Emiliania huxleyi: a pretty hardy microbe!

The oceans are an important source of atmospheric trace gases, such as Dimethyl sulfide (DMS), which originates from the enzymatic breakdown of the secondary algal metabolite Dimethylsulfoniopropionate (DMSP). DMS is believed to promote the formation of cloud- condensation nuclei (CCN) in the marine boundary layer, which may increase the earth’s albedo resulting in cooling also known as the CLAW hypothesis. The direct biological control of DMS over CCN has been questioned (Levasseur, 2013) but nevertheless DMS is still of central importance to atmospheric chemistry. Understanding the consequences of anthropogenic climate change, such as ocean acidification, on marine DMS production and its sea-to–air transfer is of principal importance. This study provides the first insight into the synergistic effects of elevated pCO₂ (associated with ocean acidification) and temperature on DMSP/DMS production and growth in Emiliania huxleyi – a globally important phytoplankton coccolithophore.

Emiliania huxleyi strain CCMP 373 was cultured in a laboratory, which formed exponentially growing, semi- continuous cultures of the high- DMS- producing haptophyte. Cultures were exposed to four different treatments: ‘ambient’, ‘future CO₂’ (+CO₂), ‘future temperature’ (+T) and ‘greenhouse’ (future CO₂ and temperature; +TCO₂). Temperature was increased from 17 (ambient) to 21°C and pCO₂ concentration was increased from 385μatm (ambient) to 1000 μatm which presents the possible increases by the year 2100. Cell growth, DMS and intracellular DMSP production was then quantified.

When exposed to 21°C, cell diameter decreased by 1.6%, but interestingly caused a 2.9% increase in growth rate based on cell number. Previous studies exposing different E. huxleyi strains to elevated CO₂ show varied responses, which highlight the high phenotypic plasticity in the species. An example of the effects this could have is shown in a study when a natural plankton assemblage from the Equatorial Pacific preferred larger diatoms to smaller Phaeocystis sp. which demonstrates the changes in nutrient cycling and competition which may result if E. huxleyi cell size changes, this is of concern as E. huxleyi is such an important food source to many zooplankton.

The results of the +CO₂ experiment suggest that DMS production may decrease 50% under future CO_2, which is an alarming decrease in the ‘climate-cooling’ DMS. However predicting the direction of future DMS production cannot be assessed from experiments that manipulate a single parameter in isolation so this result I do not think is of much importance. The results of the ‘greenhouse’ (+TCO₂) treatment suggest that future DMS production could be the same as under ambient conditions in this certain strain of E. huxleyi. This is interesting that there is no effects, but the response of E. huxleyi to changes in pCO₂ and temperature could be strain specific and further work is necessary to assess the roles of DMSP in the physiological response of algae to temperature and pCO₂ stress.

Intracellular DMSP concentrations were not significantly higher in the +CO₂ treatment, but the temperature increase from 17 to 21°C resulted in a significant increase in mean intracellular DMSP. Showing that temperature is probably the may influencing force in this experiment.

Another implication that I found interesting was that elevated pCO₂ could lessen oxidative stress, in turn decreasing DMS production. This is expected as it has been previously shown that limited pCO₂ increases oxidative stress in E. huxleyi and other phytoplankton which then leads to an increase in intracellular DMSP and DMS. Future studies to investigate the oxidative stress and DMS relationship, I feel, is the next step.

 Arnold, H. E., Kerrison, P., & Steinke, M. (2013). Interacting effects of ocean acidification and warming on growth and DMS‐production in the haptophyte coccolithophore Emiliania huxleyi. Global change biology, 19(4), 1007-1016.

Very interesting review: Levasseur, M. (2013). Impact of Arctic meltdown on the microbial cycling of sulphur. Nature Geoscience, 6(9), 691-700.

Can be found via: http://onlinelibrary.wiley.com/store/10.1111/gcb.12105/asset/gcb12105.pdf?v=1&t=i27f0r64&s=2ca0e8f1dad274c0d0e6314f97bd4c8cbbf585ec

The different defence responses of haploid and diploid Emiliania huxleyi

Marine phytoplankton are significant contributors to global primary production, as a result they are an important food source for many small ocean predators. One of the most important causes for mortality is by predation from microzooplankton (20-200mm), which is thought to be responsible for consumption of a global average of 67% of phytoplankton daily production. Although many potential defences have been suggested, there has been relatively little documentation for defences against predation in phytoplankton. Two potential avenues of research in this area include whether defences are either induced or constitutive and whether different life cycles (haploid and diploid) in phytoplankton have different defence responses. Kolb, A (2012) set out to test these two hypotheses with Emiliania huxleyi.

                                                                                         

In order to carry out this experiment, two strains (haploid and diploid) of E. huxleyi were either exposed to the ciliate predator Strombidinopsis acuminatum for 24hrs or were left naïve (no exposure). The ciliate ingestion rates of predator exposed versus naïve E. huxleyi were then compared at 3 points over 30 minutes.  The prey was considered to have a defence response when ingestion rates on naïve cells were higher than ingestion rates on predator-exposed cells.

The results showed that there was indeed an induced defence response in E. huxleyi, however, the method of this defence was not identified. The strongest candidate recognised by the author was one of a chemical nature, this included DMSP which we have previously discussed with Colin. Due to the continued feeding on other organisms with low DMSP levels in this experiment by S. acuminatum, it can be hypothesised that DMSP could act as a deterrent only when the predator is in close contact. As well as high DMS levels potentially deterring predators through being a warning of toxic acrylate, the author theorises there is the possibility for E. huxleyi to increase exudation of DMS to “play stressed”. The release of DMS can signal low nitrogen levels, so capable E. huxleyi (under low stress, adequate nutrients and high predation pressure) may be able to take advantage of the system and mimic this signal, fooling the predator to find more palatable prey. 

Diploid E. huxleyi, responsible for the large algal blooms, have been thought to be well defended against predators. However, this research suggests that there is little to no discernible induced defence. A simple idea for the lack of defence response could be down to chemicals being blocked by the calcium carbonate shell therefore limiting any signals picked up by the predator. Haploid induced defences on the other hand appeared to have a significant effect at reducing ingestion by S. acuminatum by up to 43%. This raises the question as to why the two life cycles still exist if one is clearly better at defending against predators. A simple explanation is haploid E. huxleyi are more prone to photoinhibition and therefore the diploid life cycle can maintain high growth rates in high nutrient conditions. This suggest that the two life cycles exist to take advantage of separate niches, one in oligotrophic and the other in nutrient rich waters.

The lack of an inducible defence in diploid E. huxleyi contradicts the idea of bloom forming E. huxleyi being well defended. This may be due to the lower need for a defence, with high inorganic carbon content from coccolith production, it may cause diploid E. huxleyi to have a lower nutritional value. Further research in this area may help to demonstrate the predators preferred life cycle of E. huxleyi through a simple choice test. Experiments in the field, sampling over the course of an E. huxleyi bloom may help identify how the diploid life cycle deter predators if not from an induced defence. The haploid life cycle also needs to be looked at in more detail, however, it is relatively hard to extract which remains a barrier to research. Exploring the separate life cycles in more detail will allow us to further our understanding of the key role they play in the environments and niches they inhabit, this is especially important for this species due to the large role they play in nutrient and carbon cycling in our waters.

Kolb, A. (2012). An inducible predator defense in the marine microalga Emiliania huxleyi (Prymnesiophyceae) is linked to its heteromorphic haploid-diploid life cycle.

http://cedar.wwu.edu/wwuet/211/ (Thesis)

DMSP: a bioindicator for corals?

Dimethylsulfoniopropionate (DMSP) is an organic sulphur compound which is produced by many types of phytoplankton. It plays various roles for phytoplankton and in the marine microbial world e.g.: anti predation, osmolytes and cryoprotectants.  Until recently, it was thought that phytoplankton were the only producers of this molecule, however recently it was discovered that the gastrodermis of corals symbiotic zooxanthallae contains DMSP lyase, and their symbiotic dinoflagellates contain high levels of DMSP. Researches in the past have found that stressed conditions causes an increase of reduced oxygen species accompanied by increased DMSP.  If DMSP production in corals changes in stressed conditions, it could become a bioindicator, which would be very useful with the increase of temperatures and severe weather patterns.

The study was carried out at several different stations in the Great Barrier Reef, one at the Heron Island Research Station’s aquarium complex though out a succession of winters (September) and summers (February) from 2001 to 2003.  Another base was at Nelly Bay, on the eastern coast of Magnetic island. This fringing reef has many environmental impacts: increased nutrients, suspended sediments due to dredging, nutrient run-off, sewage discharge and increased bleaching events. The studies on this fringing reef were conducted 5 months after the severe bleaching event in the summer of 1994. This site was used to compare to other reefs as “stressed”.

This study measured two different factors with Acropora intermedia, : DMSP  production between 2001-2003, and the effects of raising the seawater temperature by +2 °C measured by production DMSP and levels of cellular DMSP and  Chlorophyll a(Chl. a). They then compared this to the Magnetic Island reef (consisting of Pocillopera damicornis ) off the coast of Magnetic Island in the central Great Barrier Reef,  to study the differences between stressed an non stressed  corals. 

Acropora intermedia collected from Heron Island showed distinct seasonal cycling of DMSP with increased production during the summer, and lower in the winter. This was distinct each year despite the severe bleaching event of 2002.  The year with the largest production of DMSP was the summer months with the high sea surface temperature (SST), solar radiation and rainfall.  By increasing the water temperatures by +2 °C in the summer and winter, A. intermedia’s DMSP production increased by 45%, compared to the winter 2001 and summer  2002.  The cellular DMSP increased in the winter of 2002 and summer 2003, almost half a year after coral bleaching. The authors suggested that the increase was due to higher temperatures and high rainfall. Cellular chlorophyll a concentrations decreased from 2001-2001 (bleaching) but then increased the next summer as the coral recovered.

When comparing the Heron Bay site to the Magnetic Island site, they found that there was a highly significant correlation between cellular DMSP and chlorophyll a. This could show an adaptive response to increased numbers of reduced oxygen species which had been produced during the bleaching event, and also may have helped the corals recovery. The Increased cellular DMSP could also have been caused by changes in the symbiont community. Measurements have been made on Acropora coral from many different locations from across the Great Barrier Reef which leads to the suggestion that changes in the cellular/tissue concentration is a sensitive indicator to stress

This study suggests that DMSP is a bioindicator for all types of stress and does put forward some evidence for this theory, however there was little evidence that any of the stress that caused the DMSP to be released was manmade, it may have been mainly temperature. If this is the case, although it is interesting, how could we use this indicator? If it was affected by the sediments/ nutrient run-off, then we could use the indicator to reduce the anthropogenic pollutants in the area but we cannot change temperature. On the other hand it may be interesting to see if this effects when pathogens attack the corals, as they can use chemotaxis to follow DMSP to stressed corals which are easier to attack. 

Jones, Graham B., et al. "The effect of coral bleaching on the cellular concentration of dimethylsulphoniopropionate in reef corals." Journal of Experimental Marine Biology and Ecology 460 (2014): 19-31.

http://www.sciencedirect.com/science/article/pii/S002209811400166X

A simple centrifugation method for improving the detection of Ostreid herpesvirus-1

Ostreid herpesvirus-1 (OsHV-1) is one of the key pathogens causing summer mortalities in the Pacific oyster in Europe, Australia, New Zealand and the US. The virus affects oysters of all ages and size classes. Despite extensive studies on the virus itself, little information exists on the mechanisms of transmission and the spread of the disease in open marine environments. As a result there is little knowledge about methods to detect OsHV-1 in sea water.

It has been suggested that seawater may act as a medium in the horizontal transmission of the virus. A study by Paul-Pont et al. (2013) has led to a hypothesis about the attachment of the virus to plankton-like particles. Viruses, like other microbes, attach to particles in their environment and so their fate and transport is associated with those particles.

Methods such as ultrafiltration, tangential flow filtration, ultracentrifugation, precipitation, and adsorption of viruses onto charged membranes, have been used to concentrate viruses from water samples. However these methods are not practical when processing large numbers of, as is the case for epidemiological studies onOsHV-1. These methods also cannot be utilised in the assessment of potential virus association to particulates of varying sizes, as many of they require or result in virus dissociating from matter within the sample matrix. Evans et al. (2014) assessed and compared several simple centrifugation methods to detect OsHV-1 in seawater samples, in such way that a large number of samples can be processed efficiently and also assessed the potential for particulate attachment of the virus using a simple filtration methodology.

Low speed centrifugation of seawater at 1000 × g for 20 min, then testing the resulting pellet, improved OsHV-1 detection rates by two fold compared to the unprocessed seawater samples. Results suggest that OsHV-1 may be attached to particles large enough to be pelleted at low g-force, as well as in the form of small particles. Filtration of seawater using low protein binding filters could not be used to assess OsHV-1 particle attachment, due to interactions between particles, free virus or free viral DNA and the membranes.

For something to pellet at such low force it means the particles have to be approximately 7-12μm in diameter or larger. This suggests that as least some of the OsHV-1 virions are attached to particles large enough to be centrifuged at low speed or are present in larger viral aggregates. Viral DNA was detected equally well in supernatant after low speed centrifugation. This suggests that OsHV-1 virions are not only attached to particles, of the size mentioned above, but are also present as free viruses, viruses associated with smaller particles, or as free DNA. Microbial communities are constantly growing, changing and cycling so it can be assumed that while some viral particles are attached others may not be intact or exist free in sea water. This supports the hypothesis that the distribution and transmission of OsHV-1 in natural sea water might reflect attachment to some form of particulate matter.

This study has shown that detection by qPCR in seawater improved two fold by testing the pellet obtained by low speed centrifugation, compared to testing unprocessed seawater samples. This provides a simple method suitable for testing large numbers of water samples that will be applicable to epidemiological studies of OsHV-1. This has further application as such a simple method can be used to increase the detection of other viruses that have levels below the limits of quantitation of the qPCR. Simple filtration of seawater samples through low protein binding filters did not prove to be an appropriate method for assessing OsHV-1 association and/or attachment to particles of different sizes, probably due to dynamic interactions of the virus within the sample matrix itself as well as the filter membranes. This finding therefore needs to be explored further to identify the true nature of the viral interaction with particles as this will be critical to understanding the distribution and transmission of OsHV-1 in natural environments. Although this method of detection provides very fruitful results for OsHV-1 there was little mention if this method can be used for the detection of other viral strains. Further experiments could test this and may help reduce costs and increase efficiency of other viral research.

Evans, O., Paul-Pont, I., Hick, P., & Whittington, R. J. (2014). A simple centrifugation method for improving the detection of Ostreid herpesvirus-1 (OsHV-1) in natural seawater samples with an assessment of the potential for particulate attachment. Journal of virological methods. Oct 5;210C:59-66. doi: 10.1016/j.jviromet.2014.09.023. [Epub ahead of print]
http://www.sciencedirect.com/science/article/pii/S0166093414003784

No bacterium is an island - one strain can have a disproportionate impact on the DOC pool

One of the largest pools of carbon on Earth is marine dissolved organic carbon (DOC) generated from primary production. Bacteria play a crucial role in regulating and recycling this carbon, however, little is known about how individual bacterial strains contribute to this process. Previous studies have suggested that just a few taxa contribute to DOC degradation and Alteromonas is a taxon that has been observed to dominate microbial communities in seawater. A study by Pedler et al. (2014) seeks to quantify how a particular strain of Alteromonas (Alteromonas sp. strain Scripps Institution of Oceanography or AltSIO) consumes DOC in a coastal environment.

The study conducted a microcosm experiment, in which the survival of the AltSIO strain was tested, using competition for nutrients and grazing pressure from protists as variables. Samples were collected in San Diego, California, from a range of depths between 0-100 m. The various treatments comprised of the seawater community, pure cultures of AltSIO and AltSIO with other bacterial cultures. Parameters such as cell counts, cell concentrations and concentration of DOC over 5 days (short-term) were measured to determine how microbial cell abundances and DOC changed over time.

The study found that AltSIO could potentially degrade as much DOC as microbial assemblages with a high species diversity over the 5 days. Over longer periods, however, overall microbial diversity was important for degradation of recalcitrant DOM. The authors of the study suggested that this was due to the various types of metabolism associated with high biodiversity within the assemblages, enabling different bacteria to take advantage of the DOM pool.

AltSIO is a large, fast-growing bacterium, making it able to quickly respond to increases in substrate supply, reduced competition and grazing pressure. AltSIO is able to effectively compete against smaller bacteria for resources, regardless that smaller bacteria are theoretically better able to acquire nutrients with an increased SA/V ratio. Being a large bacterium, AltSIO is likely to be exposed to increased grazing pressure but variations in the bacterivorous grazer populations do not explain the scenario entirely. Despite this top-down control, this strain is able to persist and contribute a large amount of carbon in the environment.

These properties that enable AltSIO to rapidly take advantage of increased nutrient supply enhance its ability to degrade DOC disproportionately, compared to the total bacterial biomass and abundance within the ecosystem. Large bacteria may have a greater influence on DOM recycling than previously thought and this asymmetry has important implications for the fate of carbon, its role in the ecosystem and food webs.

This study alters our understanding of how bacterial assemblage composition influences global carbon cycling and should be considered when predicting the fate of carbon in the oceans. It would be interesting to extend this idea to other coastal regions to determine if other dominant bacteria, such as Pseudoalteromonas and Vibrio species, have this effect globally. For those who wish to study this topic in more detail, the details of the methods are in the supplementary information and I have included a link for this with the link for the paper.

Reference:
Pedler, B.E., Aluwihare, L.I., and Azam, F. (2014) Single bacterial strain capable of significant contribution to carbon cycling in the surface ocean, PNAS, 111, (20), 7202-7207.

http://www.pnas.org/content/111/20/7202.full.pdf+html

http://www.pnas.org/content/suppl/2014/04/09/1401887111.DCSupplemental