Group post: Effects of photoperiod length upon the ecophysiology of Crocosphaera watsonii

Diazotrophic phytoplankton are important contributors to the primary productivity of the world’s oceans facilitating the carbon pump and carbon sequestration in often oligotrophic regions, with an estimated global input of 100-200 Tg^-1 N y^-1. Eukaryotes such as Trichodesmium and Richelia had been previously thought as the primary open ocean N₂ fixers. However, unicellular UCYN cyanobacteria are now thought to account for ~10 % of nitrogen production in the world’s oceans.

Eco-physiology and growth dynamics of the recently characterised pelagic UCYN cyanobacteria is poorly understood with efforts focusing on temperature and nutrient concentrations found to exhibit control through enzyme kinetics and niche structure respectively. Dron et al., 2013 investigated the effect of photoperiod length (PPL) upon the carbon/nitrogen metabolism and cell cycle of the best studied culturable UCYN-B cyanobacterium Crocosphaera watsonii. It has been previously shown that 50% of C. watsonii genes exhibit a diel expression pattern. However the driver of the observed diel variation is not clear. Are cellular processes timed upon a circadian clock, or under direct control by PPL?

Continuous culture technique was employed to maintain duplicate cultures of W8501 C. watsonii in the exponential growth phase. Culture 1 was maintained under a short photoperiod (8 hr light:16 hr dark), while culture 2 was maintained under a long photoperiod (16 hr light: 8 hr dark). Irradiance followed a natural sinusoidal pattern with a maximum flux of 130 µmol quanta m^-2 s^-1 at the mid-light point. Nitrate and nitrite culture concentrations were measured every 8 hours, whilst population density and growth rates quantified every 24 hours. Nitrogen and carbon cell contents were estimated every 4 hours. DNA topology and compaction was investigated by staining DNA with Sybr-Green and quantifying the fluorescence.

Population growth (cell division) was higher for the long PPL, peaking daily at  ~10x10⁶ cells ml^-1 compared to ~6x10⁶ cells ml^-1 for the short photoperiod. Indeed cell division was so much slower for the short PPL that the dilution rate for the continuous culture had to be reduced to 0.15 day^-1 compared to 0.2 day^-1 for the long PPL.

Cell division occurred consistently in the mid-light phase for all PPLs demonstrating that C. watsonii has the ability to change the cell cycle timing to match PPL. Since N₂ fixation must occur in the dark phase it is ecologically advantageous for cell division to occur in the light-phase.

A conserved 5-6 hour time lag between the end of cell division and the onset of nitrogenase activity was identified. Most likely this time window is required to assemble and activate the enzymatic machinery for N₂ fixation, which must be separated from photosynthesis. DNA synthesis occurred in the dark phase, concomitantly with the highest rates of N₂ fixation in all three PPLs. This synchrony allows the most nitrogen to be available for DNA synthesis.

Interestingly this study identified that for C. watsonii a longer photoperiod didn’t correlate with higher carbon and nitrogen storage. Fixation and assimilation of both carbon and nitrogen was significantly lower for both the normal and long PPL compared to the short PPL. Higher carbon content at the end of the light phase may provide more energy for N₂ fixation, while higher levels of nitrogen fixation may help increase carbon fixation, both processes augmenting the other.

C. watsonii exhibited markedly different cellular energy allocation under contrasting light regimes. The physiological responses were similar under balanced and long PPLs in that rapid cell division was favoured over somatic growth (i.e. C/N storage). Conversely, under the short PPL, cell division was almost half that of the long PPL whilst favouring C/N storage and somatic growth. It is possible that this plastic response is an adaptation to changing environmental conditions, perhaps seasonal changes. The range of C. watsonii extends ~40 °N and 30 °S, with marked seasonal variability in PPL. When conditions are unfavourable, somatic growth may be favoured to increase cell stores facilitating population survival during the winter, whilst in summer when the photoperiod is long, high cell division is favoured and the population blooms. This study identified that as much as 80 % of nitrogen fixed by C. watsonii is lost to the ambient water. The reason behind this wastefulness is unknown, but is undoubtedly a vital nitrogen input into often nitrogen limited ocean waters.

This paper was a useful contribution to the study of C. watsonii and the significance of photoperiod length upon their ecophysiology. However, just a single strain of C. watsonii was investigated and it would be informative to know if different ecotypes exist within distinct water bodies. For example, would a warm water equatorial strain lack the adaptability of its poleward brothers in preference to faster proliferation rates? Given the possibility of intraspecies variability within bacteria, it would be wise to express caution when inferring the results from this study upon other species as well.

Matthew Zietz, Freya Radford, and Jack Jones

Primary reference:

Dron, A., Rabouille, S., Claquin, P., Talec, A., Raimbault, V., & Sciandra, A. (2013). Photoperiod length paces the temporal orchestration of cell cycle and carbon‐nitrogen metabolism in Crocosphaera watsonii. Environmental microbiology, 15(12), 3292-3304.

Enough sugar for a fun-gi?

Despite being ecologically successful the marine fungi are often neglected. These fungi can interact pathogenically or mutualistically with both micro- and macro- algae. As such, these fungi tend to have high metabolic rates and rapid growth. The most prevalent forms of fungi in the marine environment are filamentous ascomycetes, that said several basidiomycetes and yeast species flourish. Fungal diversity largely depends on environmental factors such as temperature and salinity. Fungi play an important role in providing nutrition for detritivores, achieved through the breakdown of POM. The high biomass of marine fungi coupled with the high cellular enzyme activity of several substrates recently observed, suggest that microscopic filamentous fungi may play a role in biogeochemical cycles. As such, this paper aimed to address how glucose metabolism (and furthermore heterotrophic activity) changes in accordance with temperature, and discuss the findings within the context of biogeochemistry and ecology.

Samples of seawater and sediment from the coast of Chile were collected, filtered, and cultured on agar (glucose, yeast extract and Emerson’s YpSs agar). Using DNA analysis, the fungal species were identified as, Penicillium decumbens, P. chrysogenum (water column) and Acremonium strictum, Fusarium fujikuroi and F. sporotrichioides (sediment). Each of these species was diluted with an OD concentration of ~0.85, subject to temperatures of 9, 13 and 20°C with 5g L^-1 of glucose, sampled daily for 7 days and  the respiration (respirometer and Optode sensors) and biomass measured using microscopy, epifluorescence and ATP content measured (experiment 1). Growth potential was then assessed with fungi subject glucose concentrations of 0.001, 0.01, 0.1 and 1 g L^-1 (experiment 2).

As anticipated, growth rates were greatest at higher temperatures (20°C) with 55-153 µg C hypha day^-1, growth at 9°C was significantly reduced with 21-51 µg C hypha day^-1 observed. Growth at 13°C was intermediate. Of the five species studied, no significant differences in growth at 0.001 and 0.01 g L^-1 glucose were observed, here carbon biomass reached 0.32 mg C and 0.45 mg C at 9 and 20°C, respectively. Growth of marine fungal species at 9 and 20°C and the four glucose concentrations all showed similar trends, with growth increasing until a plateau at the highest glucose concentration. Generally, oxygen consumption increased with increasing temperature. The variation because of body size was examined by comparing other fungal species, and their relative oxygen consumption; only 2; only 24% of the variation could be accounted for.

This investigation is particularly interesting in the perspective of climate change. The interaction of temperature and oxygen consumption is a fairly well studied subject in marine invertebrates, thus comparing these findings with those for other taxa and discussing these findings in the context of Portner’s aerobic scope hypothesis would be very interesting. As aforementioned in previous posts, all aspects of science need further investigation, however alongside viruses, I think marine fungi need more attention. Their role in biogeochemical cycles, symbioses and nutrient cycling are not known. 

Marcelo E. Fuentes, Renato A. Quiñones, Marcelo H. Gutiérrez, Silvio Pantoja (2014), Effects of temperature and glucose concentration on the growth and respiration of fungal species isolated from a highly productive coastal upwelling ecosystem. Fungal Ecology.13:135-149.

Viruses in the mucus layer of scleractinian corals

Viruses attract increasing interest from environmental microbiologists seeking to understand their function and role in coral health. Viruses can cause profound changes in the commensal prokaryotic and eukaryotic communities, which may lead to coral disfunctioning or diseases, or can conversely act as a natural phage therapy for diseased scleractinians by removing bacterial pathogen. There is a lack of basic ecological information regarding viruses, such as in situ abundance in corals, their variability among the large diversity of scleractinian species, their colonization processes and the ecological links with their hosts. In this study by Nguyen-Kim et al., (2014), the specific objectives were to determine the concentrations of coral-associated viruses in the superficial mucus layer of seven different scleractinian species collected in a coral reef of Whale Island (Vietnam), compare

their abundance with that measured in the surrounding water, and investigate the potential links between viral distribution in coral and the abundance, physiology and diversity of their bacterial hosts.

On average, the concentrations of viruses and bacteria were, respectively, 17- and two-fold higher in the mucus than in the surrounding water. The examination of bacterial community composition also showed remarkable differences between mucus and water samples. The percentage of active respiring cells was nearly threefold higher in mucus than in water. Interestingly, a positive and highly significant correlation was observed between the proportion of active cells and viral abundance in the mucus, suggesting that the metabolism of the bacterial associates is probably a strong determinant of the distribution of viruses within the coral holobiont.

Recently, high virus-to-prokaryote ratios (VPRs) have also been recorded in the mucus layer of different aquatic organisms, including corals, and this has become the foundation of a novel paradigm, called the bacteriophage-adherence-to-mucus (BAM) model. This model  demonstrates that the mucus inhabiting viruses constitutes an efficient biological barrier against bacterial pathogen colonization. According to the BAM model, the great abundance of phages in the mucus layer can be explained by the high adhesive affinities between phages and the mucinproteins of the mucus. As the result of the lytic action of such pathogens, the model predicts that bacterial abundance in mucus will be thus reduced, and conversely that of viruses will increase, unavoidably leading to an increase of the VPR. Dissolved organic matter composition has been demonstrated to shape bacterial diversity, and mucus provides a large abundance of particular substrates that select for a community composed of specialists (likely symbionts). The availability of substrates is expected to select for different functions and composition of the host communities, and therefore to also select for their specific phage communities.

In biotopes with high virus concentrations, such as in coral mucus, one might thus expect high host diversity according to the ‘kill the winner’ hypothesis. However, the absence of correlation between viral abundance and the DGGE diversity indices within the data set does not support this postulate. One of the reasons might be because DDGE method only reveals the diversity of the most dominant members of the prokaryotic community, and that most of the diversity is probably located in the rarest biosphere. Another reason would be that the bacterial community comprised few competitive specialists that account for most of the diversity. Furthermore lysogeny might also be more important than expected in coral mucus, which could explain the absence of clear links between the abundance of free viral particles and diversity indices of their host.

Coral mucus, given its unique physicochemical characteristics and sticking properties, can be regarded as a highly selective biotope for a specialized symbiotic microbial life associated with highly abundant viruses. The physiological state of the prokaryotic associates was seemingly the best determinant of viral abundance in the mucus, suggesting that most of the coral-associated viruses are phages, which might be produced by the most active fraction of the cells, through a lytic pathway. Further studies are now needed to make an accurate evaluation of lytic and lysogenic mediated infections to get better insights into the effective role of viruses in the coral microbiological balance. The results of the present study open new perspectives for the investigation of these understudied viral particles in reef environments. Temporal and spatial variability in coral communities may result in changes in relationships between viruses and bacteria so a wider range of sampling sites and dates need to be included to give a better understanding of coral microbial communities.

Nguyen‐Kim, H., Bouvier, T., Bouvier, C., Doan‐Nhu, H., Nguyen‐Ngoc, L., Rochelle‐Newall, E., ... & Bettarel, Y. (2014). High occurrence of viruses in the mucus layer of scleractinian corals. Environmental Microbiology Reports, 6(6), 675-682.

http://onlinelibrary.wiley.com/doi/10.1111/1758-2229.12185/pdf

A tool for understanding the distribution of methanogens in the deep sub-seafloor

Very little is known about the distribution of Methanogenic archaea (methanogens) in the sub-seafloor despite methane in marine methane hydrates being mostly of microbial origin. Methanogens are the final step in anaerobic biodegradation of organic matter in sediments, producing methane as a by-product. Understanding the distribution of methanogens is therefore imperative to understand the methane hydrate formation process. Isopranl glycerol ether lipids are unique to arcahaea and have been used as biomarkers in many studies. One group, the archaeal polar lipids, have also been used to show the presence of living rather than fossil archaeal biomass. However, the reliability of this technique had been called into question with compounds used at present being more stable than originally thought. One potentially more reliable technique is using archaeal lipids containing a tertiary –OH group. Due to the labile nature of this tertiary alcohol it is thought to better represent recent archaeal activity. Oba et. al. (2014) set out to investigate whether the use of different archaeal lipids can more reliably estimate the distributions and populations of methanogens in two sites in the Nankai Trench.

Cores were collected from two borehole sites and samples were frozen immediately, the outer 10mm was then removed to limit contamination. The zones of concentrated methane hydrates were distributed >100m below the seafloor and only found in the sandstone layers. The sediment core samples were then analysed for total organic carbon content using a Yanako MT-5 CHN analyser and lipids were extracted with the Bligh and Dyer method, separated and identified by gas-chromatography.

Hydroxyarchaeols are specific to methanogens and anaerobic methanotropic bacteria (ANME) and archaeol is ever-present in methanogens and widespread in other archaea. Their co-occurrence therefore does not necessarily mean they are from same biologically source. However, the results from this study strongly suggest that the polar lipids in the marine sediment were derived from a common producer. Furthermore, by comparing the carbon isotope values and depth profiles of hydroxyarcaeols and archaeols it strongly suggests that methanogens are the source and not ANMEs. Due to the strong correlations between two types of hydroxyarchaeol the candidate clades of methanogens are believed to be Methanoloccales and Methanosarcinales. The use of these archaeal lipids is therefore good potential contender for assessing the distribution of methanogens in sub-seafloor sediments. However, considering the small proportion of methanogens in prokaryotic communities (<1%) there seemed to be a higher than expected concentration of sn-2-hydroxyarchaeol, suggesting detected levels were mostly fossil. This questions the validity of this lipid as a biomarker, however, the life expectancy of this lipid is thought to be very short relative to geological timescale. This therefore means the depositional age of deep sub-seafloor sediments is much older than that of the life expectancy of this lipid, making this lipid a valid biomarker for in situ methanogens.

The use of hydroxyarchaeols and archaeols has been demonstrated here to be an effective tool as the use of a biomarker for methanogens in deep sub-seafloor sedmients. This will allow further research to study the processes of methane formation, such as insight into specific methongens involved and the causes of any fluxes in methane production as seen in other methanogen communities. It also has some relevance to global warming, increasing methane in the atmosphere is seen as a cause for concern. With the marine ecosystem contributing around 10% to global atmospheric methane concentrations, understanding the methanogen communities in the deep sub-seafloor could become a valuable area of research. This paper is a good starting block for potential research into the deep sub-seafloor methanogen communities and provides some good preliminary insight. However, it may be worth conducting analyses such as PCR or FISH on core samples as well as the analysis carried out here to be more certain about the conclusions made.

Oba, M., Sakata, S., & Fujii, T. (2014). Archaeal polar lipids in subseafloor sediments from the Nankai Trough: Implications for the distribution of methanogens in the deep marine subsurface. Organic Geochemistry.

http://www.sciencedirect.com/science/article/pii/S0146638014002861

Missing viruses!

Viruses have a major role in the evolution, ecology and mortality of marine ecosystems. Only in the last few decades it has been realised that the viruses in the ocean may not be dominated by DNA bacteriophages. It has been suggested that RNA viruses are extremely important in marine processes and that they have been understudied. Since the first reports of the RNA virus infecting Heterossigma akashiwo, there have been many other cases of RNA viruses found in the oceans. These RNA viruses are found with molecular surveys using primers that target RNA-dependant RNA polymerase genes and are now classified into several families including Picornavirales and Reoviridae. Metagenomic surveys show that Picornaviads seem to dominate the marine RNA viruses however, few isolates are so far available.

Up until now there have been technical difficulties with quantifying virus populations due to stain sensitivity making it hard to detect small genome single stranded viruses. This study addresses for the first time, whether RNA viruses have a substantial contribution to the overall virus population by taking a different approach and estimating the relative abundance of RNA viruses from their masses in sea water.

Samples for this study were taken from Kane’ohe Bay, Hawaii and were taken straight to the lab to be filtered through 0.22µm filter. The samples of viral communities were concentrated using iron flocculation and buoyancy density gradients. The nucleic acid was then removed from the samples using fraction analysis. Samples for RNA analysis were treated with DNAase to avoid a non-specific signal and fractions were pooled to obtain a more accurate RNA estimate and overestimation was avoided using a flourmetric assay and comparing to a control of purified known RNA virus. A metagenomic analysis was used to confirm the quantity and composition of RNA viruses in the samples.

When buoyancy was accounted for and fractions of nucleotides taken out that were thought to not be of virus origin, it was seen that there was a higher percentage of RNA than DNA viruses in the samples. The metagenomics analysis found that over half of these were most similar to known eukaryote–infecting RNA viruses, 50-57% of which were single stranded viruses mainly in the Picornvirales order. Only 0.02–1.2% were unknown double stranded RNA viruses. These findings suggest that the eukaryotic-infecting viruses are as abundant as the well-studied bacteriophages. If these RNA viruses are as dominant as this data suggests, it will have a huge impact towards our understanding of plankton ecology.
There was however, limitation in the experiment due to the use of a 0. 22µm filter and the relatively small range of virus buoyancy tested as this resulted in a large number of viruses still being unaccounted for in the samples. This shows that this study is just a first step into really understanding the magnitude of RNA viruses in the oceans. It shows that current virus detection methods may be needed for adjustment to account for even the smallest viruses, and that this is an area in which work needs to be undertaken to understand further the role of viruses in marine plankton ecology.

Steward, G. F., Culley, A. I., Mueller, J. A., Wood-Charlson, E. M., Belcaid, M., and Poisson, G. (2013). Are we missing half of the viruses in the ocean? ISME J. 7, 672–679.

Microbes associated with coral mucus and their potential role as an antibiotic

It is a well-known fact that there has been a significant decrease in coral populations worldwide (Harvell et al., 1999).

A study done by Ritchie (2006), looked at the mucus layer formed by the Elkhorn coral Acropora palmata. This species is highly susceptible to environmental stressors. The mucus layer is known to protect the corals from stressors in such ways as presenting a physical barrier, removal of microbes via ingestion and sloughing etc. However, little is known about the use of this mucus layer as a protector against disease.

This study by Ritchie (2006) looked at whether the coral mucus layer gives coral antibiotic resistance due to the bacteria associated with it providing antibiotic activity. An experimental approach was also used to look at the potential of mucus as a selection medium for coral symbionts. A symbiont in this case refers to bacteria that benefits from the coral mucus, whilst also benefitting the coral.

12 colonies of A.palmata were used. Bacteria samples were taken from the mucus and then diluted in seawater, plated on glycerol artificial seawater agar and kept at 24°C. Potentially invasive microbes were introduced which were known to be related to coral disease. Antibiotic producing corals were selected for and mucus associated bacteria were then used to test for the production of anti-bacterial compounds.

A.palmata has been documented as one of the worst affected corals worldwide, now listed as a threatened species. The study done by Ritchie (2006) shows the mucus from A.palmata can provide antibiotic resistance against gram positive and negative bacteria, a variety of microbes and from a pathogen associated with white pox disease. A.palmata associate with bacteria that can produce some sort of antibiotic resistance, showing that microbes play a key role in the protective properties of the mucus. It also has a role in the control of the associated microbes. A very interesting result showed that mucus collected at periods of increased sea surface temperature showed a decrease in antibiotic activity, suggesting temporal variability in mucus protection against stressors (Ritchie 2006).

This variability in antibiotic resistance due to temporal and spatial variability would be a very interesting subject for future work to look into (Ritchie, 2006). This is obviously a very important and current topic due to corals becoming subject to immediate environmental stressors. Therefore it would be very interesting to look at how the antibiotic activity varies under different conditions in order to predict possible effects on natural populations. The fact that the environmental stressors are only going to become more intense only makes this subject of more importance.

Ritchie,K. (2006) Regulation of microbial populations by coral surface mucus and mucus-associated bacteria. Marine Ecology Progress Series. 322 , 1-14.

Harvell, C., Kim, K., Burkholder, J., Colwell, R., Epstein, P., Grimes, D., Hoffmann, E., Lipp, E., Osterhaus, A., Overstreet, R., Porter, J., Smith, G., Vasta, G. (1999). Emerging Marine Diseases: Climate Links and Anthropogenic Factors. Science. 285, 1505-1510.

Pollution monitoring using Photobacterium phosphoreum

Detection of marine contaminants is a very important factor in pollution management in which biological indicators can play a big part. Bioluminescence in microbes can provide a rapid visible indicator of this contamination making it a cheap and easily reproducible method of detection. This is possible as the production of luciferase coding for bioluminescence in marine bacteria can be switched off or suppressed in response to environmental pollution. Photobacterium phosphoreum is a bacterium regularly used to detect marine pollution in commercial systems, normally done by recording bioluminescence in discontinuous culture. However, this study looks at the importance of maintaining a continuous culture and immobilising the bacterium in order to measure light output using fibre optics.

The first aim of the experiment was to evaluate bioluminescence retention in immobilized cells. This was done by comparing immobilized suspensions of the culture in NaCl solution and alginate to a control of mobilized culture on Difco agar. These samples were all then stored at 4, -20 and -80ᵒC using glycerol as a cryoprotectant, recording bioluminescence weekly using a luminescent spectrophotometer allowing luciferase activity to be expressed as relative light units (RLU). This showed that luminescence is directly proportional to viable cell number in the culture making it an appropriate measure of the toxic effect of various chemicals. However there was limitation with the detection limits of instrumentation used meaning outside a 10³-10⁶ cells per ml range the relationship was non-linear and harder to detect. At 4ᵒC the NaCl solutions only retained their bioluminescence for 2 weeks, compared to the alginate cultures which retained light emission for up to 4 weeks without significant decline. At -20ᵒC and -80ᵒC the cultures in alginate also retained their bioluminescence for a significant amount of time. At -20ᵒC there was no improvement of light retention compared to 4ᵒC, however at -80ᵒC there was significant improvement in light retention. This is thought to be due to the fact that P. phosphoreum is a temperature sensitive bacterium, preferring ambient to low temperatures. At these lower temperatures it goes into a state of cryogenic protection, which protects the cells and their metabolic function, including the process of bioluminescence.

Other immobilization matrices were also tested by having their bioluminescence measured every hour whilst in storage, these included agarose, low melting point agarose and polyacrylsmide. Agarose and low melting point agarose lost significant bioluminescence almost immediately suggesting the gelling materials may have had a harmful effect on the bacteria. Whilst polyacrylsmide lost all bioluminescence straight away making it an unsuitable medium for this method.

The solidifying agents strontium chloride and calcium chloride were also tested for use as immobilizing the cell cultures. Calcium chloride was shown to be an insufficient solidifying agent, whilst Strontium chloride performed well, allowing it to be used in subsequent experiments involving the testing of toxic substances.

The second aim of the experiment was to put this method into practice and determine the effects of reference toxic chemicals including; Pb(NO₃)₂, NaAsO₂, NiCl₂, CdCl₂, HgCI₂, SDS and pentachlorophenol, on the bioluminescence of P. phosphoreum. Each chemical was tested using the optimised immobilization method, using alginate-glycerol media stroed at -80ᵒC. This involved using cultures in a flow- through cell method to achieve a semi-continuous culture, allowing for conditions to be controlled, including oxygen supply, pH and the concentrations of reference chemicals in the system.

 It was found that P. phosphoreum was still sensitive to all reference chemicals. However, when compared to mobile cultures the toxic effects were seen to be decreased as there was less contact between the toxic chemicals and the cells in cultures where the cells could not move around. Immobilization is thought to stabilize the biological activity in the culture which aids the use of this technique as a biosensor or pollution monitoring probe.

This experiment provides a great qualitative method for testing toxic chemicals on P. phosphoreum which allows for rapid and fairly reliable detection of chemicals in the environment. There are several factors such as the detection limits of instruments and the slight difference in sensitivity between mobilized and immobilized cells, which need to be accounted for when using this method. However, this is a step forward in monitoring and detecting marine pollution and its effects in the environment.

Chun U.H., Simonov N., Chen Y., and Britz M.L. (1996) Continuous pollution monitoring using Photobacterium phosphoreum. Resources, Conservation and Recycling, 18, 25–4.

Oculina patagonica; evidence in support of the coral holobiont and probiotic hypotheses

Vibrio shiloi is known as a causative agent in the bleaching of the coral Oculina patagonica in the Eastern Mediterranean. Bleaching events are correlated with high water temperatures in the summer of ~30°C with up to 80% of O. patagonica colonies bleaching. However, since 2002 widespread resistance to V. shiloi has been observed for O. patagonica although the species has continued to bleach on a seasonal pattern since. Mills et al., 2013 conducted a study to ascertain firstly, the current bleaching cause for O. patagonica, and secondly, the mechanism that O. patagonica became resistant to V. shiloi. Corals as all invertebrates lack as adaptive immune system with the ability to produce antibodies, so what was the mechanism for this resistance?

Several prevalent bacterial strains were isolated (culture dependent methods) from healthy O. patagonica colonies in order to test for the presence of causative bleaching and V. shiloi protective bacteria. DNA was extracted from pure colonies and 16S rRNA sequences identified using GenBank. Isolated bacterium EM1 was found to be a strain of Vibrio coralliiyticus (99.8 % identity), whilst bacterium EM3 was found to be a new candidate species/genus with only 94.8 % similarity to Vibrio hepatarius. Cross-streaking and liquid culture experiments identified that bacterium EM3 produced an extracellular diffuse inhibitor that prevented growth of V. shiloi specifically, but had no effect upon bacterium EM1.

O. patagonica fragments collected from reefs in the Eastern Mediterranean were subject to three experimental laboratory treatments. Treatment 1 (colonies n=28) induced bleaching through gradual temperature increase to mimic summer environmental conditions. The temperature was raised to 31 °C causing 86 % visual coral bleaching. The second treatment (n=34) mimiced the first, gradually increasing water temperatures, but at 28 °C a 24hr antibiotic treatment (nalidixic acid, chosen for its effectiveness against coral associated bacteria) was administered. The water temperature was subsequently raised to 31 °C. Only 29 % visual bleaching was observed, whilst zooxanthellae counts were 5.3 fold higher than temperature induced bleaching without antibiotic treatment. In the third treatment independent addition of V. shiloi (coral colonies n=12) and bacterium EM1 (coral colonies n=6) to antibiotic treated O. patagonica coral fragments at 31 °C increased visual bleaching to 83 % and reduced zooxanthellae counts by 50 and 75 % respectively.

This study demonstrated that antibiotics can be protective against heat-induced bleaching for Oculina patagonica. Traditionally bleaching has been thought to be mediated by reactive oxygen species ‘leakage’ from the decoupling of photosynthesis in the zooxanthellae host. Since antibiotics would have little effect upon the rate of photosynthesis and ROS production, alternative causes for bleaching must be sought. The coral holobiont and probiotic hypothesis fits well with the results from this study.

The coral probiotic hypothesis is a recent advent of the holobiont hypothesis and postulates that beneficial bacteria acquired by the coral host help defend against infection, disease and bleaching. In this way corals can rapidly ‘adapt’ to changing environmental conditions. It is likely that excessively high temperatures act as a structuring mechanism upon the associated microbial community of the coral leading to an increase in expression of virulence genes and bleaching. Vibrio species are known coral pathogens, whilst in this study inoculation of O. patagonica with V. coralliiyticus induced bleaching in 5 out of 6 antibiotic treated corals. Antibiotic treatment in this study reduced water Vibrio spp. counts 670-fold, correlating with a significant reduction in bleaching, implicating vibrio species such as V. coralliiyticus as important bleaching mediators under stressed conditions.

Interestingly, the diffuse antimicrobial produced by bacteria EM3 seems to explain the ability of O. patagonica to resist V. shiloi infection as observed in the Eastern Mediterranean since ~2002. The incorporation of this strain into the coral holobiont with its antimicrobial action may confer a fitness advantage to O. patagonica in the Eastern Mediterranean sea. However the exact nature of this proposed antimicrobial is unknown and deserves further investigation. It is also unknown how the coral acquired strain EM3, presumably from the water column. Due to the specific nature of host/microbial association and temporal and spatial variation in microbial water column communities it would be informative to discover how ubiquitous strain EM3 is.

This study and its discovery of causative bleaching bacteria and protective bleaching bacteria is an effective step forward in the illumination of coral health, increasingly important in the 21^st centuary as we try to understand and conserve our ocean’s rainforests. I would like to see further study look at the comparative actions and interactions between the traditional ROS mediated bleaching hypothesis and the more recent hypothesis of microbial mediation. Likely their action is not independent, but synergistic. 

Reference:

Mills, E., Shechtman, K., Loya, Y., & Rosenberg, E. (2013). Bacteria appear to play important roles both causing and preventing the bleaching of the coral Oculina patagonica. MEPS, 489, 155-162.

Coral associated bacteria and their potential role in the sulfur cycle.

Marine bacteria play a central role in the degradation of DMSP to DMS and acrylic acid. This DMS that is produced is important in areas such as cloud formation. It has recently been found that some scleractinian corals contain high levels of DMSP and DMS, associated to the symbiotic algae they possess. This raises questions about the role that coral reefs play in the cycling of sulfur (Raina et al., 2009).

Most of the DMSP produced in coral ecosystems is released to the surrounding area. It has been estimated that other types of bacteria degrade 50-80% of the DMS produced, however that pathways and degradation are poorly understood. The coral associated with bacterial communities are very diverse.

A study done by Raina et al., (2009) investigates the potential of DMSP, DMS and acrylic acid as a driver of coral associated microbial community. They used these compounds as a sole carbon source to isolate bacteria from 2 coral species and then compared these to coral associated microbiota.

The coral species used by Raina et al., (2009) were Acropora millepora and Montipora aequiluberulata. They collected 3 colonies of each species from the Great Barrier Reef. 3 types of sample were taken from these corals; these included tissue slurry, mucus slurry and crushed skeletal material. There were 5 replicates for each of these sample types for both species. As mentioned earlier, the carbon sources used were DMSP, DMS and acrylic acid. The control used was made up of basal medium and the carbon source only. DNA extraction was carried out on single strain liquid cultures using an extraction kit and then PCR carried out to amplify bacteria and DMSP (Raina et al., 2009).

4 genera of bacteria, Roseobacter, Spongiobacter, Vibrio and Alteromonas, isolated on agar possessing either DMSP or DMS as a carbon source made up the majority of clones from coral tissue and mucus. M.aequiluberulata was cotained 28% Roseobacter sp, whilst A.millepora contained 59% Spongiobacter genus. Vibrio species were associated with DMS and acrylic acid enrichment and were often found in coral mucus. This finding suggests that they may be a natural part of coral ecosystems. Results gained show that DMSP, DMS and acrylic acid can be used as a nutrient source for bacteria associated with coral reefs (Raina et al., 2009).

There is some evidence for species-specificity, however this is poorly understood. Overall, the potential of DMS, DMSP and acrylic acid as a role in structuring coral microbe communities is an area that has been little investigated (Raina et al., 2009).

This study is a very good starting point for investigating bacteria that can metabolise the sulfur compounds previously mentioned. It would be interesting for further studies to be carried out in order to get a more complete spectrum of the bacteria that can metabolise these compounds. Raina et al., (2009) sees this study as being a possible perquisite for looking into coral health.

Raina, JB., Tapiolas, D., Willis, B., Bourne, D.. (2009). Coral-Associated Bacteria and Their Role in the Biogeochemical Cycling of Sulfur. Applied and Environmental Microbiology. 75, 3492-3501

  

Molecular probing on Pfiesteria piscicida to assess variability.

Pfiesteria piscida is a marine dinoflagellate related to toxic algal blooms. They pose threats to areas such as public health and natural resources. They’re also related to large-scale fish kills, which are well documented, being recorded since 1991. They’re particularly well documented in the North Carolina Albermale-Pam-Lico estuaries. In some cases they have caused deaths of one billion fish in a single event. These will have clear ecological and economic impacts, as well as possible threats to humans (Conye et al., 2001)

There are high levels of P.piscicida found along the Atlantic coast of the US. These estuaries in the mid-Atlantic are at a particularly high risk of toxic blooms. Therefore, the levels of P.piscicida need to be correctly assessed in order to develop monitoring strategies of the dinoflagellates. Monitoring strategies are in place, using such techniques as light microscopy. However, these methods aren’t sensitive or accurate enough aren’t species-specific and are unable to detect numbers. With monitoring of such importance, more sensitive techniques are needed.

A study done by Conye et al., 2007, used 2 different techniques for detection and enumeration of P.piscicida in the Delaware inland bays and Pokomoke River. Samples were collected from shallow water estuaries in this area. DNA was then extracted from each water sample. The samples were cultured in f/2 medium. The first technique used on the samples was denaturing gradient gel electrophoresis (DGGE). This technique was performed as in Muyzer et al., 1993. The second technique used to quantitatively enumerate samples was PCR fluorescent fragment detection (PCR-FFD). This was done by firstly doing PCR amplification, followed by detection of HEX-labeled PCR products on an ABI prism 310 genetic analyzer using genescan software (Conye et al., 2009). Positive PCR products were shown by retention time during electropheresis, and quantified by peak area (Conye et al., 2009).

The DGCE confirmed that the spread of P.piscicida in the mid-Atlantic estuaries. On top of the already known toxic strain, 5 other strains were also identified, which wasn’t an unexpected result. They also found that the use of PCR-FFD had a huge increase in sensitivity in comparison to microscope techniques, up to 1000 times more so.

The study by Conye et al., 2007 demonstrated the utility of PCR-FFD by doing a diel study in relation to other physical, chemical and biological factors. It indicates that P.piscicida is present in low levels in channels in highly turbid conditions and in areas of high water exchange, not just in surface waters. Overall they state P.piscicida as a minor but important part of the phytoplankton.

As a whole, I see this study as being a huge help to the identification of the dinoflagellates responsible for harmful algal blooms. This is also of huge importance due to the impacts that these algal blooms have, especially in such areas as human health and fish kills. It can then be linked to other aspects of algal blooms, in areas such as global warming and its effects on bloom severity and frequency. So the techniques used in this study have definite potential when looking at bloom forming dinoflagellates, and further research may be done that looks at particular areas within harmful algal blooms.

Coyne, K., Hutchins, D., Hare, C., Cary, S. (2001). Assessing temporal and spatial variability in Pfiesteria piscicida distributions using molecular probing techniques. Aquatic Microbial Ecology. 24, 275-285

Muyzer ,G., De Waal, E., Uitterlinden, A.. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.. Applied and Environmental Microbiology. 59, 695-700

Polyamory in Corals - An adaptive mechanism to environmental changes

Obligate mutualistic symbiosis occurs when the host must possess symbionts in order to survive. The most studied symbiosis of this type is the one between reef building corals and dinoflagellates, but how much do we actually know? What happens in the symbiosis between dinoflagellate and coral throughout the hosts ontogenesis is rarely studied yet important for a better understanding of the host-symbiont relationship.

There are two ways in how symbionts are transmitted from one generation to the next, both having positive and negative aspects for the host. In vertical transmission, the symbiont is directly transferred from the host parent to the offspring. Due to physiological differences between the symbionts clades (light and temperature sensitivity), which symbiont the offspring inherit may affect the hosts survivorship in different habitats. The diversity of symbiont inheritance may vary between and even within a single parent. Vertical transmission secures the offspring having symbionts, however, these may not be advantageous if the offspring lives in an environment that differs from that of their parent. In opposite, in horizontal symbiont transmission the offspring must acquire the symbiont form the environment which might be advantageous in a new environment but could also mean that beneficial symbionts gained in the last generation would have been lost.

In presence of global warming, neither vertical nor horizontal symbiont alone provides mechanism for the adaptation of an obligate symbiosis to the changing environment. This may be the reason for loss of whole ecosystems. Only combining both acquisition strategies may provide mechanisms for adaptation, Byler et al. (2013) therefore investigated whether or not both symbiont transmission modes could occur in an eukaryote invertebrate obligate symbiosis using the coral Stylophora pistillata and its symbiotic dinoflagellate Symbiodinium. For the purpose of this study, samples were collected from two depths, shallow and deep. The Symbiodinium genetic identity in S. pistillata adults, their released planulae and juvenile colonies were examined using technics such as DNA Extraction, amplification, denaturing-gradient gel electrophoresis and a real-time PCR to detect both abundant and possible low-level symbiont populations. 

As S. pistillata planulae contained only one Symbiodinium type while some of the juvenile colonies at shallow and deep depths harboured mixed symbioses, this study provides evidence for the possibility of both symbiont acquisition modes, horizontal and vertical. This is a great start point but further investigations are needed as there is no evidence that juveniles that acquired symbionts from the environment actually survive to adulthood. Recording the whole coral life-cycle would be a challenge, considering corals being such a slow growing organism. However, if it was possible that coral juveniles that show both vertical and horizontal symbiont transmission maintain advantageous symbionts into adulthood and later on inherited them to their offspring, novel Symbiodinium could be maintained over generations. Such adaptation to the environment as an evolutionary mechanism can potentially establish novel host symbiont combinations that may be advantageous during changing environmental conditions. Even though only 35% of coral species vertically transmit Symbiodinium, these species belong to several widely distributed dominant coral genera. The future of coral ecosystems may not be as depressing as we thought but in the end we are still talking about corals, sparsely sexual reproducing and painfully slow growing in a fast-paced world.

Byler K. A., Carmi-Veal M., Fine M. & Goulet T. L. (2013) Multiple Symbiont Acquisition Strategies as an Adaptive Mechanism in the Coral Stylophora pistillata. PLoS ONE 8(39): e59596. doi:10.1371/journal.pone.0059596

Different coral species share a small core bacterial community within highly diverse communities

The diverse and complex communities of coral-associated bacteria that form part of the coral holobiont play an important role in coral health, disease and the tolerance of abiotic stress. Currently there is much contradiction and inconsistency between studies regarding the specificity of coral-associated assemblages. Some studies report that bacterial communities are specific to the species of coral; others describe bacterial communities that were influenced more by location than coral species. Other studies report that both coral species and location are equally important. Clearly, further investigation is warranted to investigate the specificity of coral-associated bacterial assemblages globally.

This study by Li et al. (2013) investigated the bacterial communities associated with three dominant coral species in the South China Sea, with the aim of characterising bacteria communities that were specific to particular coral species and those that were common to all three coral species. The study looked at coral species Porites lutea, Galaxea fascicularis and Acropora millepora as these were dominant in the South China Sea. Coral reefs within the South China Sea occupy a similar area to the Great Barrier Reef and have both a comparable latitudinal range and biodiversity.

Samples of the three coral species along with seawater were collected from the Luhuitou fringing reef, China at a depth of 3-5 m using a punch and hammer. Samples were then filtered, DNA extracted and the 16S rRNA gene was targeted for PCR amplification. Quantified sequences were grouped into operational taxonomic units (OTUs) with a minimum of 97% similarity. The analysis of pyrosequencing libraries combined with barcoded PCR primers highlighted that bacterial assemblages associated with the three coral species were more diverse than previously thought and three new bacterial phyla were discovered. Bacterial communities associated with A. millepora differed from those of P. lutea and G. fascicular, which had more similar bacterial communities. In addition, all three coral community types differed from those found in seawater.

Between the three coral species there were only 22 97% OTUs that were shared and these were found in the following groups: Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Chloroflexi, Actinobacteria, Acidobacteria plus an uncategorized bacterial group. The proportions of these phyla differed between the three coral species. Additionally, the study detected potential nitrogen-fixing bacteria and bacteria involved in the degradation of DMSP and DMS. This has important implications in oligotrophic waters where many corals reside. There was also an abundance of Actinobacteria in coral samples compared to seawater. This phylum is known to produce a range of antibacterial compounds which may offer protection against pathogens.

Bacteria associated with the corals examined in this study were highly diverse and different from seawater bacterial communities, yet a small core bacterial community may be shared by different coral species. Coral morphology may have a role in microbial diversity as previous reports showed that mound-forming corals had higher coral-associated bacterial diversity than branch-forming corals such as A. millepora, as supported by this study. Furthermore, bacterial communities in the South China Sea associated with A. millepora and P. lutea were different from those found in the Great Barrier Reef and Indo-Pacific reefs. The authors report that these differences may be due to the different environmental conditions at separate locations rather than species-specific differences.

I feel that this paper has downplayed the implications of the study’s discovery in terms of the importance of characterising the bacterial communities associated with corals and that the overall message was slightly unclear when considering the arguments of other similar studies. This study has however helped support Rohwer’s theory that corals may harbour specific and conserved bacterial populations. This has implications for the health and stability of corals, particularly with the threat of declining coral reef ecosystems globally.

Reference:

Li, J., Chen, Q., Zhang, S., Huang, H., Yang, J. Tian, X.P. and Long, L.J. (2013) Highly heterogeneous bacterial communities associated with the South China Sea reef corals Porites lutea, Galaxea fascicularis and Acropora millepora, PLOS One, 8, (8), 1-8.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0071301

You need me, man, I don't need you; what causes a macro-algae to change its microbes?

The model system that was used in this paper was Phyllospora comosa and its microbial assemblages due to their disappearance off the coast of Sydney, Australia. Campbell et al. (2014) focussed on the effect that this spatial change can have on the microbial biofilms that can be key to the health and development of the macroalgae. Disruption to their normal habitat may have detrimental effects to allowing the macroalgae to thrive if the microbe communities cannot survive in different conditions. The decreasing number of this species was linked to sewage pollution in the metropolitan area of Sydney.

The authors discuss how functional redundancy can occur in large colonies with high species diversity. This is the process where the phylotypes in the biofilm can perform core functions that the host may need. This is particularly beneficial for the host because it doesn’t have to be picky with its microorganisms. Any microorganisms that can perform a certain function like, for example, nitrogen-fixing, can be utilised by the algae which means that disruption to the biofilm is not necessarily an issue. This is a far cry from specific host-bacterial symbioses which, if disturbed, can be detrimental to both parties. With massive amounts of ocean and coastal degradation more prevalent in the recent years than ever before such as warming oceans, lower pH and increasing pollution, the macroalgal populations can be severely devastated. The authors referred to a paper that showed evidence of variability within the biofilm on the surface of Ulva australis. However, the assemblage show conservation of functional gene profiles within these communities, among different samples. It was suggested that the green alga required its microbe association in providing key functions. This would mean that any disruption would have negative impacts on the macroalgae if those interactions weren’t re-established quickly.

Samples of two extant populations of P. comosa on the outer limits of Sydney (donor habitats) were transplanted into reef habitats that exhibited physical similarity closer to Sydney where the macroalgae wasn’t found (recipient habitats). Forty adults were collected randomly at the same depth (1-2m) from 28^th February to 9^th May, 2011. Individuals were randomly allocated to one of three treatments; transplanted individuals (moved to a recipient site from their original donor site), disturbed individuals (removed from donor site and treated the same as transplanted individuals but placed back in the same donor site) and translocated individuals (same treatment as the other two but placed in the alternate donor site). The controls were randomly selected individuals but were not handled or disturbed. After two months, a blade was taken from each sample and their surface biofilms were sampled. A second experiment was started on 9^th August, 2011. They altered it slightly by taking algae from both donor populations and transplanting them to all recipient sites to allow comparison of algae from different sources at the same place. DNA fingerprint analysis was done on bacterial assemblages and PCR amplified the 16S rRNA genes of each sample using the community DNA as a template. 

The bacterial TRFLP fingerprints were different in both structure and composition but these features differed in accordance with the origin of the sample in the first experiment.  In the second experiment, the TRFLP differed across all the treatments from the samples from both different donor populations. Although where these differences occurred could not be determined through pairwise comparisons. The results showed that the biofilms were more affected by local conditions than the type of habitat they occurred in. They suggested that simply moving the algae to a different habitat caused a change in their microbial communities. However they also found evidence for host-specificity where algae moved from one site to another did not change its assemblages to the same found in undisturbed individuals at the same site. They also report how communities on transplanted individuals that were placed in the same location but collected from different sample sites, still had different communities after five months (NB. The phrasing used in this sentence is difficult to understand so this is what I inferred from what had been written).

Overall, there is a lack of evidence concerning consistency in algae and their microbial communities with regards to its environment. The authors referenced a paper that showed consistent results regarding species-specificity in the context that their method was not sufficient to provide a clear enough result. The authors concluded that the system implemented by this algal species was a type of ‘competitive lottery model’ and that Phyllospora may just require a set of functions rather than species. That could be why individuals may keep their original microbial assemblages as they have the necessary functions accounted for. Individuals that may change their assemblages may be subject to microbial competition from the microbes present in the environment that the algae has been moved to. They theorised that the environment in Sydney may be causing the failure of regrowth of Phyllospora but no evidence has been found to suggest that this might be the case. With lowering rates of pollution in the area than before, they suggested that the algae might be able to recolonize. This is supported by another study referenced in this paper that Phyllospora is sensitive to high levels of nutrients brought about by the dumping of high concentrations of sewage in that area.

This paper is key to understanding the environmental impact human activity can have on the population levels of different species. By understanding the effects these activities have, safer alternatives can be implemented to prevent ecological impacts like this from occurring. There would even be methods to reverse such effects such as with the lowering sewage concentrations which might allow the algae to recolonize. The triggers that cause individuals from a population to alter their microorganisms is an interesting facet of not just macroalgal biology, but also provides a greater comprehension of symbioses between a host and its symbiont.

Ref: Campbell, A. H., Marzinelli, E. M., Gelber, J. & Steinberg, P. D. (2014) Spatial variability of microbial assemblages associated with a dominant habitat-forming seaweed. Front. Microbiol. (5) 737.

doi:10.3389/fmicb.2014.00737