. Nature 326, 655–661 (1987).
This paper introduced the CLAW hypothesis proposing the link between marine biota and climate. 

Found on: http://www.nature.com/nature/journal/v480/n7375/full/nature10580.html%3FWT.ec_id%3DNATURE-20111201

The story of jellyfish taking over the world and microbes ruling the waves

After handing in my dissertation about the effects of hypoxia on jellyfish growth and behaviour, I was a little sad to say goodbye to my gelatinous friends. However, after doing some research I realized that there is no need to say goodbye – luckily microbes are everywhere and associated with everything, and so jellyfish are no exception.  Bacterial communities have been first associated with ctenophores in Florida. Different ctenophore genera were found to contain a unique micro-biota (Daniels and Breitbart 2012), with Alphaproteobacteria, Gammaproteobacteria and Becteroidetes most abundant. There have also been several associations between scyphozoans and microbes. Biofilms with certain bacteria, induce planula larvae settlement in scyphozoan (Vibrio sp. found to induce metamorphosis of Cassiopea sp.). Jellyfish biomass is also highly bio-available to jellyfish associated and or free living heterotrophic bacteria, and bacteria thrive in the DOC released by live animals. Jellyfish could be therefore a suitable substrate for specific bacteria community. 

A recent study (Hao et al. 2014) identified how planktonic bacterial communities respond to the dissolved organic matter released by two species of live jellyfish.  This was done by a combination of ARISA fingerprinting and CARD-FISH analysis and the impacts on bacterial abundance were investigated by flow cytometry. The DOM released by live jellyfish stimulated bacterial communities and induced large changes in bacterial community composition. Gammaproteobacteria dominating the community conducted with Cyanea lamarckii, Bacteroidetes, decreased at the beginning and recovered at the end of the experiment. Gammaproteobacteria and Bacteroidetes dominating the community within Chrysaora hysoscella, were present in equal amounts throughout the experiment. Differences in bacterial community composition and succession indicate, that the DOM released by different jellyfish genera might consist of a variety of compounds, which are species specific. However there is need for chemical characterization of the DOM pools to establish a linkage between certain taxa and specific carbon compounds. 

Although there is room for a lot more work, I think this is a great study in a very important topic! Here it was clearly shown that bacterioplankton communities are not only influenced by dead jellyfish biomass but also strongly impacted by DOM released from metabolic processes of live animals. Findings suggest that jellyfish derived organic matter may function as a newly discovered trophic pathway for organic matter from the benthic environment to pelagic food chains in marine ecosystems. Fundamental transformations in the biogeochemical functioning associated with jellyfish blooms, accompanying a channel of Carbon towards bacterial CO₂ production and away from higher trophic levels can potentially alter the food web dramatically. There is concern of anthropogenic-induced increase of jellyfish blooms; this topic should therefore be given way more attention in the future.

Hao WJ, Wichels A, Fuchs B, Gerdts G (2014) Bacterial communities respond to the excretion of DOM relseased by live jellyfish. PhD Thesis, Universität Bremen.

Predicting how human disease causing Vibrio species will react to climate change.

  

Turner et al. (2014) investigated how seasonal changes in temperature and plankton abundance affect the prevalence of Vibrio parahaemolyticus, V. vulnificus and V. cholerae and subpopulations that harbour clinically associated genes. These Vibrio species are Gram-negative bacilli autochthonous to marine, estuarine and freshwater environments and a few environmental strains are capable of causing illness in humans most commonly indirectly through eating shellfish. Understanding the specific interactions between Vibrio subpopulations and seasonally predictable changes in biotic and abiotic factors can offer critical information needed to better understand changes in risk from environmental exposure.

For a period of one year, surface water and plankton samples (63-200μm and >200 μm) and environmental variables (surface water temperature and salinity) were collected bi-monthly from 12 commercial or public oyster harvesting sites along the South Atlantic Bight of coastal Georgia, USA. DNA samples were taken from water samples and concentrated plankton samples. PCR primers were then used to detect bacteria and clinically associated genes (Table 1). PCR was used to investigate the detection frequency of species-specific gene targets (i.e. present in all strains of a species), instead of culture-based methods. The benefits of using this technique instead include increased sensitivity (i.e. reduced false negatives) and reduced analysis times, which may outweigh risks of false-positive detection. 

V. parahaemolyticus, V. vulnificus and V. cholerae were frequently detected in bulk water and plankton net tow samples. Among all samples (n=210) V. parahaemolyticus was detected significantly more than V. vulnificus, which was detected significantly more than V. cholerae. V. vulnificus and V. cholera targets were only detected when water temperature exceeded >15 °C, however V. parahaemolyticus targets were detected in all water samples even when they dropped below 10 °C. Salinity had little effect on detection of Vibrio, only the ORF8 region of  V. parahaemolyticus was correlated with an increase in salinity. All Vibrio species increased in detection when there were more of an abundance of of chitinous zooplankton (copepods and decapods) and that of chitin- producing diatoms.

The results of this study suggest that some Vibrio species may exhibit a stronger chitin affinity compared with the general Vibrio community and may indicate some competitive advantage for a commensal lifestyle. Combined increases in temperature and shifts in plankton abundance may facilitate an expanded seasonal and geographic range of Vibrio species. In light of climate change predictions, which include increases in sea surface temperature as well as shifts in the range and composition of marine plankton communities, these results may inform future estimates of exposure risk and serve a baseline for future investigations. It would have been useful for the authors to explain the known distributions of the Vibrio  and zoo/phyto-plankton species that were found in this study, as this could then predict which routes and regions these species will colonise and cause problems in. 

Referance: Turner, J. W., Malayil, L., Guadagnoli, D., Cole, D., & Lipp, E. K. (2014). Detection of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae with respect to seasonal fluctuations in temperature and plankton abundance.Environmental microbiology, 16(4), 1019-1028.

Can be found at: http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12246/full

Metabolic plasticity - why I fell for microbes

It is widely accepted that abiotic stress causes unfavourable shift in the coral holobiont. Metagenomics are a relatively cheap technique, which enables extensive community analyses. Changes in bacterial taxa associated with stress enable predictions to be made regarding “healthy” and “disease-related” assemblages. However, the functional role of bacteria within the holobiont is less well understood, and although bacterial taxa can be quantified, the outcome of the new community remains unclear.

Vega-Turber et al (2009) sequentially sequenced the microbiota of corals over a 64-hour period, during exposure to four abiotic stressors: temperature, dissolve organic carbon (DOC), nutrients; nitrogen and phosphorus, and pH. The sequences were annotated for known functional genes and classified into a hierarchical system with three levels.

All abiotic stressors resulted in an increase, at least two-fold, of genes encoding virulence, fatty acid catabolism/ anabolism, and RNA and protein metabolism. Yet at the lower hierarchical level, virulence varied between stressor. During temperature stress the function of virulence genes were related to invasion and intracellular resistance, whereas the virulence function in nutrient treatment was related to toxin and antibiotic production. This might be explained by the difference in bacterial taxa between stress treatments. Temperature stress resulted in a significant decrease in Cyanobacteria, Firmicutes, and Symbiondinium, but had a large increase in fungi. Whereas, in pH stress, the community decreased in Fusobacteria, and Cyanobacteria increased.

The complexities associated with determining the function of bacteria within the holobiont is evident. Changes in abiotic conditions can result in changes in genetic expression, highlighting the bacterial plasticity, which I find fascinating. The implications of synergistic effects of abiotic stress could be severe, and is likely to be why severely degraded reefs struggle to recover.

This study was highly interesting, and as this aspect of bacteria is so foreign to the large majority of people, more studies like this will contribute hugely to the understanding of the holobiont. I think it would be interesting to do a similar study, but on a healthy coral, as everyone is fascinated with how genetic expression changes according to stress, but have not considered how it changes temporally.

It may have been more appropriate to use lower temperatures however, as here only used an increase of 5°C, which is not comparable to changes in natural ecosystems.

Thurber, R. V., Willner‐Hall, D., Rodriguez‐Mueller, B., Desnues, C., Edwards, R. A., Angly, F., ... & Rohwer, F. (2009). Metagenomic analysis of stressed coral holobionts. Environmental Microbiology, 11(8), 2148-2163.